The commonly used electron donor 2,6-dichlorophenolindophenol also serves as an efficient electron acceptor for Photosystem I

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Petrova, Georgy E. Milanovsky, Ilya A. Volkhin, Marina A. Kozuleva, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7917984/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Photosynthesis Research → Version 1 posted 9 You are reading this latest preprint version Abstract The sustainable functioning of the purified photosystem I (PSI) pigment-protein complex requires exogenous redox-mediators, facilitating the primary electron donor P 700 + reduction and terminal acceptor [F A /F B ] − oxidation. The redox couple ascorbate/2,6-dichlorophenolindophenol (Asc/DCPIP) was shown to be more efficient, than the couple Asc/ N,N,N',N' -tetramethyl- p -phenylenediamine (TMPD) both in photosynthetic studies and in biohybrid photovoltaic devices. We investigated the interactions of DCPIP with purified cyanobacterial PSI in the presence of Asc excess under laser flash excitation. Here we show that DCPIP, in contrast to TMPD, competes efficiently as an electron acceptor with the backward electron transfer from [F A /F B ] − to P 700 + even at micromolar concentrations, indicating accumulation of the oxidized DCPIP under aerobic conditions in the presence of Asc excess. The reduction of P 700 + includes contributions both from reduced DCPIP and semiquinone DCPIP •− . The rates of P 700 + reduction and [F A /F B ] − oxidation by DCPIP demonstrate complex pH-dependencies, related to changes in protonation state of the mediator and probably to redistribution of electron density between the terminal cofactors F A and F B . The rate constants of the electron transfer from Asc, DCPIP and TMPD to P 700 + and of the electron outflow from [F A /F B ] − to the oxidized forms of these compounds are estimated by kinetic modeling. The obtained data reveal thermodynamic, kinetic and electrostatic factors responsible for the high DCPIP efficiency as electron donor and acceptor for PSI. Photosystem I Electron transfer Midpoint redox potentials Artificial electron acceptor Redox-mediator Biohybrid photovoltaic device Kinetic modeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Photosystem I (PSI) is the transmembrane pigment-protein complex of photosynthetic electron transport chain of oxygenic phototrophs. PSI complexes of cyanobacteria Synechocystis sp. PCC 6803 are trimeric with each monomer comprising 11 subunits (Malavath et al., 2018 ). The homologous core subunits, PsaA and PsaB, bind most of the electron-transfer cofactors (Fig. 1 A). Three pairs of chlorophyll a molecules of the reaction center perform photochemical charge separation, producing oxidized chlorophyll a dimer P 700 + . Two phylloquinone molecules in the pseudo-symmetrical branches of redox-cofactors A and B (A 1A /A 1B ) and an intersubunit iron-sulfur 4Fe-4S cluster F X facilitate electron transfer to the acceptor side of PSI complex, which consists of two iron-sulfur 4Fe-4S clusters F A /F B bound to the external hydrophilic subunit PsaC. PSI represents an efficient photovoltaic unit, generating the lowest reduction potential (<–1 V vs NHE) in biological systems with a high quantum efficiency. These properties of PSI complexes make it promising for integration in biohybrid photovoltaic devices (Nguyen and Bruce, 2014 ; Teodor and Bruce, 2020 ). Isolated PSI complexes retain the ability to generate light-dependent electric potential. In the absence of the exogenous acceptors and donors, the final radical pair state P 700 + [F A /F B ] – decays within ~ 200 ms via backward electron transfer, followed by charge recombination (Vassiliev et al., 1997 ; Makita et al., 2015 ; Petrova et al., 2017 ). Therefore the rapid outflow of electrons from terminal [F A /F B ] – clusters and the fast reduction of P 700 + are essential factors contributing to photoefficiency in photovoltaic devices based on PSI (Goyal et al., 2022 ; Szewczyk et al., 2022 ). The highly efficient reduction of the native low-potential electron acceptor ferredoxin (Fd) is the main function of PSI in vivo (rate constant ~ 10 8 M − 1 s − 1 (Setif and Bottin, 1994 )). The reduced Fd in turn serves as an electron donor for NADP + and downstream metabolic reactions, such as Calvin-Benson cycle, nitrogen and sulfur assimilation. Besides Fd, in vivo the PSI complexes are capable of reducing oxidized forms of ascorbate (Asc) (Forti and Ehrenheim, 1993 ; Trubitsin et al., 2014 ) and molecular oxygen (Kozuleva et al., 2021 , 2014 ). Among artificial electron acceptors methyl viologen (MV) is the most widely used (Nguyen and Bruce, 2014 ). The rate constant ( k ) of the [F A /F B ] – oxidation by MV is considerably high (~ 10 7 M − 1 s − 1 ), while the k value of MV oxidation by O 2 is almost two orders of magnitude higher (8×10 8 M − 1 s − 1 ) (Farrington et al., 1973 ), which increases probability of the side reaction with O 2 in case of MV application in photovoltaic devices (Passantino et al., 2020 ). The fraction of PSI complexes in which an electron escaped to the exogenous acceptor requires P 700 + reduction by exogenous electron donor. In vivo it is provided by hydrophilic electron carriers plastocyanin (Pc) and cytochrome c 6 (in cyanobacteria) (Hippler and Drepper, 2006 ; Kovalenko et al., 2011 ). In vitro reduced forms of redox mediators, such as Asc, 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N' -tetramethyl- p -phenylenediamine (TMPD) are commonly used as electron donors for P 700 + . Asc in combination with DCPIP or TMPD is utilized in photovoltaic devices, as well as in studies of electron transfer in thylakoid membranes and in purified PSI complexes (Fig. 1 B). The reduction of P 700 + by millimolar concentrations of Asc occurs within tens of seconds (Mano et al., 2004 ). P 700 + reduction is enhanced by the addition of micromolar concentrations of DCPIP or TMPD with characteristic time up to tens – hundreds of milliseconds. The redox-mediators are maintained in a reduced state in the presence of excess of Asc. TMPD is shown to be a less efficient electron donor to PSI, than DCPIP (Gourovskaya et al., 1997 ; Petrova et al., 2018 ). Yet, even the latter is far from reaching the efficiency of natural electron donor Pc, which reduces P 700 + in the time range of tens of microseconds (Hervás et al., 1994 ; Mamedov et al., 2001 ). Asc and DCPIP are two-electron redox-mediators, while TMPD is capable of one-electron transition (Fig. 1 B). Oxidized forms of Asc, TMPD and DCPIP may act as electron acceptors for PSI (Izawa, 1980 ). Acceptor properties of the oxidized form of TMPD (TMPD •+ , Wurster’s blue) and oxidized DCPIP (DCPIP OX ) are widely used in the studies of redox enzymes (Jahn et al., 2020 ; Loktyushkin et al., 2021 ). The reactions of the redox-couples Asc/DCPIP or Asc/TMPD with the acceptor side of PSI have not been studied well enough yet. Asc, when used as the only redox mediator, is shown to be able to oxidize the acceptor side of PSI, yet it is still unknown which of two forms – monodehydroascorbate radical (Asc •− ) or dehydroascorbate (DHA) – serves as the main acceptor (Trubitsin et al., 2014 ). Dry TMPD retain its reduced state, but in solution under aerobic conditions it gradually oxidizes. This allowed to investigate acceptor properties of TMPD •+ in the absence of Asc, using TMPD as an exclusive donor. TMPD •+ was shown to function as an electron acceptor for PSI even under laser flash excitation conditions (Hiyama and Ke, 1971 ). However, upon addition of Asc excess this effect disappeared, apparently because TMPD had been converted to the reduced form (Hiyama and Ke, 1971 ). DCPIP, on the contrary, is initially oxidized, and Asc is required to maintain DCPIP mostly in the reduced form (DCPIP RED ). In the case of Asc/DCPIP redox couple, oxidized forms of both mediators may accept electrons from the acceptor side of PSI. Their equilibrium concentrations are still unknown, yet under continuous illumination, it was shown that DCPIP OX was present in sufficient concentration to compete for electrons with MV on the acceptor side of the PSI (Petrova et al., 2018 ). The product of this reaction – semiquinone form of DCPIP (DCPIP •− ) – is highly reactive: in the presence of molecular oxygen it is quickly oxidized, thereby mediating electron transfer between the acceptor side of PSI and O 2 molecule, likewise MV (Dvoranová et al., 2015 ; Marchanka and Gastel, 2012 ; Petrova et al., 2018 ). Under laser flash excitation conditions, it was shown that the amount of PSI complexes reduced by an exogenous electron donor increases with growing DCPIP concentration, yet, the concentration of oxidized forms of Asc increased simultaneously (Hiyama and Ke, 1971 ; Vassiliev et al., 1997 ). Thus the role of redox-mediator DCPIP in the oxidation of terminal cofactors of PSI in combination with Asc requires further investigations (Ciesielski et al., 2010 ; Goyal et al., 2022 ). DCPIP was shown to be a more efficient component of the PSI-based photovoltaic devices, than TMPD (Chen et al., 1992 ). This effect can be related to a better donor efficiency of DCPIP or to the enhancement of the outflow of electrons from PSI by DCPIP. The redox properties of DCPIP might be affected by the deoxygenation of the medium or by the change in pH since DCPIP molecule has two protonation sites with pK values in physiological pH range (Diebler, 1963 ; Tonomura et al., 1978 ). In this work, we used laser absorption spectroscopy to examine the efficiencies of the donor and acceptor reactions of the PSI complexes from cyanobacteria Synechocystis sp. PCC 6803 with DCPIP and TMPD in the presence of Asc excess. The effects of the O 2 depletion and pH change on the efficiency of the reactions were estimated. The results were used to develop kinetic model of electron transfer processes on the donor and acceptor sides of PSI in order to describe the observed reactions quantitatively. Material and methods Sample preparation Cells of cyanobacteria Synechocystis sp. PCC 6803 were grown in BG-11N medium at room temperature under fluorescent lights at ∼40 µmol of photon m − 2 s − 1 . Trimeric PSI particles were purified as described in (Johnson et al., 2000 ) with some changes. In brief, the thylakoid membranes were solubilized in the presence of 1% n-dodecyl-β-D-maltoside, solubilized material was fractionized via centrifugation on linear sucrose density gradients for 3 h at 140,000 g in a VTi50 vertical rotor (acquired as a part of Moscow State University Development Program). The lower green band containing the trimeric PSI complexes was collected, dialyzed, concentrated to 3–4 mg Chl/ml, and stored at − 80°C. Transient optical spectroscopy Flash-induced millisecond absorption changes were monitored at a wavelength of 820 nm in order to observe P 700 + signal decay in PSI samples. A frequency-doubled Quantel Nd:YAG laser (pulse half-width, 12 ns; flash intensity, 20 mJ) provided saturating actinic flashes at 532 nm, while a Spinder and Hoyer DC25A laser diode (wavelength, 820 nm) was used as a measuring light source. Measurements were performed in standard 1-cm optical path quartz cuvette. The assay medium contained PSI at chlorophyll concentration of 50 µg/ml, 50 mM HEPES-NaOH buffer, pH 7.5 (unless otherwise stated), 10 mM sodium ascorbate and varying concentrations of redox-mediators DCPIP or TMPD. The samples before measurements were incubated in the dark for 10 min. The final kinetic curves were obtained by averaging 64 sample curves. Laser flashes were given with the interval of 10 s to 2 min, depending on the concentration of the redox-mediator. When the measurements were performed under anaerobic conditions, the additions of 10 mM glucose, glucose oxidase (100 U ml − 1 ) and catalase (100 U ml − 1 ) were used. Before measurements the samples were incubated for 10 minutes in the dark under nitrogen flow. During the measurement, the cuvette remained under a transparent polycarbonate cap in a nitrogen environment. The kinetics did not change, which indicated that anaerobic conditions were maintained at the measurement course. In the case of the pH-dependence measurements, a mixture of MES (5 mM pH 5.6), HEPES-NaOH (5 mM pH 7.5) and Tris-HCl (5 mM pH 8.2) buffers was used in order to avoid effects related to the change of buffer (Morlock et al., 2022 ). The pH was adjusted with 1 N NaOH to the values of 5.6, 6.4, 7, 7.5, 8.2 and 8.6. Asc at 10 mM and DCPIP at 15 µM concentration were added to the sample before the measurements. Data analysis The obtained kinetic was analyzed using laboratory-developed scripts for Matlab R2023a [MATLAB: 9.14.0, The MathWorks Inc., Natick, Massachusetts (2023). Available at: https://www.mathworks.com/ ]. Each kinetic curve was approximated by a sum of exponential functions and non-decaying residual by using nonlinear minimization. In most cases a good approximation required only two exponents, which corresponded to the P 700 + reduction by i) the backward electron transfer (lifetime τ 1 and amplitude A 1 ) and ii) exogenous electron donors (lifetime τ 2 and amplitude A 2 ). If the approximation of the backward electron transfer kinetics required more than one exponential component, their total amplitude and weighted average lifetime was calculated. Kinetic modeling Electron transfer reactions after the flash-induced charge separation and electron transfer to the terminal iron-sulfur clusters (resulting in formation of the P 700 + [F A /F B ] – state) involve recombination within the PSI complex and its interaction with exogenous redox-mediators. These reactions can be described by the kinetic scheme depicted in the Fig. 2 . Electron from the terminal 4Fe–4S clusters [F A /F B ] – can either undergo the recombination to P 700 + with a reaction rate constant k r or escape to exogenous electron acceptors in the medium – to Asc in the oxidized state (at the constant concentration of 10 mM) with the rate constant k a0 or to the oxidized DCPIP/TMPD (at variable concentration) with the rate constant k a . Similarly, P 700 + can be reduced through one of three reactions – backwards electron transfer from [F A /F B ] – , electron donation from either Asc in the reduced state with the rate constant k d0 or from the reduced DCPIP/TMPD with the rate constant k d0 . Redox transitions of the kinetic system in Fig. 2 can be described with a set of linear differential equations. Solving these equations (see Supplementary information), the kinetics of P 700 + decay can be represented by a sum of two exponential functions with the amplitudes A 1 and A 2 and the characteristic times τ 1 and τ 2 . These parameters depend on concentrations of electron donors (the constant concentration donor d 0 and the variable concentration donor d ) and acceptors (the constant concentration acceptor a 0 and the variable concentration acceptor a ): [ P 700 + ] = A 1 •exp(-τ 1 /t) + A 2 •exp(-τ 2 /t) (1) A 1 = k r /(k r +k a •a + k a0 •a 0 ) (2) A 2 = (k a •a + k a0 •a 0 )/(k r +k a •a + k a0 •a 0 ) (3) 1/τ 1 = k d •d + k d0 •d 0 + k a •a + k a0 •a 0 + k r (4) 1/τ 2 = k d •d + k d0 •d 0 (5) The obtained representation of P 700 + kinetics as a sum of two exponential functions mirrors the bi-exponential deconvolution described above. Using the obtained apparent lifetimes τ 1 and τ 2 , and amplitudes A 1 and A 2 at different concentrations of the exogenous redox mediator (DCPIP or TMPD), the reaction rate constants k d0 , k d , k a0 , k a and k r were found using equations (1–5) as a fitting functions of the redox mediators concentrations d , d 0 , a and a 0 . Results The redox-reactions on the donor and acceptor sides of PSI with redox-mediators Laser flash-induced P 700 + photo-oxidation reveals a broad absorption increase in the near-infrared spectral region centered at 820 nm. The signal decays due to the fast backward electron transfer within PSI from the terminal [F A /F B ] – clusters to P 700 + or due to the P 700 + reduction by exogenous electron donors. The rate of the latter component depends on concentration of the exogenous donor, while its amplitude depends on the efficiency of exogenous acceptors (Vassiliev et al., 1997 ; Petrova et al., 2017 ; Milanovsky et al., 2017 ). Figure 3 – 4 represent kinetics of the P 700 + reduction in the presence of 0.5–50 µM of DCPIP or TMPD under aerobic or anaerobic conditions at the constant Asc concentration of 10 mM. Biexponential approximations of the kinetics are shown by circles. The dependencies of lifetimes ( τ 1 and τ 2 ) and amplitudes ( A 1 and A 2 ) of two exponential components on the redox-mediators concentrations (dots) and their approximation by the kinetic model (lines) are plotted in panels B and C, correspondingly. The parameters of biexponential decompositions are summarized in the Tables S1.1–S1.4. At the lowest DCPIP concentration (red curve in Fig. 3 A) τ 1 was defined to be ~ 70 ms, the complete P 700 + reduction by the redox-mediator took more than > 2•10 3 ms (the 7.6•10 3 ms lifetime was approximately estimated by the exponential decomposition). The contribution of the faster component A 1 accounted for ~ 80% of the decay, indicating that under these conditions P 700 + was reduced preferentially via the backward electron transfer pathway. With increasing DCPIP concentration the slower phase underwent gradual acceleration up to τ 2 = 170 ms at 50 µM (close circles in Fig. 3 B). The P 700 + reduction by DCPIP accelerated by a factor of ~ 45 in the studied concentration range. At the same time, the τ 1 value remained unchanged at the DCPIP concentrations of 0.5–15 µM, decreasing to 24 ms at the highest DCPIP concentration (open circles in Fig. 3 B). The observed decrease indicates that the apparent τ 1 is a complex parameter, which depends not only on the backward electron transfer rate constant, but also on the reaction rate of PSI interaction with exogenous donors and acceptors. Simultaneously, the contribution of the slower component A 2 increased, accounting for ~ 60% of the decay at 50 µM DCPIP (closed circles in Fig. 3 C). This reveals predominance of the exogenous electron acceptor reduction by [F A /F B ] – over the backward electron transfer. Both DCPIP and Asc in oxidized states might accept electron from PSI under experimental conditions (Trubitsin et al., 2014 ; Petrova et al., 2018 ). In order to estimate the Asc contribution, we monitored changes in the P 700 + reduction kinetics upon TMPD concentration increase in the presence of 10 mM Asc (Fig. 4 A). The slower component of the biexponential fit accelerated from τ 2 = 6•10 3 ms at 0.5 µM TMPD to 5.4•10 2 ms at 50 µM TMPD, while τ 1 remained unchanged. The slower kinetic component accelerated by a factor of ~ 10 in the investigated concentration range of TMPD, and the minimal τ 2 value was 3 times higher in the case of TMPD compared to DCPIP indicating a lower donor efficiency of TMPD. The relative contributions of the two kinetic components remained stable in the investigated TMPD concentration range (Fig. 4 C). Some increase in the slower component contribution was observed only at 50 µM TMPD. From this observation we may conclude that neither TMPD nor Asc under aerobic conditions can efficiently accept electrons from PSI acceptor side in the investigated concentration range. The contribution of the slower kinetic component increased only upon addition of DCPIP, indicating the specificity of the effect for DCPIP. The effects of the anaerobic conditions on the PSI interactions with redox-mediators As described in “Introduction”, the oxidation of DCPIP •− by molecular oxygen is highly favorable, which provides regeneration of DCPIP OX and enhances the electron outflow from PSI under aerobic conditions. We estimated the contribution of the O 2 -dependent DCPIP OX effect on the kinetics of Р 700 + reduction by monitoring the DCPIP concentration dependency of A 2 contribution upon O 2 depletion (Fig. 3 D–F, Table S1 .3). Indeed, under anaerobic conditions the relative amplitude A 2 increased to a less extent, than under aerobic conditions: the highest A 2 value did not exceed 50% (compare panels C and F in Fig. 3 ). In the case of TMPD, anaerobic conditions did not affect A 2 , which remained stable upon the increase in the mediator concentration (Fig. 4 D–F). This once again confirms the assumption that TMPD •+ under conditions of experiment did not accept electron from the acceptor side of PSI complex, while DCPIP OX did. Moreover, the efficiency of DCPIP OX reduction depends significantly on the rate of the DCPIP •− reoxidation, which is much higher under aerobic conditions. Surprisingly, in the case of DCPIP, anaerobic conditions also noticeably affected τ 2 values (see Tables S1.1 and S1.3), which were on average ~ 2 times slower under anaerobic conditions, than in the presence of O 2 in the concentration range studied. One may conclude that regeneration of the DCPIP form capable of reducing P 700 + was suppressed under anaerobic conditions too. The pH effects on the PSI interaction with redox-mediators The pH-dependence of the P 700 + decay obtained on the sample containing 10 mM Asc and 15 µM DCPIP shows the decrease in the τ 2 and simultaneous increase in the A 2 value at high pH (Fig. 5, Table 1 ). Figure 5 Р 700 + signal decay kinetics changes upon pH change in the presence of 15 µM of DCPIP and 10 mM Asc: transient absorbance traces at 820 nm (solid lines), the result of biexponential fitting (circles) and the plot of the slower exponential component ( A ); the pH-dependence of the contributions ( B ) of the exponential components (circles). Lines in panel B show the approximation of the experimental data with Nernst equation The value of τ 1 remained stable except for a slight decrease at a high pH which most probably was caused by a complex nature of the parameter, see “Kinetic modeling” section for the details. The inflection point in the pH-dependence of the τ 2 value was observed around pH 7, which coincides with the pK of DCPIP RED . The higher donor capacity of deprotonated DCPIP RED could be explained by the electrostatic attraction between negatively charged mediator and P 700 + . At the same time, the observed increase in the A 2 value upon the alkalization of the medium indicates growing outflow of electron to the exogenous redox-mediator (Fig. 5B). This could not be explained by the growing electrostatic attraction of the [F A /F B ] − and the electron acceptor since DCPIP OX is also charged negatively. The fitting of the A 2 (pH) curve by Nernst equation revealed the pK value of 7.54, which is significantly higher than pK = 6 of DCPIP OX (Gibbs et al., 1925 ; Tonomura et al., 1978 ; Loktyushkin et al., 2021 ). Besides the protonation state of DCPIP, the efficiency of the PSI interaction with DCPIP depends on the other factors, such as protonation state of the amino acids of the protein. We will consider some possible explanations in the Discussion. Table 1 Characteristic times and amplitudes of exponential components corresponding to charge recombination and reduction of P 700 + by the external electron donor at 15 µM DCPIP at different pH levels pH characteristic time, ms amplitude, % τ 1 τ 2 A 1 A 2 5.6 63 > 4000 68 32 6.4 97 > 4000 63 37 7 56 2300 51 49 7.5 54 1670 42 58 8.2 42 1060 35 65 8.6 34 960 35 65 Kinetic modeling The results of the multiexponential decomposition were used to obtain a numerical solution of the kinetic model describing electron transfer processes depicted at Fig. 2 . Within the model, both donor and acceptor sides of PSI interact with two kinds of the electron donors/acceptors with constant or variable concentrations (having different reaction rate constants). Analytical solution of the model, described above by equations (1–5), corresponds to the experimentally observed concentration dependence of PSI transient absorption kinetics: the exponential components amplitudes A 1 and A 2 depend on the rate constants of the electron transfer to the exogenous acceptor ( k a , k a0 ) and backward electron transfer ( k r ) reactions; the τ 1 value depends not only on the rate constant of the backward electron transfer ( k r ), but is also affected by the rate constants of the reactions on the donor ( k d , k d0 ) and acceptor ( k a , k a0 ) sides of the PSI complex; the τ 2 value depends on the rate constants of the donor reactions ( k d , k d0 ). Each experimental series differing by redox mediator (DCPIP/TMPD) and oxygen presence (aerobic/anaerobic) was fitted independently except of the k r parameter, which was set as 8.7 s − 1 . This value corresponds to the charge recombination of P 700 + [F A /F B ] − ion-radical pair with characteristic time 115 ms, matching the literature values (Vassiliev et al., 1997 ; Makita and Hastings, 2015 ; Milanovsky et al., 2017 ). The fitting of the apparent lifetimes and amplitudes of P 700 + decay with the model is shown in Fig. 3 – 4 (panels B, C, E and F) by solid lines. The rate constants of the reactions normalized to the total concentration of the redox mediators (Asc, DCPIP and TMPD) are presented in Table 2 . Table 2 Apparent rate constants of PSI interaction with exogenous redox-mediators, according to the fitting of experimental data, in aerobic and anaerobic conditions. See Fig. 2 for the legend k d , M − 1 s − 1 k a , M − 1 s − 1 k d0 , M − 1 s − 1 k a0 , M − 1 s − 1 DCPIP +O 2 1.3•10 5 2.1•10 5 8.6 270 −O 2 5.9•10 4 10 5 20 220 TMPD +O 2 4.1•10 4 3.7•10 4 15 360 −O 2 2.2•10 4 4.7•10 4 21 270 According to the Table 2 , DCPIP is ~ 3 times more efficient as an electron donor and ~ 6 times more efficient as an electron acceptor, than TMPD. Both k d and k a of DCPIP decrease by a factor of 2 upon O 2 depletion, pointing to the complex nature of both reactions. The k a value of TMPD was not sensitive to the O 2 presence, while its donor activity noticeably decreased under anaerobic conditions. We do not expect significant decrease in the TMPD RED concentration under anaerobic conditions. It is more likely that the observed decrease in k d (TMPD) is an artifact, related to the low donor reaction rate under conditions of the experiment (see Discussion for the details). The acceptor efficiency of Asc is by an order of magnitude higher than its donor activity according to Table 2 (compare the value of k a0 and k d0 ). This is consistent with the data showing that direct P 700 + reduction by Asc is insignificant compared to its acceptor activity in vivo (Tóth et al., 2009 ). The values of k a0 and k d0 were relatively stable in all experiment series except for the measurements in the presence of DCPIP under aerobic conditions, where k d0 was about twofold higher. This might indicate the significant increase in the concentration of the electron donating form of Asc under these conditions. Discussion Redox-mediators, such as Asc, DCPIP or TMPD, are commonly used in the electron transfer studies of purified PSI, PSII complexes and bacterial reaction centers. Among these mediators, DCPIP was proven to be remarkably more efficient component of the PSI-based photovoltaic devices (Nguyen and Bruce, 2014 ). The redox chemistry of both Asc and DCPIP, which comprise a redox-couple, is quite complex, which creates unavoidable side-reactions, such as reduction of DCPIP OX by terminal [F A /F B ] – clusters and the subsequent O 2 -dependent reoxidation of DCPIP •− , which has been detected under continuous illumination (Petrova et al., 2018 ). In the present work we investigated DCPIP interaction with the donor and acceptor sides of PSI complexes under conditions of flash-induced electron transfer in the presence of Asc excess. Comparing the efficiencies of DCPIP and TMPD interaction with PSI on the acceptor side under aerobic and anaerobic conditions, we demonstrated that it is DCPIP OX , and not Asc, which is the main exogenic electron acceptor for PSI. This fact is noteworthy since in previous works, the slow DCPIP-mediated P 700 + reduction was attributed to the account of the fraction of PSI complexes reducing directly O 2 (Vassiliev et al., 1998 ; Petrova et al., 2017 ; Milanovsky et al., 2017 ). The acceptor efficiency of the mediators in the present work was studied by monitoring how an increase in DCPIP/TMPD concentration affected the relative fraction of PSI complexes in which electron escaped to an external acceptor. We discovered that under laser flash excitation, DCPIP, unlike Asc and TMPD, is capable of accepting electron from PSI at a total concentration of 5 µM, noticeably lessening the contribution of charge recombination. Kinetic model gave an apparent k a for this reaction of 2.9•10 5 M − 1 s − 1 , which is almost an order of magnitude higher than the apparent k a (TMPD). Table 3 The values of the midpoint redox-potentials (E m ) of the natural and artificial electron donors and acceptors for PSI Redox couple E m , mV MV 2+ /MV + −456 (Orgill et al., 2015 ) Asc •− /DA −200 (Sapper et al., 1982 ) DCPIP OX /DCPIP •− −(155–190) (Petrova et al., 2018 ) О 2 /О 2 •− −155 (Asada and Nakano, 1978 ) AscН − /DA +(60–100) (Trubitsin et al., 2014 ) DCPIP OX /DCPIP RED +(217–290) (Gibbs et al., 1925 ; Dvoranová et al., 2015 ) TMPD •+ /TMPD + 260 (Dutton, 1978 ) AscН − /Asc •− + 320 (Sapper et al., 1982 ) DCPIP •− /DCPIP RED +(415–450) (Petrova et al., 2018 ) However, redox mediators DCPIP and TMPD can serve as electron acceptors to PSI only in their oxidized states, which amount to a small fraction of the total redox mediator concentration (< 1% in the presence of excess Asc (Passantino et al., 2020 )). Assuming redox potential of the medium, maintained by Asc, as +(60–100) mV (Trubitsin et al., 2014 ), we can calculate the relative fraction of these forms according to the Nernst equation. Various estimates of midpoint potential E m (DCPIP OX /DCPIP RED ) put it between + 217 and + 290 mV, and the value for E m (TMPD OX /TMPD RED ) is + 260 mV (see Table 3 ). Thus, the fraction of both DCPIP OX and TMPD OX can be estimated as not exceeding 1%. In order to have an opportunity to compare the donor and acceptor efficiencies more correctly, we adjusted apparent k a values from Table 2 for the concentrations of the DCPIP OX and TMPD OX . The adjusted estimates are provided in Table 4 . The value of k a (TMPD) = 3.6•10 6 M − 1 s − 1 is similar to the one, obtained earlier in the experiments, where TMPD acceptor function was investigated in the absence of Asc (Hiyama and Ke, 1971 ). The apparent rate constants of the donor reactions, k d , were elucidated from the lifetimes of the slow sub-second kinetic phase of P 700 + reduction using the kinetic model. Under aerobic conditions the estimated values of k d for DCPIP and TMPD as electron donors were 1.3•10 5 and 4.1•10 4 M − 1 s − 1 , respectively, which were remarkably close to those reported in the literature for the steady-state conditions ( k d (DCPIP) = 1.9•10 5 M − 1 s − 1 and k d (TMPD) = 3•10 4 M − 1 s − 1 ) (Fujii et al., 1990 ). Table 4 Estimated rates constants of DCPIP and TMPD redox forms interactions with PSI as donor ( k d ) or acceptor ( k a ) k d , M − 1 s − 1 k a , M − 1 s − 1 DCPIP +O 2 > 7.2•10 6* , 5.9•10 4** > 2.9•10 7 −O 2 5.9•10 4** > 10 7 TMPD +O 2 4.1•10 4 > 3.6•10 6 −O 2 2.2•10 4 > 4.5•10 6 * DCPIP •− ** DCPIP RED TMPD efficiency as electron donor decreased approximately two-fold upon oxygen removal from the reaction medium, as seen from Table 2 . This is unexpected as TMPD should not react with molecular oxygen under experimental conditions and, hence, no changes in its redox properties were intended. It should be noted that the estimated value of k d (TMPD –O2 ) is the lowest of all values in Table 3 , and, according to this estimate, only at the highest concentration of TMPD (50 µM) the reaction rate of its electron donation to P 700 + is comparable to the same reaction with Asc (in a much higher concentration of 10 mM). As seen from Fig. 3 – 4 and Eq. (5), the τ 2 dependence on the redox-mediator concentration has a hyperbolic form. In the case of TMPD in contrast to DCPIP we observe the far-left side of this hyperbola, with a very slight dependence on the TMPD concentration. Hence, we expect the accuracy of k d (TMPD –O2 ) estimation to be very low, and the observed difference between TMPD donor activity under aerobic and anaerobic conditions seems unreliable. At the same time the observed apparent k d (DCPIP) decrease from 1.3•10 5 M − 1 s − 1 to 5.9•10 4 M − 1 s − 1 upon O 2 depletion most probably indicates the decrease in the concentration of the P 700 + -reducing form under anaerobic conditions, since the change in conditions induced the shift from the linear to the hyperbolic section of the concentration dependency. This affirms DCPIP •− contribution to the donor reaction. In this case the apparent k d = 1.3•10 5 M − 1 s − 1 value may be considered as a weighted average of true k d values for DCPIP RED (~ 5.9•10 4 M − 1 s − 1 ) and DCPIP •− . The relative concentration of DCPIP •− can be assumed to be equal or less than that for DCPIP OX , 7.2•10 6 M − 1 s − 1 (Table 4 ). This value is several orders of magnitude higher compared to TMPD (Table 4 ), which is consistent with the earlier reported data showing lower efficiency of TMPD as an electron donor (Gourovskaya et al., 1997 ; Petrova et al., 2018 ). Under anaerobic conditions only DCPIP RED functions as an electron donor, showing k d closer to k d (TMPD). The values presented in Table 4 imply that under aerobic conditions DCPIP operates as an efficient electron donor due to the contribution of DCPIP •− . It might be produced from DCPIP RED as a result of its oxidation by molecular oxygen. DCPIP •− is a highly reactive species due to the negative E m of the DCPIP •− /DCPIP OX redox couple. The estimated E m value for DCPIP •− /DCPIP OX transition is − 155/–190 mV, while DCPIP RED /DCPIP •− has a much higher value of + 415/+450 mV (Table 3 ) being almost equipotential to P 700 /P 700 + . Free energy (ΔG) of the P 700 + reduction by DCPIP •− is about − 600/–650 meV, making this reaction highly favorable (Fig. 6 ). Another implicit factor, which might contribute to the decrease in the DCPIP •− concentration under anaerobic conditions, is retardation of the DCPIP OX reduction. Due to the negative E m of DCPIP OX /DCPIP •− only the reduction of DCPIP OX by Asc •− is thermodynamically favorable, which has ΔG = − 50 meV (Fig. 6 ) and the high rate constant of 9.5•10 5 M − 1 s − 1 (Iyanagi et al., 1985 ; Marchanka and Gastel, 2012 ). Yet, under anaerobic conditions AscН − /Asc •− transition is arrested causing rapid decrease in the Asc •− concentration (Trubitsin et al., 2014 ). Besides the ΔG value and concentrations of the reduced forms, the efficiency of P 700 + reduction depends on the electrostatic interactions with the electron donor, which could be modulated by the pH of the medium. According to our observations, the donor efficiency of DCPIP increases above pH 7 (Table 1 ), which coincides with the pK value of the DCPIP RED (Tonomura et al., 1978 ), while the pK of DCPIP •− is unknown. Yet, both deprotonated DCPIP RED and DCPIP •− are charged negatively, which should enhance P 700 + reduction, while TMPD RED is neutral, making its interaction with P 700 + less favorable. The higher rate constant of P 700 + reduction by DCPIP vs TMPD is thermodynamically explicable, while higher acceptor efficiency of DCPIP OX compared to TMPD •+ could not be easily explained. ΔG of the TMPD •+ reduction by [F A /F B ] – is − 450 meV higher, than for the analogous reaction with DCPIP OX . Electrostatic attraction of the positively charged TMPD •+ to [F A /F B ] – is also advantageous for the interaction. Yet, the oxidation of TMPD RED by O 2 has high positive ΔG (+ 415 meV) slowing down its oxidation under aerobic conditions. TMPD •+ reduction by Asc is thermodynamically favorable process, since it is characterized by ΔG of − 370 meV. A completely different situation is observed in the presence of DCPIP, since the fast reoxidation of DCPIP •− and much less negative ΔG of the reaction of DCPIP OX with Asc •− most probably promote DCPIP OX accumulation in the medium. This is not in line with the previously supposed lack of the DCPIP OX in the presence of Asc excess (Szewczyk et al., 2020 ). The acceptor efficiency of DCPIP OX , as well as its interaction with the donor side of the complex exhibited pH-dependency. The pK value of the acceptor reaction observed in the experiment was 7.5 (Fig. 5B), while the pK value of for DCPIP OX is 5.7. Therefore, the observed pH dependence could not be explained by a more efficient interaction of the acceptor side of PSI with the deprotonated form of DCPIP OX . Yet, this value corresponds nicely to the earlier reported data. First of all, the pH-dependence of the O 2 uptake rate in suspension, containing PSI, Asc and DCPIP, had maximum at pH 7.8 under steady-state conditions (Petrova et al., 2018 ). The highest photocurrent density in some PSI-DCPIP based photovoltaic device was achieved at similar pH values of the medium (Passantino et al., 2020 ). According to the literature, the pK value of DCPIP can change significantly upon immobilization in an acetate cellulose film covering a carbon electrode, reaching 7.4 (Florou et al., 2000 ). In the case of the donor reaction, the pK of DCPIP RED and the pK value observed in the experiment are in a good agreement, indicating that DCPIP RED bound on the donor side of PSI apparently does not exhibit the pK shift. Yet, it cannot be ruled out that the pK value of DCPIP upon interaction with PSI may differ from those in solution. On the other hand, an increase in pH may affect the protonation state and charge of the surface amino acids of PSI. Deprotonation of the amino acid residues, leading to the appearance of additional surface negative charges, should, on the contrary, hinder the binding of the DCPIP OX molecule to the PsaC subunit. However, appearance of additional negative charges upon the pH increase also affects the E m values of the F A and F B iron-sulfur clusters. The electrostatic interactions in PSI can be taken into account by using operating redox potentials values (Ptushenko et al., 2008 ). Comparison of the operating E m of F A and F B (–479 mV and − 539 mV, respectively) shows that this difference is mostly due to the effect of the charged amino acid groups of the PsaC subunit. Their electrostatic contributions are + 98 mV for the F A cluster proximal to the PSI core and + 35 mV for the distal F B cluster, which results in final electron density shifting towards the F A cluster. If we assume that this asymmetry decreases upon increasing pH above pK = 7.5, the iron-sulfur clusters F A and F B may become almost equipotential at alkaline pH. Compared to the neutral pH conditions, this will lead to the redistribution of the electron density in favor of the distal F B cluster, decreasing the effective distance of electron transfer to the exogenous acceptor, which can significantly accelerate this reaction. The hypothesis of electron density redistribution between the iron-sulfur clusters F A and F B upon pH changes explains not only the enhancement of the acceptor properties of DCPIP with increasing pH reported previously (Petrova et al., 2018 ) and observed in the present work, but also overcomes the contradiction between the kinetic data showing that at pH 7.8–8.3 the electron density is shifted towards the iron-sulfur cluster F B (Fujii et al., 1990 ; Vassiliev et al., 1998 ; Mamedov et al., 1998 ), and the fact that the redox potential of the iron-sulfur cluster F B is lower than that of F A . The results presented here are in line with the previously published data, including the lower performance of the photovoltaic devices using Asc-TMPD redox couple in comparison with the other redox-mediators (Chen et al., 1992 ). That is why TMPD, unlike DCPIP, is rarely used in PSI based photovoltaic cells. We propose that it is related to the ability of DCPIP to mediate both reduction of P 700 + and outflow of electrons from F A /F B – in the presence of Asc, decreasing backward electron transfer in the complex. This explains the appearance of a large-amplitude response in PSI-based photovoltaic cells containing 50 µM DCPIP and 10 mM Asc (Zaspa et al., 2022 ). The use of the natural electron donor cytochrome c 6 and the efficient artificial acceptor MV instead of DCPIP made it possible to increase the duration of light-dependent generation of electric potential (Zaspa et al., 2022 ). The rate constant of electron transfer from [F A /F B ] – to MV is 10 7 M − 1 s − 1 (Hiyama and Ke, 1971 ), which is comparable to the value of k a (DCPIP), presented in Table 4 . This is in line with the data obtained previously under continuous illumination, where DCPIP OX efficiently competed for electrons from PSI with MV (Petrova et al., 2018 ). The rate constant of P 700 + reduction by cytochrome c 6 (10 7 M − 1 s − 1 (Hervás et al., 1994 )) is comparable to the k d (DCPIP •− ) and two orders of magnitude higher than k d (DCPIP RED ). Yet unstable DCPIP •− could not be accumulated in sufficient concentration to provide efficient P 700 + reduction at micromolar total DCPIP concentration. That is why millimolar concentrations of redox mediators were required to remove the limitation of the overall electron transfer rate on the donor side of PSI when using MV. Conclusions Thus, concluding, DCPIP as electron acceptor is capable of competing with backward electron transfer in PSI complexes. Accepting electrons from [F A /F B ] – , it mediates O 2 reduction by PSI even under single flash excitation conditions. This reaction maintains DCPIP OX concentration high enough to provide leading role of DCPIP in electron outflow from PSI even in the presence of Asc excess. High donor capacity of DCPIP is ensured by DCPIP •− /DCPIP OX transition due to a high negative ΔG of the reaction with P 700 + . TMPD, being one-electron mediator, in the presence of Asc is not accumulated in oxidized state and cannot provide an efficient oxidation of the terminal PSI cofactors. The efficiency of the P 700 + reduction depends on the electrostatic attraction with negatively charged deprotonated reduced DCPIP form. The efficiency of the electron transfer from PSI to external acceptors appeared to be sensitive to the distribution of the electron density between iron-sulfur clusters F A and F B , which could be modulated by pH. Abbreviations Photosystem I (PSI), Special pair of chlorophyll molecules (P 700 ), Ferredoxin (Fd), Ascorbate (Asc), Methyl viologen (MV), Molecular oxygen (O 2­ ), Plastocyanin (Pc), 2,6-dichlorophenolindophenol (DCPIP), N,N,N',N' -tetramethyl- p -phenylenediamine (TMPD), Monodehydroascorbate radical (Asc •− ), Dehydroascorbate (DHA), Midpoint potential (E m ), Free energy (ΔG) Declarations Ethics and consent to participate Not applicable. Competing interests The authors declare no competing interests. Funding This research was supported by the Russian Science Foundation Grant 23-74-00025 Author Contribution A.P., M.K. and A.S. developed experiment design and wrote the manuscript; A.P. and I.V. conducted experiments; G.M., D.Ch. and A.P. analyzed the data and performed kinetic modeling. All authors reviewed the manuscript. Acknowledgement The authors wish to thank Andrey A. Zaspa and Arseny V. Aybush for the technical support and Mahir D. Mamedov for valuable discussions. 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Supplementary Files 2025PetrovaSI.docx Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Photosynthesis Research → Version 1 posted Editorial decision: Revision requested 24 Nov, 2025 Reviews received at journal 17 Nov, 2025 Reviews received at journal 01 Nov, 2025 Reviewers agreed at journal 29 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 24 Oct, 2025 Submission checks completed at journal 23 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Petrova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYDCCA1CajyGxgeEDkMHGTqwWNqAWxhkgBjPxWhIYmHlALEJa+G4ffvbg55478mzsyY2fbX5tk+djZmD88DEHtxbJc2nmhj3Pnhm28Txsls7tu23YxszALDlzG24tBmcYzCR4DhxmbJNIbJDO7bnNCNTCxsyLVwv7N8k/Bw7bA7U0/7bsuW1PhBYeM2mgLYlALW3SDD9uJxLUInmGp9xY5sDhZKBf2ix7G24ntzEzNuP1C98Z9m0P3xw4bNvPnv74xo8/t23ntzcf/PARjxYGUIzAAWMbmGzAqx5VC8MfQopHwSgYBaNgJAIAxUtUAeaVQuQAAAAASUVORK5CYII=","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":true,"prefix":"","firstName":"Anastasia","middleName":"A.","lastName":"Petrova","suffix":""},{"id":538411649,"identity":"b18ed3ec-ac66-48a2-8145-90385e69b6e0","order_by":1,"name":"Georgy E. Milanovsky","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Georgy","middleName":"E.","lastName":"Milanovsky","suffix":""},{"id":538411650,"identity":"f1a3b0e9-054d-4c93-923f-2cd25149cb55","order_by":2,"name":"Ilya A. Volkhin","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Ilya","middleName":"A.","lastName":"Volkhin","suffix":""},{"id":538411651,"identity":"64177349-6416-4964-ac04-19ca1457b7be","order_by":3,"name":"Marina A. Kozuleva","email":"","orcid":"","institution":"Institute of Basic Biological Problems of the Russian Academy of Sciences, Pushchino Scientific Center for biological Research of the Russian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"A.","lastName":"Kozuleva","suffix":""},{"id":538411652,"identity":"f5ca06ae-473b-432a-b366-95619bec7d48","order_by":4,"name":"Dmitry A. Cherepanov","email":"","orcid":"","institution":"Russian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dmitry","middleName":"A.","lastName":"Cherepanov","suffix":""},{"id":538411653,"identity":"1643bfd6-03b5-47e1-a974-618e41f06620","order_by":5,"name":"Alexey Yu. Semenov","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Alexey","middleName":"Yu.","lastName":"Semenov","suffix":""}],"badges":[],"createdAt":"2025-10-22 08:13:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7917984/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7917984/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11120-025-01190-1","type":"published","date":"2025-12-18T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95291133,"identity":"70d44836-d671-4dba-a066-366aa12bf289","added_by":"auto","created_at":"2025-11-06 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11:01:56","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177844,"visible":true,"origin":"","legend":"","description":"","filename":"d65baf5562fc491eb0985d6b1b46b1c21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/6721b155f3bd67fe9159aca5.xml"},{"id":95314287,"identity":"3c4ec364-0d50-4208-a933-340f8f6a9cb2","added_by":"auto","created_at":"2025-11-06 15:52:39","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185632,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/8ecddbd7bf7a272ba73cff7e.html"},{"id":95291119,"identity":"59daaf30-dac0-42eb-85ae-43ea727c5246","added_by":"auto","created_at":"2025-11-06 11:01:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":381366,"visible":true,"origin":"","legend":"\u003cp\u003eElectron transfer events in PSI (white solid arrows), charge recombination (dashed arrows) and an interaction of PSI with exogenous electron acceptor and donor (black solid arrows) (A); exogenous redox-mediators DCPIP, TMPD and Asc, which are capable of reducing or oxidizing PSI cofactors (B)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/3116257562ab03bdc04c766e.png"},{"id":95291137,"identity":"7d27672a-151a-48a1-8365-1100830d4ec7","added_by":"auto","created_at":"2025-11-06 11:01:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13894,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic scheme of electron transfer in PSI after charge separation, used to model the experimental kinetics of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction. Forward electron transfer from P\u003csub\u003e700\u003c/sub\u003e to [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e] was not considered within the model due to its ultra‑fast time scale\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/9dddce913824bcafdd123e3b.jpeg"},{"id":95314686,"identity":"dd8cb1d1-5dcd-43df-8266-288bcc3097e2","added_by":"auto","created_at":"2025-11-06 15:53:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":906404,"visible":true,"origin":"","legend":"\u003cp\u003eР\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e signal decay changes upon DCPIP concentration increase under aerobic (panels A–C) and anaerobic (panels D–F) conditions: transient absorbance traces at 820 nm (solid lines), the result of biexponential fitting (circles) and the plot of the slower exponential component (dashed lines) (A, D); the dependence of the lifetimes (B, E) and amplitudes (C, F) of the two exponential components on DCPIP concentration. Circles in panels B, C, E and F represent the result of the exponential deconvolution of the kinetics, while solid lines display model prediction of these parameters.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/795eda8913642331b9aaaac7.png"},{"id":95291126,"identity":"46012cf6-20bf-479c-9a8a-b9bc732ca94a","added_by":"auto","created_at":"2025-11-06 11:01:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":916091,"visible":true,"origin":"","legend":"\u003cp\u003eР\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e signal decay changes upon TMPD concentration increase under aerobic (panels A–C) and anaerobic (panels D–F) conditions: transient absorbance traces at 820 nm (solid lines), the result of biexponential fitting (circles) and the plot of the slower exponential component (dashed lines) (A, D); the dependence of the lifetimes (B, E) and amplitudes (C, F) of the two exponential components on TMPD concentration. Circles in panels B, C, E and F represent the result of the exponential deconvolution of the kinetics, while solid lines display model prediction of these parameters.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/a273add8f643551755705645.png"},{"id":95291122,"identity":"dbd6de69-2f5b-456e-a719-286262a3f6b5","added_by":"auto","created_at":"2025-11-06 11:01:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208211,"visible":true,"origin":"","legend":"\u003cp\u003eР\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e signal decay kinetics changes upon pH change in the presence of 15 μM of DCPIP and 10 mM Asc: transient absorbance traces at 820 nm (solid lines), the result of biexponential fitting (circles) and the plot of the slower exponential component (\u003cstrong\u003eA\u003c/strong\u003e); the pH-dependence of the contributions (\u003cstrong\u003eB\u003c/strong\u003e) of the exponential components\u003cstrong\u003e \u003c/strong\u003e(circles). Lines in panel \u003cstrong\u003eB\u003c/strong\u003e show the approximation of the experimental data with Nernst equation\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/c0d051c305f104360d9fef2f.png"},{"id":95291120,"identity":"b2d66eeb-682d-4426-bede-17f373e1f62a","added_by":"auto","created_at":"2025-11-06 11:01:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":201323,"visible":true,"origin":"","legend":"\u003cp\u003eThe general energetic scheme of the interaction of PSI with DCPIP and TMPD under aerobic conditions. Check the text for the detailed explanation\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/03e986623f1d9ba40799b6e3.png"},{"id":98814051,"identity":"8d9416ea-4481-4bff-9a51-d892ccc92546","added_by":"auto","created_at":"2025-12-22 16:10:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3829484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/14037893-a3e9-441f-8aa3-bc0d2de70129.pdf"},{"id":95291136,"identity":"5638954a-0110-4c64-82e4-757b1b5661ad","added_by":"auto","created_at":"2025-11-06 11:01:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25188,"visible":true,"origin":"","legend":"","description":"","filename":"2025PetrovaSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7917984/v1/f8bd20aa5e1caed6847e6056.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The commonly used electron donor 2,6-dichlorophenolindophenol also serves as an efficient electron acceptor for Photosystem I","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhotosystem I (PSI) is the transmembrane pigment-protein complex of photosynthetic electron transport chain of oxygenic phototrophs. PSI complexes of cyanobacteria \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 are trimeric with each monomer comprising 11 subunits (Malavath et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The homologous core subunits, PsaA and PsaB, bind most of the electron-transfer cofactors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Three pairs of chlorophyll \u003cem\u003ea\u003c/em\u003e molecules of the reaction center perform photochemical charge separation, producing oxidized chlorophyll \u003cem\u003ea\u003c/em\u003e dimer P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. Two phylloquinone molecules in the pseudo-symmetrical branches of redox-cofactors \u003cem\u003eA\u003c/em\u003e and \u003cem\u003eB\u003c/em\u003e (A\u003csub\u003e1A\u003c/sub\u003e/A\u003csub\u003e1B\u003c/sub\u003e) and an intersubunit iron-sulfur 4Fe-4S cluster F\u003csub\u003eX\u003c/sub\u003e facilitate electron transfer to the acceptor side of PSI complex, which consists of two iron-sulfur 4Fe-4S clusters F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e bound to the external hydrophilic subunit PsaC. PSI represents an efficient photovoltaic unit, generating the lowest reduction potential (\u0026lt;\u0026ndash;1 V \u003cem\u003evs\u003c/em\u003e NHE) in biological systems with a high quantum efficiency. These properties of PSI complexes make it promising for integration in biohybrid photovoltaic devices (Nguyen and Bruce, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Teodor and Bruce, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIsolated PSI complexes retain the ability to generate light-dependent electric potential. In the absence of the exogenous acceptors and donors, the final radical pair state P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e[F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003edecays within ~\u0026thinsp;200 ms \u003cem\u003evia\u003c/em\u003e backward electron transfer, followed by charge recombination (Vassiliev et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Makita et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore the rapid outflow of electrons from terminal [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e clusters and the fast reduction of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e are essential factors contributing to photoefficiency in photovoltaic devices based on PSI (Goyal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Szewczyk et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe highly efficient reduction of the native low-potential electron acceptor ferredoxin (Fd) is the main function of PSI \u003cem\u003ein vivo\u003c/em\u003e (rate constant\u0026thinsp;~\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Setif and Bottin, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1994\u003c/span\u003e)). The reduced Fd in turn serves as an electron donor for NADP\u003csup\u003e+\u003c/sup\u003e and downstream metabolic reactions, such as Calvin-Benson cycle, nitrogen and sulfur assimilation. Besides Fd, \u003cem\u003ein vivo\u003c/em\u003e the PSI complexes are capable of reducing oxidized forms of ascorbate (Asc) (Forti and Ehrenheim, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Trubitsin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and molecular oxygen (Kozuleva et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Among artificial electron acceptors methyl viologen (MV) is the most widely used (Nguyen and Bruce, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The rate constant (\u003cem\u003ek\u003c/em\u003e) of the [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e oxidation by MV is considerably high (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while the \u003cem\u003ek\u003c/em\u003e value of MV oxidation by O\u003csub\u003e2\u003c/sub\u003e is almost two orders of magnitude higher (8\u0026times;10\u003csup\u003e8\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Farrington et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1973\u003c/span\u003e), which increases probability of the side reaction with O\u003csub\u003e2\u003c/sub\u003e in case of MV application in photovoltaic devices (Passantino et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe fraction of PSI complexes in which an electron escaped to the exogenous acceptor requires P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by exogenous electron donor. \u003cem\u003eIn vivo\u003c/em\u003e it is provided by hydrophilic electron carriers plastocyanin (Pc) and cytochrome c\u003csub\u003e6\u003c/sub\u003e (in cyanobacteria) (Hippler and Drepper, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kovalenko et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e reduced forms of redox mediators, such as Asc, 2,6-dichlorophenolindophenol (DCPIP) and \u003cem\u003eN,N,N',N'\u003c/em\u003e-tetramethyl-\u003cem\u003ep\u003c/em\u003e-phenylenediamine (TMPD) are commonly used as electron donors for P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. Asc in combination with DCPIP or TMPD is utilized in photovoltaic devices, as well as in studies of electron transfer in thylakoid membranes and in purified PSI complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The reduction of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e by millimolar concentrations of Asc occurs within tens of seconds (Mano et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction is enhanced by the addition of micromolar concentrations of DCPIP or TMPD with characteristic time up to tens \u0026ndash; hundreds of milliseconds. The redox-mediators are maintained in a reduced state in the presence of excess of Asc. TMPD is shown to be a less efficient electron donor to PSI, than DCPIP (Gourovskaya et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Yet, even the latter is far from reaching the efficiency of natural electron donor Pc, which reduces P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the time range of tens of microseconds (Herv\u0026aacute;s et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Mamedov et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Asc and DCPIP are two-electron redox-mediators, while TMPD is capable of one-electron transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Oxidized forms of Asc, TMPD and DCPIP may act as electron acceptors for PSI (Izawa, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Acceptor properties of the oxidized form of TMPD (TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e, Wurster\u0026rsquo;s blue) and oxidized DCPIP (DCPIP\u003csup\u003eOX\u003c/sup\u003e) are widely used in the studies of redox enzymes (Jahn et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Loktyushkin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The reactions of the redox-couples Asc/DCPIP or Asc/TMPD with the acceptor side of PSI have not been studied well enough yet. Asc, when used as the only redox mediator, is shown to be able to oxidize the acceptor side of PSI, yet it is still unknown which of two forms \u0026ndash; monodehydroascorbate radical (Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) or dehydroascorbate (DHA) \u0026ndash; serves as the main acceptor (Trubitsin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Dry TMPD retain its reduced state, but in solution under aerobic conditions it gradually oxidizes. This allowed to investigate acceptor properties of TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e in the absence of Asc, using TMPD as an exclusive donor. TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e was shown to function as an electron acceptor for PSI even under laser flash excitation conditions (Hiyama and Ke, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). However, upon addition of Asc excess this effect disappeared, apparently because TMPD had been converted to the reduced form (Hiyama and Ke, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDCPIP, on the contrary, is initially oxidized, and Asc is required to maintain DCPIP mostly in the reduced form (DCPIP\u003csup\u003eRED\u003c/sup\u003e). In the case of Asc/DCPIP redox couple, oxidized forms of both mediators may accept electrons from the acceptor side of PSI. Their equilibrium concentrations are still unknown, yet under continuous illumination, it was shown that DCPIP\u003csup\u003eOX\u003c/sup\u003e was present in sufficient concentration to compete for electrons with MV on the acceptor side of the PSI (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The product of this reaction \u0026ndash; semiquinone form of DCPIP (DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) \u0026ndash; is highly reactive: in the presence of molecular oxygen it is quickly oxidized, thereby mediating electron transfer between the acceptor side of PSI and O\u003csub\u003e2\u003c/sub\u003e molecule, likewise MV (Dvoranov\u0026aacute; et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Marchanka and Gastel, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Under laser flash excitation conditions, it was shown that the amount of PSI complexes reduced by an exogenous electron donor increases with growing DCPIP concentration, yet, the concentration of oxidized forms of Asc increased simultaneously (Hiyama and Ke, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Vassiliev et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Thus the role of redox-mediator DCPIP in the oxidation of terminal cofactors of PSI in combination with Asc requires further investigations (Ciesielski et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Goyal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDCPIP was shown to be a more efficient component of the PSI-based photovoltaic devices, than TMPD (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). This effect can be related to a better donor efficiency of DCPIP or to the enhancement of the outflow of electrons from PSI by DCPIP. The redox properties of DCPIP might be affected by the deoxygenation of the medium or by the change in pH since DCPIP molecule has two protonation sites with pK values in physiological pH range (Diebler, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; Tonomura et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). In this work, we used laser absorption spectroscopy to examine the efficiencies of the donor and acceptor reactions of the PSI complexes from cyanobacteria \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 with DCPIP and TMPD in the presence of Asc excess. The effects of the O\u003csub\u003e2\u003c/sub\u003e depletion and pH change on the efficiency of the reactions were estimated. The results were used to develop kinetic model of electron transfer processes on the donor and acceptor sides of PSI in order to describe the observed reactions quantitatively.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSample preparation\u003c/h2\u003e\u003cp\u003eCells of cyanobacteria \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 were grown in BG-11N medium at room temperature under fluorescent lights at \u0026sim;40 \u0026micro;mol of photon m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Trimeric PSI particles were purified as described in (Johnson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) with some changes. In brief, the thylakoid membranes were solubilized in the presence of 1% n-dodecyl-β-D-maltoside, solubilized material was fractionized \u003cem\u003evia\u003c/em\u003e centrifugation on linear sucrose density gradients for 3 h at 140,000 g in a VTi50 vertical rotor (acquired as a part of Moscow State University Development Program). The lower green band containing the trimeric PSI complexes was collected, dialyzed, concentrated to 3\u0026ndash;4 mg Chl/ml, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTransient optical spectroscopy\u003c/h3\u003e\n\u003cp\u003eFlash-induced millisecond absorption changes were monitored at a wavelength of 820 nm in order to observe P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e signal decay in PSI samples. A frequency-doubled Quantel Nd:YAG laser (pulse half-width, 12 ns; flash intensity, 20 mJ) provided saturating actinic flashes at 532 nm, while a Spinder and Hoyer DC25A laser diode (wavelength, 820 nm) was used as a measuring light source. Measurements were performed in standard 1-cm optical path quartz cuvette. The assay medium contained PSI at chlorophyll concentration of 50 \u0026micro;g/ml, 50 mM HEPES-NaOH buffer, pH 7.5 (unless otherwise stated), 10 mM sodium ascorbate and varying concentrations of redox-mediators DCPIP or TMPD. The samples before measurements were incubated in the dark for 10 min. The final kinetic curves were obtained by averaging 64 sample curves. Laser flashes were given with the interval of 10 s to 2 min, depending on the concentration of the redox-mediator.\u003c/p\u003e\u003cp\u003eWhen the measurements were performed under anaerobic conditions, the additions of 10 mM glucose, glucose oxidase (100 U ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and catalase (100 U ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were used. Before measurements the samples were incubated for 10 minutes in the dark under nitrogen flow. During the measurement, the cuvette remained under a transparent polycarbonate cap in a nitrogen environment. The kinetics did not change, which indicated that anaerobic conditions were maintained at the measurement course.\u003c/p\u003e\u003cp\u003eIn the case of the pH-dependence measurements, a mixture of MES (5 mM pH 5.6), HEPES-NaOH (5 mM pH 7.5) and Tris-HCl (5 mM pH 8.2) buffers was used in order to avoid effects related to the change of buffer (Morlock et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The pH was adjusted with 1 N NaOH to the values of 5.6, 6.4, 7, 7.5, 8.2 and 8.6. Asc at 10 mM and DCPIP at 15 \u0026micro;M concentration were added to the sample before the measurements.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe obtained kinetic was analyzed using laboratory-developed scripts for Matlab R2023a [MATLAB: 9.14.0, The MathWorks Inc., Natick, Massachusetts (2023). Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mathworks.com/\u003c/span\u003e\u003cspan address=\"https://www.mathworks.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. Each kinetic curve was approximated by a sum of exponential functions and non-decaying residual by using nonlinear minimization. In most cases a good approximation required only two exponents, which corresponded to the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by i) the backward electron transfer (lifetime \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and amplitude \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e) and ii) exogenous electron donors (lifetime \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e and amplitude \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e). If the approximation of the backward electron transfer kinetics required more than one exponential component, their total amplitude and weighted average lifetime was calculated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eKinetic modeling\u003c/h3\u003e\n\u003cp\u003eElectron transfer reactions after the flash-induced charge separation and electron transfer to the terminal iron-sulfur clusters (resulting in formation of the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e[F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e state) involve recombination within the PSI complex and its interaction with exogenous redox-mediators. These reactions can be described by the kinetic scheme depicted in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eElectron from the terminal 4Fe\u0026ndash;4S clusters [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e can either undergo the recombination to P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e with a reaction rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e or escape to exogenous electron acceptors in the medium \u0026ndash; to Asc in the oxidized state (at the constant concentration of 10 mM) with the rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e or to the oxidized DCPIP/TMPD (at variable concentration) with the rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e. Similarly, P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e can be reduced through one of three reactions \u0026ndash; backwards electron transfer from [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e, electron donation from either Asc in the reduced state with the rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e or from the reduced DCPIP/TMPD with the rate constant \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eRedox transitions of the kinetic system in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e can be described with a set of linear differential equations. Solving these equations (see Supplementary information), the kinetics of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e decay can be represented by a sum of two exponential functions with the amplitudes \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e and the characteristic times \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e. These parameters depend on concentrations of electron donors (the constant concentration donor \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e and the variable concentration donor \u003cem\u003ed\u003c/em\u003e) and acceptors (the constant concentration acceptor \u003cem\u003ea\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e and the variable concentration acceptor \u003cem\u003ea\u003c/em\u003e):\u003c/p\u003e\u003cp\u003e[\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e700\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e]\u0026thinsp;\u003cem\u003e=\u0026thinsp;A\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;exp(-τ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/t)\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;exp(-τ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/t)\u003c/em\u003e (1)\u003c/p\u003e\u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e=\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/(k\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e+k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u0026thinsp;+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e)\u003c/em\u003e (2)\u003c/p\u003e\u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= (k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u0026thinsp;+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e)/(k\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e+k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u0026thinsp;+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e)\u003c/em\u003e (3)\u003c/p\u003e\u003cp\u003e\u003cem\u003e1/τ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e=\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;d\u0026thinsp;+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;d\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u0026thinsp;+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;a\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e (4)\u003c/p\u003e\u003cp\u003e\u003cem\u003e1/τ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e=\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;d\u0026thinsp;+\u0026thinsp;k\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026bull;d\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e (5)\u003c/p\u003e\u003cp\u003eThe obtained representation of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e kinetics as a sum of two exponential functions mirrors the bi-exponential deconvolution described above. Using the obtained apparent lifetimes \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e, and amplitudes \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e at different concentrations of the exogenous redox mediator (DCPIP or TMPD), the reaction rate constants \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e were found using equations (1\u0026ndash;5) as a fitting functions of the redox mediators concentrations \u003cem\u003ed\u003c/em\u003e, \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ea\u003c/em\u003e and \u003cem\u003ea\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eThe redox-reactions on the donor and acceptor sides of PSI with redox-mediators\u003c/h2\u003e\u003cp\u003eLaser flash-induced P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e photo-oxidation reveals a broad absorption increase in the near-infrared spectral region centered at 820 nm. The signal decays due to the fast backward electron transfer within PSI from the terminal [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e clusters to P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e or due to the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by exogenous electron donors. The rate of the latter component depends on concentration of the exogenous donor, while its amplitude depends on the efficiency of exogenous acceptors (Vassiliev et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Milanovsky et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e represent kinetics of the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction in the presence of 0.5\u0026ndash;50 \u0026micro;M of DCPIP or TMPD under aerobic or anaerobic conditions at the constant Asc concentration of 10 mM. Biexponential approximations of the kinetics are shown by circles. The dependencies of lifetimes (\u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e) and amplitudes (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e) of two exponential components on the redox-mediators concentrations (dots) and their approximation by the kinetic model (lines) are plotted in panels B and C, correspondingly. The parameters of biexponential decompositions are summarized in the Tables S1.1\u0026ndash;S1.4.\u003c/p\u003e\u003cp\u003eAt the lowest DCPIP concentration (red curve in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e was defined to be ~\u0026thinsp;70 ms, the complete P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by the redox-mediator took more than \u0026gt;\u0026thinsp;2\u0026bull;10\u003csup\u003e3\u003c/sup\u003e ms (the 7.6\u0026bull;10\u003csup\u003e3\u003c/sup\u003e ms lifetime was approximately estimated by the exponential decomposition). The contribution of the faster component \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e accounted for ~\u0026thinsp;80% of the decay, indicating that under these conditions P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was reduced preferentially \u003cem\u003evia\u003c/em\u003e the backward electron transfer pathway.\u003c/p\u003e\u003cp\u003eWith increasing DCPIP concentration the slower phase underwent gradual acceleration up to \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;170 ms at 50 \u0026micro;M (close circles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by DCPIP accelerated by a factor of ~\u0026thinsp;45 in the studied concentration range. At the same time, the \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e value remained unchanged at the DCPIP concentrations of 0.5\u0026ndash;15 \u0026micro;M, decreasing to 24 ms at the highest DCPIP concentration (open circles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The observed decrease indicates that the apparent \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e is a complex parameter, which depends not only on the backward electron transfer rate constant, but also on the reaction rate of PSI interaction with exogenous donors and acceptors.\u003c/p\u003e\u003cp\u003eSimultaneously, the contribution of the slower component \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e increased, accounting for ~\u0026thinsp;60% of the decay at 50 \u0026micro;M DCPIP (closed circles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This reveals predominance of the exogenous electron acceptor reduction by [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e over the backward electron transfer. Both DCPIP and Asc in oxidized states might accept electron from PSI under experimental conditions (Trubitsin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to estimate the Asc contribution, we monitored changes in the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction kinetics upon TMPD concentration increase in the presence of 10 mM Asc (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The slower component of the biexponential fit accelerated from \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6\u0026bull;10\u003csup\u003e3\u003c/sup\u003e ms at 0.5 \u0026micro;M TMPD to 5.4\u0026bull;10\u003csup\u003e2\u003c/sup\u003e ms at 50 \u0026micro;M TMPD, while \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e remained unchanged. The slower kinetic component accelerated by a factor of ~\u0026thinsp;10 in the investigated concentration range of TMPD, and the minimal \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e value was 3 times higher in the case of TMPD compared to DCPIP indicating a lower donor efficiency of TMPD.\u003c/p\u003e\u003cp\u003eThe relative contributions of the two kinetic components remained stable in the investigated TMPD concentration range (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Some increase in the slower component contribution was observed only at 50 \u0026micro;M TMPD. From this observation we may conclude that neither TMPD nor Asc under aerobic conditions can efficiently accept electrons from PSI acceptor side in the investigated concentration range. The contribution of the slower kinetic component increased only upon addition of DCPIP, indicating the specificity of the effect for DCPIP.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe effects of the anaerobic conditions on the PSI interactions with redox-mediators\u003c/h3\u003e\n\u003cp\u003eAs described in \u0026ldquo;Introduction\u0026rdquo;, the oxidation of DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e by molecular oxygen is highly favorable, which provides regeneration of DCPIP\u003csup\u003eOX\u003c/sup\u003e and enhances the electron outflow from PSI under aerobic conditions. We estimated the contribution of the O\u003csub\u003e2\u003c/sub\u003e-dependent DCPIP\u003csup\u003eOX\u003c/sup\u003e effect on the kinetics of Р\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e reduction by monitoring the DCPIP concentration dependency of \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e contribution upon O\u003csub\u003e2\u003c/sub\u003e depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;F, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.3). Indeed, under anaerobic conditions the relative amplitude \u003cem\u003eA\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e increased to a less extent, than under aerobic conditions: the highest \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e value did not exceed 50% (compare panels C and F in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the case of TMPD, anaerobic conditions did not affect \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e, which remained stable upon the increase in the mediator concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;F). This once again confirms the assumption that TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e under conditions of experiment did not accept electron from the acceptor side of PSI complex, while DCPIP\u003csup\u003eOX\u003c/sup\u003e did. Moreover, the efficiency of DCPIP\u003csup\u003eOX\u003c/sup\u003e reduction depends significantly on the rate of the DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e reoxidation, which is much higher under aerobic conditions.\u003c/p\u003e\u003cp\u003eSurprisingly, in the case of DCPIP, anaerobic conditions also noticeably affected \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e values (see Tables S1.1 and S1.3), which were on average\u0026thinsp;~\u0026thinsp;2 times slower under anaerobic conditions, than in the presence of O\u003csub\u003e2\u003c/sub\u003e in the concentration range studied. One may conclude that regeneration of the DCPIP form capable of reducing P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was suppressed under anaerobic conditions too.\u003c/p\u003e\n\u003ch3\u003eThe pH effects on the PSI interaction with redox-mediators\u003c/h3\u003e\n\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe pH-dependence of the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e decay obtained on the sample containing 10 mM Asc and 15 \u0026micro;M DCPIP shows the decrease in the \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e and simultaneous increase in the \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e value at high pH (Fig.\u0026nbsp;5, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Р\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e signal decay kinetics changes upon pH change in the presence of 15 \u0026micro;M of DCPIP and 10 mM Asc: transient absorbance traces at 820 nm (solid lines), the result of biexponential fitting (circles) and the plot of the slower exponential component (\u003cb\u003eA\u003c/b\u003e); the pH-dependence of the contributions (\u003cb\u003eB\u003c/b\u003e) of the exponential components (circles). Lines in panel \u003cb\u003eB\u003c/b\u003e show the approximation of the experimental data with Nernst equation\u003c/p\u003e\u003cp\u003eThe value of \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e remained stable except for a slight decrease at a high pH which most probably was caused by a complex nature of the parameter, see \u0026ldquo;Kinetic modeling\u0026rdquo; section for the details. The inflection point in the pH-dependence of the \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e value was observed around pH 7, which coincides with the pK of DCPIP\u003csup\u003eRED\u003c/sup\u003e. The higher donor capacity of deprotonated DCPIP\u003csup\u003eRED\u003c/sup\u003e could be explained by the electrostatic attraction between negatively charged mediator and P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. At the same time, the observed increase in the \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e value upon the alkalization of the medium indicates growing outflow of electron to the exogenous redox-mediator (Fig.\u0026nbsp;5B). This could not be explained by the growing electrostatic attraction of the [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e and the electron acceptor since DCPIP\u003csup\u003eOX\u003c/sup\u003e is also charged negatively. The fitting of the \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e(pH) curve by Nernst equation revealed the pK value of 7.54, which is significantly higher than pK\u0026thinsp;=\u0026thinsp;6 of DCPIP\u003csup\u003eOX\u003c/sup\u003e (Gibbs et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1925\u003c/span\u003e; Tonomura et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Loktyushkin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides the protonation state of DCPIP, the efficiency of the PSI interaction with DCPIP depends on the other factors, such as protonation state of the amino acids of the protein. We will consider some possible explanations in the Discussion.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristic times and amplitudes of exponential components corresponding to charge recombination and reduction of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e by the external electron donor at 15 \u0026micro;M DCPIP at different pH levels\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003echaracteristic time, ms\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eamplitude, %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eτ\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eτ\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eA\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;4000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6.4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;4000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7.5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1670\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8.2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e960\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eKinetic modeling\u003c/h2\u003e\u003cp\u003eThe results of the multiexponential decomposition were used to obtain a numerical solution of the kinetic model describing electron transfer processes depicted at Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Within the model, both donor and acceptor sides of PSI interact with two kinds of the electron donors/acceptors with constant or variable concentrations (having different reaction rate constants). Analytical solution of the model, described above by equations (1\u0026ndash;5), corresponds to the experimentally observed concentration dependence of PSI transient absorption kinetics:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003ethe exponential components amplitudes \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e depend on the rate constants of the electron transfer to the exogenous acceptor (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e) and backward electron transfer (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e) reactions;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ethe \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e value depends not only on the rate constant of the backward electron transfer (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e), but is also affected by the rate constants of the reactions on the donor (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e) and acceptor (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e) sides of the PSI complex;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ethe \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e value depends on the rate constants of the donor reactions (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eEach experimental series differing by redox mediator (DCPIP/TMPD) and oxygen presence (aerobic/anaerobic) was fitted independently except of the \u003cem\u003ek\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e parameter, which was set as 8.7 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This value corresponds to the charge recombination of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e[F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e ion-radical pair with characteristic time 115 ms, matching the literature values (Vassiliev et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Makita and Hastings, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Milanovsky et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The fitting of the apparent lifetimes and amplitudes of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e decay with the model is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (panels B, C, E and F) by solid lines. The rate constants of the reactions normalized to the total concentration of the redox mediators (Asc, DCPIP and TMPD) are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eApparent rate constants of PSI interaction with exogenous redox-mediators, according to the fitting of experimental data, in aerobic and anaerobic conditions. See Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for the legend\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e, M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e, M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e, M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e, M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eDCPIP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.3\u0026bull;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.1\u0026bull;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e270\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.9\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e220\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eTMPD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.1\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.7\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e360\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.2\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.7\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e270\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAccording to the Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, DCPIP is ~\u0026thinsp;3 times more efficient as an electron donor and ~\u0026thinsp;6 times more efficient as an electron acceptor, than TMPD. Both \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e of DCPIP decrease by a factor of 2 upon O\u003csub\u003e2\u003c/sub\u003e depletion, pointing to the complex nature of both reactions. The \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e value of TMPD was not sensitive to the O\u003csub\u003e2\u003c/sub\u003e presence, while its donor activity noticeably decreased under anaerobic conditions. We do not expect significant decrease in the TMPD\u003csup\u003eRED\u003c/sup\u003e concentration under anaerobic conditions. It is more likely that the observed decrease in \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(TMPD) is an artifact, related to the low donor reaction rate under conditions of the experiment (see Discussion for the details).\u003c/p\u003e\u003cp\u003eThe acceptor efficiency of Asc is by an order of magnitude higher than its donor activity according to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (compare the value of \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e). This is consistent with the data showing that direct P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by Asc is insignificant compared to its acceptor activity \u003cem\u003ein vivo\u003c/em\u003e (T\u0026oacute;th et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The values of \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea0\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e were relatively stable in all experiment series except for the measurements in the presence of DCPIP under aerobic conditions, where \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed0\u003c/em\u003e\u003c/sub\u003e was about twofold higher. This might indicate the significant increase in the concentration of the electron donating form of Asc under these conditions.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRedox-mediators, such as Asc, DCPIP or TMPD, are commonly used in the electron transfer studies of purified PSI, PSII complexes and bacterial reaction centers. Among these mediators, DCPIP was proven to be remarkably more efficient component of the PSI-based photovoltaic devices (Nguyen and Bruce, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The redox chemistry of both Asc and DCPIP, which comprise a redox-couple, is quite complex, which creates unavoidable side-reactions, such as reduction of DCPIP\u003csup\u003eOX\u003c/sup\u003e by terminal [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e clusters and the subsequent O\u003csub\u003e2\u003c/sub\u003e-dependent reoxidation of DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, which has been detected under continuous illumination (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present work we investigated DCPIP interaction with the donor and acceptor sides of PSI complexes under conditions of flash-induced electron transfer in the presence of Asc excess. Comparing the efficiencies of DCPIP and TMPD interaction with PSI on the acceptor side under aerobic and anaerobic conditions, we demonstrated that it is DCPIP\u003csup\u003eOX\u003c/sup\u003e, and not Asc, which is the main exogenic electron acceptor for PSI. This fact is noteworthy since in previous works, the slow DCPIP-mediated P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction was attributed to the account of the fraction of PSI complexes reducing directly O\u003csub\u003e2\u003c/sub\u003e (Vassiliev et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Milanovsky et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe acceptor efficiency of the mediators in the present work was studied by monitoring how an increase in DCPIP/TMPD concentration affected the relative fraction of PSI complexes in which electron escaped to an external acceptor. We discovered that under laser flash excitation, DCPIP, unlike Asc and TMPD, is capable of accepting electron from PSI at a total concentration of 5 \u0026micro;M, noticeably lessening the contribution of charge recombination. Kinetic model gave an apparent \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e for this reaction of 2.9\u0026bull;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is almost an order of magnitude higher than the apparent \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e(TMPD).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe values of the midpoint redox-potentials (E\u003csub\u003em\u003c/sub\u003e) of the natural and artificial electron donors and acceptors for PSI\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRedox couple\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE\u003csub\u003em\u003c/sub\u003e, mV\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMV\u003csup\u003e2+\u003c/sup\u003e/MV\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;456 (Orgill et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e/DA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;200 (Sapper et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1982\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDCPIP\u003csup\u003eOX\u003c/sup\u003e/DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;(155\u0026ndash;190) (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eО\u003csub\u003e2\u003c/sub\u003e/О\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;155 (Asada and Nakano, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1978\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAscН\u003csup\u003e\u0026minus;\u003c/sup\u003e/DA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+(60\u0026ndash;100) (Trubitsin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDCPIP\u003csup\u003eOX\u003c/sup\u003e/DCPIP\u003csup\u003eRED\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+(217\u0026ndash;290) (Gibbs et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1925\u003c/span\u003e; Dvoranov\u0026aacute; et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e/TMPD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u0026thinsp;260 (Dutton, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1978\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAscН\u003csup\u003e\u0026minus;\u003c/sup\u003e/Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u0026thinsp;320 (Sapper et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1982\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e/DCPIP\u003csup\u003eRED\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+(415\u0026ndash;450) (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHowever, redox mediators DCPIP and TMPD can serve as electron acceptors to PSI only in their oxidized states, which amount to a small fraction of the total redox mediator concentration (\u0026lt;\u0026thinsp;1% in the presence of excess Asc (Passantino et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)). Assuming redox potential of the medium, maintained by Asc, as +(60\u0026ndash;100) mV (Trubitsin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), we can calculate the relative fraction of these forms according to the Nernst equation. Various estimates of midpoint potential E\u003csub\u003em\u003c/sub\u003e(DCPIP\u003csup\u003eOX\u003c/sup\u003e/DCPIP\u003csup\u003eRED\u003c/sup\u003e) put it between +\u0026thinsp;217 and +\u0026thinsp;290 mV, and the value for E\u003csub\u003em\u003c/sub\u003e(TMPD\u003csup\u003eOX\u003c/sup\u003e/TMPD\u003csup\u003eRED\u003c/sup\u003e) is +\u0026thinsp;260 mV (see Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, the fraction of both DCPIP\u003csup\u003eOX\u003c/sup\u003e and TMPD\u003csup\u003eOX\u003c/sup\u003e can be estimated as not exceeding 1%. In order to have an opportunity to compare the donor and acceptor efficiencies more correctly, we adjusted apparent \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values from Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for the concentrations of the DCPIP\u003csup\u003eOX\u003c/sup\u003e and TMPD\u003csup\u003eOX\u003c/sup\u003e. The adjusted estimates are provided in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The value of \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e(TMPD)\u0026thinsp;=\u0026thinsp;3.6\u0026bull;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is similar to the one, obtained earlier in the experiments, where TMPD acceptor function was investigated in the absence of Asc (Hiyama and Ke, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe apparent rate constants of the donor reactions, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e, were elucidated from the lifetimes of the slow sub-second kinetic phase of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction using the kinetic model. Under aerobic conditions the estimated values of \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e for DCPIP and TMPD as electron donors were 1.3\u0026bull;10\u003csup\u003e5\u003c/sup\u003e and 4.1\u0026bull;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, which were remarkably close to those reported in the literature for the steady-state conditions (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(DCPIP)\u0026thinsp;=\u0026thinsp;1.9\u0026bull;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(TMPD)\u0026thinsp;=\u0026thinsp;3\u0026bull;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fujii et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEstimated rates constants of DCPIP and TMPD redox forms interactions with PSI as donor (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e) or acceptor (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e, M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e, M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eDCPIP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u0026thinsp;7.2\u0026bull;10\u003csup\u003e6*\u003c/sup\u003e,\u0026nbsp;5.9\u0026bull;10\u003csup\u003e4**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2.9\u0026bull;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.9\u0026bull;10\u003csup\u003e4**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eTMPD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.1\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;3.6\u0026bull;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;O\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.2\u0026bull;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;4.5\u0026bull;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e*\u003c/sup\u003e DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e**\u003c/sup\u003e DCPIP\u003csup\u003eRED\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTMPD efficiency as electron donor decreased approximately two-fold upon oxygen removal from the reaction medium, as seen from Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This is unexpected as TMPD should not react with molecular oxygen under experimental conditions and, hence, no changes in its redox properties were intended. It should be noted that the estimated value of \u003cem\u003ek\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e(TMPD\u003csub\u003e\u0026ndash;O2\u003c/sub\u003e) is the lowest of all values in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and, according to this estimate, only at the highest concentration of TMPD (50 \u0026micro;M) the reaction rate of its electron donation to P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is comparable to the same reaction with Asc (in a much higher concentration of 10 mM). As seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Eq.\u0026nbsp;(5), the \u003cem\u003eτ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e dependence on the redox-mediator concentration has a hyperbolic form. In the case of TMPD in contrast to DCPIP we observe the far-left side of this hyperbola, with a very slight dependence on the TMPD concentration. Hence, we expect the accuracy of \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(TMPD \u003csub\u003e\u0026ndash;O2\u003c/sub\u003e) estimation to be very low, and the observed difference between TMPD donor activity under aerobic and anaerobic conditions seems unreliable.\u003c/p\u003e\u003cp\u003eAt the same time the observed apparent \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(DCPIP) decrease from 1.3\u0026bull;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 5.9\u0026bull;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e upon O\u003csub\u003e2\u003c/sub\u003e depletion most probably indicates the decrease in the concentration of the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-reducing form under anaerobic conditions, since the change in conditions induced the shift from the linear to the hyperbolic section of the concentration dependency. This affirms DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e contribution to the donor reaction. In this case the apparent \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e = 1.3\u0026bull;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e value may be considered as a weighted average of true \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e values for DCPIP\u003csup\u003eRED\u003c/sup\u003e (~\u0026thinsp;5.9\u0026bull;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. The relative concentration of DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e can be assumed to be equal or less than that for DCPIP\u003csup\u003eOX\u003c/sup\u003e, \u0026lt;\u0026thinsp;1% of total DCPIP concentration, which gives an estimate for \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;7.2\u0026bull;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This value is several orders of magnitude higher compared to TMPD (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which is consistent with the earlier reported data showing lower efficiency of TMPD as an electron donor (Gourovskaya et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Under anaerobic conditions only DCPIP\u003csup\u003eRED\u003c/sup\u003e functions as an electron donor, showing \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e closer to \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(TMPD).\u003c/p\u003e\u003cp\u003eThe values presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e imply that under aerobic conditions DCPIP operates as an efficient electron donor due to the contribution of DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. It might be produced from DCPIP\u003csup\u003eRED\u003c/sup\u003e as a result of its oxidation by molecular oxygen. DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e is a highly reactive species due to the negative E\u003csub\u003em\u003c/sub\u003e of the DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e/DCPIP\u003csup\u003eOX\u003c/sup\u003e redox couple. The estimated E\u003csub\u003em\u003c/sub\u003e value for DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e/DCPIP\u003csup\u003eOX\u003c/sup\u003e transition is \u0026minus;\u0026thinsp;155/\u0026ndash;190 mV, while DCPIP\u003csup\u003eRED\u003c/sup\u003e/DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e has a much higher value of +\u0026thinsp;415/+450 mV (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) being almost equipotential to P\u003csub\u003e700\u003c/sub\u003e/P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. Free energy (ΔG) of the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e is about \u0026minus;\u0026thinsp;600/\u0026ndash;650 meV, making this reaction highly favorable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnother implicit factor, which might contribute to the decrease in the DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e concentration under anaerobic conditions, is retardation of the DCPIP\u003csup\u003eOX\u003c/sup\u003e reduction. Due to the negative E\u003csub\u003em\u003c/sub\u003e of DCPIP\u003csup\u003eOX\u003c/sup\u003e/DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e only the reduction of DCPIP\u003csup\u003eOX\u003c/sup\u003e by Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e is thermodynamically favorable, which has ΔG = \u0026minus;\u0026thinsp;50 meV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e) and the high rate constant of 9.5\u0026bull;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Iyanagi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Marchanka and Gastel, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Yet, under anaerobic conditions AscН\u003csup\u003e\u0026minus;\u003c/sup\u003e/Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003etransition is arrested causing rapid decrease in the Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e concentration (Trubitsin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBesides the ΔG value and concentrations of the reduced forms, the efficiency of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction depends on the electrostatic interactions with the electron donor, which could be modulated by the pH of the medium. According to our observations, the donor efficiency of DCPIP increases above pH 7 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which coincides with the pK value of the DCPIP\u003csup\u003eRED\u003c/sup\u003e (Tonomura et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1978\u003c/span\u003e), while the pK of DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e is unknown. Yet, both deprotonated DCPIP\u003csup\u003eRED\u003c/sup\u003e and DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e are charged negatively, which should enhance P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction, while TMPD\u003csup\u003eRED\u003c/sup\u003e is neutral, making its interaction with P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e less favorable.\u003c/p\u003e\u003cp\u003eThe higher rate constant of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by DCPIP \u003cem\u003evs\u003c/em\u003e TMPD is thermodynamically explicable, while higher acceptor efficiency of DCPIP\u003csup\u003eOX\u003c/sup\u003e compared to TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e could not be easily explained. ΔG of the TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e reduction by [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e is \u0026minus;\u0026thinsp;450 meV higher, than for the analogous reaction with DCPIP\u003csup\u003eOX\u003c/sup\u003e. Electrostatic attraction of the positively charged TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e to [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e is also advantageous for the interaction. Yet, the oxidation of TMPD\u003csup\u003eRED\u003c/sup\u003e by O\u003csub\u003e2\u003c/sub\u003e has high positive ΔG (+\u0026thinsp;415 meV) slowing down its oxidation under aerobic conditions. TMPD\u003csup\u003e\u0026bull;+\u003c/sup\u003e reduction by Asc is thermodynamically favorable process, since it is characterized by ΔG of \u0026minus;\u0026thinsp;370 meV. A completely different situation is observed in the presence of DCPIP, since the fast reoxidation of DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and much less negative ΔG of the reaction of DCPIP\u003csup\u003eOX\u003c/sup\u003e with Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e most probably promote DCPIP\u003csup\u003eOX\u003c/sup\u003e accumulation in the medium. This is not in line with the previously supposed lack of the DCPIP\u003csup\u003eOX\u003c/sup\u003e in the presence of Asc excess (Szewczyk et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe acceptor efficiency of DCPIP\u003csup\u003eOX\u003c/sup\u003e, as well as its interaction with the donor side of the complex exhibited pH-dependency. The pK value of the acceptor reaction observed in the experiment was 7.5 (Fig.\u0026nbsp;5B), while the pK value of for DCPIP\u003csup\u003eOX\u003c/sup\u003e is 5.7. Therefore, the observed pH dependence could not be explained by a more efficient interaction of the acceptor side of PSI with the deprotonated form of DCPIP\u003csup\u003eOX\u003c/sup\u003e. Yet, this value corresponds nicely to the earlier reported data. First of all, the pH-dependence of the O\u003csub\u003e2\u003c/sub\u003e uptake rate in suspension, containing PSI, Asc and DCPIP, had maximum at pH 7.8 under steady-state conditions (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The highest photocurrent density in some PSI-DCPIP based photovoltaic device was achieved at similar pH values of the medium (Passantino et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to the literature, the pK value of DCPIP can change significantly upon immobilization in an acetate cellulose film covering a carbon electrode, reaching 7.4 (Florou et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In the case of the donor reaction, the pK of DCPIP\u003csup\u003eRED\u003c/sup\u003e and the pK value observed in the experiment are in a good agreement, indicating that DCPIP\u003csup\u003eRED\u003c/sup\u003e bound on the donor side of PSI apparently does not exhibit the pK shift. Yet, it cannot be ruled out that the pK value of DCPIP upon interaction with PSI may differ from those in solution.\u003c/p\u003e\u003cp\u003eOn the other hand, an increase in pH may affect the protonation state and charge of the surface amino acids of PSI. Deprotonation of the amino acid residues, leading to the appearance of additional surface negative charges, should, on the contrary, hinder the binding of the DCPIP\u003csup\u003eOX\u003c/sup\u003e molecule to the PsaC subunit. However, appearance of additional negative charges upon the pH increase also affects the E\u003csub\u003em\u003c/sub\u003e values of the F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e iron-sulfur clusters. The electrostatic interactions in PSI can be taken into account by using operating redox potentials values (Ptushenko et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Comparison of the operating E\u003csub\u003em\u003c/sub\u003e of F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e (\u0026ndash;479 mV and \u0026minus;\u0026thinsp;539 mV, respectively) shows that this difference is mostly due to the effect of the charged amino acid groups of the PsaC subunit. Their electrostatic contributions are +\u0026thinsp;98 mV for the F\u003csub\u003eA\u003c/sub\u003e cluster proximal to the PSI core and +\u0026thinsp;35 mV for the distal F\u003csub\u003eB\u003c/sub\u003e cluster, which results in final electron density shifting towards the F\u003csub\u003eA\u003c/sub\u003e cluster. If we assume that this asymmetry decreases upon increasing pH above pK\u0026thinsp;=\u0026thinsp;7.5, the iron-sulfur clusters F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e may become almost equipotential at alkaline pH. Compared to the neutral pH conditions, this will lead to the redistribution of the electron density in favor of the distal F\u003csub\u003eB\u003c/sub\u003e cluster, decreasing the effective distance of electron transfer to the exogenous acceptor, which can significantly accelerate this reaction. The hypothesis of electron density redistribution between the iron-sulfur clusters F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e upon pH changes explains not only the enhancement of the acceptor properties of DCPIP with increasing pH reported previously (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and observed in the present work, but also overcomes the contradiction between the kinetic data showing that at pH 7.8\u0026ndash;8.3 the electron density is shifted towards the iron-sulfur cluster F\u003csub\u003eB\u003c/sub\u003e (Fujii et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Vassiliev et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mamedov et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and the fact that the redox potential of the iron-sulfur cluster F\u003csub\u003eB\u003c/sub\u003e is lower than that of F\u003csub\u003eA\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eThe results presented here are in line with the previously published data, including the lower performance of the photovoltaic devices using Asc-TMPD redox couple in comparison with the other redox-mediators (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). That is why TMPD, unlike DCPIP, is rarely used in PSI based photovoltaic cells. We propose that it is related to the ability of DCPIP to mediate both reduction of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and outflow of electrons from F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e in the presence of Asc, decreasing backward electron transfer in the complex. This explains the appearance of a large-amplitude response in PSI-based photovoltaic cells containing 50 \u0026micro;M DCPIP and 10 mM Asc (Zaspa et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The use of the natural electron donor cytochrome c\u003csub\u003e6\u003c/sub\u003e and the efficient artificial acceptor MV instead of DCPIP made it possible to increase the duration of light-dependent generation of electric potential (Zaspa et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The rate constant of electron transfer from [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e to MV is 10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Hiyama and Ke, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1971\u003c/span\u003e), which is comparable to the value of \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e(DCPIP), presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. This is in line with the data obtained previously under continuous illumination, where DCPIP\u003csup\u003eOX\u003c/sup\u003e efficiently competed for electrons from PSI with MV (Petrova et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The rate constant of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction by cytochrome c\u003csub\u003e6\u003c/sub\u003e (10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Herv\u0026aacute;s et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e)) is comparable to the \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) and two orders of magnitude higher than \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e(DCPIP\u003csup\u003eRED\u003c/sup\u003e). Yet unstable DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e could not be accumulated in sufficient concentration to provide efficient P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction at micromolar total DCPIP concentration. That is why millimolar concentrations of redox mediators were required to remove the limitation of the overall electron transfer rate on the donor side of PSI when using MV.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThus, concluding, DCPIP as electron acceptor is capable of competing with backward electron transfer in PSI complexes. Accepting electrons from [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026ndash;\u003c/sup\u003e, it mediates O\u003csub\u003e2\u003c/sub\u003e reduction by PSI even under single flash excitation conditions. This reaction maintains DCPIP\u003csup\u003eOX\u003c/sup\u003e concentration high enough to provide leading role of DCPIP in electron outflow from PSI even in the presence of Asc excess. High donor capacity of DCPIP is ensured by DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e/DCPIP\u003csup\u003eOX\u003c/sup\u003e transition due to a high negative ΔG of the reaction with P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. TMPD, being one-electron mediator, in the presence of Asc is not accumulated in oxidized state and cannot provide an efficient oxidation of the terminal PSI cofactors. The efficiency of the P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction depends on the electrostatic attraction with negatively charged deprotonated reduced DCPIP form. The efficiency of the electron transfer from PSI to external acceptors appeared to be sensitive to the distribution of the electron density between iron-sulfur clusters F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e, which could be modulated by pH.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePhotosystem I (PSI), Special pair of chlorophyll molecules (P\u003csub\u003e700\u003c/sub\u003e), Ferredoxin (Fd), Ascorbate (Asc), Methyl viologen (MV), Molecular oxygen (O\u003csub\u003e2\u0026shy;\u003c/sub\u003e), Plastocyanin (Pc), 2,6-dichlorophenolindophenol (DCPIP), \u003cem\u003eN,N,N\u0026apos;,N\u0026apos;\u003c/em\u003e-tetramethyl-\u003cem\u003ep\u003c/em\u003e-phenylenediamine (TMPD), Monodehydroascorbate radical (Asc\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e), Dehydroascorbate (DHA), Midpoint potential (E\u003csub\u003em\u003c/sub\u003e), Free energy (\u0026Delta;G)\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cb\u003eEthics and consent to participate\u003c/b\u003e Not applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the Russian Science Foundation Grant 23-74-00025\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.P., M.K. and A.S. developed experiment design and wrote the manuscript; A.P. and I.V. conducted experiments; G.M., D.Ch. and A.P. analyzed the data and performed kinetic modeling. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank Andrey A. Zaspa and Arseny V. Aybush for the technical support and Mahir D. Mamedov for valuable discussions.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAsada K, Nakano Y (1978) Affinity for oxygen in photoreduction of molecular oxygen and scavenging of hydrogen peroxide in spinach chloroplasts. 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Biophys J 74:2029\u0026ndash;2035. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0006-3495(98)77909-3\u003c/span\u003e\u003cspan address=\"10.1016/S0006-3495(98)77909-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZaspa A, Vitukhnovskaya L, Mamedova A, Allakhverdiev SI, Semenov A, Mamedov M (2022) Voltage generation by photosystem I complexes immobilized onto a millipore filter under continuous illumination. Int J Hydrog Energy 47:11528\u0026ndash;11538. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijhydene.2022.01.175\u003c/span\u003e\u003cspan address=\"10.1016/j.ijhydene.2022.01.175\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photosystem I, Electron transfer, Midpoint redox potentials, Artificial electron acceptor, Redox-mediator, Biohybrid photovoltaic device, Kinetic modeling","lastPublishedDoi":"10.21203/rs.3.rs-7917984/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7917984/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe sustainable functioning of the purified photosystem I (PSI) pigment-protein complex requires exogenous redox-mediators, facilitating the primary electron donor P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction and terminal acceptor [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e oxidation. The redox couple ascorbate/2,6-dichlorophenolindophenol (Asc/DCPIP) was shown to be more efficient, than the couple Asc/\u003cem\u003eN,N,N',N'\u003c/em\u003e-tetramethyl-\u003cem\u003ep\u003c/em\u003e-phenylenediamine (TMPD) both in photosynthetic studies and in biohybrid photovoltaic devices. We investigated the interactions of DCPIP with purified cyanobacterial PSI in the presence of Asc excess under laser flash excitation. Here we show that DCPIP, in contrast to TMPD, competes efficiently as an electron acceptor with the backward electron transfer from [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e to P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e even at micromolar concentrations, indicating accumulation of the oxidized DCPIP under aerobic conditions in the presence of Asc excess. The reduction of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e includes contributions both from reduced DCPIP and semiquinone DCPIP\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. The rates of P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction and [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e oxidation by DCPIP demonstrate complex pH-dependencies, related to changes in protonation state of the mediator and probably to redistribution of electron density between the terminal cofactors F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e. The rate constants of the electron transfer from Asc, DCPIP and TMPD to P\u003csub\u003e700\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and of the electron outflow from [F\u003csub\u003eA\u003c/sub\u003e/F\u003csub\u003eB\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e to the oxidized forms of these compounds are estimated by kinetic modeling. The obtained data reveal thermodynamic, kinetic and electrostatic factors responsible for the high DCPIP efficiency as electron donor and acceptor for PSI.\u003c/p\u003e","manuscriptTitle":"The commonly used electron donor 2,6-dichlorophenolindophenol also serves as an efficient electron acceptor for Photosystem I","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 11:01:50","doi":"10.21203/rs.3.rs-7917984/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T00:06:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T16:30:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-01T10:12:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162489401428357065954524217666051604881","date":"2025-10-29T07:48:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153280898175415370493561106835385104557","date":"2025-10-27T08:48:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T08:20:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T08:29:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-23T08:34:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photosynthesis Research","date":"2025-10-21T16:31:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f4c93766-f944-48e8-ab92-90d4b46f907f","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:04:22+00:00","versionOfRecord":{"articleIdentity":"rs-7917984","link":"https://doi.org/10.1007/s11120-025-01190-1","journal":{"identity":"photosynthesis-research","isVorOnly":false,"title":"Photosynthesis Research"},"publishedOn":"2025-12-18 15:57:37","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-11-06 11:01:50","video":"","vorDoi":"10.1007/s11120-025-01190-1","vorDoiUrl":"https://doi.org/10.1007/s11120-025-01190-1","workflowStages":[]},"version":"v1","identity":"rs-7917984","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7917984","identity":"rs-7917984","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}