New insights into the involvement of residue D1/V185 in Photosystem II function in Synechocystis 6803 and Thermosynechococcus vestitus

New insights into the involvement of residue D1/V185 in Photosystem II function in Synechocystis 6803 and Thermosynechococcus vestitus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article New insights into the involvement of residue D1/V185 in Photosystem II function in Synechocystis 6803 and Thermosynechococcus vestitus Alain Boussac, Julien Sellés, Miwa Sugiura, Robert L. Burnap This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5504214/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The effects of D1-V185T and D1-V185N mutations in Photosystem II (PSII) from Thermosynechococcus vestitus (formerly T. elongatus ) and Synechocystis 6803, respectively, were studied using both EPR and optical kinetics. EPR spectroscopy reveals the presence of a mixture of a S 2 state in a high spin configuration (S 2 HS ) and in a low spin configuration (S 2 LS ) in both mutants. In contrast to the S 2 HS in the wild type, the S 2 HS state in the D1-V185T mutant does not progress to the S 3 state at 198 K. This inability is likely due to alterations in the protonation state and hydrogen-bonding network around the Mn 4 CaO 5 cluster. Optical studies show that these mutations significantly affect proton release during the S 3 -to-S 0 transition. While the initial fast proton release associated with Tyr Z ● formation remains unaffected within the resolution of our measurements, the second, and slower, proton release is delayed, suggesting that the mutations disrupt the hydrogen-bonding interactions necessary for efficient deprotonation of substrate water (O6). This disruption in proton transfer also correlates with slower water exchange in the S 3 state, likely due to non-native hydrogen bonds introduced by the threonine or asparagine side chains at position 185. These findings point to a critical role of D1-V185 in regulating both proton transfer dynamics and water binding, underscoring a complex interplay between structural and functional changes in PSII. Biophysics Photosystem II D1-V185 EPR proton release S-state cycle spin state Mn4CaO5 cluster Figures Figure 1 Figure 2 Figure 3 Introduction Oxygenic photosynthesis in cyanobacteria, algae and higher plants converts solar energy into the chemical bonds of sugars and oxygen [ 1 ]. Photosystem II (PSII) begins this process by splitting water to obtain electrons in the form of reduced quinone, while generating a proton gradient and releasing O 2 . The mature PSII binds 35 chlorophylls a (Chl- a ),two pheophytins (Phe- a ), one membrane b-type cytochrome, one extrinsic c-type cytochrome (in cyanobacteria and red algae), one non-heme iron, two plastoquinones (Q A and Q B ), the Mn 4 CaO 5 cluster, 2 Cl − , 12 carotenoids and 25 lipids [ 2 , 3 ]. In the cyanobacterium Synechocystis sp. PCC 6803 a 4th extrinsic subunit, PsbQ, has also been found in addition to PsbV, PsbO and PsbU [ 4 ]. Among the 35 Chl- a , 31 are antenna Chls. When one is excited, the excitation energy is transferred to other chlorophylls until it reaches the key pigments in the photochemical reaction center: 4 Chl- a molecules, P D1 , P D2 , Chl D1 , Chl D2 and 2 Phe- a molecules, Phe D1 and Phe D2 . A few picoseconds after the formation of the excited *Chl D1 , charge separation occurs, ultimately forming the Chl D1 + Phe D1 − and then P D1 + Phe D1 − radical pair states, e.g. [ 5 , 6 ]. Formation of the Chl D1 + Phe D1 − radical pair was recently defined as a fast pathway (short-range charge-separation) in contrast with a slow pathway with P D1 P D2 as the initial donor (long-range charge separation) that would result directly in the formation of the P D1 + Phe D1 − radical pair [ 7 ]. After the charge separation, P D1 + oxidizes Tyr Z , the Tyr161 of the D1 polypeptide, which is then reduced by the Mn 4 CaO 5 cluster. The electron on Phe D1 − is then transferred to Q A , the primary quinone electron acceptor, and then to Q B , the second quinone electron acceptor. Whereas Q A can be only singly reduced under normal conditions, Q B accepts two electrons and two protons before leaving its binding site and being replaced by an oxidized Q B molecule from the membrane plastoquinone pool, see for example [ 1 , 8 – 12 ] for a non-exhaustive list of recent reviews on PSII function. The Mn 4 CaO 5 cluster, oxidized by the Tyr Z • radical formed after each charge separation, cycles through five redox states denoted Sn, where n designates the number of stored oxidizing equivalents. The S 1 state is stable in the dark, which makes S 1 the preponderant state after the decay of the S 3 and S 2 states in the dark. When the S 4 state is formed, after the 3rd flash of light given on dark-adapted PSII, two water molecules bound to the cluster are oxidized, O 2 is released and the S 0 -state is reformed, [ 13 , 14 ]. Thanks to the advent of serial femtosecond X-ray free electron laser crystallography and cryo-EM spectroscopy, structures of the Mn 4 CaO 5 cluster have been resolved in the dark-adapted S 1 state, the S 2 and S 3 states, in a redox state as close as possible to those expected taking into account the misses under flash illumination. In the S 1 state, i.e. in the Mn III 2 Mn IV 2 redox state, the Mn 4 CaO 5 structure resembles a distorted chair, including a µ-oxo-bridged cuboidal Mn 3 O 4 Ca unit with a fourth Mn attached to this core structure via two µ-oxo bridges involving the two oxygen's O4 and O5 [ 2 ]. Recently, important progress has been made in the resolution of the crystal structures in the S 2 , S 3 [ 15 – 17 ] and S 0 states [ 15 , 16 ]. Briefly, changes in the S 1 to S 2 transition more or less correspond to those expected for the valence change of the Mn4 from + III to + IV. Importantly, water molecules in the “O1” and “O4” channels, defined as such because they start from the O1 and O4 oxygens of the cluster, appeared localized slightly differently in S 2 and S 1 . In contrast, in the S 2 to S 3 transition, major structural changes have been detected together with the insertion of a 6th oxygen (named either O6 or Ox), possibly that one of W3 originally bound to the Ca site, bridging Mn1 and Ca. This oxygen is supposed to correspond to the second water substrate molecule and is close to the bridging oxygen O5 supposed to be the first water substrate molecule [ 18 ]. An important movement of the Glu189 residue would allow its carboxylate chain to make a hydrogen bond with the protonated form of the 6th oxygen in S 3 [ 16 , 17 ]. EPR data studies show the existence of multiple structural forms for each of the S 1 , S 2 , and S 3 states. The S 1 EPR signals seen with a parallel mode detection at g ~ 4.8 and g ~ 12 [ 19 – 21 ] were attributed to an orientational Jahn–Teller isomerism of the dangler Mn4 with the valence + III [ 22 ]. In S 2 , at least two EPR signals can be detected at helium temperatures. The first one has a low-spin S = 1/2 value, S 2 LS , characterized by a multiline signal (ML) made up of at least 20 lines separated by approximately 80 gauss, centered at g ~ 2.0 and spread over roughly 1800 gauss [ 23 – 25 ]. The second configuration of S 2 is a high-spin ground state, S 2 HS , with S ≥ 5/ 2. In plant PSII, S 2 HS may exhibit either a derivative-like EPR signal centered at g ~ 4.1 [ 26 , 27 ] or more complex signals at higher g values [ 28 , 29 ]. In cyanobacterial PSII, the g ~ 4.1 signal is not detected. Instead, the S 2 HS EPR signal has a derivative-like shape centered at g ~ 4.8 [ 30 , 31 ]. An influential computational study proposed that the g ~ 4.1 signal has almost the same coordination and environment as the g ~ 2.0 ML signal, but with the Mn III ion located on the dangler Mn4 in the S 2 HS state instead on the Mn1 in the S 2 LS state [ 32 ]. This valence swap would be accompanied by a moving of the oxygen O5 from a position where it links the Mn4, Mn3 and the Ca in the S 2 LS configuration to a position where it bridges the Mn1, Mn3, and Ca ions in the S 2 HS configuration resulting in the so-called closed cubane structure. However, this proposed closed cubane structure for the S 2 HS configuration has never been observed by XFEL studies of PSII, either from plants or cyanobacteria, in conditions where the S 2 HS is known to be trapped at low temperatures. For cyanobacteria, this result is not surprising since in this PSII the g ~ 4.1 signal has never been observed until now. Yet, it remains possible that the proposed configuration is a short-lived transient that the time-resolved studies have not captured due to limited post-flash sampling frequency. On the other hand, other computational works [ 33 – 35 ] led to a different model, that, starting from the S 2 LS configuration, the protonation of O4 would lead to an S = 5/2 ground state when W1, one of the two water molecules with W2 bound to the dangler Mn4, is present as an aquo ligand. The further deprotonation of W1 to form a hydroxo ligand would then give rise to an S = 7/2 ground state [ 33 – 35 ]. It was further proposed that the form S = 7/2 was required to progress to S 3 . Importantly, the S 2 HS form detected at g ~ 4.8 form, i.e. the S = 7/2 form, that corresponds to an open cubane structure in [ 33 – 35 ], is able to progress to S 3 at low temperatures [ 30 , 31 ], whereas in plant PSII the g ~ 4.1 state cannot [ 36 ]. The S 3 -state also exhibits some heterogeneities based on EPR analysis. Most centers exhibit a spin S = 3 ground state [ 37 – 39 ]. In this S = 3 configuration, the four Mn ions of the cluster have an Mn IV formal oxidation state with an octahedral ligation sphere in an open cubane structure [ 39 ]. In this model, the dangler Mn IV ( S = 3/2) is antiferromagnetically coupled to the open cubane motif Mn IV 3 with a total spin value S = 9/2. The remaining centers are EPR invisible, e.g. [ 40 ]. A third S 3 configuration with a broadened S 3 signal was identified with ELDOR-detected NMR (EDNMR) in the presence of glycerol [ 41 , 42 ] and in PSII/Sr [ 43 ]. Although in [ 43 ] the authors did not completely rule out the presence of a closed cubane, five-coordinate S 3 form, at the origin of this EPR signal, they favored a perturbation of the coordination environment at Mn4 and/or Mn3 in an open cubane S 3 structure induced by glycerol. With X-and Q-band EPR experiments performed in the S 3 -state of plant PSII, both in the perpendicular and parallel modes, a high-spin, S = 6, was proposed to coexist with the S = 3 configuration. This S = 6 form was attributed to a form of S 3 without O6/Ox bound and with the Mn IV 3 part of the cluster in ferromagnetic interaction with the unsaturated dangler Mn IV [ 42 ]. These two forms of S 3 are, however, not detected by X-band EPR, so it seems unlikely that they correspond to the EPR invisible S 3 mentioned above. Indeed, these new S 3 signals described in [ 41 , 42 ] are detectable in the presence of glycerol and methanol, whereas the formation of the (S 2 Tyr Z ● )′ state upon a near-IR illumination in the centers in S 3 defined as EPR invisible is inhibited in the presence of glycerol (and in the presence of methanol in plant PSII) [ 44 , 45 ]. In cyanobacterial PSII/Sr [ 43 ], a proportion of centers exhibited a pulsed W-band field-swept S 3 spectrum much broader than in PSII/Ca. This signal was proposed to be present in centers containing a 5-coordinate Mn ion in centers in which no water binding event takes place during the S 2 to S 3 transition. It was therefore proposed that, in these centers, the oxidation event would precede the water binding. Computational analyses also suggested heterogeneities in S 3 [ 46 – 48 ] with also a S = 6 spin value [ 48 ]. None of the heterogeneities described above were detected in the crystallographic structures of S 2 and S 3 known to date (see references above). In addition, recent high-energy resolution fluorescence detected X-ray Absorption Spectroscopy together with QM calculations ruled out the presence of either peroxo or oxo/oxyl level intermediate to explain the S 3 heterogeneity [ 49 ]. It is quite possible that some of the structural differences that cause the differences identified in EPR are too small to be detectable given the resolution of the crystallographic data. This at least shows, if it were necessary, that spectroscopy remains an indispensable complement to crystallography. The EPR data summarized above describes a static view of the trapped configurations. Kinetically, it is well documented that the transition from S 2 to S 3 involved at least two phases. The fastest phase, with a t 1/2 ≤ 25 µs, is attributed to a proton transfer/release. This fast phase precedes the electron transfer from S 2 to the Tyr Z ● occurring with a t 1/2 ≤ 300 µs [ 50 – 54 ], and the binding of O6/Ox to the Mn1, e.g. [ 55 ]. It was proposed that the fast phase could correspond to the release of a proton in an intermediate step S 2 LS Tyr Z ● → S 2 HS Tyr Z ● before the S 2 HS Tyr Z ● → S 3 S = 3 transition occurs [ 30 ]. The existence of intermediate states in the S 2 to S 3 transition was tracked by following, at room temperature, the structural changes in the S 2 to S 3 transition in the µs to ms time-range after the 2nd flash [ 17 , 55 ]. No indication was found for a closed cubane intermediate. However, there was no conclusion on the spin state of the intermediate forms of S 2 able to progress to S 3 . By definition, an intermediate state has a low concentration that makes its detection difficult and thus, the question of the existence of a high spin intermediate state in the S 2 to S 3 transition remains. It is possible that the S 2 HS corresponds to a subtle structural/tautomeric intermediate that nonetheless mediates proton release during the S 2 LS Tyr Z ● → S 2 HS Tyr Z ● as a prerequisite to the formation of the S 3 S = 3 state. It has been suggested that the fast phase observed in this transition corresponds to a proton release/movement associated with the formation of a S 2 HS state [ 30 , 36 ]. If this is correct, we would expect to detect a change in the flash pattern of the proton release in conditions in which the S 2 HS EPR signal is the flash-induced state. This was indeed the case since we have kinetically detected a proton release in PSII/Ca and PSII/Sr at pH 6.0 and 7.0, knowing that at pH 7.0, in PSII/Sr in contrast to PSII/Ca, half of the centers exhibit the S 2 HS signal at g ~ 4.8 able to progress to S 3 at low temperatures [ 36 ]. Independently of the pH effect and Ca/Sr exchange, several mutations, the list being too long for making it here, in the first and second coordination sphere of the Mn 3 O 4 Ca unit strongly perturb the water splitting process. One of these mutants is the D1/V185N mutant in Synechocystis 6803 [ 56 – 59 ]. The D1/V185 residue is located adjacent to the cluster near three potential substrate candidates (W2, W3 and O5). It may also block or regulate the access to the open coordination site at Mn1. The D1/V185N mutation in Synechocystis sp. PCC 6803 slows down the release of O 2 from 1.2 ms to about 27 ms halftime at 27°C. In addition, the rate of exchange for the slower exchangeable water substrate molecule, Ws, was increased in the S 1 and S 2 states in the D1/V185N mutant, while both Wf, the faster exchangeable water substrate molecule, and Ws exchange rates were decreased in the S 3 state in this mutant [ 59 ]. In contrast to the situation in the V185T mutant in Synechocystis 6803 in which the oxygen release kinetics was hardly affected (from 1.2 ms to 1.5 ms) [ 57 ], the D1/V185T mutation in T. vestitus resulted in similar phenotype to those in the D1/V185N in Synechocystis sp. PCC 6803 [ 60 ]. In the D1-V185T mutant in T. vestitus we found that the S 2 state was mostly present in a high spin configuration [ 60 ] although a modified multiline was also observed. This observation triggered us to probe the proton release in the S 1 to S 2 transition in the context of the model mentioned above where the proton released in the S 2 to S 3 transition occurs in the S 1 Tyr Z ● →S 2 HS Tyr Z ● transition prior to the S 2 HS Tyr Z ● to S 3 transition [ 30 , 36 ]. One of the problem here is that measuring the proton release kinetics by using a dye as we did in [ 60 ] required an extensive washing of the PSII samples to remove all the buffers, a treatment that has deleterious effects on the particularly fragile D1/V185T mutant. In so doing, we observed a rapid phase of proton release into the bulk, the kinetics of which was close, and difficult to distinguish from that observed following oxidation of Tyr Z at pH 6.3 in Mn-depleted PSII. To get around this problem, we also measured the electrochromic band shifts induced by each of the flashes in a sequence that is an alternative way to follow the movement of the charges in and around the Mn 4 CaO 5 cluster [ 36 , 54 ]. As the µs components kinetics in the P 680 + reduction reflect the progressive shift to the left of the equilibrium P 680 + Tyr Z ↔ P 680 Tyr Z ● resulting from slower proton release/movements [ 61 – 63 ], we also measured the P 680 + decay in the D1/V185T mutant in T. vestitus . Recently [ 59 ], it was reported that the high spin EPR signal in the S 2 state of the D1/V185N PSII in Synechocystis 6803 was not detectable. We report here, in an independent EPR experiment, that the S 2 HS EPR signal in the D1/V185N PSII from Synechocystis 6803 is clearly present. This combined with the retardation in the S n state transition reaction kinetics, now provides a consistent picture of the effects mutations at this second sphere location and reinforces criticality of the H-bonding network in in mediating the catalysis of water oxidation. Materials and methods Purification of the D1-V185N mutant from Synechocystis 6803 was previously described in [ 56 ]. The D1 protein is PsbA2 in this mutant strain. Purification of the D1/V185T mutant from T. vestitus was previously described in [ 60 ]. The D1/V185T mutant has been constructed in the psbA 3 gene so that the wild type PSII is that one with PsbA3. Time-resolved absorption changes measurements were performed with a lab-built spectrophotometer [ 64 ] slightly modified as described in detail in [ 65 ]. For the ΔI/I measurements the samples were diluted in 1 M betaine, 15 mM CaCl 2 , 15 mM MgCl 2 , and 40 mM MES (pH 6.5). PSII samples were dark-adapted for ~ 1 h at room temperature (20–22°C) before the addition of 0.1 mM phenyl p –benzoquinone (PPBQ) dissolved in dimethyl sulfoxide. The chlorophyll concentration of all the samples was ~ 25 µg of Chl mL − 1 . After the measurements, the ΔI/I values were normalized to exactly A 673 = 1.75, that is very close to 25 µg Chl mL − 1 . The measurements with the dye bromocresol purple were done as previously reported after the removal of Mes [ 36 ]. X-band cw-EPR spectra were recorded with a Bruker Elexsys 500 X-band spectrometer equipped with a standard ER 4102 (Bruker) X-band resonator, a Bruker teslameter, an Oxford Instruments cryostat (ESR 900) and an Oxford ITC504 temperature controller. The samples, at ~ 1.35 mg Chl mL − 1 , were dark-adapted for 1 h on ice then the samples were frozen in the dark (without any addition) to 198 K in a dry-ice/ethanol bath and then transferred into liquid N 2 (77 K). Prior to recording the spectra, the samples were degassed at 198 K. The S 2 state was induced by illumination with an 800 W tungsten lamp filtered by water and infrared cut-off filters for approximately 5–10 s at 198 K in a non‑silvered Dewar in ethanol cooled with dry ice. Results As the interpretation of the data strongly depends on the presence of an S 2 HS state in the D1/V185N mutant in Synechocystis 6803, something that was contested in [ 59 ], we will firstly show the EPR spectra recorded in this strain. Figure 1 compares the spectra in the PSII from Synechocystis 6803 WT (Panel A), and from the S 6803 D1/V185N-PSII (Panel B). The black spectra were recorded in the dark-adapted samples, the red spectra after continuous illumination at 198 K for 5–10 s, and the blue spectra are the light- minus -dark difference spectra. In the WT sample (Panel A) the light-induced state exhibits a normal S 2 LS multiline signal similar to that induced in plant PSII and cyanobacterial PSII [ 10 ]. In contrast, in the D1/V185N PSII (Panel B), the shape and the amplitude of the multiline signal differs significantly from that one in the WT PSII. The S 2 multiline signal in the D1/V185N sample is very similar, if not identical, to the S 2 multiline signal in the D1/V185T mutant in T. vestitus [ 60 ]. Importantly, a large S 2 HS EPR signal, centered at ~ 1700 gauss, was induced by the illumination at 198 K in the D1/V185N mutant. This S 2 HS EPR signal is also virtually identical to the S 2 HS EPR signal detected in the D1/V185T mutant in T. vestitus [ 60 ]. In the black and red spectra other signals are detected. The g z and g y of Cyt c 550 are detected at ~ 2300 and ~ 3000 gauss, respectively. The two small negative signals in the blue spectra, between ~ 800 and ~ 1000 gauss, correspond to the negative non-heme iron signal. This means that the non-heme iron was oxidized in a proportion of centers in the dark-adapted state and reduced by the Q A − formed by the illumination at 198 K [ 66 ]. The large narrow signal at around 1600 gauss is due to contaminating Fe 3+ . In both samples, the g ~ 1.9 form of the Q A − Fe 2+ Q B signal (~ 3700 gauss) and the Q A − Fe 2+ Q B − signal at g ~ 1.6 (~ 4400 gauss) are detected. The change in their relative proportion is likely due to a different proportion of the Q A Fe 2+ Q B − state in the dark adapted samples. In the black spectra, the Q A Fe 2+ Q B − signal is possibly present at ~ 4200 gauss and indeed larger in the mutant, i.e. [ 67 ]. In conclusion, an unequivocally similar S 2 HS EPR signal is formed in the D1/V185N PSII from Synechocystis 6803 and the D1/V185T PSII mutant from T. vestitus mutant [ 60 ]. Knowing with certainty that both mutants exhibit an S 2 HS state at pH 6.5, the question about a proton release into the bulk in the S 1 to an S 2 state with such S 2 HS EPR properties should be addressed again. Firstly by measuring the proton/release using the dye bromocresol purple. Secondly by recording the electrochromic band-shifts in the Soret region of the P D1 absorption spectrum at 440 nm. Indeed, this measurement takes into account both the proton uptake/release and the electron transfer events, e.g. [ 54 , 68 ] and references therein. In addition, unlike the situation with the dyes, the electrochromism is not contaminated by the protonation/deprotonation events coupled to the reduction/oxidation of the non-heme iron. For the removal of the contributions due to the reduction of Q A the ΔI/I at 424 nm was also measured. Indeed, the electrochromism due to Q A − equally contributes at 440 nm and 424 nm [ 68 ]. These measurements can be compared to the proton uptake/release kinetics again measured with bromocresol purple as in [ 60 ]. Panels A and B in Fig. 2 shows the absorbance changes of bromocresol purple in PsbA3-PSII and PsbA3/V185T-PSII, respectively, both from T. vestitus . An uptake of protons by PSII induces an increase in the ΔI/I at 575 nm, and a release of protons into the bulk by PSII induces a decrease of the ΔI/I. After the first flash (black points), there was a large increase in the absorption in both samples. This increase is due to the proton uptake following the reduction of the non-heme iron [ 36 ]. In PsbA3-PSII (Panel A) there is no detectable proton release associated with the S 1 to S 2 transition as already seen in such a PsbA3-PSII [ 36 ]. This is consistent with a large body of experiments indicating the absence of proton release in this transition, and with a 1-0-1-2 pattern of proton release for the S 0 to S 1 , S 1 to S 2 , S 2 to S 3 and S 3 to S 0 transitions, reviewed in [ 36 , 56 ]. In contrast with our previous study [ 60 ], however, the decrease in the ΔI/I in the 10 µs to 100 µs time range is very small, suggesting that a proton release, if any, occurs in a very low proportion of the centers. This lack of proton release is very likely due to the use of a PsbA3/V185T-PSII sample less damaged by the washing steps compared to the earlier study. Additionally, proton uptake is detected after each flash as previously reported [ 36 ] explaining the drift in the signal (not subtracted in Panels A and B) observed on each flash. The proton release kinetics in the PsbA3/V185T-PSII in the S 2 to S 3 transition (red points) and in the S 0 to S 1 transition (green points) appears hardly affected when compared to PsbA3-PSII (~ 30–40 µs, and ~ 100–200 µs, respectively). In the S 3 to S 0 transition, the release of the first proton is also hardly affected in the mutant. In contrast, the release of the second proton is strongly slowed down with a kinetics similar to the O 2 release (~ 20–30 ms) [ 56 , 60 ]. Panel C in Fig. 2 shows the kinetics of the 440 nm- minus -424 nm difference in the PsbA3/V185T-PSII from T. vestitus . The results can be compared to those obtained in PsbA3-PSII that have been described in details previously [ 36 , 54 ]. After the first flash (black points), the kinetics that corresponds to the S 1 Tyr Z ● to the S 2 Tyr Z transition is slightly slowed down from 10–20 µs to 50–60 µs in the PsbA3/V185T mutant. After the second flash (red points) the kinetics is biphasic with a fast phase ( t 1/2 ~ 10–15 µs) that we have attributed to a proton movement coupled to the S 2 LS Tyr Z ● to S 2 HS Tyr Z ● transition, and the slow phase was attributed to the S 2 HS Tyr Z ● to the S 3 Tyr Z transition [ 36 ]. In the PsbA3/V185T-PSII mutant the two phases are not really differentiated, which makes the interpretation difficult even if this result could suggest a slowing down of the proton release in the S 2 to S 3 transition. In the PsbA3-PSII, after the fourth flash, the kinetics is also biphasic with the fast phase corresponding to the electron transfer in the S 0 to S 1 transition ( t 1/2 ~ 10–15 µs), which precedes the proton release with a t 1/2 ~ 200 µs [ 36 , 50 , 54 ]. In the PsbA3/V185T-PSII, after the fourth flash (green points), although the two phases are also difficult to be detected due to a less good signal-to-noise ratio, and an increase miss parameter (see however below), the two phases seem to have more or less the same kinetics. The big difference between Panel A and Panel B is the slow phase after the third flash (blue points) with a t 1/2 ~ 20–30 ms in D1/V185T-PSII, which is similar to that of the proton release in this sample. After the 4th flash the slow decay certainly originates from a residual S 3 to S 0 transition due to the misses but we have no clear explanation for the slow drift after the 1st and 2nd flash since the redox events at the non-heme iron do not contribute in the electrochromism of P D1 . Each event, such as a change in the kinetics of electron and proton motion, should affect the P 680 + Tyr Z ↔ P 680 Tyr Z ● equilibrium and therefore the P 680 + decay kinetics. Panels A, B, C, and D in Fig. 3 show the P 680 + reduction kinetics measured at 432 nm in PsbA3-PSII (black), and PsbA3/V185T-PSII (red) after the 1st flash (Panel A), the 2nd flash (Panel B), the 3rd flash (Panel C) and the 4th flash (Panel D). In all the S n states, the decay of P 680 + in the PsbA3/V185T mutant is significantly slower both in the ns time-domain corresponding to the pure electron transfer step, and the µs time-domain where the electron transfer(s) is(are) coupled to proton movements. The P 680 + Tyr Z ↔ P 680 Tyr Z ● is therefore shifted to the left in the PsbA3/V185T mutant when compared to the PsbA3-PSII in all the S n states [ 61 , 63 ] and we expect that some of the proton movements coupled to the electron transfer are also slowed down. Panel E in Fig. 3 shows the DI/I measured 20 ns after each flash of the sequence in the PsbA3-PSII (black) and in the PsbA3/V185T-PSII (red). Due to the slower decay of P 680 + in the S 2 and S 3 states [ 69 ] than in the S 0 and S 1 states we obtain an oscillation with the period of four. Since in all the S n states the decay of P 680 + is faster in PsbA3-PSII than in PsbA3/V185T-SII, the DI/I values in PsbA3-PSII, 20 ns after the flashes, are smaller (less negative) in all the S n states. However, the oscillations are similar in both samples, which indicates that in the PsbA3/V185T-PSII used here the miss parameter is not significantly larger than in the PsbA3-PSII. Discussion One of the main results shown in this work is the presence of a S 2 HS in the D1/V185N-PSII from Synechocystis 6803, with EPR characteristics strikingly similar to those observed in the D1/V185T-PSII mutant from T. vestitus [ 60 ]. The discrepancy between the EPR spectra shown here and those in [ 59 ] is likely attributable to baseline distortions in the low-field region of the spectra in [ 59 ]. Such distortions hinder the clear detection of the S 2 HS signal. By addressing this technical issue, we demonstrate the consistent appearance of the S 2 HS state in these mutants, reinforcing the idea that the D1/V185 residue influences the high-spin/low-spin equilibrium of the S₂ state in a similar manner across different cyanobacterial species and supports a consistent mechanism by which a perturbation of this site modulates the still enigmatic S 2 HS state in PSII. Because these are substitutions of a hydrophobic with polar residues, they introduce H-bonding potential at a location adjacent to the insertion point of substrate water O 6 /O x that arrives at the open coordination position of Mn1 during the S 2 → S 3 transition. Thus, the mutations almost certainly exert their effects by introducing aberrant H-bonding interactions that compete and thus perturb the native H-bond network, which has been evolutionarily optimized to enhance catalytic efficiency. The inferred mutational perturbation of native H-bonding may thus be at the basis of some of the observed EPR and kinetic alterations as discussed below. It has been shown that the equilibrium between the S 2 LS and S 2 HS states is pH dependent [ 30 ], with a pK a ~ 8.3 for the native Mn 4 CaO 5 cluster and a significantly shifted pK a of ~ 7.0 for the Mn₄SrO₅ cluster [ 36 ]. Alkaline conditions are thought to mimic the charged state of the Tyr Z ● , emulating the electrostatic proton ejection regime that facilitates progression through the Kok cycle [ 30 , 36 , 50 , 51 , 70 , 71 ]. Furthermore, there is some evidence that the Mn 4 CaO 5 must pass through an S 2 HS intermediate state before forming the S 3 state. For example, the S 2 HS state formed by the room temperature flash illumination could progress to S 3 under further continuous illumination at 198 K. Similarly, samples advanced to the S₂ LS state upon illumination at 198 K subsequently form the S 2 HS state when warmed to room temperature, enabling progression to the S₃ state under further illumination at 198 K. These observations have led to the tentative conclusion that the S 2 HS state is a necessary intermediate during the S 2 → S 3 transition. The lowered pK a of the Mn₄SrO₅ cluster explains why the S 2 HS to S₃ transition can occur in a significant proportion of centers even at pH 6.5. Interestingly, D1/V185T-PSII also exhibits a high proportion of centers with an S₂ HS signal at pH 6.5, suggesting that these centers could progress to the S₃ state under illumination at 198 K. However, this progression is not at all evident in D1/V185T-PSII with a Mn 4 CaO 5 cluster, as shown in spectrum a of Fig. 9 in [ 60 ]. In contrast, in D1/V185T-PSII with a Mn 4 SrO 5 cluster, the effect of the 198 K illumination was more pronounced (spectrum b in Fig. 9 in [ 60 ]). However, the S 2 HS EPR signal that disappeared in D1/V185T-PSII with a Mn 4 SrO 5 cluster upon the 198 K illumination had a shape similar to that in the wild type PSII (with both Ca and Sr in the cluster). This observation suggests some heterogeneity in the EPR properties of the S 2 HS state in D1/V185T-PSII with a Mn 4 SrO 5 cluster. Importantly, since the illumination at 198 K results in the formation of the S 2 HS and S 2 LS states in both species, it seems likely that each of these two states arises from two structurally different S 1 states. The previously mentioned possibility [ 60 ] for a S 2 HS to S 3 transition occurring at 198 K in D1/V185T-PSII seemed to be supported by the detection of a proton release on the first flash, see [ 36 ]. However, it is shown here that this proton release does not occurs in active D1/V185T-PSII but rather as originating from the oxidation of Tyr Z in a minor sub-population Mn-depleted centers in the D1/V185T-PSII used for proton release experiment in [ 60 ]. It can now be concluded that the S 2 HS state in D1/V185T-PSII, with EPR properties different from those in wild type PSII, is unable to progress to S 3 at 198 K, most likely because its protonation state differs. The S 2 HS EPR signal in the D1/V185T-PSII from T. vestitus , and the D1/V185N-PSII from Synechocystis 6803, have a shape reminiscent of the spectrum induced by near-infrared illumination of the S 2 HS state at high pH in PsbA3-PSII see Fig. 3 in [ 72 ]. Presently, there is no explanation of the near-infrared illumination however this similarity could suggest that the two states only differ by small structural changes, such as shifts in the H-bonding and proton tautomer configurations. Functionally significant hydrogen bonding changes due to mutations at D1/V185 likely affect the stabilization and dynamics of substrate water molecules in the S 3 state based upon previous findings [ 56 , 58 , 59 ]. The slowdowns of both the lag and oxygen release phase during the S 3 to S 0 transition have been attributed to changes in the H-bond network that correspond to a suboptimal configuration of reactants and an associated entropic penalty for crossing the activation barrier of the transition state [ 56 ]. The observation of slower water exchange in S 3 [ 59 ] has been suggested to be the result of the stabilization of water molecules due to altered H-bonding around the inserted substrate water, O 6 /O x . However, the mechanism of substrate exchange remains to be elucidated. As noted, D1/V185T and D1/V185N mutations replace the non-polar valine side chain with threonine or asparagine, which can introduce non-native hydrogen bonds to the putative O 6 /O x substrate and with water molecules ( e.g. W3). This disruption likely affects both water binding and proton release. The findings presented here underscore the central role of the H-bond network in modulating the LS-to-HS transition and subsequent steps in the Kok cycle. In Ca-PSII, the LS-to-HS transition is characterized by cooperative interactions among multiple protonatable groups, as evidenced by the best-fit value of n ~ 4.4 [ 30 ], although only one proton is ultimately released to the bulk solution [ 36 ]. Here, we can speculate that this cooperativity lowers the transition state barrier for proton release rather than stabilizing the deprotonated HS state itself. According to this view, the well-organized H-bond network facilitates a collective reorganization that creates an energetically favorable pathway for the LS-to-HS transition, ensuring efficient coupling of proton dynamics with substrate water insertion at the Mn₄CaO₅ cluster. This may provide a view of some shared characteristics of proton transfer during both the S 2 → S 3 and S 3 → S 0 transitions. This S 2 → S 3 transition also coincides with the critical insertion of W3—initially coordinated to Ca²⁺—into the open coordination site on Mn1. This step is crucial for positioning W3 to become Ox, one of the two substrate waters for the water oxidation reaction. The timing and coordination of W3 insertion and its deprotonation is expected to be strongly influenced by the surrounding H-bond network. In the LS state, the network stabilizes W3 on Ca²⁺ in a protonated form, maintaining readiness for the subsequent catalytic steps. Upon insertion into Mn1 following the LS-to-HS transition, the reorganization of the H-bond network facilitates the deprotonation of W3, with the cooperative action lowering the activation barrier for this step and ensuring the system is primed for subsequent oxidation chemistry. Similarly, simulations in [ 73 ] emphasize that concerted proton shuffling between O6 and the waters W3 and W2, along with the extremely wide preorganization of an extended hydrogen bond network, is mandatory for Tyr Z ● reduction in the S 3 → S 0 transition. While a similar proton cooperativity to that seen in the formation of the S 2 HS state, such a cooperative transition on the HBN and its disruption by mutations or Sr-substitution could also account, for the slowdown of the S 3 → S 0 transitions and help explain the increase in the entropic penalty and H/D effects previously observed for V185N. This insight aligns with the idea that efficient catalysis requires not just local H-bond interactions but a globally integrated network capable of synchronizing proton and water dynamics across the system. Disruptions to this network, as seen in D1/V185 mutations and Sr²⁺ substitutions, likely hinder the necessary concerted movements, leading to slowed transitions and impaired water oxidation. Consistent with this interpretation, we observe that the proton release during the S 3 → S 0 transition in the D1/V185 mutants was primarily affected by the second of the two protons released during that transition. In contrast, the first proton release, which occurs rapidly following the formation of the tyrosine radical, Tyr Z ● , remains largely unaffected. This initial fast proton release is proposed to be linked to the electrostatic impact of uncompensated charge formation upon the effects of the formation of Tyr Z OH-His190 → Tyr Z O ● -H + His190 [ 70 , 71 ]. Functionally, this is proposed to occur to prepare Asp61D1 as a proton acceptor for the slower, second proton release from substrate water O6 [ 70 , 71 , 73 , 74 ]. The transient electric field during the Tyr Z O ● -H + His190 state is calculated to promote proton transfer from W1 to Asp61, inducing rearrangements in the cluster of residues D1-Asp61, D1-Glu65, and D2-Glu312 into a configuration that facilitates rapid proton egress to the bulk [ 70 , 71 , 74 ]. The rapid exit of this proton is likely gated, preventing back-reactions. Once deprotonated, the cluster forms the base that accepts the proton from substrate water O6, facilitating the final electron transfer leading to O 2 formation [ 73 ]. The fact that the fast proton release remains unaffected by the mutation, while the second proton release is delayed, is consistent with the D1/V185 residue being distant from the fast proton release vector but positioned near the substrate water (O6), where deprotonation occurs. Thus, the delayed second proton release in the mutants is attributed to the disruption of the hydrogen-bonding network around O6, which slows the proton transfer pathway necessary for its deprotonation and the final stages of water oxidation. Abbreviations Photosystem II PSII Chl chlorophyll Chl D1 /Chl D2 monomeric Chl on the D1 or D2 side,respectively MES 2-( N -morpholino) ethanesulfonic acid P 680 primary electron donor P D1 and P D2 individual Chl on the D1 or D2 side,respectively,which constitute a pair of Chl with partially overlapping aromatic rings Phe D1 and Phe D2 pheophytin on the D1 or D2 side,respectively PPBQ phenyl p –benzoquinone Q A primary quinone acceptor Q B secondary quinone acceptor Tyr Z the tyrosine 161 of D1 acting as the electron donor to P 680 WT*3 T. elongatus mutant strain deleted psbA 1 and psbA 2 genes and with a His-tag on the carboxy terminus of CP43. EPR,Electron Paramagnetic Resonance. ML,multiline. Declarations Acknowledgements This work has been in part supported by (i) the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05, (ii) the Labex Dynamo (ANR-11-LABX-0011-01), (iii) the JSPS-KAKENHI Grant in Scientific Research on Innovative Areas JP17H064351, JSPS-KAKENHI Grant 21H02447 and a grant from the US- National Science Foundation (MCB1716408). One of the PSII preparations used in this work was done by T. Tibiletti.. References Shevela D, Kern JF, Govindjee G, Messinger J (2023) Solar energy conversion by photosystem II: principles and structures. Photosynth Res 156:279–307. https://doi.org/10.1007/s11120-022-00991-y Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Crystal structure of oxygen evolving Photosystem II at a resolution of 1.9 angstrom. 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Phys Chem Chem Phys 6:4838–4843. https://doi.org/10.1039/b407656g Christen G, Renger G (1999) The role of hydrogen bonds for the multiphasic P 680 + reduction by Y Z in photosystem II with intact oxygen evolution capacity: analysis of kinetic H/D isotope exchange effects. Biochemistry 38:2068–2077. https://doi.org/10.1021/bi982188t Béal D, Rappaport F, Joliot P (1999) A new high-sensitivity 10-ns time-resolution spectrophotometric technique adapted to in vivo analysis of the photosynthetic apparatus. Rev Sci Instrum 70:202–207. https://doi.org/10.1063/1.1149566 Sugiura M, Taniguchi T, Tango N, Nakamura M, Sellés J, Boussac A (2021) Probing the role of arginine 323 of the D1 protein in photosystem II function. Physiol Plant 171:183–199. https://doi.org/10.1111/ppl.13115 Zimmermann J-L, Rutherford AW (1986) Photoreductant-induced oxidation of Fe 2+ in the electron-acceptor complex of Photosystem II. Biochim Biophys Acta 851:416–423. https://doi.org/10.1016/0005-2728(86)90078-2 Fufezan C, Zhang C, Krieger-Liszkay A, Rutherford AW (2005) Secondary quinone in Photosystem II of Thermosynechococcus elongatus : Semiquinone – Iron EPR signals and temperature dependence of electron transfer. Biochemistry 44:12780–12789. https://doi.org/10.1021/bi051000k Rappaport F, Lavergne J (1991) Proton release during successive oxidation steps of the photosynthetic water oxidation process - stoichiometries and pH-dependence. Biochemistry 30:10004–10012. https://doi.org/10.1021/bi00105a027 Brettel K, Schlodder E, Witt HT (1984) Nanosecond reduction kinetics of photooxidized chlorophyll-a II (P-680) in single flashes as a probe for the electron pathway, H + -release and charge accumulation in the O 2 -evolving complex. Biochim Biophys Acta 766:403–415. https://doi.org/10.1016/0005-2728(84)90256-1 Allgöwer F, Gamiz-Hernandez AP, Rutherford AW, Kaila VRI (2022) Molecular principles of redox-coupled protonation dynamics in Photosystem II. J Am Chem Soc 144:7171–7180. https://doi.org/10.1021/jacs.1c13041 Allgöwer F, Pöverlein MC, Rutherford AW, Kaila VRI, BioRxiV https://doi.org/10.1101/2024.07.03.602004 Boussac A (2019) Temperature dependence of the high-spin S 2 to S 3 transition in Photosystem II: Mechanistic consequences, Biochim. Biophys Acta 1860:508–519. https://doi.org/10.1016/j.bbabio.2019.05.001 Dau H, Greife P (2023) Applicability of transition state theory to the (proton-coupled) electron transfer in photosynthetic water oxidation with emphasis on the entropy of activation. Inorganics 11:389. https://doi.org/10.3390/inorganics11100389 Noguchi T (2024) Mechanism of proton transfer through the D1-E65/D2-E312 gate during photosynthetic water oxidation, J. Phys. Chem. B 128 1866 – 1875. https://doi.org/10.1021/acs.jpcb.3c07787 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5504214","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381372519,"identity":"7d7b2a64-156c-4893-b7c0-7debf0b874ec","order_by":0,"name":"Alain Boussac","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYDACdgaGA2AEBhUWRGhhRtFyRgJEMjaASB48WhjgWhjbiNDC38z88HABwx05g/PHH374OU9C3py9/fmDD0ARexxaJA6zGRyewfDM2OBGjrFk7zYJw509ZwwbQSI4HXaYweAwD8PhxA03eBikGbdJMG64kcPYDBLpwaFD/jD7B4iW88cf/2acI2G/4Ub6w+Y/DIfrcWkBWgG15UCCmTRjgwTQugTDZqDtCbgcZniYpwCo65mx5I0cM8ueYxLJIL/M7DE4bNhzALsWuePtmz/zVNyR4wM67MaPGhvb7eztDz78qDgsz96Ay/9g52GwDbArJKx9FIyCUTAKRgEIAAB5z2BN4ss/DQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3441-3861","institution":"I2BC, CEA/CNRS","correspondingAuthor":true,"prefix":"","firstName":"Alain","middleName":"","lastName":"Boussac","suffix":""},{"id":381372520,"identity":"2b9d5ab4-a548-4ad1-8631-e08f53390d66","order_by":1,"name":"Julien Sellés","email":"","orcid":"https://orcid.org/0000-0001-9262-8257","institution":"Institut de Biologie Physico-Chimique, UMR CNRS 7141 and Sorbonne Université","correspondingAuthor":false,"prefix":"","firstName":"Julien","middleName":"","lastName":"Sellés","suffix":""},{"id":381372521,"identity":"8d5afd9c-dc04-497c-a479-78e57fb5306a","order_by":2,"name":"Miwa Sugiura","email":"","orcid":"https://orcid.org/0000-0002-4232-5941","institution":"Proteo-Science Research Center, Department of Chemistry, Graduate School of Science and Technology, Ehime University","correspondingAuthor":false,"prefix":"","firstName":"Miwa","middleName":"","lastName":"Sugiura","suffix":""},{"id":381372522,"identity":"0937f0de-2ada-417d-ab53-85c1f2d24979","order_by":3,"name":"Robert L. Burnap","email":"","orcid":"https://orcid.org/0000-0001-6715-5961","institution":"Department of Microbiology and Molecular Genetics, Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"L.","lastName":"Burnap","suffix":""}],"badges":[],"createdAt":"2024-11-22 11:23:28","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5504214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5504214/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69805164,"identity":"008987d7-61da-4b2f-95c2-77302e34914c","added_by":"auto","created_at":"2024-11-25 11:41:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15102,"visible":true,"origin":"","legend":"\u003cp\u003eEPR spectra recorded at 8.6 K in WT PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 (Panel A) and D1/V185N PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 (Panel B). The black spectra were recorded in dark-adapted PSII. The red spectra were recorded after an illumination at 198 k for 5-10 s. The blue spectra are the “light”-\u003cem\u003eminus\u003c/em\u003e-“dark” difference spectra. Instrument settings: [Chl] ~ 1.35 mg /ml; modulation amplitude, 25 G; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. The unresolved spectral region at g ~ 2 (~ 3390 gauss) corresponds to the saturated signal from Tyr\u003csub\u003eD\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Diapositive1.png","url":"https://assets-eu.researchsquare.com/files/rs-5504214/v1/0bf717c0686a9736d8d7faba.png"},{"id":69805524,"identity":"51544c0b-1843-4327-868f-aded272a1480","added_by":"auto","created_at":"2024-11-25 11:49:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23307,"visible":true,"origin":"","legend":"\u003cp\u003eTime-courses of the absorption changes of bromocresol purple at 575 nm in PsbA3-PSII (Panel A) and PsbA3/V185T-PSII (Panel B) from \u003cem\u003eT. vestitus\u003c/em\u003e after the 1\u003csup\u003est\u003c/sup\u003e flash (black points), the 2\u003csup\u003end\u003c/sup\u003e flash (red points), the 3\u003csup\u003erd\u003c/sup\u003e flash (blue points), and the 4\u003csup\u003eth\u003c/sup\u003e flash (green points). Panel C; time-courses of the absorption change differences 440 nm-\u003cem\u003eminus\u003c/em\u003e-424 nm after the first flash (black), the second flash (red), the third flash (blue), and the fourth flash (green) given to dark-adapted PsbA3/V185T-PSII in the presence of 100 μM PPBQ with flashes spaced 400 ms apart.\u003c/p\u003e","description":"","filename":"Diapositive2.png","url":"https://assets-eu.researchsquare.com/files/rs-5504214/v1/d7de806716ca9ce6d9311135.png"},{"id":69805167,"identity":"265c160f-bb62-4b78-a5d8-8f0e767f94ab","added_by":"auto","created_at":"2024-11-25 11:41:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32609,"visible":true,"origin":"","legend":"\u003cp\u003eKinetics of flash-induced absorption changes measured at 432 nm in PsbA3-PSII (black), and PsbA3/V185T-PSII (red) from \u003cem\u003eT. vestitus\u003c/em\u003e after the 1\u003csup\u003est\u003c/sup\u003e flash (Panel A), the 2\u003csup\u003end\u003c/sup\u003e flash (Panel B), the 3\u003csup\u003erd\u003c/sup\u003e flash (Panel C) and the 4\u003csup\u003eth\u003c/sup\u003e flash (Panel D). The amplitudes of the kinetics were normalized to OD673nm = 1.75 (a Chl concentration of ~ 25 μg/ml). 100 μM PPBQ was added before the measurements were taken. Panel E shows the amplitude of the DI/I at 20 ns, and at 432 nm, after each flash of the sequence with flashes spaced 400 ms apart.\u003c/p\u003e","description":"","filename":"Diapositive3.png","url":"https://assets-eu.researchsquare.com/files/rs-5504214/v1/9521366ef24d9a0e2729d58e.png"},{"id":69806612,"identity":"664da221-86ef-4170-8895-28f78ccea85a","added_by":"auto","created_at":"2024-11-25 11:57:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":775882,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5504214/v1/08959700-4e78-4d48-94f5-607543fb119d.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eNew insights into the involvement of residue D1/V185 in Photosystem II function in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSynechocystis 6803\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThermosynechococcus vestitus\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOxygenic photosynthesis in cyanobacteria, algae and higher plants converts solar energy into the chemical bonds of sugars and oxygen [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Photosystem II (PSII) begins this process by splitting water to obtain electrons in the form of reduced quinone, while generating a proton gradient and releasing O\u003csub\u003e2\u003c/sub\u003e. The mature PSII binds 35 chlorophylls \u003cem\u003ea\u003c/em\u003e (Chl-\u003cem\u003ea\u003c/em\u003e),two pheophytins (Phe-\u003cem\u003ea\u003c/em\u003e), one membrane b-type cytochrome, one extrinsic c-type cytochrome (in cyanobacteria and red algae), one non-heme iron, two plastoquinones (Q\u003csub\u003eA\u003c/sub\u003e and Q\u003csub\u003eB\u003c/sub\u003e), the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster, 2 Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, 12 carotenoids and 25 lipids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the cyanobacterium \u003cem\u003eSynechocystis sp.\u003c/em\u003e PCC 6803 a 4th extrinsic subunit, PsbQ, has also been found in addition to PsbV, PsbO and PsbU [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the 35 Chl-\u003cem\u003ea\u003c/em\u003e, 31 are antenna Chls. When one is excited, the excitation energy is transferred to other chlorophylls until it reaches the key pigments in the photochemical reaction center: 4 Chl-\u003cem\u003ea\u003c/em\u003e molecules, P\u003csub\u003eD1\u003c/sub\u003e, P\u003csub\u003eD2\u003c/sub\u003e, Chl\u003csub\u003eD1\u003c/sub\u003e, Chl\u003csub\u003eD2\u003c/sub\u003e and 2 Phe-\u003cem\u003ea\u003c/em\u003e molecules, Phe\u003csub\u003eD1\u003c/sub\u003e and Phe\u003csub\u003eD2\u003c/sub\u003e. A few picoseconds after the formation of the excited *Chl\u003csub\u003eD1\u003c/sub\u003e, charge separation occurs, ultimately forming the Chl\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003ePhe\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and then P\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003ePhe\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e radical pair states, \u003cem\u003ee.g.\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Formation of the Chl\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003ePhe\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e radical pair was recently defined as a fast pathway (short-range charge-separation) in contrast with a slow pathway with P\u003csub\u003eD1\u003c/sub\u003eP\u003csub\u003eD2\u003c/sub\u003e as the initial donor (long-range charge separation) that would result directly in the formation of the P\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003ePhe\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e radical pair [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter the charge separation, P\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e oxidizes Tyr\u003csub\u003eZ\u003c/sub\u003e, the Tyr161 of the D1 polypeptide, which is then reduced by the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster. The electron on Phe\u003csub\u003eD1\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e is then transferred to Q\u003csub\u003eA\u003c/sub\u003e, the primary quinone electron acceptor, and then to Q\u003csub\u003eB\u003c/sub\u003e, the second quinone electron acceptor. Whereas Q\u003csub\u003eA\u003c/sub\u003e can be only singly reduced under normal conditions, Q\u003csub\u003eB\u003c/sub\u003e accepts two electrons and two protons before leaving its binding site and being replaced by an oxidized Q\u003csub\u003eB\u003c/sub\u003e molecule from the membrane plastoquinone pool, see for example [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] for a non-exhaustive list of recent reviews on PSII function. The Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster, oxidized by the Tyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e\u0026bull;\u003c/sup\u003e radical formed after each charge separation, cycles through five redox states denoted Sn, where n designates the number of stored oxidizing equivalents. The S\u003csub\u003e1\u003c/sub\u003e state is stable in the dark, which makes S\u003csub\u003e1\u003c/sub\u003e the preponderant state after the decay of the S\u003csub\u003e3\u003c/sub\u003e and S\u003csub\u003e2\u003c/sub\u003e states in the dark. When the S\u003csub\u003e4\u003c/sub\u003e state is formed, after the 3rd flash of light given on dark-adapted PSII, two water molecules bound to the cluster are oxidized, O\u003csub\u003e2\u003c/sub\u003e is released and the S\u003csub\u003e0\u003c/sub\u003e-state is reformed, [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThanks to the advent of serial femtosecond X-ray free electron laser crystallography and cryo-EM spectroscopy, structures of the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster have been resolved in the dark-adapted S\u003csub\u003e1\u003c/sub\u003e state, the S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e states, in a redox state as close as possible to those expected taking into account the misses under flash illumination. In the S\u003csub\u003e1\u003c/sub\u003e state, \u003cem\u003ei.e.\u003c/em\u003e in the Mn\u003csup\u003eIII\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003eMn\u003csup\u003eIV\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e redox state, the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e structure resembles a distorted chair, including a \u0026micro;-oxo-bridged cuboidal Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCa unit with a fourth Mn attached to this core structure \u003cem\u003evia\u003c/em\u003e two \u0026micro;-oxo bridges involving the two oxygen's O4 and O5 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recently, important progress has been made in the resolution of the crystal structures in the S\u003csub\u003e2\u003c/sub\u003e, S\u003csub\u003e3\u003c/sub\u003e [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and S\u003csub\u003e0\u003c/sub\u003e states [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Briefly, changes in the S\u003csub\u003e1\u003c/sub\u003e to S\u003csub\u003e2\u003c/sub\u003e transition more or less correspond to those expected for the valence change of the Mn4 from +\u0026thinsp;III to +\u0026thinsp;IV. Importantly, water molecules in the \u0026ldquo;O1\u0026rdquo; and \u0026ldquo;O4\u0026rdquo; channels, defined as such because they start from the O1 and O4 oxygens of the cluster, appeared localized slightly differently in S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e1\u003c/sub\u003e. In contrast, in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition, major structural changes have been detected together with the insertion of a 6th oxygen (named either O6 or Ox), possibly that one of W3 originally bound to the Ca site, bridging Mn1 and Ca. This oxygen is supposed to correspond to the second water substrate molecule and is close to the bridging oxygen O5 supposed to be the first water substrate molecule [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. An important movement of the Glu189 residue would allow its carboxylate chain to make a hydrogen bond with the protonated form of the 6th oxygen in S\u003csub\u003e3\u003c/sub\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEPR data studies show the existence of multiple structural forms for each of the S\u003csub\u003e1\u003c/sub\u003e, S\u003csub\u003e2\u003c/sub\u003e, and S\u003csub\u003e3\u003c/sub\u003e states. The S\u003csub\u003e1\u003c/sub\u003e EPR signals seen with a parallel mode detection at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.8 and \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;12 [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] were attributed to an orientational Jahn\u0026ndash;Teller isomerism of the dangler Mn4 with the valence\u0026thinsp;+\u0026thinsp;III [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn S\u003csub\u003e2\u003c/sub\u003e, at least two EPR signals can be detected at helium temperatures. The first one has a low-spin \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1/2 value, S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e, characterized by a multiline signal (ML) made up of at least 20 lines separated by approximately 80 gauss, centered at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;2.0 and spread over roughly 1800 gauss [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The second configuration of S\u003csub\u003e2\u003c/sub\u003e is a high-spin ground state, S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e, with \u003cem\u003eS\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;5/ 2. In plant PSII, S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e may exhibit either a derivative-like EPR signal centered at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.1 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] or more complex signals at higher \u003cem\u003eg\u003c/em\u003e values [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In cyanobacterial PSII, the \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.1 signal is not detected. Instead, the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal has a derivative-like shape centered at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.8 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn influential computational study proposed that the \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.1 signal has almost the same coordination and environment as the \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;2.0 ML signal, but with the Mn\u003csup\u003eIII\u003c/sup\u003e ion located on the dangler Mn4 in the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state instead on the Mn1 in the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e state [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This valence swap would be accompanied by a moving of the oxygen O5 from a position where it links the Mn4, Mn3 and the Ca in the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e configuration to a position where it bridges the Mn1, Mn3, and Ca ions in the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e configuration resulting in the so-called closed cubane structure. However, this proposed closed cubane structure for the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e configuration has never been observed by XFEL studies of PSII, either from plants or cyanobacteria, in conditions where the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e is known to be trapped at low temperatures. For cyanobacteria, this result is not surprising since in this PSII the \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.1 signal has never been observed until now. Yet, it remains possible that the proposed configuration is a short-lived transient that the time-resolved studies have not captured due to limited post-flash sampling frequency.\u003c/p\u003e \u003cp\u003eOn the other hand, other computational works [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] led to a different model, that, starting from the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e configuration, the protonation of O4 would lead to an \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5/2 ground state when W1, one of the two water molecules with W2 bound to the dangler Mn4, is present as an aquo ligand. The further deprotonation of W1 to form a hydroxo ligand would then give rise to an \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7/2 ground state [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It was further proposed that the form \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7/2 was required to progress to S\u003csub\u003e3\u003c/sub\u003e. Importantly, the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e form detected at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;4.8 form, \u003cem\u003ei.e.\u003c/em\u003e the \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7/2 form, that corresponds to an open cubane structure in [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], is able to progress to S\u003csub\u003e3\u003c/sub\u003e at low temperatures [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], whereas in plant PSII the g\u0026thinsp;~\u0026thinsp;4.1 state cannot [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe S\u003csub\u003e3\u003c/sub\u003e-state also exhibits some heterogeneities based on EPR analysis. Most centers exhibit a spin \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 ground state [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 configuration, the four Mn ions of the cluster have an Mn\u003csup\u003eIV\u003c/sup\u003e formal oxidation state with an octahedral ligation sphere in an open cubane structure [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this model, the dangler Mn\u003csup\u003eIV\u003c/sup\u003e (\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3/2) is antiferromagnetically coupled to the open cubane motif Mn\u003csup\u003eIV\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e with a total spin value \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9/2. The remaining centers are EPR invisible, \u003cem\u003ee.g.\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A third S\u003csub\u003e3\u003c/sub\u003e configuration with a broadened S\u003csub\u003e3\u003c/sub\u003e signal was identified with ELDOR-detected NMR (EDNMR) in the presence of glycerol [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and in PSII/Sr [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Although in [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] the authors did not completely rule out the presence of a closed cubane, five-coordinate S\u003csub\u003e3\u003c/sub\u003e form, at the origin of this EPR signal, they favored a perturbation of the coordination environment at Mn4 and/or Mn3 in an open cubane S\u003csub\u003e3\u003c/sub\u003e structure induced by glycerol. With X-and Q-band EPR experiments performed in the S\u003csub\u003e3\u003c/sub\u003e-state of plant PSII, both in the perpendicular and parallel modes, a high-spin, \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6, was proposed to coexist with the \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 configuration. This \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 form was attributed to a form of S\u003csub\u003e3\u003c/sub\u003e without O6/Ox bound and with the Mn\u003csup\u003eIV\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e part of the cluster in ferromagnetic interaction with the unsaturated dangler Mn\u003csup\u003eIV\u003c/sup\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These two forms of S\u003csub\u003e3\u003c/sub\u003e are, however, not detected by X-band EPR, so it seems unlikely that they correspond to the EPR invisible S\u003csub\u003e3\u003c/sub\u003e mentioned above. Indeed, these new S\u003csub\u003e3\u003c/sub\u003e signals described in [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] are detectable in the presence of glycerol and methanol, whereas the formation of the (S\u003csub\u003e2\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e)\u0026prime; state upon a near-IR illumination in the centers in S\u003csub\u003e3\u003c/sub\u003e defined as EPR invisible is inhibited in the presence of glycerol (and in the presence of methanol in plant PSII) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn cyanobacterial PSII/Sr [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], a proportion of centers exhibited a pulsed W-band field-swept S\u003csub\u003e3\u003c/sub\u003e spectrum much broader than in PSII/Ca. This signal was proposed to be present in centers containing a 5-coordinate Mn ion in centers in which no water binding event takes place during the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition. It was therefore proposed that, in these centers, the oxidation event would precede the water binding. Computational analyses also suggested heterogeneities in S\u003csub\u003e3\u003c/sub\u003e [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] with also a \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 spin value [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNone of the heterogeneities described above were detected in the crystallographic structures of S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e known to date (see references above). In addition, recent high-energy resolution fluorescence detected X-ray Absorption Spectroscopy together with QM calculations ruled out the presence of either peroxo or oxo/oxyl level intermediate to explain the S\u003csub\u003e3\u003c/sub\u003e heterogeneity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is quite possible that some of the structural differences that cause the differences identified in EPR are too small to be detectable given the resolution of the crystallographic data. This at least shows, if it were necessary, that spectroscopy remains an indispensable complement to crystallography.\u003c/p\u003e \u003cp\u003eThe EPR data summarized above describes a static view of the trapped configurations. Kinetically, it is well documented that the transition from S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e involved at least two phases. The fastest phase, with a \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e \u0026le; 25 \u0026micro;s, is attributed to a proton transfer/release. This fast phase precedes the electron transfer from S\u003csub\u003e2\u003c/sub\u003e to the Tyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e occurring with a \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e \u0026le; 300 \u0026micro;s [\u003cspan additionalcitationids=\"CR51 CR52 CR53\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and the binding of O6/Ox to the Mn1, \u003cem\u003ee.g.\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. It was proposed that the fast phase could correspond to the release of a proton in an intermediate step S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e \u0026rarr; S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e before the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e \u0026rarr; S\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u003c/sup\u003e transition occurs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The existence of intermediate states in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition was tracked by following, at room temperature, the structural changes in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition in the \u0026micro;s to ms time-range after the 2nd flash [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. No indication was found for a closed cubane intermediate. However, there was no conclusion on the spin state of the intermediate forms of S\u003csub\u003e2\u003c/sub\u003e able to progress to S\u003csub\u003e3\u003c/sub\u003e. By definition, an intermediate state has a low concentration that makes its detection difficult and thus, the question of the existence of a high spin intermediate state in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition remains. It is possible that the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e corresponds to a subtle structural/tautomeric intermediate that nonetheless mediates proton release during the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e \u0026rarr; S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e as a prerequisite to the formation of the S\u003csub\u003e3\u003c/sub\u003e \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 state. It has been suggested that the fast phase observed in this transition corresponds to a proton release/movement associated with the formation of a S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. If this is correct, we would expect to detect a change in the flash pattern of the proton release in conditions in which the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal is the flash-induced state. This was indeed the case since we have kinetically detected a proton release in PSII/Ca and PSII/Sr at pH 6.0 and 7.0, knowing that at pH 7.0, in PSII/Sr in contrast to PSII/Ca, half of the centers exhibit the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e signal at g\u0026thinsp;~\u0026thinsp;4.8 able to progress to S\u003csub\u003e3\u003c/sub\u003e at low temperatures [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIndependently of the pH effect and Ca/Sr exchange, several mutations, the list being too long for making it here, in the first and second coordination sphere of the Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCa unit strongly perturb the water splitting process. One of these mutants is the D1/V185N mutant in \u003cem\u003eSynechocystis\u003c/em\u003e 6803 [\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The D1/V185 residue is located adjacent to the cluster near three potential substrate candidates (W2, W3 and O5). It may also block or regulate the access to the open coordination site at Mn1. The D1/V185N mutation in \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 slows down the release of O\u003csub\u003e2\u003c/sub\u003e from 1.2 ms to about 27 ms halftime at 27\u0026deg;C. In addition, the rate of exchange for the slower exchangeable water substrate molecule, Ws, was increased in the S\u003csub\u003e1\u003c/sub\u003e and S\u003csub\u003e2\u003c/sub\u003e states in the D1/V185N mutant, while both Wf, the faster exchangeable water substrate molecule, and Ws exchange rates were decreased in the S\u003csub\u003e3\u003c/sub\u003e state in this mutant [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In contrast to the situation in the V185T mutant in \u003cem\u003eSynechocystis\u003c/em\u003e 6803 in which the oxygen release kinetics was hardly affected (from 1.2 ms to 1.5 ms) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], the D1/V185T mutation in \u003cem\u003eT. vestitus\u003c/em\u003e resulted in similar phenotype to those in the D1/V185N in \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the D1-V185T mutant in \u003cem\u003eT. vestitus\u003c/em\u003e we found that the S\u003csub\u003e2\u003c/sub\u003e state was mostly present in a high spin configuration [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] although a modified multiline was also observed. This observation triggered us to probe the proton release in the S\u003csub\u003e1\u003c/sub\u003e to S\u003csub\u003e2\u003c/sub\u003e transition in the context of the model mentioned above where the proton released in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition occurs in the S\u003csub\u003e1\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e\u0026rarr;S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e transition prior to the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e to S\u003csub\u003e3\u003c/sub\u003e transition [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. One of the problem here is that measuring the proton release kinetics by using a dye as we did in [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] required an extensive washing of the PSII samples to remove all the buffers, a treatment that has deleterious effects on the particularly fragile D1/V185T mutant. In so doing, we observed a rapid phase of proton release into the bulk, the kinetics of which was close, and difficult to distinguish from that observed following oxidation of Tyr\u003csub\u003eZ\u003c/sub\u003e at pH 6.3 in Mn-depleted PSII. To get around this problem, we also measured the electrochromic band shifts induced by each of the flashes in a sequence that is an alternative way to follow the movement of the charges in and around the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the \u0026micro;s components kinetics in the P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction reflect the progressive shift to the left of the equilibrium P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e \u0026harr; P\u003csub\u003e680\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e resulting from slower proton release/movements [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], we also measured the P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e decay in the D1/V185T mutant in \u003cem\u003eT. vestitus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRecently [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], it was reported that the high spin EPR signal in the S\u003csub\u003e2\u003c/sub\u003e state of the D1/V185N PSII in \u003cem\u003eSynechocystis\u003c/em\u003e 6803 was not detectable. We report here, in an independent EPR experiment, that the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal in the D1/V185N PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 is clearly present. This combined with the retardation in the S\u003csub\u003en\u003c/sub\u003e state transition reaction kinetics, now provides a consistent picture of the effects mutations at this second sphere location and reinforces criticality of the H-bonding network in in mediating the catalysis of water oxidation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePurification of the D1-V185N mutant from \u003cem\u003eSynechocystis 6803\u003c/em\u003e was previously described in [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The D1 protein is PsbA2 in this mutant strain. Purification of the D1/V185T mutant from \u003cem\u003eT. vestitus\u003c/em\u003e was previously described in [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The D1/V185T mutant has been constructed in the \u003cem\u003epsbA\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e gene so that the wild type PSII is that one with PsbA3.\u003c/p\u003e \u003cp\u003eTime-resolved absorption changes measurements were performed with a lab-built spectrophotometer [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] slightly modified as described in detail in [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. For the ΔI/I measurements the samples were diluted in 1 M betaine, 15 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 15 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 40 mM MES (pH 6.5). PSII samples were dark-adapted for ~\u0026thinsp;1 h at room temperature (20\u0026ndash;22\u0026deg;C) before the addition of 0.1 mM phenyl \u003cem\u003ep\u003c/em\u003e\u0026ndash;benzoquinone (PPBQ) dissolved in dimethyl sulfoxide. The chlorophyll concentration of all the samples was ~\u0026thinsp;25 \u0026micro;g of Chl mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After the measurements, the ΔI/I values were normalized to exactly \u003cem\u003eA\u003c/em\u003e673\u0026thinsp;=\u0026thinsp;1.75, that is very close to 25 \u0026micro;g Chl mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The measurements with the dye bromocresol purple were done as previously reported after the removal of Mes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eX-band cw-EPR spectra were recorded with a Bruker Elexsys 500 X-band spectrometer equipped with a standard ER 4102 (Bruker) X-band resonator, a Bruker teslameter, an Oxford Instruments cryostat (ESR 900) and an Oxford ITC504 temperature controller. The samples, at ~\u0026thinsp;1.35 mg Chl mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, were dark-adapted for 1 h on ice then the samples were frozen in the dark (without any addition) to 198 K in a dry-ice/ethanol bath and then transferred into liquid N\u003csub\u003e2\u003c/sub\u003e (77 K). Prior to recording the spectra, the samples were degassed at 198 K. The S\u003csub\u003e2\u003c/sub\u003e state was induced by illumination with an 800 W tungsten lamp filtered by water and infrared cut-off filters for approximately 5\u0026ndash;10 s at 198 K in a non‑silvered Dewar in ethanol cooled with dry ice.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAs the interpretation of the data strongly depends on the presence of an S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state in the D1/V185N mutant in \u003cem\u003eSynechocystis\u003c/em\u003e 6803, something that was contested in [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], we will firstly show the EPR spectra recorded in this strain. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e compares the spectra in the PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 WT (Panel A), and from the \u003cem\u003eS\u003c/em\u003e 6803 D1/V185N-PSII (Panel B). The black spectra were recorded in the dark-adapted samples, the red spectra after continuous illumination at 198 K for 5\u0026ndash;10 s, and the blue spectra are the light-\u003cem\u003eminus\u003c/em\u003e-dark difference spectra.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the WT sample (Panel A) the light-induced state exhibits a normal S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e multiline signal similar to that induced in plant PSII and cyanobacterial PSII [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, in the D1/V185N PSII (Panel B), the shape and the amplitude of the multiline signal differs significantly from that one in the WT PSII. The S\u003csub\u003e2\u003c/sub\u003e multiline signal in the D1/V185N sample is very similar, if not identical, to the S\u003csub\u003e2\u003c/sub\u003e multiline signal in the D1/V185T mutant in \u003cem\u003eT. vestitus\u003c/em\u003e [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Importantly, a large S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal, centered at ~\u0026thinsp;1700 gauss, was induced by the illumination at 198 K in the D1/V185N mutant. This S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal is also virtually identical to the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal detected in the D1/V185T mutant in \u003cem\u003eT. vestitus\u003c/em\u003e [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the black and red spectra other signals are detected. The \u003cem\u003eg\u003c/em\u003e\u003csub\u003ez\u003c/sub\u003e and \u003cem\u003eg\u003c/em\u003e\u003csub\u003ey\u003c/sub\u003e of Cyt\u003cem\u003ec\u003c/em\u003e\u003csub\u003e550\u003c/sub\u003e are detected at ~\u0026thinsp;2300 and ~\u0026thinsp;3000 gauss, respectively. The two small negative signals in the blue spectra, between ~\u0026thinsp;800 and ~\u0026thinsp;1000 gauss, correspond to the negative non-heme iron signal. This means that the non-heme iron was oxidized in a proportion of centers in the dark-adapted state and reduced by the Q\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e formed by the illumination at 198 K [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The large narrow signal at around 1600 gauss is due to contaminating Fe\u003csup\u003e3+\u003c/sup\u003e. In both samples, the \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;1.9 form of the Q\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eFe\u003csup\u003e2+\u003c/sup\u003eQ\u003csub\u003eB\u003c/sub\u003e signal (~\u0026thinsp;3700 gauss) and the Q\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eFe\u003csup\u003e2+\u003c/sup\u003eQ\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e signal at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;~\u0026thinsp;1.6 (~\u0026thinsp;4400 gauss) are detected. The change in their relative proportion is likely due to a different proportion of the Q\u003csub\u003eA\u003c/sub\u003eFe\u003csup\u003e2+\u003c/sup\u003eQ\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e state in the dark adapted samples. In the black spectra, the Q\u003csub\u003eA\u003c/sub\u003eFe\u003csup\u003e2+\u003c/sup\u003eQ\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e signal is possibly present at ~\u0026thinsp;4200 gauss and indeed larger in the mutant, \u003cem\u003ei.e.\u003c/em\u003e [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, an unequivocally similar S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal is formed in the D1/V185N PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803 and the D1/V185T PSII mutant from \u003cem\u003eT. vestitus\u003c/em\u003e mutant [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKnowing with certainty that both mutants exhibit an S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state at pH 6.5, the question about a proton release into the bulk in the S\u003csub\u003e1\u003c/sub\u003e to an S\u003csub\u003e2\u003c/sub\u003e state with such S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR properties should be addressed again. Firstly by measuring the proton/release using the dye bromocresol purple. Secondly by recording the electrochromic band-shifts in the Soret region of the P\u003csub\u003eD1\u003c/sub\u003e absorption spectrum at 440 nm. Indeed, this measurement takes into account both the proton uptake/release and the electron transfer events, \u003cem\u003ee.g.\u003c/em\u003e [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] and references therein. In addition, unlike the situation with the dyes, the electrochromism is not contaminated by the protonation/deprotonation events coupled to the reduction/oxidation of the non-heme iron. For the removal of the contributions due to the reduction of Q\u003csub\u003eA\u003c/sub\u003e the ΔI/I at 424 nm was also measured. Indeed, the electrochromism due to Q\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e equally contributes at 440 nm and 424 nm [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. These measurements can be compared to the proton uptake/release kinetics again measured with bromocresol purple as in [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePanels A and B in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the absorbance changes of bromocresol purple in PsbA3-PSII and PsbA3/V185T-PSII, respectively, both from \u003cem\u003eT. vestitus\u003c/em\u003e. An uptake of protons by PSII induces an increase in the ΔI/I at 575 nm, and a release of protons into the bulk by PSII induces a decrease of the ΔI/I. After the first flash (black points), there was a large increase in the absorption in both samples. This increase is due to the proton uptake following the reduction of the non-heme iron [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In PsbA3-PSII (Panel A) there is no detectable proton release associated with the S\u003csub\u003e1\u003c/sub\u003e to S\u003csub\u003e2\u003c/sub\u003e transition as already seen in such a PsbA3-PSII [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This is consistent with a large body of experiments indicating the absence of proton release in this transition, and with a 1-0-1-2 pattern of proton release for the S\u003csub\u003e0\u003c/sub\u003e to S\u003csub\u003e1\u003c/sub\u003e, S\u003csub\u003e1\u003c/sub\u003e to S\u003csub\u003e2\u003c/sub\u003e, S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e to S\u003csub\u003e0\u003c/sub\u003e transitions, reviewed in [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In contrast with our previous study [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], however, the decrease in the ΔI/I in the 10 \u0026micro;s to 100 \u0026micro;s time range is very small, suggesting that a proton release, if any, occurs in a very low proportion of the centers. This lack of proton release is very likely due to the use of a PsbA3/V185T-PSII sample less damaged by the washing steps compared to the earlier study. Additionally, proton uptake is detected after each flash as previously reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] explaining the drift in the signal (not subtracted in Panels A and B) observed on each flash. The proton release kinetics in the PsbA3/V185T-PSII in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition (red points) and in the S\u003csub\u003e0\u003c/sub\u003e to S\u003csub\u003e1\u003c/sub\u003e transition (green points) appears hardly affected when compared to PsbA3-PSII (~\u0026thinsp;30\u0026ndash;40 \u0026micro;s, and ~\u0026thinsp;100\u0026ndash;200 \u0026micro;s, respectively). In the S\u003csub\u003e3\u003c/sub\u003e to S\u003csub\u003e0\u003c/sub\u003e transition, the release of the first proton is also hardly affected in the mutant. In contrast, the release of the second proton is strongly slowed down with a kinetics similar to the O\u003csub\u003e2\u003c/sub\u003e release (~\u0026thinsp;20\u0026ndash;30 ms) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePanel C in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the kinetics of the 440 nm-\u003cem\u003eminus\u003c/em\u003e-424 nm difference in the PsbA3/V185T-PSII from \u003cem\u003eT. vestitus\u003c/em\u003e. The results can be compared to those obtained in PsbA3-PSII that have been described in details previously [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. After the first flash (black points), the kinetics that corresponds to the S\u003csub\u003e1\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e to the S\u003csub\u003e2\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e transition is slightly slowed down from 10\u0026ndash;20 \u0026micro;s to 50\u0026ndash;60 \u0026micro;s in the PsbA3/V185T mutant. After the second flash (red points) the kinetics is biphasic with a fast phase (\u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e ~ 10\u0026ndash;15 \u0026micro;s) that we have attributed to a proton movement coupled to the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e to S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e transition, and the slow phase was attributed to the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e to the S\u003csub\u003e3\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e transition [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the PsbA3/V185T-PSII mutant the two phases are not really differentiated, which makes the interpretation difficult even if this result could suggest a slowing down of the proton release in the S\u003csub\u003e2\u003c/sub\u003e to S\u003csub\u003e3\u003c/sub\u003e transition. In the PsbA3-PSII, after the fourth flash, the kinetics is also biphasic with the fast phase corresponding to the electron transfer in the S\u003csub\u003e0\u003c/sub\u003e to S\u003csub\u003e1\u003c/sub\u003e transition (\u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e ~ 10\u0026ndash;15 \u0026micro;s), which precedes the proton release with a \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e ~ 200 \u0026micro;s [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the PsbA3/V185T-PSII, after the fourth flash (green points), although the two phases are also difficult to be detected due to a less good signal-to-noise ratio, and an increase miss parameter (see however below), the two phases seem to have more or less the same kinetics. The big difference between Panel A and Panel B is the slow phase after the third flash (blue points) with a \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e ~ 20\u0026ndash;30 ms in D1/V185T-PSII, which is similar to that of the proton release in this sample. After the 4th flash the slow decay certainly originates from a residual S\u003csub\u003e3\u003c/sub\u003e to S\u003csub\u003e0\u003c/sub\u003e transition due to the misses but we have no clear explanation for the slow drift after the 1st and 2nd flash since the redox events at the non-heme iron do not contribute in the electrochromism of P\u003csub\u003eD1\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eEach event, such as a change in the kinetics of electron and proton motion, should affect the P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e \u0026harr; P\u003csub\u003e680\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e equilibrium and therefore the P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e decay kinetics. Panels A, B, C, and D in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show the P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e reduction kinetics measured at 432 nm in PsbA3-PSII (black), and PsbA3/V185T-PSII (red) after the 1st flash (Panel A), the 2nd flash (Panel B), the 3rd flash (Panel C) and the 4th flash (Panel D). In all the S\u003csub\u003en\u003c/sub\u003e states, the decay of P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the PsbA3/V185T mutant is significantly slower both in the ns time-domain corresponding to the pure electron transfer step, and the \u0026micro;s time-domain where the electron transfer(s) is(are) coupled to proton movements. The P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003eTyr\u003csub\u003eZ\u003c/sub\u003e \u0026harr; P\u003csub\u003e680\u003c/sub\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e is therefore shifted to the left in the PsbA3/V185T mutant when compared to the PsbA3-PSII in all the S\u003csub\u003en\u003c/sub\u003e states [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and we expect that some of the proton movements coupled to the electron transfer are also slowed down.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePanel E in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the DI/I measured 20 ns after each flash of the sequence in the PsbA3-PSII (black) and in the PsbA3/V185T-PSII (red). Due to the slower decay of P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e states [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] than in the S\u003csub\u003e0\u003c/sub\u003e and S\u003csub\u003e1\u003c/sub\u003e states we obtain an oscillation with the period of four. Since in all the S\u003csub\u003en\u003c/sub\u003e states the decay of P\u003csub\u003e680\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is faster in PsbA3-PSII than in PsbA3/V185T-SII, the DI/I values in PsbA3-PSII, 20 ns after the flashes, are smaller (less negative) in all the S\u003csub\u003en\u003c/sub\u003e states. However, the oscillations are similar in both samples, which indicates that in the PsbA3/V185T-PSII used here the miss parameter is not significantly larger than in the PsbA3-PSII.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOne of the main results shown in this work is the presence of a S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e in the D1/V185N-PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803, with EPR characteristics strikingly similar to those observed in the D1/V185T-PSII mutant from \u003cem\u003eT. vestitus\u003c/em\u003e [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The discrepancy between the EPR spectra shown here and those in [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] is likely attributable to baseline distortions in the low-field region of the spectra in [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Such distortions hinder the clear detection of the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e signal. By addressing this technical issue, we demonstrate the consistent appearance of the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state in these mutants, reinforcing the idea that the D1/V185 residue influences the high-spin/low-spin equilibrium of the S₂ state in a similar manner across different cyanobacterial species and supports a consistent mechanism by which a perturbation of this site modulates the still enigmatic S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state in PSII. Because these are substitutions of a hydrophobic with polar residues, they introduce H-bonding potential at a location adjacent to the insertion point of substrate water O\u003csub\u003e6\u003c/sub\u003e/O\u003csub\u003ex\u003c/sub\u003e that arrives at the open coordination position of Mn1 during the S\u003csub\u003e2\u003c/sub\u003e \u0026rarr; S\u003csub\u003e3\u003c/sub\u003e transition. Thus, the mutations almost certainly exert their effects by introducing aberrant H-bonding interactions that compete and thus perturb the native H-bond network, which has been evolutionarily optimized to enhance catalytic efficiency. The inferred mutational perturbation of native H-bonding may thus be at the basis of some of the observed EPR and kinetic alterations as discussed below.\u003c/p\u003e \u003cp\u003eIt has been shown that the equilibrium between the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e and S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e states is pH dependent [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with a pK\u003csub\u003ea\u003c/sub\u003e ~ 8.3 for the native Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster and a significantly shifted pK\u003csub\u003ea\u003c/sub\u003e of ~\u0026thinsp;7.0 for the Mn₄SrO₅ cluster [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Alkaline conditions are thought to mimic the charged state of the Tyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e, emulating the electrostatic proton ejection regime that facilitates progression through the Kok cycle [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Furthermore, there is some evidence that the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e must pass through an S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e intermediate state before forming the S\u003csub\u003e3\u003c/sub\u003e state. For example, the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state formed by the room temperature flash illumination could progress to S\u003csub\u003e3\u003c/sub\u003e under further continuous illumination at 198 K. Similarly, samples advanced to the S₂\u003csup\u003eLS\u003c/sup\u003e state upon illumination at 198 K subsequently form the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state when warmed to room temperature, enabling progression to the S₃ state under further illumination at 198 K. These observations have led to the tentative conclusion that the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state is a necessary intermediate during the S\u003csub\u003e2\u003c/sub\u003e \u0026rarr; S\u003csub\u003e3\u003c/sub\u003e transition.\u003c/p\u003e \u003cp\u003eThe lowered pK\u003csub\u003ea\u003c/sub\u003e of the Mn₄SrO₅ cluster explains why the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e to S₃ transition can occur in a significant proportion of centers even at pH 6.5. Interestingly, D1/V185T-PSII also exhibits a high proportion of centers with an S₂\u003csup\u003eHS\u003c/sup\u003e signal at pH 6.5, suggesting that these centers could progress to the S₃ state under illumination at 198 K. However, this progression is not at all evident in D1/V185T-PSII with a Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster, as shown in spectrum \u003cem\u003ea\u003c/em\u003e of Fig.\u0026nbsp;9 in [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In contrast, in D1/V185T-PSII with a Mn\u003csub\u003e4\u003c/sub\u003eSrO\u003csub\u003e5\u003c/sub\u003e cluster, the effect of the 198 K illumination was more pronounced (spectrum \u003cem\u003eb\u003c/em\u003e in Fig.\u0026nbsp;9 in [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]). However, the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal that disappeared in D1/V185T-PSII with a Mn\u003csub\u003e4\u003c/sub\u003eSrO\u003csub\u003e5\u003c/sub\u003e cluster upon the 198 K illumination had a shape similar to that in the wild type PSII (with both Ca and Sr in the cluster). This observation suggests some heterogeneity in the EPR properties of the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state in D1/V185T-PSII with a Mn\u003csub\u003e4\u003c/sub\u003eSrO\u003csub\u003e5\u003c/sub\u003e cluster. Importantly, since the illumination at 198 K results in the formation of the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e and S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e states in both species, it seems likely that each of these two states arises from two structurally different S\u003csub\u003e1\u003c/sub\u003e states.\u003c/p\u003e \u003cp\u003eThe previously mentioned possibility [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] for a S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e to S\u003csub\u003e3\u003c/sub\u003e transition occurring at 198 K in D1/V185T-PSII seemed to be supported by the detection of a proton release on the first flash, see [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, it is shown here that this proton release does not occurs in active D1/V185T-PSII but rather as originating from the oxidation of Tyr\u003csub\u003eZ\u003c/sub\u003e in a minor sub-population Mn-depleted centers in the D1/V185T-PSII used for proton release experiment in [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It can now be concluded that the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state in D1/V185T-PSII, with EPR properties different from those in wild type PSII, is unable to progress to S\u003csub\u003e3\u003c/sub\u003e at 198 K, most likely because its protonation state differs.\u003c/p\u003e \u003cp\u003eThe S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e EPR signal in the D1/V185T-PSII from \u003cem\u003eT. vestitus\u003c/em\u003e, and the D1/V185N-PSII from \u003cem\u003eSynechocystis\u003c/em\u003e 6803, have a shape reminiscent of the spectrum induced by near-infrared illumination of the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state at high pH in PsbA3-PSII see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e in [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Presently, there is no explanation of the near-infrared illumination however this similarity could suggest that the two states only differ by small structural changes, such as shifts in the H-bonding and proton tautomer configurations.\u003c/p\u003e \u003cp\u003eFunctionally significant hydrogen bonding changes due to mutations at D1/V185 likely affect the stabilization and dynamics of substrate water molecules in the S\u003csub\u003e3\u003c/sub\u003e state based upon previous findings [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The slowdowns of both the lag and oxygen release phase during the S\u003csub\u003e3\u003c/sub\u003e to S\u003csub\u003e0\u003c/sub\u003e transition have been attributed to changes in the H-bond network that correspond to a suboptimal configuration of reactants and an associated entropic penalty for crossing the activation barrier of the transition state [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The observation of slower water exchange in S\u003csub\u003e3\u003c/sub\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] has been suggested to be the result of the stabilization of water molecules due to altered H-bonding around the inserted substrate water, O\u003csub\u003e6\u003c/sub\u003e/O\u003csub\u003ex\u003c/sub\u003e. However, the mechanism of substrate exchange remains to be elucidated. As noted, D1/V185T and D1/V185N mutations replace the non-polar valine side chain with threonine or asparagine, which can introduce non-native hydrogen bonds to the putative O\u003csub\u003e6\u003c/sub\u003e/O\u003csub\u003ex\u003c/sub\u003e substrate and with water molecules (\u003cem\u003ee.g.\u003c/em\u003e W3). This disruption likely affects both water binding and proton release.\u003c/p\u003e \u003cp\u003eThe findings presented here underscore the central role of the H-bond network in modulating the LS-to-HS transition and subsequent steps in the Kok cycle. In Ca-PSII, the LS-to-HS transition is characterized by cooperative interactions among multiple protonatable groups, as evidenced by the best-fit value of n\u0026thinsp;~\u0026thinsp;4.4 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], although only one proton is ultimately released to the bulk solution [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, we can speculate that this cooperativity lowers the transition state barrier for proton release rather than stabilizing the deprotonated HS state itself. According to this view, the well-organized H-bond network facilitates a collective reorganization that creates an energetically favorable pathway for the LS-to-HS transition, ensuring efficient coupling of proton dynamics with substrate water insertion at the Mn₄CaO₅ cluster.\u003c/p\u003e \u003cp\u003eThis may provide a view of some shared characteristics of proton transfer during both the S\u003csub\u003e2\u003c/sub\u003e \u0026rarr; S\u003csub\u003e3\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e \u0026rarr; S\u003csub\u003e0\u003c/sub\u003e transitions. This S\u003csub\u003e2\u003c/sub\u003e \u0026rarr; S\u003csub\u003e3\u003c/sub\u003e transition also coincides with the critical insertion of W3\u0026mdash;initially coordinated to Ca\u0026sup2;⁺\u0026mdash;into the open coordination site on Mn1. This step is crucial for positioning W3 to become Ox, one of the two substrate waters for the water oxidation reaction. The timing and coordination of W3 insertion and its deprotonation is expected to be strongly influenced by the surrounding H-bond network. In the LS state, the network stabilizes W3 on Ca\u0026sup2;⁺ in a protonated form, maintaining readiness for the subsequent catalytic steps. Upon insertion into Mn1 following the LS-to-HS transition, the reorganization of the H-bond network facilitates the deprotonation of W3, with the cooperative action lowering the activation barrier for this step and ensuring the system is primed for subsequent oxidation chemistry. Similarly, simulations in [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] emphasize that concerted proton shuffling between O6 and the waters W3 and W2, along with the extremely wide preorganization of an extended hydrogen bond network, is mandatory for Tyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e reduction in the S\u003csub\u003e3\u003c/sub\u003e \u0026rarr; S\u003csub\u003e0\u003c/sub\u003e transition. While a similar proton cooperativity to that seen in the formation of the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state, such a cooperative transition on the HBN and its disruption by mutations or Sr-substitution could also account, for the slowdown of the S\u003csub\u003e3\u003c/sub\u003e \u0026rarr; S\u003csub\u003e0\u003c/sub\u003e transitions and help explain the increase in the entropic penalty and H/D effects previously observed for V185N. This insight aligns with the idea that efficient catalysis requires not just local H-bond interactions but a globally integrated network capable of synchronizing proton and water dynamics across the system. Disruptions to this network, as seen in D1/V185 mutations and Sr\u0026sup2;⁺ substitutions, likely hinder the necessary concerted movements, leading to slowed transitions and impaired water oxidation.\u003c/p\u003e \u003cp\u003eConsistent with this interpretation, we observe that the proton release during the S\u003csub\u003e3\u003c/sub\u003e \u0026rarr; S\u003csub\u003e0\u003c/sub\u003e transition in the D1/V185 mutants was primarily affected by the second of the two protons released during that transition. In contrast, the first proton release, which occurs rapidly following the formation of the tyrosine radical, Tyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e, remains largely unaffected. This initial fast proton release is proposed to be linked to the electrostatic impact of uncompensated charge formation upon the effects of the formation of Tyr\u003csub\u003eZ\u003c/sub\u003eOH-His190 \u0026rarr; Tyr\u003csub\u003eZ\u003c/sub\u003eO\u003csup\u003e●\u003c/sup\u003e-H\u003csup\u003e+\u003c/sup\u003eHis190 [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Functionally, this is proposed to occur to prepare Asp61D1 as a proton acceptor for the slower, second proton release from substrate water O6 [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The transient electric field during the Tyr\u003csub\u003eZ\u003c/sub\u003eO\u003csup\u003e●\u003c/sup\u003e-H\u003csup\u003e+\u003c/sup\u003eHis190 state is calculated to promote proton transfer from W1 to Asp61, inducing rearrangements in the cluster of residues D1-Asp61, D1-Glu65, and D2-Glu312 into a configuration that facilitates rapid proton egress to the bulk [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The rapid exit of this proton is likely gated, preventing back-reactions. Once deprotonated, the cluster forms the base that accepts the proton from substrate water O6, facilitating the final electron transfer leading to O\u003csub\u003e2\u003c/sub\u003e formation [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The fact that the fast proton release remains unaffected by the mutation, while the second proton release is delayed, is consistent with the D1/V185 residue being distant from the fast proton release vector but positioned near the substrate water (O6), where deprotonation occurs. Thus, the delayed second proton release in the mutants is attributed to the disruption of the hydrogen-bonding network around O6, which slows the proton transfer pathway necessary for its deprotonation and the final stages of water oxidation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePhotosystem II\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePSII\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echlorophyll\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChl\u003csub\u003eD1\u003c/sub\u003e/Chl\u003csub\u003eD2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonomeric Chl on the D1 or D2 side,respectively\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMES\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-(\u003cem\u003eN\u003c/em\u003e-morpholino) ethanesulfonic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003csub\u003e680\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprimary electron donor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003csub\u003eD1\u003c/sub\u003e and P\u003csub\u003eD2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eindividual Chl on the D1 or D2 side,respectively,which constitute a pair of Chl with partially overlapping aromatic rings\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePhe\u003csub\u003eD1\u003c/sub\u003e and Phe\u003csub\u003eD2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epheophytin on the D1 or D2 side,respectively\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPBQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephenyl \u003cem\u003ep\u003c/em\u003e\u0026ndash;benzoquinone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQ\u003csub\u003eA\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprimary quinone acceptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQ\u003csub\u003eB\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esecondary quinone acceptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTyr\u003csub\u003eZ\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethe tyrosine 161 of D1 acting as the electron donor to P\u003csub\u003e680\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT*3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eT. elongatus\u003c/em\u003e mutant strain deleted \u003cem\u003epsbA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003epsbA\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e genes and with a His-tag on the carboxy terminus of CP43. EPR,Electron Paramagnetic Resonance. ML,multiline.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work has been in part supported by (i) the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05, (ii) the Labex Dynamo (ANR-11-LABX-0011-01), (iii) the JSPS-KAKENHI Grant in Scientific Research on Innovative Areas JP17H064351, JSPS-KAKENHI Grant 21H02447 and a grant from the US- National Science Foundation (MCB1716408). One of the PSII preparations used in this work was done by T. Tibiletti..\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShevela D, Kern JF, Govindjee G, Messinger J (2023) Solar energy conversion by photosystem II: principles and structures. 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B 128 1866\u0026thinsp;\u0026ndash;\u0026thinsp;1875. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jpcb.3c07787\u003c/span\u003e\u003cspan address=\"10.1021/acs.jpcb.3c07787\" 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":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Photosystem II, D1-V185, EPR, proton release, S-state cycle, spin state, Mn4CaO5 cluster","lastPublishedDoi":"10.21203/rs.3.rs-5504214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5504214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe effects of D1-V185T and D1-V185N mutations in Photosystem II (PSII) from \u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e (formerly \u003cem\u003eT. elongatus\u003c/em\u003e) and \u003cem\u003eSynechocystis\u003c/em\u003e 6803, respectively, were studied using both EPR and optical kinetics. EPR spectroscopy reveals the presence of a mixture of a S\u003csub\u003e2\u003c/sub\u003e state in a high spin configuration (S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e) and in a low spin configuration (S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eLS\u003c/sup\u003e) in both mutants. In contrast to the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e in the wild type, the S\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eHS\u003c/sup\u003e state in the D1-V185T mutant does not progress to the S\u003csub\u003e3\u003c/sub\u003e state at 198 K. This inability is likely due to alterations in the protonation state and hydrogen-bonding network around the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster. Optical studies show that these mutations significantly affect proton release during the S\u003csub\u003e3\u003c/sub\u003e-to-S\u003csub\u003e0\u003c/sub\u003e transition. While the initial fast proton release associated with Tyr\u003csub\u003eZ\u003c/sub\u003e\u003csup\u003e●\u003c/sup\u003e formation remains unaffected within the resolution of our measurements, the second, and slower, proton release is delayed, suggesting that the mutations disrupt the hydrogen-bonding interactions necessary for efficient deprotonation of substrate water (O6). This disruption in proton transfer also correlates with slower water exchange in the S\u003csub\u003e3\u003c/sub\u003e state, likely due to non-native hydrogen bonds introduced by the threonine or asparagine side chains at position 185. These findings point to a critical role of D1-V185 in regulating both proton transfer dynamics and water binding, underscoring a complex interplay between structural and functional changes in PSII.\u003c/p\u003e","manuscriptTitle":"New insights into the involvement of residue D1/V185 in Photosystem II function in Synechocystis 6803 and Thermosynechococcus vestitus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 11:40:57","doi":"10.21203/rs.3.rs-5504214/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63a24726-e7dd-4c9d-ac0e-a5a8973a26ca","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40621914,"name":"Biophysics"}],"tags":[],"updatedAt":"2024-11-25T11:40:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 11:40:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5504214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5504214","identity":"rs-5504214","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}