Nanosecond Time-Resolved Infrared Spectroscopy for the Study of Electron Transfer in Photosystem I

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Mäusle, Neva Agarwala, Viktor G. Eichmann, Holger Dau, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2898981/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jul, 2023 Read the published version in Photosynthesis Research → Version 1 posted 8 You are reading this latest preprint version Abstract Microsecond time-resolved step-scan FTIR difference spectroscopy was used to study photosystem I (PSI) from Thermosynechococcus vestitus BP-1 ( T. vestitus , formerly known as T. elongatus ) at 77 K. Photoaccumulated (P700 + – P700) FTIR difference spectra were also obtained for PSI from T. vestitus at both 77 and 293 K. The FTIR difference spectra are presented here for the first time. To greatly extend upon these FTIR studies nanosecond time-resolved infrared difference spectroscopy was also used to study PSI from T. vestitus at 293 K. Nanosecond infrared spectroscopy has never been used to study PSI samples at physiological temperatures, and here it is shown that such an approach has great value as it allows a direct probe of electron transfer down both branches in PSI. In PSI at 293 K, the infrared flash-induced absorption changes indicate electron transfer down the A- and B-branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies. These time constants are associated with forward electron transfer from A 1 – to F X on the B- and A-branches, respectively. At several infrared wavelengths flash-induced absorption changes at 293 K recover in tens to hundreds of milliseconds. The dominant decay phase is characterized by a lifetime of 128 ms. These millisecond changes are assigned to radical pair recombination reactions, with the changes being associated primarily with P700 + rereduction. This conclusion follows from the observation that the millisecond infrared spectrum is very similar to the photoaccumulated (P700 + – P700) FTIR difference spectrum. Photosynthesis Photosystem I Thermosynechococcus vestitus BP-1 nanosecond time-resolved infrared spectroscopy electron transfer A1 P700 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION In photosynthesis solar energy is harvested and used to synthesize chemical products that are ultimately the source of most of the food and fuels consumed by humanity today (Walker 1993 ). In oxygen evolving organisms two photosystems, called photosystem (PS) I and II 1 , capture and convert solar energy independently, but cooperatively (Barber 1992 ; Walker 1993 ). The solar conversion reactions occur in a centralized pigment-protein unit called a reaction center. In the reaction center, light is used to drive electrons, via a series of acceptors, across a biological membrane (the thylakoid membrane). This light-induced separation of charge is the basic mechanism underlying solar energy capture in all photosynthetic organisms. In this manuscript we focus on electron transfer (ET) in isolated cyanobacterial PSI complexes from Thermosynechococcus vestitus BP-1 (previously known as Thermosynechococcus elongatus BP-1). PSI consists of 11–13 protein subunits, many of which have been characterized (Chitnis et al. 1995 ; Golbeck 1995 ; Pakrasi 1995 ). The ET cofactors are bound to the PsaA and PsaB membrane-spanning protein subunits (Fig. 1 A) (Fromme and Grotjohann 2006 ). The terminal ET cofactors, F A and F B , are bound to the stromal PsaC subunit (Fromme and Grotjohann 2006 ). The organization of the bound ET cofactors is outlined in Fig. 1 B. The cofactor organization is near identical in PSI from plants, algae and cyanobacteria (Ben-Shem et al. 2003 ; Jolley et al. 2005 ; Malavath et al. 2018 ; Mazor et al. 2015 ; Mazor et al. 2014 ; Qin et al. 2015a ; Qin et al. 2015b ). In isolated PSI particles, following light excitation, the P700 + A 1 – radical pair state is formed within ~ 50 picoseconds (Hastings et al. 1995 ; Hastings et al. 1994 ). In PSI there are two cofactor branches with approximate C 2 symmetry (Fig. 1 B), referred to as the A– and B–branch. In this manuscript the A branch refers to the side in which the pigment in the A 1 binding site is bound to the PsaA protein subunit (A 1A in Fig. 1 ). ET can occur down both branches from P700 to F x (Fig. 1 B) (Makita and Hastings 2015 ; Redding and van der Est 2006 ). Both the A 1A and A 1B pigments in PSI are phylloquinone (PhQ) molecules, in a very similar protein environment (Brettel 1997 ; Golbeck 1992 ; Golbeck and Bryant 1991 ; Srinivasan and Golbeck 2009 ). In cyanobacterial PSI at RT, forward ET from A 1 – to F X proceeds biphasically with time constants of ~ 25 and ~ 250 nanoseconds (Fig. 1 B). The “fast” and “slow” time constants were observed in visible transient absorption spectroscopy experiments and are thought to be associated with ET from A 1B – and A 1A – to F X , respectively (Agalarov and Brettel 2003 ; Makita and Hastings 2015 ; Makita et al. 2015 ; Redding and van der Est 2006 ; Schlodder et al. 1998 ; van der Est 2006 ). At RT, ET from F X – to F A and then on to F B occurs on a tens to hundreds of nanosecond timescale (Byrdin et al. 2006 ). In the absence of added electron acceptors, the P700 + F A/B – state recombines in 50–150 milliseconds (Golbeck and Bryant 1991 ). The F A/B terminology is used to indicate that we do not specifically distinguish between the F A and F B states. In cyanobacterial PSI, forward ET from A 1 – to F X diminishes as the temperature is lowered, and is replaced by a P700 + A 1A – recombination reaction, which is characterized by a time constant of ~ 300 microseconds at 77 K (Makita and Hastings 2015 ; Makita and Hastings 2016a ; Makita and Hastings 2016b ; Makita et al. 2015 ; Schlodder et al. 1998 ). On the one hand, since ET recombination is essentially unidirectional down the A-branch at 77 K, spectral analysis is simplified. On the other hand, however, in microsecond time-resolved step-scan (TRSS) FTIR difference spectroscopy (DS) studies of PSI at 77 K, the bands associated with the P700 + and A 1 – states decay with the same time constant, which negates the possibility of using the temporal evolution of bands to distinguish their origin. At ~ 293 K (room temperature, RT), P700 + and A 1 – decay on different timescales, allowing the possibility of assigning spectroscopic signatures (bands) based on their temporal evolution, thus the time resolution itself allows for a new approach to infrared spectral band identification and assignment. At RT, the P700 + A 1A – /P700 + A 1B – states decay in ~ 25/250 ns, respectively, and can potentially be studied using TRSS FTIR DS. However, noise levels near or below 1x10 –5 in OD units (0.1 mOD) are required (Hastings 2001 ). At present, such TRSS FTIR DS measurements are challenging (Hage et al. 1996 ; Hastings 2001 ; Rodig and Siebert 1999 ), and have only been demonstrated for protein systems such as bacteriorhodopsin, that display very intense absorption difference features (Hage et al. 1996 ; Siebert et al. 1982 ; Weidlich and Siebert 1993 ), or for CO-myoglobin/hemoglobin which displays intense absorption changes at frequencies where absorption changes due to water and protein are minimal (in the 1900–2100 cm –1 region) (Hu et al. 1996 ; Plunkett et al. 1995 ). Recently, we have studied photosystem II using TRSS FTIR DS, but this work required data acquisition over a period of several months [Greife et al., Nature in press ], which is unacceptably long for most studies. TRSS FTIR DS is advantageous as it allows the collection of time-resolved data over a broad spectral region. The downside is that sensitivity is limited, especially on nanosecond timescales (Hastings 2001 ; Rammelsberg et al. 1997 ). An alternative approach is to undertake nanosecond time resolved infrared (TRIR) DS measurements at single frequencies using intense IR light sources. Quantum cascade lasers (QCL) can provide such high intensity IR light. Previously, QCLs were thought to be disadvantageous because of limited spectral coverage. However, we demonstrate here with our QCL setup that TR data can be collected with high spectral resolution covering the entire 1310–1890 cm –1 region. One advantage in investigation of PSI is that it is a highly robust and stable protein that can be subjected to literally thousands of laser flashes without any appreciable degradation or damage. This allows us to undertake many separate single wavelength experiments covering the entire spectral region, with considerable signal averaging at each wavelength, allowing high signal to noise ratio at each of the wavelengths in the experiment. In this manuscript we have used a QCL based pump-probe spectrometer to undertake the first nanosecond TRIR DS studies of PSI complexes in the physiological temperature range (23°C). Such measurements have allowed us to probe absorption changes associated with single molecular bonds of the phylloquinone molecules on both the A and B branch as they undergo ET. To complement and aid in the analysis of the TRIR data, photoaccumulated (P700 + – P700) FTIR DS were obtained at 77 and 295 K and (P700 + A 1 – – P700A 1 ) TRSS FTIR DS were obtained at 77 K. Time resolved and photoaccumulated FTIR DS for PSI from T. vestitus have also never been presented previously. [1] Abbreviations: C=O, carbonyl; Chl a , chlorophyll a ; DAS, decay associated spectrum; DS, difference spectra/spectrum/spectroscopy; ET, electron transfer; FTIR, Fourier transform-infrared; H-bond, hydrogen bond; IRF, instrument response function; ms, ms, ns, micro, milli, and nano-second; NQ, 1,4-naphthoquinone; PhQ, phylloquinone (2-methyl-3-phytyl-1,4-naphthoqiuinone); PSI, photosystem I; PSII, photosystem II; Thermosynechococcus vestitus BP-1, T. vestitus ; TRIR, time-resolved infrared; TRSS, time-resolved step-scan. MATERIALS AND METHODS Trimeric PSI particles from T. vestitus were prepared by first binding His-tagged PSII complexes to a nickel resin (Mäusle et al. 2020 ) and applying the PSI-enriched flow through to a Toyopearl DEAE-650S column as previously described (Kölsch et al. 2018 ). The eluted PSI fractions were concentrated using an Amicon Stirred Cell with a Synder LY membrane (Sterlitech, USA), frozen in liquid nitrogen and stored at -80°C. Photoaccumulated (P700 + − P700) FTIR DS (at both RT and 77 K) and TRSS FTIR DS (at 77 K), all at 4 cm –1 spectral resolution, were collected as described previously (Agarwala et al. 2023 ; Makita and Hastings 2020 ). PSI samples for single-frequency TRIR DS experiments were prepared in a similar manner: After thawing on ice, the sample was diluted in 50 mM TRIS buffer (pH 8), and 0.04% b-DM, such that a chlorophyll concentration of 0.2 mg/ml was reached. The sample was centrifuged at 300,000 g for three hours at 4°C. A CaF 2 window was prepared with a ring of vacuum grease and a 15 µm spacer. Two artificial electron donors (0.1 µl of 20 µM PMS and 0.1 µl of 20 mM sodium ascorbate) were pipetted onto the CaF 2 window, before adding a small amount of the soft PSI pellet and carefully mixing with a spatula. A second CaF 2 window was placed onto the first, tightly sealing the sample in between. TRIR DS measurements were performed using the previously described experimental setup (Mäusle et al. 2020 ) with some modifications: 1. The intensity of the pulsed 532 nm Nd:YAG laser is now regulated by a motorized attenuator (Eksma Optics, Vilnius, Lithuania). 2. The QCL system was replaced by a newer model (MIRcat-QT-Z-2300) with improved optical properties (wavenumber accuracy < 1 cm –1 , covering the entire 1310–1890 cm –1 spectral range with improved pointing stability) (Daylight Solutions, San Diego, CA). For each desired wavelength, a flash sequence of two dark measurements (no flashes), followed by five saturating excitation flashes (~ 0.1 mJ/mm 2 ) and a final flash with higher intensity (~ 0.3 mJ/mm 2 ) was applied repeatedly to a single sample spot. The IR signal was measured from 20 ms before and up to 800 ms after each excitation flash. The difference in signal between the low and high excitation flashes was used to calculate a sample heating artefact, which was fit by a sum of exponentials and subtracted from the data (Fig. S1 ). A pulse generator was used for triggering the excitation laser and data acquisition. The timing of the excitation flash was determined by recording each flash with a photodiode. The sample chamber was flushed with dry air and the temperature was set at 23 ± 2°C. At each frequency the results obtained for 600–6000 laser flashes were averaged. The data was then subjected to a global analysis procedure as follows: Transient IR absorption changes were fitted to a sum of exponential functions plus a constant [ \(y\left(t,\nu \right)= {y}_{o}+{\sum }_{i}{A}_{i,{\nu }}{e}^{-t/{\tau }_{i}}\) ] using a least-squares approach implemented in Python 3.7 (Python Software Foundation, Delaware, USA). For the fits on the nanosecond timescale, the exponential function was convolved with an approximate instrument response function (a Gaussian with standard width of 17 ns) as illustrated in Fig. S2. For results obtained by an alternative approach, see Fig. S3 and S4. As is standard in these types of global analysis fitting procedures, the time constants were constrained to be the same at each wavelength, while the amplitudes (preexponential factors) were allowed to vary. RESULTS Figure 2 A shows photoaccumulated (P700 + – P700) FTIR DS collected at 295 K for PSI from T. vestitus in the 3800–1200 cm –1 region. The dark minus dark spectrum indicates the noise level in the experiment as well as the zero-absorbance line. As is relatively well known (Breton 2001 ; Nabedryk et al. 2000 ) and is clearly seen in Fig. 2 A, P700 + displays several very broad, positive, electronic absorption bands throughout the 3800–1200 cm –1 region. This broad positive absorption difference overlies the IR difference bands in the 1800–1400 cm –1 region (Fig. 2 B), essentially “pushing” the whole spectrum (of IR difference bands) more positive. Figure 2 C shows microsecond TRSS (P700 + A 1 – – P700A 1 ) FTIR DS obtained using T. vestitus PSI samples at 77 K ( blue ). As has been described (Hastings 2015 ) this TRSS FTIR DS is the average of nine spectra, collected in consecutive 6 µs increments following a laser flash. This spectrum can therefore be considered as an averaged spectrum obtained over a ~ 54 µs time-period. Photoaccumulated (P700 + – P700) FTIR DS at both 77 ( red ) and 298 K ( black ) are also shown in Fig. 2 C for comparison. Both the TRSS and photoaccumulated FTIR DS for PSI from T. vestitus are presented here for the first time. Although we call the RT photoaccumulated FTIR DS in Fig. 2 A (P700 + – P700) FTIR DS, it is strictly speaking a (P700 + F A/B – – P700F A/B ) FTIR DS. The 77 K photoaccumulated FTIR DS is also strictly speaking a (P700 + F X – – P700F X ) FTIR DS. In the latter case this is because P700 + F A/B – formation, which occurs in ~ 35% of the PSI complexes, is irreversible at 77 K (Schlodder et al. 1998 ), and signals associated with P700 + F A/B – will not contribute in our experiments that involve repetitive illumination. However, P700 + F X – recombination can occur at 77 K, with a half lifetime of 5-100 ms, in ~ 20% of the PSI complexes (Schlodder et al. 1998 ). Under repetitive bouts of continuous illumination at 77 K the P700 + F X – state will be the dominant photoaccumulated species. P700 + A 1 – is unlikely to contribute, as it decays in ~ 300 µs at 77 K making it difficult to photoaccumulate. By adding a variety of electron donors and acceptors, and by studying PSI samples that lack either F A/B (Breton et al. 1999 ; Hastings and Sivakumar 2001 ), or F A/B and F X (Hastings and Sivakumar 2001 ), it has been determined that the iron sulfur clusters do not contribute significantly in (P700 + F A/B – – P700F A/B ) FTIR DS in the 1800–1200 cm –1 region (Fe-S vibrations are expected below ~ 500 cm –1 (Chu et al. 2000 ; Chu et al. 1999 )), and this is the justification for calling these photoaccumulated spectra (P700 + – P700) FTIR DS. Nonetheless one might expect electrochromic effects from protein or pigment species near the reduced iron sulfur clusters to contribute to some degree in photoaccumulated FTIR DS. In a more recent study of PSI site directed mutants we did suggest possible small contributions to photoaccumulated FTIR DS from modes that might be impacted by F X reduction (Agarwala et al. 2020 ). The 77 K (P700 + A 1 – – P700A 1 ) TRSS FTIR DS in Fig. 2 C displays positive bands at 1567, 1511, 1495 and 1415 cm –1 , which are absent in the 77 K photoaccumulated (P700 + – P700) FTIR DS. These bands are therefore associated with A 1 – . The bands at 1495 and 1415 cm –1 are well known to be due to stretching vibrations of the C 1 \(\stackrel{⃛}{-}\) O and C 4 \(\stackrel{⃛}{-}\) O groups of the phyllosemiquinone anion in the A 1A binding site (Hastings 2015 ; Rohani et al. 2019 ). Given the similarity in the frequencies of the bands in the (P700 + – P700) FTIR DS at 295 and 77 K (Fig. 2 C), it is likely that the phyllosemiquinone anion bands will also be at similar frequencies at 77 and 295 K. We therefore expect to observe TRIR absorption changes at ~ 1495 and ~ 1415 cm –1 associated with A 1A reduction and recovery at RT. In addition, phylloquinone in the A 1B binding site is structurally very similar to that on the A-side, and it is likely that the B-side phyllosemiquinone will also display absorption near 1494 and 1415 cm –1 at both 77 K and 298 K. Both the (P700 + A 1A – – P700A 1A ) and (P700 + – P700) FTIR DS in Fig. 2 C display peaks around 1482, 1458 and 1431 cm –1 , so probing absorption changes at these frequencies (in TRIR measurements at RT) will likely result in little or no nanosecond TR absorption changes associated with forward ET from A 1 – , but a long-lived (millisecond) contribution due to P700 + is expected. RT flash-induced absorption changes at 1415, 1430, 1482, 1494, 1510, 1534, 1542 and 1679 cm –1 , on a nanosecond to second timescale, are shown in Fig. 3 . The kinetic traces in Fig. 3 A are shown on a linear timescale, from 0-2000 ns. In Fig. 3 B the same kinetic traces are shown on a logarithmic timescale, from 2x10 –3 − 8x10 3 ms. All the transients exhibit nanosecond kinetic phases (Fig. 3 A). The absorption changes are relatively constant on microsecond timescales, and decay back to zero on a hundreds of milliseconds timescale (Fig. 3 B). Transient data at all the wavelengths indicated in Fig. 3 were subjected to a global analysis fitting procedure outlined in the materials section and the supplementary information section. The transient kinetics were analyzed in the 0–5 µs time range. From this fitting procedure it is found that the data is best described by a function consisting of two exponential components (and a constant, non-decaying component). The fitted functions are also shown in Fig. 3 and are characterized by lifetimes of 33 and 364 ns (see Table S1 in the supplementary information section). The time constants vary slightly when only a subset of the listed transients are included in the global fitting procedure ( not shown ). Figure 5 shows millisecond TRIR DS extracted from the data collected using the QCL based spectrometer at RT. This data was produced by scanning the QCL output (in 2 cm –1 increments) and collecting transients for 6 laser flashes at each wavenumber (300 flashes for wavenumbers below 1550 cm − 1 ). In this mode relatively low-sensitivity kinetic data was acquired across the entire 1770–1380 cm –1 region. The signal to noise ratio for the data at each wavelength, especially in the ns region, is significantly poorer than that shown in Fig. 3 . However, by applying a spectral smoothing algorithm (three neighboring wavenumbers are averaged and assigned to the wavenumber in the middle, resulting in an effective resolution of 6 cm –1 ), and averaging each of the spectra obtained over a given time interval, a high sensitivity TRIR absorption difference spectrum is obtained. Figure 5 ( black curve ) shows the flash-induced TRIR DS resulting from the averaging all 220 spectra collected in the 0.1–1 ms time window, a time range in which absorption changes are minimal (Fig. 3 ). We also globally fitted the eight kinetic traces in Fig. 3 A, in the 1-800 ms time range, and found that at least five exponential components are needed for a satisfactory fit for the decay back to zero (as judged by the residuals). Time constants extracted are 1.3, 11, 46, 128 and 444 ms, with the 128 ms component being the dominant phase. Instead of averaging the different spectral blocks derived in this QCL wavelength scanning experiment, we could just directly globally analyze the entire data block (with or without fixed time constants that were derived from fitting the data in Fig. 3 B). From this analysis a decay associated spectrum (DAS) associated with the ~ 128 ms phase is obtained, and this DAS is also shown in Fig. 5 . This 128 ms DAS is essentially identical to the average of all the spectra in the 0.1–1 ms range. It is also identical to the average of the spectra obtained in the 1–800 ms range (not shown). Also shown in Fig. 5 is a photoaccumulated (P700 + – P700) FTIR DS obtained at RT, employing 4 cm –1 spectral resolution ( green ) (from Fig. 2 ). The similarity between the photoaccumulated FTIR DS and the different TRIR DS is obvious, except for the intensity of the positive peak near 1655 cm –1 , which is more pronounced in the TRIR DS. The origin of this difference in the TRIR and FTIR DS is not clear at present. There are other very small differences in the TRIR and FTIR DS in Fig. 5 , for example the absorption change near 1735 cm –1 , which may relate to the different spectral resolutions in the two experiments. DISCUSSION Nanosecond transient absorption spectroscopy, with 480 nm light, is often used to probe forward ET from A 1 – to F X in PSI at RT (Agalarov and Brettel 2003 ; Badshah et al. 2018 ). The positive absorption change at 480 nm is due to an electrochromic effect on a chlorophyll pigment (likely A 0 ) caused by the electron residing on the A 1 pigment (Bautista et al. 2005 ). The absorption changes at 480 nm were shown to decay biphasically with time constants of 11 and 340 ns at 295 K in PSI from Synechocystis sp. 6803 (Agalarov and Brettel 2003 ). The two time constants have been shown to be consistent with ET down the B and A branches in PSI (Guergova-Kuras et al. 2001 ). Since absorption changes on the nanosecond timescale are associated with forward ET from A 1 – to F X , and changes that do not decay/grow on a nanosecond-microsecond timescale are associated mainly with P700 or P700 + (as changes associated with F X /F X – are minimal in the 1800 − 1200 cm –1 region), we can separate IR absorption contributions from the different pigments based on their time evolution. For the 1494 cm –1 kinetic, the 33 and 364 ns phases have large amplitudes and are well resolved (Fig. 3 A and 4 A). The 1494 cm –1 band in the TRSS FTIR DS at 77 K (Fig. 2 C) is due to the C 1 \(\stackrel{⃛}{-}\) O group of PhQ – in the A 1A binding site (Rohani et al. 2019 ). For the 1494 cm –1 absorption change, 72% of the positive, flash-induced absorption change decays on the nanosecond timescale, with 40 and 32% of the amplitude being associated with the 33 and 364 ns time constants, respectively (Fig. 4 B, Table S1 ). A long-lived positive feature contributing to 28% of the initial absorption change remains following the nanosecond kinetics. As indicated above, the A 1 , A 2 and y 0 relative contributions (outlined in Fig. 4 B) are proxies for relative contributions from A 1B – , A 1A – and P700 + . Therefore, at 1494 cm –1 , A 1A – , A 1B – and P700 + all have a positive absorption change. This observation also follows from the photoaccumulated and TRSS FTIR DS in Fig. 2 C. The kinetic at 1510 cm –1 exhibits similar absorption changes to the one at 1494 cm –1 (Fig. 4 B), but with a less pronounced 364 ns phase (Fig. 4 A). A positive band is also observed at 1511 cm –1 in the microsecond TRSS FTIR DS at 77 K, with an intensity that is also considerably lower than that at 1494 cm − 1 . The similarity in the relative amplitudes of the nanosecond phases at the two wavelengths at RT, with that in the TRSS FTIR DS at 77 K, suggests that the bands at 1511 and 1494 cm − 1 at 77 K likely corresponds to bands at similar wavenumber at RT, and that the 33 and 364 ns phases at 1510 cm –1 are likely due to A 1B – and A 1A – , respectively (as is the case for the 1494 cm − 1 band). The 33 and 364 ns phases in the kinetic at 1510 cm − 1 at RT are likely associated with quinone ring C \(\stackrel{⃛}{-}\) C modes of A 1B – and A 1A – , respectively. It is well known that semiquinone ring C \(\stackrel{⃛}{-}\) C modes have higher frequency vibrations than C \(\stackrel{⃛}{-}\) O modes (Bauscher and Mantele 1992 ; Bauscher et al. 1990 ). The absorption changes at 1679 cm –1 displays an intense 33 ns phase (46%), with smaller contributions from the 364 ns (33%) and non-decaying (21%) phases. The non-decaying phase of the 1679 cm –1 kinetic is weakly negative, as is the amplitude in the photoaccumulated FTIR DS in Fig. 2 B, C. So, the 1679 cm –1 kinetic indicates that during ET from A 1 – to F X , the absorption change is initially positive and the nanosecond bioenergetics lead to a negative absorption change that is due to P700 + . The species responsible for these nanosecond phases at 1679 cm − 1 are not clear at present. They could be associated with amide I vibrations associated with amino acids near A 1 , or with keto carbonyl modes of the A –1 or A 0 pigments (Sivakumar et al. 2005 ). This is a topic that will be further investigated in the future. The flash-induced absorption increase at 1482 cm –1 occurs within the time resolution of the instrument, followed by a fast 33 ns decay (Fig. 3 B, black ) with little further absorption changes on the hundreds of nanoseconds to microseconds timescale (the 364 ns phase is of very low amplitude (Fig. 4 B)). The 33 ns and non-decaying phases account for 34 and 61% of the absorption difference signal at 1482 cm –1 (Fig, 4B and Table S1 ), respectively. However, there is considerable uncertainty in the amplitude for the 33 ns phase. The fact that we observe both positive and negative amplitudes for the 33 ns phase at the different wavelengths (Table S1 ) confirms that this phase is not an instrumental artefact. However, the 33 ns phase is close to the temporal resolution limits of the instrument (estimated to be 17 ns), and the amplitudes of this phase at the different wavelengths correspondingly have large error (see discussion of this topic in the supplementary information section). At 1482 cm –1 , given that there is a 33 ns phase and essentially no 364 ns phase (Table S1 ), it is unlikely that the 33 ns phase is associated with molecular groups of A 1B – itself, because then a corresponding 364 ns phase might also be expected near 1482 cm –1 associated with A 1A – . Note that we presume that A 1B – and A 1A – have absorption bands at similar frequencies, as they do at 1494 and 1415 cm –1 . What the 33 ns phase at 1482 cm –1 is due to is not clear, possibly a protein group between A 1B and F X . Since the 33 ns feature at 1482 cm –1 should reflect B-branch and not A-branch ET, we speculate that we have uncovered a spectral observable indicating an asymmetry in A and B branch ET process. This type of asymmetry between the fast and slow phases may potentially be observed at other wavelengths, such as at 1542 cm –1 , where the 33 ns phase is of much lower amplitude than the 364 ns phase (Fig. 4 B). However, this latter difference may also reflect a branch-utilization asymmetry. In cyanobacterial PSI the A/B branching ratio is roughly 80/20 (Makita and Hastings 2015 ), respectively. So, the differences in the amplitudes of the two phases at 1542 cm –1 may reflect this branch-utilization asymmetry. From the data in Fig. 3 A, it is clear that the 33 ns phase is very poorly resolved at 1542 cm –1 , and also at 1534 cm –1 , and it is probably unwise to make assertions based on the amplitude of the 33 ns phase at these frequencies. In Fig. 3 B, following the decay of the nanosecond phases (from ~ 5 µs onwards) a pronounced positive/negative absorption change is observed at 1542/1534 cm –1 , respectively, that subsequently decays with a time constant of ~ 128 ms. From Table S1 (and Fig. 3 A), the non-decaying phase has an amplitude of + 3.17/-1.95 at 1542/1534 cm –1 , respectively. In contrast, the TRIR DS in Fig. 5 indicates a higher negative amplitude at 1534 cm –1 compared to the corresponding positive amplitude at 1542 cm –1 . The solution to this issue is to recognize that the broad positive absorption of the P700 + state (Fig. 2 A, B) also decays in ~ 128 ms. In Fig. 2 B one observes that the negative signal at 1534 cm –1 , relative to the zero line, is considerably less than the positive amplitude of the signal at 1542 cm –1 (relative to the zero line), and this amplitude ratio is in line with the millisecond-TRIR kinetic data in Fig. 3 A. Although the absorption difference features in the kinetics in Fig. 3 appear well resolved, the 33 ns kinetic phase is close to the instrumental time resolution. The instrumental time resolution is determined mostly by the response of the IR detector with integrated preamplifier (10 MHz cut-off frequency) and the sampling rate of the A/D converter, where the smallest possible bin size is 15 ns. In our global analysis curve-fitting procedures, the instrument response function (IRF) is well modelled by a Gaussian function with standard width of 17 ns (see discussion in the supplementary information section). Given the 17 ns IRF, and the limited data sampling, the amplitudes of the 33 ns phase at the different wavenumbers is difficult to pinpoint with high precision. Nevertheless, the 33 ns phase is clearly discernable at many wavelengths (Fig. 3 A) and is not artefactual. The exact time constant and amplitudes of the 33 ns phase at the different wavelengths are poorly determined, however, depending on the details of the fitting procedure (see the supplementary information section). This is an important consideration. For example, if the 33 and 364 ns phases in the kinetic at 1494 cm –1 are due to forward ET from A 1B – and A 1A – , one might expect the 364 ns phase to exhibit a similar, or up to a two-to-four times larger amplitude, than the 33 ns phase, simply because the branching ratio in PSI is expected to be in the 50/50 to 20/80 range (Redding and van der Est 2006 ). This is clearly not found to be the case for the 1494 cm –1 kinetic data (Fig. 4 B). Using an alternate fitting procedure, where zero time for the kinetics is manually adjusted, and the instrument response function is not included in the fitting, the amplitude of the 33 ns phase relative to the 364 ns phase can fall to ~ 40/60 (see discussion in the supplementary information section). So, caution should be exercised at present on building thesis based on the amplitude of the 33 ns phase in the various kinetics. On the other hand, it may be the case that the amplitude of the 33 ns phase is truly larger than the 364 ns at 1494 cm –1 (as indicated in Fig. 4 B), and if this is the case it could mean that an alternative model of ET needs to be considered, such as the radical pair equilibrium model (Santabarbara et al. 2019 ). A final answer may have to await the production of kinetic data with improved temporal resolution. In the analysis of the millisecond-TRIR data in Fig. 3 A we found that several exponential components are needed to accurately fit the data on the millisecond timescale. This is in line with previous reports of multi-exponential P700 + recombination kinetics that were probed at 820 nm (Vassiliev et al. 1997 ). In these previous studies DCPIP was used as an electron acceptor, while here we used ascorbate mediated by PMS as an electron donor to P700 + . In either case the conclusion is that the bioenergetics of radical pair recombination in isolated PSI samples is complex. Attempting to fully disentangle the millisecond bioenergetics is beyond the scope of this manuscript. In spite of the complex millisecond TR bioenergetics, however, Fig. 5 demonstrates that the dominant millisecond DAS (or the average of spectra collected over 0.1–1 ms) agrees well with the photoaccumulated (P700 + – P700) FTIR DS. The origin of the extra intense positive feature near 1650 cm –1 in the TRIR DS is unclear at present. It may be related to intense amide I and water absorption of the samples used in the TRIR experiments. CONCLUSIONS Microsecond TRSS FTIR DS at 77 K for PSI from T. vestitus are presented here for the first time. Photoaccumulated (P700 + - P700) FTIR DS for PSI from T. vestitus at both 77 and 295 K, are also presented here for the first time. To greatly extend upon this work PSI from T. vestitus was also studied using nanosecond TRIR DS at 295 K. This approach allowed us to probe ET down both branches in PSI. We show that in PSI at 295 K ET down the A and B branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies. Declarations ACKNOWLEDGEMENTS This work was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences (award number DE-SC-0017937 to GH) and the Deutsche Forschungsgemeinschaft (DFG) (Emmy Noether program; NU 421/1 to DJN; CRC1078, project A4 to HD). One of the coauthors (VGE) tragically passed away unexpectedly during the course of this work. We dedicate this manuscript to his memory. References Agalarov R, Brettel K (2003) Temperature dependence of biphasic forward electron transfer from the phylloquinone(s) A1 in photosystem I: only the slower phase is activated. Biochim Biophys Acta 1604:7-12 Agarwala N, Makita H, Hastings G (2023) Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1864:148918 Agarwala N, Makita H, Luo L, Xu W, Hastings G (2020) Reversible inhibition and reactivation of electron transfer in photosystem I. Photosynthesis Research Badshah SL et al. (2018) Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1859:42-55 Barber J (1992) The Photosystems : structure, function, and molecular biology vol 11. Topics in photosynthesis. Elsevier Science Publishers, Amsterdam, New York Bauscher M, Mantele W (1992) Electrochemical and Infrared-Spectroscopic Characterization of Redox Reactions of p-Quinones. J Phys Chem 96:11101-11108 Bauscher M, Nabedryk E, Bagley K, Breton J, Mäntele W (1990) Investigation of models for photosynthetic electron acceptors: Infrared spectroelectrochemistry of ubiquinone and its anions. 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In: Golbeck JH (ed) Photosystem I: The Light Driven Plastocyanin:Ferredoxin Oxidoreductase., vol 24. Advances in Photosynthesis and Respiration vol 24, vol 24. Springer, Dordrecht, pp 47-69 Golbeck J (1992) Structure and Function of Photosystem I. In: Annu. Rev. Plant Physiol. Plant Mol. Biol., vol 43. pp 293-324 Golbeck J (1995) Photosystem I in Cyanobacteria. In: Bryant D (ed) Advances in Photosynthesis. Molecular Biology of Cyanobacteria. Kluwer Academic Publishers,, pp 311-360 Golbeck J, Bryant D (1991) Photosystem I. In: Current topics in bioenergetics, vol 16. Academic Press, New York, pp 83-175 Guergova-Kuras M, Boudreaux B, Joliot A, Joliot P, Redding K (2001) Evidence for two active branches for electron transfer in photosystem I. Proc Natl Acad Sci U S A 98:4437-4442 Hage W, Kim M, Frei H, Mathies RA (1996) Protein dynamics in the bacteriorhodopsin photocycle: A nanosecond step-scan FTIR investigation of the KL to L transition. 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Biochem 40:3681-3689 Hu X, Frei H, Spiro TG (1996) Nanosecond step-scan FTIR spectroscopy of hemoglobin: ligand recombination and protein conformational changes. Biochemistry 35:13001-13005 Jolley C, Ben-Shem A, Nelson N, Fromme P (2005) Structure of plant photosystem I revealed by theoretical modeling. The Journal of biological chemistry 280:33627-33636 Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauss N (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 angstrom resolution. Nature 411:909-917 Kölsch A et al. (2018) Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus. J Biol Chem 293:9090-9100 Makita H, Hastings G (2015) Directionality of electron transfer in cyanobacterial photosystem I at 298 and 77 K. Febs Lett 589:1412-1417 Makita H, Hastings G (2016a) Modeling electron transfer in photosystem I. 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Biochimica et Biophysica Acta (BBA) - Bioenergetics 1859:645-654 Mäusle SM, Abzaliyeva A, Greife P, Simon PS, Perez R, Zilliges Y, Dau H (2020) Activation energies for two steps in the S2 -> S3 transition of photosynthetic water oxidation from time-resolved single-frequency infrared spectroscopy. J Chem Phys 153:215101 Mazor Y, Borovikova A, Nelson N (2015) The structure of plant photosystem I super-complex at 2.8 Å resolution. Elife 4:e07433 Mazor Y, Nataf D, Toporik H, Nelson N (2014) Crystal structures of virus-like photosystem I complexes from the mesophilic cyanobacterium Synechocystis PCC 6803. Elife 3 Nabedryk E, Schulz C, Muh F, Lubitz W, Breton J (2000) Heterodimeric versus homodimeric structure of the primary electron donor in Rhodobacter sphaeroides reaction centers genetically modified at position M202. Photochem Photobiol 71:582-588 Pakrasi H (1995) Genetic Analysis of the Form and Function of Photosystem I and Photosystem-II. In: Annu. Rev. Genet., vol 29. pp 755-756 Plunkett SE, Chao JL, Tague TJ, Palmer RA (1995) Time-Resolved Step-Scan Ft-Ir Spectroscopy of the Photodynamics of Carbonmonoxymyoglobin. Applied Spectroscopy 49:702-708 Qin XC, Suga M, Kuang TY, Shen JR (2015a) Structural basis for energy transfer pathways in the plant PSI-LHCI supercomplex. Science 348:989-995 Qin XC et al. (2015b) Isolation and characterization of a PSI-LHCI super-complex and its sub-complexes from a siphonaceous marine green alga, Bryopsis Corticulans. Photosynthesis Research 123:61-76 Rammelsberg R, Hessling B, Chorongiewski H, Gerwert K (1997) Molecular reaction mechanisms of proteins monitored by nanosecond step-scan FT-IR difference spectroscopy. Applied Spectroscopy 51:558-562 Redding K, van der Est A (2006) The Directionality of Electron Transport in Photosystem I. In: Golbeck J (ed) Photosystem I: The Light Driven Plastocyanin:Ferredoxin Oxidoreductase. Springer, Dordrecht, pp 413-437 Rodig C, Siebert F (1999) Errors and artifacts in time-resolved step-scan FT-IR spectroscopy. Applied Spectroscopy 53:893-901 Rohani L, Makita H, Levitz A, Henary M, Hastings G (2019) Calculated vibrational properties of semiquinones in the A 1 binding site in photosystem I. Biochim et Biophys Acta-Bioenergetics 1860:699-707 Santabarbara S, Casazza AP, Hastings G (2019) Modelling electron transfer in photosystem I: limits and perspectives. Physiologia plantarum 166:73-87 Schlodder E, Falkenberg K, Gergeleit M, Brettel K (1998) Temperature dependence of forward and reverse electron transfer from A 1 - , the reduced secondary electron acceptor in photosystem I. Biochem 37:9466-9476 Siebert F, Mantele W, Kreutz W (1982) Evidence For The Protonation Of Two Internal Carboxylic Groups During The Photocycle Of Bacteriorhodopsin. FEBS Lett 141:82-87 Sivakumar V, Wang R, Hastings G (2005) A(1) reduction in intact cyanobacterial photosystem I particles studied by time-resolved step-scan Fourier transform infrared difference spectroscopy and isotope labeling. Biochemistry 44:1880-1893 Srinivasan N, Golbeck JH (2009) Protein-cofactor interactions in bioenergetic complexes: The role of the A(1A) and A(1B) phylloquinones in Photosystem I. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1787:1057-1088 van der Est A (2006) Electron Transfer Involving Phylloquinone in Photosystem I. In: Golbeck J (ed) Photosystem I: The Light Driven Plastocyanin:Ferredoxin Oxidoreductase. Advances in Photosynthesis and Respiration vol 24, vol 24. Springer, Dordrecht, pp 387-411 Vassiliev IR, Jung YS, Mamedov MD, Semenov A, Golbeck JH (1997) Near-IR absorbance changes and electrogenic reactions in the microsecond-to-second time domain in Photosystem I. Biophys J 72:301-315 Walker D (1993) Energy, plants and man. 2nd edn. Oxygraphics, Brighton, East Sussex : Mill Valley, CA Weidlich O, Siebert F (1993) Time-Resolved Step-Scan Ft-Ir Investigations of the Transition from Kl to L in the Bacteriorhodopsin Photocycle - Identification of Chromophore Twists by Assigning Hydrogen-out-of-Plane (Hoop) Bending Vibrations. Applied Spectroscopy 47:1394-1400 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationFinal.docx Cite Share Download PDF Status: Published Journal Publication published 07 Jul, 2023 Read the published version in Photosynthesis Research → Version 1 posted Editorial decision: Major revision 08 Jun, 2023 Reviews received at journal 16 May, 2023 Reviewers agreed at journal 09 May, 2023 Reviewers agreed at journal 08 May, 2023 Reviewers invited by journal 07 May, 2023 Editor assigned by journal 07 May, 2023 Submission checks completed at journal 06 May, 2023 First submitted to journal 05 May, 2023 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-2898981","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":197895102,"identity":"9f95a057-9b1b-4bda-a992-734e3d59b0e9","order_by":0,"name":"Sarah M. Mäusle","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"M.","lastName":"Mäusle","suffix":""},{"id":197895106,"identity":"60f1986a-3597-48d6-934c-bcef22c25cc1","order_by":1,"name":"Neva Agarwala","email":"","orcid":"","institution":"Georgia State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Neva","middleName":"","lastName":"Agarwala","suffix":""},{"id":197895109,"identity":"60058c88-e15d-4d11-b399-7a6e570f5df4","order_by":2,"name":"Viktor G. Eichmann","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Viktor","middleName":"G.","lastName":"Eichmann","suffix":""},{"id":197895113,"identity":"560b46cb-fdc9-497f-8a34-76422d4ea358","order_by":3,"name":"Holger Dau","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Holger","middleName":"","lastName":"Dau","suffix":""},{"id":197895118,"identity":"d36216ad-344a-4681-ab6d-adf992b7e350","order_by":4,"name":"Dennis J. Nürnberg","email":"","orcid":"","institution":"Freie Universität Berlin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dennis","middleName":"J.","lastName":"Nürnberg","suffix":""},{"id":197895119,"identity":"6d9b7cf3-58ff-4588-8abf-453b24021b4f","order_by":5,"name":"Gary Hastings","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACCRBhAMTsDWA+YwNRWg6AtPAcIEkLmJFApBb56ObHnz8U3LGbP/P55c88DDayGw4Q0GJ455iZxAGDZ8kbbueUSfMwpBkT1jIjwQzol8PJBtI5acw8DIcTidCS/vkDSIv8zDPJQIf9J6xFXiLHAOiww3YMN9gPAB12gLAWA4mcMokzBocTDM7ksEnOMUg2nknQlhnpmz9U/DlsL99+/PGHNxV2sn0EbYEqSGxg4DGAJANCQL4BQtsDU8wDItSPglEwCkbBSAQAKNZJ5TWOuKwAAAAASUVORK5CYII=","orcid":"","institution":"Georgia State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Gary","middleName":"","lastName":"Hastings","suffix":""}],"badges":[],"createdAt":"2023-05-05 15:59:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2898981/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2898981/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11120-023-01035-9","type":"published","date":"2023-07-07T21:30:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36748988,"identity":"9a77a5c3-64ed-413e-a771-a88837a46634","added_by":"auto","created_at":"2023-05-09 16:19:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":489992,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cartoon depicting the PSI complex embedded in the thylakoid membrane. ET paths between cofactors are indicated. Protein subunits are labeled A-X. (B) Arrangement of ET cofactors in PSI. Figure produced using the 2.5 Å crystal structure of PSI from \u003cem\u003eT\u003c/em\u003e.\u003cem\u003e vestitus\u003c/em\u003e (Jordan et al. 2001). Cofactor hydrocarbon tails have been truncated. Arrows indicate ET routes along with associated time constants. Cyt c\u003csub\u003e6\u003c/sub\u003e is cytochrome c\u003csub\u003e6\u003c/sub\u003e, A\u003csub\u003e-1B\u003c/sub\u003e, A\u003csub\u003e-1A, \u003c/sub\u003eA\u003csub\u003e0B\u003c/sub\u003e and A\u003csub\u003e0A\u003c/sub\u003e are chlorophylls, A\u003csub\u003e1B\u003c/sub\u003e and A\u003csub\u003e1A\u003c/sub\u003e are phylloquinones, F\u003csub\u003eX\u003c/sub\u003e, F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e are iron-sulfur clusters and Fd is ferredoxin.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/7d19b4764b0fc0ea02ed1a46.png"},{"id":36748987,"identity":"371f7c72-e163-40f7-b786-4107647cdaf9","added_by":"auto","created_at":"2023-05-09 16:19:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":434878,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR DS for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e: Photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e – P700) FTIR DS at RT in the (\u003cstrong\u003eA\u003c/strong\u003e) 4000 – 1200 and (\u003cstrong\u003eB\u003c/strong\u003e) 1775 – 1400 cm\u003csup\u003e–1\u003c/sup\u003e regions. The “flat line” is a “dark – dark” FTIR DS that indicates the noise level in the experiment, as well as the zero-absorbance line. (\u003cstrong\u003eC\u003c/strong\u003e) Microsecond time-resolved (P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e–\u003c/sup\u003e – P700A\u003csub\u003e1A\u003c/sub\u003e) FTIR DS at 77 K (\u003cem\u003eblue\u003c/em\u003e). This spectrum is the average of nine spectra collected in 6 ms increments following a laser flash. Photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e – P700) FTIR DS for PSI at 77 (\u003cem\u003ered\u003c/em\u003e) and 293 K (\u003cem\u003eblack\u003c/em\u003e) are also shown in C (redrawn from B). Spectra were scaled to the 1718(+)/1700(–) cm\u003csup\u003e–1\u003c/sup\u003e difference band. The absorbance scale in (C) is for the TR FTIR DS.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/6e2cdd4f37bd3de0be7c65cb.png"},{"id":36748991,"identity":"ff58e74c-d1be-4e52-a318-b60379893896","added_by":"auto","created_at":"2023-05-09 16:19:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":554969,"visible":true,"origin":"","legend":"\u003cp\u003eTransient absorption changes at 1415 (\u003cem\u003egrey\u003c/em\u003e), 1430 (\u003cem\u003eblue\u003c/em\u003e), 1482 (\u003cem\u003eblack\u003c/em\u003e), 1494 (\u003cem\u003ered\u003c/em\u003e), 1510 (\u003cem\u003eyellow\u003c/em\u003e), 1534 (\u003cem\u003edark green\u003c/em\u003e), 1542 (\u003cem\u003elight green\u003c/em\u003e) and 1679 (\u003cem\u003epurple\u003c/em\u003e) cm\u003csup\u003e-1\u003c/sup\u003e obtained following 532 nm laser flash excitation of PSI samples at room temperature (~293 K). The data around zero (\u003cem\u003eblack\u003c/em\u003e) illustrates the noise level in the experiment and is associated with transient data collected at 1482 cm\u003csup\u003e-1\u003c/sup\u003e, in the absence of laser flash excitation. (A) A linear plot showing the absorption changes up to 2 µs after excitation. (B) A semi-logarithmic plot of the same data showing the absorption changes from 2 µs to 800 ms after excitation. The data at all six wavelengths, between 0 - 5 µs, is fitted simultaneously to a function consisting of two exponential components and a constant, convolved with the IRF. The time constants but not the pre-exponential amplitudes are constrained to be the same at each wavenumber. The fitted functions are shown in (A) and (B).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/f59d6820ffb21b88c9724d83.png"},{"id":36749437,"identity":"197cd298-8053-4ee4-88fb-77727b7e6c8c","added_by":"auto","created_at":"2023-05-09 16:27:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":294016,"visible":true,"origin":"","legend":"\u003cp\u003eResults from global analysis of the TRIR data in Fig. 3A. (A) Amplitudes, A\u003csub\u003e1\u003c/sub\u003e and A\u003csub\u003e2\u003c/sub\u003e, of the 33 and 364 ns exponential components, and the amplitude of the non-decaying component (\u003cem\u003ey\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e), for each wavenumber. (B) Chart showing the relative contribution of each of the three components for each wavenumber. A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e and y\u003csub\u003e0\u003c/sub\u003e relative contributions are proxies for contributions from A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e–\u003c/sup\u003e, A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e–\u003c/sup\u003e and P700\u003csup\u003e+\u003c/sup\u003e, respectively.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/500d88772531d2401a146fe9.png"},{"id":36749720,"identity":"ffe4c3ce-911c-43f8-bc5a-a9568817ad59","added_by":"auto","created_at":"2023-05-09 16:35:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":251291,"visible":true,"origin":"","legend":"\u003cp\u003eTRIR DS obtained by averaging the flash-induced transients between 0.1 – 1 ms (\u003cem\u003eblack\u003c/em\u003e). Decay associated spectrum of the 128 ms phase obtained by globally fitting TRIR kinetics is also shown (\u003cem\u003ered\u003c/em\u003e). For comparison a photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e – P700) FTIR DS obtained for similarly prepared and mounted PSI samples at RT is also shown (\u003cem\u003egreen\u003c/em\u003e). This FTIR DS is also shown in Fig. 2.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/85a7efabb05c613308aaf693.png"},{"id":44732539,"identity":"f3858073-b5d3-4d60-a73a-a762918617d1","added_by":"auto","created_at":"2023-10-16 21:55:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1703754,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/fc2c2a59-664e-4ae6-b173-ab0cfb9b9a42.pdf"},{"id":36749436,"identity":"90c77843-fb79-4252-ae61-04df19103718","added_by":"auto","created_at":"2023-05-09 16:27:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":595169,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-2898981/v1/a7ae8082f019cc17122cfa7d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanosecond Time-Resolved Infrared Spectroscopy for the Study of Electron Transfer in Photosystem I","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn photosynthesis solar energy is harvested and used to synthesize chemical products that are ultimately the source of most of the food and fuels consumed by humanity today (Walker \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In oxygen evolving organisms two photosystems, called photosystem (PS) I and II\u003csup\u003e1\u003c/sup\u003e, capture and convert solar energy independently, but cooperatively (Barber \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Walker \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The solar conversion reactions occur in a centralized pigment-protein unit called a reaction center. In the reaction center, light is used to drive electrons, via a series of acceptors, across a biological membrane (the thylakoid membrane). This light-induced separation of charge is the basic mechanism underlying solar energy capture in all photosynthetic organisms.\u003c/p\u003e \u003cp\u003eIn this manuscript we focus on electron transfer (ET) in isolated cyanobacterial PSI complexes from \u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e BP-1 (previously known as \u003cem\u003eThermosynechococcus elongatus\u003c/em\u003e BP-1). PSI consists of 11\u0026ndash;13 protein subunits, many of which have been characterized (Chitnis et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Golbeck \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Pakrasi \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The ET cofactors are bound to the PsaA and PsaB membrane-spanning protein subunits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) (Fromme and Grotjohann \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The terminal ET cofactors, F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e, are bound to the stromal PsaC subunit (Fromme and Grotjohann \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The organization of the bound ET cofactors is outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. The cofactor organization is near identical in PSI from plants, algae and cyanobacteria (Ben-Shem et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jolley et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Malavath et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mazor et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mazor et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn isolated PSI particles, following light excitation, the P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e radical pair state is formed within ~\u0026thinsp;50 picoseconds (Hastings et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Hastings et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). In PSI there are two cofactor branches with approximate C\u003csub\u003e2\u003c/sub\u003e symmetry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), referred to as the A\u0026ndash; and B\u0026ndash;branch. In this manuscript the A branch refers to the side in which the pigment in the A\u003csub\u003e1\u003c/sub\u003e binding site is bound to the PsaA protein subunit (A\u003csub\u003e1A\u003c/sub\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). ET can occur down both branches from P700 to F\u003csub\u003ex\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (Makita and Hastings \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Redding and van der Est \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth the A\u003csub\u003e1A\u003c/sub\u003e and A\u003csub\u003e1B\u003c/sub\u003e pigments in PSI are phylloquinone (PhQ) molecules, in a very similar protein environment (Brettel \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Golbeck \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Golbeck and Bryant \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Srinivasan and Golbeck \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In cyanobacterial PSI at RT, forward ET from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e proceeds biphasically with time constants of ~\u0026thinsp;25 and ~\u0026thinsp;250 nanoseconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The \u0026ldquo;fast\u0026rdquo; and \u0026ldquo;slow\u0026rdquo; time constants were observed in visible transient absorption spectroscopy experiments and are thought to be associated with ET from A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e, respectively (Agalarov and Brettel \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Makita and Hastings \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Makita et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Redding and van der Est \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Schlodder et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; van der Est \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). At RT, ET from F\u003csub\u003eX\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eA\u003c/sub\u003e and then on to F\u003csub\u003eB\u003c/sub\u003e occurs on a tens to hundreds of nanosecond timescale (Byrdin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In the absence of added electron acceptors, the P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eA/B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e state recombines in 50\u0026ndash;150 milliseconds (Golbeck and Bryant \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). The F\u003csub\u003eA/B\u003c/sub\u003e terminology is used to indicate that we do not specifically distinguish between the F\u003csub\u003eA\u003c/sub\u003e and F\u003csub\u003eB\u003c/sub\u003e states.\u003c/p\u003e \u003cp\u003eIn cyanobacterial PSI, forward ET from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e diminishes as the temperature is lowered, and is replaced by a P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e recombination reaction, which is characterized by a time constant of ~\u0026thinsp;300 microseconds at 77 K (Makita and Hastings \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Makita and Hastings \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Makita and Hastings \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e; Makita et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Schlodder et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). On the one hand, since ET recombination is essentially unidirectional down the A-branch at 77 K, spectral analysis is simplified. On the other hand, however, in microsecond time-resolved step-scan (TRSS) FTIR difference spectroscopy (DS) studies of PSI at 77 K, the bands associated with the P700\u003csup\u003e+\u003c/sup\u003e and A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e states decay with the same time constant, which negates the possibility of using the temporal evolution of bands to distinguish their origin. At ~\u0026thinsp;293 K (room temperature, RT), P700\u003csup\u003e+\u003c/sup\u003e and A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e decay on different timescales, allowing the possibility of assigning spectroscopic signatures (bands) based on their temporal evolution, thus the time resolution itself allows for a new approach to infrared spectral band identification and assignment.\u003c/p\u003e \u003cp\u003eAt RT, the P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e/P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e states decay in ~\u0026thinsp;25/250 ns, respectively, and can potentially be studied using TRSS FTIR DS. However, noise levels near or below 1x10\u003csup\u003e\u0026ndash;5\u003c/sup\u003e in OD units (0.1 mOD) are required (Hastings \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). At present, such TRSS FTIR DS measurements are challenging (Hage et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Hastings \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rodig and Siebert \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), and have only been demonstrated for protein systems such as bacteriorhodopsin, that display very intense absorption difference features (Hage et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Siebert et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Weidlich and Siebert \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), or for CO-myoglobin/hemoglobin which displays intense absorption changes at frequencies where absorption changes due to water and protein are minimal (in the 1900\u0026ndash;2100 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region) (Hu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Plunkett et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Recently, we have studied photosystem II using TRSS FTIR DS, but this work required data acquisition over a period of several months [Greife et al., Nature \u003cem\u003ein press\u003c/em\u003e], which is unacceptably long for most studies.\u003c/p\u003e \u003cp\u003eTRSS FTIR DS is advantageous as it allows the collection of time-resolved data over a broad spectral region. The downside is that sensitivity is limited, especially on nanosecond timescales (Hastings \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rammelsberg et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). An alternative approach is to undertake nanosecond time resolved infrared (TRIR) DS measurements at single frequencies using intense IR light sources. Quantum cascade lasers (QCL) can provide such high intensity IR light. Previously, QCLs were thought to be disadvantageous because of limited spectral coverage. However, we demonstrate here with our QCL setup that TR data can be collected with high spectral resolution covering the entire 1310\u0026ndash;1890 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region. One advantage in investigation of PSI is that it is a highly robust and stable protein that can be subjected to literally thousands of laser flashes without any appreciable degradation or damage. This allows us to undertake many separate single wavelength experiments covering the entire spectral region, with considerable signal averaging at each wavelength, allowing high signal to noise ratio at each of the wavelengths in the experiment.\u003c/p\u003e \u003cp\u003eIn this manuscript we have used a QCL based pump-probe spectrometer to undertake the first nanosecond TRIR DS studies of PSI complexes in the physiological temperature range (23\u0026deg;C). Such measurements have allowed us to probe absorption changes associated with single molecular bonds of the phylloquinone molecules on both the A and B branch as they undergo ET. To complement and aid in the analysis of the TRIR data, photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS were obtained at 77 and 295 K and (P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700A\u003csub\u003e1\u003c/sub\u003e) TRSS FTIR DS were obtained at 77 K. Time resolved and photoaccumulated FTIR DS for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e have also never been presented previously.\u003c/p\u003e\n\u003cp\u003e[1]\u003cstrong\u003eAbbreviations:\u003c/strong\u003e C=O, carbonyl; Chl \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003ea\u003c/em\u003e; DAS, decay associated spectrum; DS, difference spectra/spectrum/spectroscopy; ET, electron transfer; FTIR, Fourier transform-infrared; H-bond, hydrogen bond; IRF, instrument response function;\u0026nbsp;ms, ms, ns, micro, milli, and nano-second; NQ, 1,4-naphthoquinone; PhQ, phylloquinone (2-methyl-3-phytyl-1,4-naphthoqiuinone); PSI, photosystem I; PSII, photosystem II; \u003cem\u003eThermosynechococcus vestitus\u0026nbsp;\u003c/em\u003eBP-1, \u003cem\u003eT. vestitus\u003c/em\u003e; TRIR, time-resolved infrared; TRSS, time-resolved step-scan.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eTrimeric PSI particles from \u003cem\u003eT. vestitus\u003c/em\u003e were prepared by first binding His-tagged PSII complexes to a nickel resin (M\u0026auml;usle et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and applying the PSI-enriched flow through to a Toyopearl DEAE-650S column as previously described (K\u0026ouml;lsch et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The eluted PSI fractions were concentrated using an Amicon Stirred Cell with a Synder LY membrane (Sterlitech, USA), frozen in liquid nitrogen and stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003ePhotoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026minus; P700) FTIR DS (at both RT and 77 K) and TRSS FTIR DS (at 77 K), all at 4 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e spectral resolution, were collected as described previously (Agarwala et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Makita and Hastings \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PSI samples for single-frequency TRIR DS experiments were prepared in a similar manner: After thawing on ice, the sample was diluted in 50 mM TRIS buffer (pH 8), and 0.04% b-DM, such that a chlorophyll concentration of 0.2 mg/ml was reached. The sample was centrifuged at 300,000 g for three hours at 4\u0026deg;C. A CaF\u003csub\u003e2\u003c/sub\u003e window was prepared with a ring of vacuum grease and a 15 \u0026micro;m spacer. Two artificial electron donors (0.1 \u0026micro;l of 20 \u0026micro;M PMS and 0.1 \u0026micro;l of 20 mM sodium ascorbate) were pipetted onto the CaF\u003csub\u003e2\u003c/sub\u003e window, before adding a small amount of the soft PSI pellet and carefully mixing with a spatula. A second CaF\u003csub\u003e2\u003c/sub\u003e window was placed onto the first, tightly sealing the sample in between.\u003c/p\u003e \u003cp\u003eTRIR DS measurements were performed using the previously described experimental setup (M\u0026auml;usle et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with some modifications: 1. The intensity of the pulsed 532 nm Nd:YAG laser is now regulated by a motorized attenuator (Eksma Optics, Vilnius, Lithuania). 2. The QCL system was replaced by a newer model (MIRcat-QT-Z-2300) with improved optical properties (wavenumber accuracy\u0026thinsp;\u0026lt;\u0026thinsp;1 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, covering the entire 1310\u0026ndash;1890 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e spectral range with improved pointing stability) (Daylight Solutions, San Diego, CA).\u003c/p\u003e \u003cp\u003eFor each desired wavelength, a flash sequence of two dark measurements (no flashes), followed by five saturating excitation flashes (~\u0026thinsp;0.1 mJ/mm\u003csup\u003e2\u003c/sup\u003e) and a final flash with higher intensity (~\u0026thinsp;0.3 mJ/mm\u003csup\u003e2\u003c/sup\u003e) was applied repeatedly to a single sample spot. The IR signal was measured from 20 ms before and up to 800 ms after each excitation flash. The difference in signal between the low and high excitation flashes was used to calculate a sample heating artefact, which was fit by a sum of exponentials and subtracted from the data (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA pulse generator was used for triggering the excitation laser and data acquisition. The timing of the excitation flash was determined by recording each flash with a photodiode. The sample chamber was flushed with dry air and the temperature was set at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C.\u003c/p\u003e \u003cp\u003eAt each frequency the results obtained for 600\u0026ndash;6000 laser flashes were averaged. The data was then subjected to a global analysis procedure as follows: Transient IR absorption changes were fitted to a sum of exponential functions plus a constant [\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(y\\left(t,\\nu \\right)= {y}_{o}+{\\sum }_{i}{A}_{i,{\\nu }}{e}^{-t/{\\tau }_{i}}\\)\u003c/span\u003e\u003c/span\u003e] using a least-squares approach implemented in Python 3.7 (Python Software Foundation, Delaware, USA). For the fits on the nanosecond timescale, the exponential function was convolved with an approximate instrument response function (a Gaussian with standard width of 17 ns) as illustrated in Fig. S2. For results obtained by an alternative approach, see Fig. S3 and S4. As is standard in these types of global analysis fitting procedures, the time constants were constrained to be the same at each wavelength, while the amplitudes (preexponential factors) were allowed to vary.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS collected at 295 K for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e in the 3800\u0026ndash;1200 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region. The dark minus dark spectrum indicates the noise level in the experiment as well as the zero-absorbance line. As is relatively well known (Breton \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Nabedryk et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and is clearly seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, P700\u003csup\u003e+\u003c/sup\u003e displays several very broad, positive, \u003cem\u003eelectronic absorption bands\u003c/em\u003e throughout the 3800\u0026ndash;1200 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region. This broad positive absorption difference overlies the IR difference bands in the 1800\u0026ndash;1400 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), essentially \u0026ldquo;pushing\u0026rdquo; the whole spectrum (of IR difference bands) more positive. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC shows microsecond TRSS (P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700A\u003csub\u003e1\u003c/sub\u003e) FTIR DS obtained using \u003cem\u003eT. vestitus\u003c/em\u003e PSI samples at 77 K (\u003cem\u003eblue\u003c/em\u003e). As has been described (Hastings \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) this TRSS FTIR DS is the average of nine spectra, collected in consecutive 6 \u0026micro;s increments following a laser flash. This spectrum can therefore be considered as an averaged spectrum obtained over a\u0026thinsp;~\u0026thinsp;54 \u0026micro;s time-period. Photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS at both 77 (\u003cem\u003ered\u003c/em\u003e) and 298 K (\u003cem\u003eblack\u003c/em\u003e) are also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC for comparison. Both the TRSS and photoaccumulated FTIR DS for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e are presented here for the first time.\u003c/p\u003e \u003cp\u003eAlthough we call the RT photoaccumulated FTIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS, it is strictly speaking a (P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eA/B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700F\u003csub\u003eA/B\u003c/sub\u003e) FTIR DS. The 77 K photoaccumulated FTIR DS is also strictly speaking a (P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eX\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700F\u003csub\u003eX\u003c/sub\u003e) FTIR DS. In the latter case this is because P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eA/B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e formation, which occurs in ~\u0026thinsp;35% of the PSI complexes, is irreversible at 77 K (Schlodder et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and signals associated with P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eA/B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e will not contribute in our experiments that involve repetitive illumination. However, P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eX\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e recombination can occur at 77 K, with a half lifetime of 5-100 ms, in ~\u0026thinsp;20% of the PSI complexes (Schlodder et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Under repetitive bouts of continuous illumination at 77 K the P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eX\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e state will be the dominant photoaccumulated species. P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e is unlikely to contribute, as it decays in ~\u0026thinsp;300 \u0026micro;s at 77 K making it difficult to photoaccumulate.\u003c/p\u003e \u003cp\u003eBy adding a variety of electron donors and acceptors, and by studying PSI samples that lack either F\u003csub\u003eA/B\u003c/sub\u003e (Breton et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Hastings and Sivakumar \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), or F\u003csub\u003eA/B\u003c/sub\u003e and F\u003csub\u003eX\u003c/sub\u003e (Hastings and Sivakumar \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), it has been determined that the iron sulfur clusters do not contribute significantly in (P700\u003csup\u003e+\u003c/sup\u003eF\u003csub\u003eA/B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700F\u003csub\u003eA/B\u003c/sub\u003e) FTIR DS in the 1800\u0026ndash;1200 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region (Fe-S vibrations are expected below ~\u0026thinsp;500 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (Chu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Chu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e)), and this is the justification for calling these photoaccumulated spectra (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS. Nonetheless one might expect electrochromic effects from protein or pigment species near the reduced iron sulfur clusters to contribute to some degree in photoaccumulated FTIR DS. In a more recent study of PSI site directed mutants we did suggest possible small contributions to photoaccumulated FTIR DS from modes that might be impacted by F\u003csub\u003eX\u003c/sub\u003e reduction (Agarwala et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 77 K (P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700A\u003csub\u003e1\u003c/sub\u003e) TRSS FTIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC displays positive bands at 1567, 1511, 1495 and 1415 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, which are absent in the 77 K photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS. These bands are therefore associated with A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e. The bands at 1495 and 1415 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e are well known to be due to stretching vibrations of the C\u003csub\u003e1\u003c/sub\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{⃛}{-}\\)\u003c/span\u003e\u003c/span\u003eO and C\u003csub\u003e4\u003c/sub\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{⃛}{-}\\)\u003c/span\u003e\u003c/span\u003eO groups of the phyllosemiquinone anion in the A\u003csub\u003e1A\u003c/sub\u003e binding site (Hastings \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rohani et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Given the similarity in the frequencies of the bands in the (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS at 295 and 77 K (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), it is likely that the phyllosemiquinone anion bands will also be at similar frequencies at 77 and 295 K. We therefore expect to observe TRIR absorption changes at ~\u0026thinsp;1495 and ~\u0026thinsp;1415 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e associated with A\u003csub\u003e1A\u003c/sub\u003e reduction and recovery at RT. In addition, phylloquinone in the A\u003csub\u003e1B\u003c/sub\u003e binding site is structurally very similar to that on the A-side, and it is likely that the B-side phyllosemiquinone will also display absorption near 1494 and 1415 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e at both 77 K and 298 K.\u003c/p\u003e \u003cp\u003eBoth the (P700\u003csup\u003e+\u003c/sup\u003eA\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026ndash; P700A\u003csub\u003e1A\u003c/sub\u003e) and (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC display peaks around 1482, 1458 and 1431 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, so probing absorption changes at these frequencies (in TRIR measurements at RT) will likely result in little or no nanosecond TR absorption changes associated with forward ET from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, but a long-lived (millisecond) contribution due to P700\u003csup\u003e+\u003c/sup\u003e is expected.\u003c/p\u003e \u003cp\u003eRT flash-induced absorption changes at 1415, 1430, 1482, 1494, 1510, 1534, 1542 and 1679 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, on a nanosecond to second timescale, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The kinetic traces in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA are shown on a linear timescale, from 0-2000 ns. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB the same kinetic traces are shown on a logarithmic timescale, from 2x10\u003csup\u003e\u0026ndash;3\u003c/sup\u003e \u0026minus;\u0026thinsp;8x10\u003csup\u003e3\u003c/sup\u003e ms. All the transients exhibit nanosecond kinetic phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The absorption changes are relatively constant on microsecond timescales, and decay back to zero on a hundreds of milliseconds timescale (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTransient data at all the wavelengths indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e were subjected to a global analysis fitting procedure outlined in the materials section and the supplementary information section. The transient kinetics were analyzed in the 0\u0026ndash;5 \u0026micro;s time range. From this fitting procedure it is found that the data is best described by a function consisting of two exponential components (and a constant, non-decaying component). The fitted functions are also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e and are characterized by lifetimes of 33 and 364 ns (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in the supplementary information section). The time constants vary slightly when only a subset of the listed transients are included in the global fitting procedure (\u003cem\u003enot shown\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows millisecond TRIR DS extracted from the data collected using the QCL based spectrometer at RT. This data was produced by scanning the QCL output (in 2 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e increments) and collecting transients for 6 laser flashes at each wavenumber (300 flashes for wavenumbers below 1550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In this mode relatively low-sensitivity kinetic data was acquired across the entire 1770\u0026ndash;1380 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region. The signal to noise ratio for the data at each wavelength, especially in the ns region, is significantly poorer than that shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. However, by applying a spectral smoothing algorithm (three neighboring wavenumbers are averaged and assigned to the wavenumber in the middle, resulting in an effective resolution of 6 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), and averaging each of the spectra obtained over a given time interval, a high sensitivity TRIR absorption difference spectrum is obtained. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (\u003cem\u003eblack curve\u003c/em\u003e) shows the flash-induced TRIR DS resulting from the averaging all 220 spectra collected in the 0.1\u0026ndash;1 ms time window, a time range in which absorption changes are minimal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also globally fitted the eight kinetic traces in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, in the 1-800 ms time range, and found that at least five exponential components are needed for a satisfactory fit for the decay back to zero (as judged by the residuals). Time constants extracted are 1.3, 11, 46, 128 and 444 ms, with the 128 ms component being the dominant phase.\u003c/p\u003e \u003cp\u003eInstead of averaging the different spectral blocks derived in this QCL wavelength scanning experiment, we could just directly globally analyze the entire data block (with or without fixed time constants that were derived from fitting the data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). From this analysis a decay associated spectrum (DAS) associated with the ~\u0026thinsp;128 ms phase is obtained, and this DAS is also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. This 128 ms DAS is essentially identical to the average of all the spectra in the 0.1\u0026ndash;1 ms range. It is also identical to the average of the spectra obtained in the 1\u0026ndash;800 ms range (not shown). Also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e is a photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS obtained at RT, employing 4 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e spectral resolution (\u003cem\u003egreen\u003c/em\u003e) (from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The similarity between the photoaccumulated FTIR DS and the different TRIR DS is obvious, except for the intensity of the positive peak near 1655 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, which is more pronounced in the TRIR DS. The origin of this difference in the TRIR and FTIR DS is not clear at present. There are other very small differences in the TRIR and FTIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, for example the absorption change near 1735 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, which may relate to the different spectral resolutions in the two experiments.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eNanosecond transient absorption spectroscopy, with 480 nm light, is often used to probe forward ET from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e in PSI at RT (Agalarov and Brettel \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Badshah et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The positive absorption change at 480 nm is due to an electrochromic effect on a chlorophyll pigment (likely A\u003csub\u003e0\u003c/sub\u003e) caused by the electron residing on the A\u003csub\u003e1\u003c/sub\u003e pigment (Bautista et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The absorption changes at 480 nm were shown to decay biphasically with time constants of 11 and 340 ns at 295 K in PSI from \u003cem\u003eSynechocystis\u003c/em\u003e sp. 6803 (Agalarov and Brettel \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The two time constants have been shown to be consistent with ET down the B and A branches in PSI (Guergova-Kuras et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince absorption changes on the nanosecond timescale are associated with forward ET from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e, and changes that do not decay/grow on a nanosecond-microsecond timescale are associated mainly with P700 or P700\u003csup\u003e+\u003c/sup\u003e (as changes associated with F\u003csub\u003eX\u003c/sub\u003e/F\u003csub\u003eX\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e are minimal in the 1800\u0026thinsp;\u0026minus;\u0026thinsp;1200 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e region), we can separate IR absorption contributions from the different pigments based on their time evolution.\u003c/p\u003e \u003cp\u003eFor the 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e kinetic, the 33 and 364 ns phases have large amplitudes and are well resolved (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e band in the TRSS FTIR DS at 77 K (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) is due to the C\u003csub\u003e1\u003c/sub\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{⃛}{-}\\)\u003c/span\u003e\u003c/span\u003eO group of PhQ\u003csup\u003e\u0026ndash;\u003c/sup\u003e in the A\u003csub\u003e1A\u003c/sub\u003e binding site (Rohani et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For the 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e absorption change, 72% of the positive, flash-induced absorption change decays on the nanosecond timescale, with 40 and 32% of the amplitude being associated with the 33 and 364 ns time constants, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A long-lived positive feature contributing to 28% of the initial absorption change remains following the nanosecond kinetics. As indicated above, the A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e and y\u003csub\u003e0\u003c/sub\u003e relative contributions (outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) are proxies for relative contributions from A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and P700\u003csup\u003e+\u003c/sup\u003e. Therefore, at 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and P700\u003csup\u003e+\u003c/sup\u003e all have a positive absorption change. This observation also follows from the photoaccumulated and TRSS FTIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003eThe kinetic at 1510 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e exhibits similar absorption changes to the one at 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), but with a less pronounced 364 ns phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). A positive band is also observed at 1511 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in the microsecond TRSS FTIR DS at 77 K, with an intensity that is also considerably lower than that at 1494 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The similarity in the relative amplitudes of the nanosecond phases at the two wavelengths at RT, with that in the TRSS FTIR DS at 77 K, suggests that the bands at 1511 and 1494 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 77 K likely corresponds to bands at similar wavenumber at RT, and that the 33 and 364 ns phases at 1510 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e are likely due to A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, respectively (as is the case for the 1494 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band). The 33 and 364 ns phases in the kinetic at 1510 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at RT are likely associated with quinone ring C\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{⃛}{-}\\)\u003c/span\u003e\u003c/span\u003eC modes of A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, respectively. It is well known that semiquinone ring C\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{⃛}{-}\\)\u003c/span\u003e\u003c/span\u003eC modes have higher frequency vibrations than C\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{⃛}{-}\\)\u003c/span\u003e\u003c/span\u003eO modes (Bauscher and Mantele \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Bauscher et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1990\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe absorption changes at 1679 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e displays an intense 33 ns phase (46%), with smaller contributions from the 364 ns (33%) and non-decaying (21%) phases. The non-decaying phase of the 1679 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e kinetic is weakly negative, as is the amplitude in the photoaccumulated FTIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C. So, the 1679 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e kinetic indicates that during ET from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e, the absorption change is initially positive and the nanosecond bioenergetics lead to a negative absorption change that is due to P700\u003csup\u003e+\u003c/sup\u003e. The species responsible for these nanosecond phases at 1679 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are not clear at present. They could be associated with amide I vibrations associated with amino acids near A\u003csub\u003e1\u003c/sub\u003e, or with keto carbonyl modes of the A\u003csub\u003e\u0026ndash;1\u003c/sub\u003e or A\u003csub\u003e0\u003c/sub\u003e pigments (Sivakumar et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This is a topic that will be further investigated in the future.\u003c/p\u003e \u003cp\u003eThe flash-induced absorption increase at 1482 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e occurs within the time resolution of the instrument, followed by a fast 33 ns decay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cem\u003eblack\u003c/em\u003e) with little further absorption changes on the hundreds of nanoseconds to microseconds timescale (the 364 ns phase is of very low amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB)). The 33 ns and non-decaying phases account for 34 and 61% of the absorption difference signal at 1482 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (Fig, 4B and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively. However, there is considerable uncertainty in the amplitude for the 33 ns phase. The fact that we observe both positive and negative amplitudes for the 33 ns phase at the different wavelengths (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) confirms that this phase is not an instrumental artefact. However, the 33 ns phase is close to the temporal resolution limits of the instrument (estimated to be 17 ns), and the amplitudes of this phase at the different wavelengths correspondingly have large error (see discussion of this topic in the supplementary information section).\u003c/p\u003e \u003cp\u003eAt 1482 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, given that there is a 33 ns phase and essentially no 364 ns phase (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), it is unlikely that the 33 ns phase is associated with molecular groups of A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e itself, because then a corresponding 364 ns phase might also be expected near 1482 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e associated with A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e. Note that we presume that A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e have absorption bands at similar frequencies, as they do at 1494 and 1415 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhat the 33 ns phase at 1482 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e is due to is not clear, possibly a protein group between A\u003csub\u003e1B\u003c/sub\u003e and F\u003csub\u003eX\u003c/sub\u003e. Since the 33 ns feature at 1482 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e should reflect B-branch and not A-branch ET, we speculate that we have uncovered a spectral observable indicating an asymmetry in A and B branch ET process.\u003c/p\u003e \u003cp\u003eThis type of asymmetry between the fast and slow phases may potentially be observed at other wavelengths, such as at 1542 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, where the 33 ns phase is of much lower amplitude than the 364 ns phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, this latter difference may also reflect a branch-utilization asymmetry. In cyanobacterial PSI the A/B branching ratio is roughly 80/20 (Makita and Hastings \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), respectively. So, the differences in the amplitudes of the two phases at 1542 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e may reflect this branch-utilization asymmetry. From the data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, it is clear that the 33 ns phase is very poorly resolved at 1542 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, and also at 1534 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, and it is probably unwise to make assertions based on the amplitude of the 33 ns phase at these frequencies.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, following the decay of the nanosecond phases (from ~\u0026thinsp;5 \u0026micro;s onwards) a pronounced positive/negative absorption change is observed at 1542/1534 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, respectively, that subsequently decays with a time constant of ~\u0026thinsp;128 ms. From Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the non-decaying phase has an amplitude of +\u0026thinsp;3.17/-1.95 at 1542/1534 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, respectively. In contrast, the TRIR DS in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e indicates a higher negative amplitude at 1534 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e compared to the corresponding positive amplitude at 1542 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e. The solution to this issue is to recognize that the broad positive absorption of the P700\u003csup\u003e+\u003c/sup\u003e state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B) also decays in ~\u0026thinsp;128 ms. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB one observes that the negative signal at 1534 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, relative to the zero line, is considerably less than the positive amplitude of the signal at 1542 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (relative to the zero line), and this amplitude ratio is in line with the millisecond-TRIR kinetic data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eAlthough the absorption difference features in the kinetics in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e appear well resolved, the 33 ns kinetic phase is close to the instrumental time resolution. The instrumental time resolution is determined mostly by the response of the IR detector with integrated preamplifier (10 MHz cut-off frequency) and the sampling rate of the A/D converter, where the smallest possible bin size is 15 ns. In our global analysis curve-fitting procedures, the instrument response function (IRF) is well modelled by a Gaussian function with standard width of 17 ns (see discussion in the supplementary information section). Given the 17 ns IRF, and the limited data sampling, the amplitudes of the 33 ns phase at the different wavenumbers is difficult to pinpoint with high precision. Nevertheless, the 33 ns phase is clearly discernable at many wavelengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and is not artefactual. The exact time constant and amplitudes of the 33 ns phase at the different wavelengths are poorly determined, however, depending on the details of the fitting procedure (see the supplementary information section). This is an important consideration. For example, if the 33 and 364 ns phases in the kinetic at 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e are due to forward ET from A\u003csub\u003e1B\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and A\u003csub\u003e1A\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, one might expect the 364 ns phase to exhibit a similar, or up to a two-to-four times larger amplitude, than the 33 ns phase, simply because the branching ratio in PSI is expected to be in the 50/50 to 20/80 range (Redding and van der Est \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This is clearly not found to be the case for the 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e kinetic data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Using an alternate fitting procedure, where zero time for the kinetics is manually adjusted, and the instrument response function is not included in the fitting, the amplitude of the 33 ns phase relative to the 364 ns phase can fall to ~\u0026thinsp;40/60 (see discussion in the supplementary information section). So, caution should be exercised at present on building thesis based on the amplitude of the 33 ns phase in the various kinetics.\u003c/p\u003e \u003cp\u003eOn the other hand, it may be the case that the amplitude of the 33 ns phase is truly larger than the 364 ns at 1494 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), and if this is the case it could mean that an alternative model of ET needs to be considered, such as the radical pair equilibrium model (Santabarbara et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A final answer may have to await the production of kinetic data with improved temporal resolution.\u003c/p\u003e \u003cp\u003eIn the analysis of the millisecond-TRIR data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA we found that several exponential components are needed to accurately fit the data on the millisecond timescale. This is in line with previous reports of multi-exponential P700\u003csup\u003e+\u003c/sup\u003e recombination kinetics that were probed at 820 nm (Vassiliev et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In these previous studies DCPIP was used as an electron acceptor, while here we used ascorbate mediated by PMS as an electron donor to P700\u003csup\u003e+\u003c/sup\u003e. In either case the conclusion is that the bioenergetics of radical pair recombination in isolated PSI samples is complex. Attempting to fully disentangle the millisecond bioenergetics is beyond the scope of this manuscript. In spite of the complex millisecond TR bioenergetics, however, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrates that the dominant millisecond DAS (or the average of spectra collected over 0.1\u0026ndash;1 ms) agrees well with the photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR DS. The origin of the extra intense positive feature near 1650 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in the TRIR DS is unclear at present. It may be related to intense amide I and water absorption of the samples used in the TRIR experiments.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eMicrosecond TRSS FTIR DS at 77 K for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e are presented here for the first time. Photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e - P700) FTIR DS for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e at both 77 and 295 K, are also presented here for the first time. To greatly extend upon this work PSI from \u003cem\u003eT. vestitus\u003c/em\u003e was also studied using nanosecond TRIR DS at 295 K. This approach allowed us to probe ET down both branches in PSI. We show that in PSI at 295 K ET down the A and B branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis work was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences (award number DE-SC-0017937 to GH) and the Deutsche Forschungsgemeinschaft (DFG) (Emmy Noether program; NU 421/1 to DJN; CRC1078, project A4 to HD). One of the coauthors (VGE) tragically passed away unexpectedly during the course of this work. We dedicate this manuscript to his memory.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgalarov R, Brettel K (2003) Temperature dependence of biphasic forward electron transfer from the phylloquinone(s) A1 in photosystem I: only the slower phase is activated. Biochim Biophys Acta 1604:7-12\u003c/li\u003e\n\u003cli\u003eAgarwala N, Makita H, Hastings G (2023) Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1864:148918\u003c/li\u003e\n\u003cli\u003eAgarwala N, Makita H, Luo L, Xu W, Hastings G (2020) Reversible inhibition and reactivation of electron transfer in photosystem I. Photosynthesis Research\u003c/li\u003e\n\u003cli\u003eBadshah SL et al. 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Applied Spectroscopy 47:1394-1400\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photosynthesis, Photosystem I, Thermosynechococcus vestitus BP-1, nanosecond time-resolved infrared spectroscopy, electron transfer, A1, P700","lastPublishedDoi":"10.21203/rs.3.rs-2898981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2898981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrosecond time-resolved step-scan FTIR difference spectroscopy was used to study photosystem I (PSI) from \u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e BP-1 (\u003cem\u003eT. vestitus\u003c/em\u003e, formerly known as \u003cem\u003eT. elongatus\u003c/em\u003e) at 77 K. Photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR difference spectra were also obtained for PSI from \u003cem\u003eT. vestitus\u003c/em\u003e at both 77 and 293 K. The FTIR difference spectra are presented here for the first time. To greatly extend upon these FTIR studies nanosecond time-resolved infrared difference spectroscopy was also used to study PSI from \u003cem\u003eT. vestitus\u003c/em\u003e at 293 K. Nanosecond infrared spectroscopy has never been used to study PSI samples at physiological temperatures, and here it is shown that such an approach has great value as it allows a direct probe of electron transfer down both branches in PSI. In PSI at 293 K, the infrared flash-induced absorption changes indicate electron transfer down the A- and B-branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies. These time constants are associated with forward electron transfer from A\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e to F\u003csub\u003eX\u003c/sub\u003e on the B- and A-branches, respectively. At several infrared wavelengths flash-induced absorption changes at 293 K recover in tens to hundreds of milliseconds. The dominant decay phase is characterized by a lifetime of 128 ms. These millisecond changes are assigned to radical pair recombination reactions, with the changes being associated primarily with P700\u003csup\u003e+\u003c/sup\u003e rereduction. This conclusion follows from the observation that the millisecond infrared spectrum is very similar to the photoaccumulated (P700\u003csup\u003e+\u003c/sup\u003e \u0026ndash; P700) FTIR difference spectrum.\u003c/p\u003e","manuscriptTitle":"Nanosecond Time-Resolved Infrared Spectroscopy for the Study of Electron Transfer in Photosystem I","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-09 16:19:12","doi":"10.21203/rs.3.rs-2898981/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-06-08T22:02:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-05-16T04:53:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0608e085-c24b-4129-bc98-22ebe50a4bc4","date":"2023-05-10T03:04:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"396a671b-e78f-4d6c-982a-f82c8a0f5d4f","date":"2023-05-08T08:07:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-07T21:19:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-07T07:35:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-05-06T09:08:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photosynthesis Research","date":"2023-05-05T15:56:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1e2c9a7f-1a17-4578-94c1-cd94f06892f6","owner":[],"postedDate":"May 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:41:18+00:00","versionOfRecord":{"articleIdentity":"rs-2898981","link":"https://doi.org/10.1007/s11120-023-01035-9","journal":{"identity":"photosynthesis-research","isVorOnly":false,"title":"Photosynthesis Research"},"publishedOn":"2023-07-07 21:30:27","publishedOnDateReadable":"July 7th, 2023"},"versionCreatedAt":"2023-05-09 16:19:12","video":"","vorDoi":"10.1007/s11120-023-01035-9","vorDoiUrl":"https://doi.org/10.1007/s11120-023-01035-9","workflowStages":[]},"version":"v1","identity":"rs-2898981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2898981","identity":"rs-2898981","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}