Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

How Aqueous Solvation Impacts the Frequencies and Intensities of Infrared Absorption Bands in Flavin: The Quest for a Suitable Solvent Model.

FTIR spectroscopy accompanied by quantum chemical simulations can reveal important information about molecular structure and intermolecular interactions in the condensed phase. Simulations typically account for the solvent either through cluster quantum mechanical (QM) models, polarizable continuum models (PCM), or hybrid quantum mechanical/molecular mechanical (QM/MM) models. Recently, we studied the effect of aqueous solvent interactions on the vibrational frequencies of lumiflavin, a minimal flavin model, using cluster QM and PCM models. Those models successfully reproduced the relative frequencies of four prominent stretching modes of flavin's isoalloxazine ring in the diagnostic 1450-1750 cm-1 range but poorly reproduced the relative band intensities. Here, we extend our studies on this system and account for solvation through a series of increasingly sophisticated models. Only by combining elements of QM clusters, QM/MM, and PCM approaches do we obtain an improved agreement with the experiment. The study sheds light more generally on factors that can impact the computed frequencies and intensities of IR bands in solution.

Nanosecond time-resolved infrared spectroscopy for the study of electron transfer in photosystem I.

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. In addition, photoaccumulated (P700+-P700) FTIR difference spectra were obtained at both 77 and 293 K. The FTIR difference spectra are presented here for the first time. To extend upon these FTIR studies nanosecond time-resolved infrared difference spectroscopy was also used to study PSI from T. vestitus at 296 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 296 K, the infrared flash-induced absorption changes indicate electron transfer down the B- and A-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 A1- to FX on the B- and A-branches, respectively. At several infrared wavelengths flash-induced absorption changes at 296 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.

Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site 2: Identification of neutral state quinone bands.

Time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study photosystem I (PSI) from Synechocystis sp. PCC 6803 with four high-potential, 1,4-naphthoquinones (NQs) incorporated into the A1 binding site. The incorporated quinones are 2-chloro-NQ (2ClNQ), 2-bromo-NQ (2BrNQ), 2,3-dichloro-NQ (Cl2NQ), and 2,3-dibromo-NQ (Br2NQ). For completeness 2-methyl-NQ (2MNQ) was also incorporated and studied. Previously, PSI with the same quinones incorporated were studied in the, so-called, anion spectral region between 1550 and 1400 cm-1 (Agarwala et al. in Biochim Biophys Acta 1864(1):148918, 2023). Here we focus on spectra in the previously unexplored 1400-1200 cm-1 spectral region. In this region several bands are identified and assigned to the neutral state of the incorporated quinones. This is important as identification of neutral state quinone bands in the regular 1700-1600 cm-1 region has proven difficult in the past. For neutral PhQ in PSI a broad, intense band appears at ~ 1300 cm-1. For the symmetric di-substituted NQs (Cl2NQ/Br2NQ) a single intense neutral state band is found at ~ 1280/1269 cm-1, respectively. For both mono-substituted NQs, 2ClNQ and 2BrNQ, however, two neutral state bands are observed at ~ 1280 and ~ 1250 cm-1, respectively. These observations from time-resolved spectra agree well with conclusions drawn from absorption spectra of the NQs in THF, which are also presented here. Density functional theory based vibrational frequency calculations were undertaken allowing an identification of the normal modes associated with the neutral state quinone bands.

Calculated vibrational properties of pigments in protein binding sites 2: Semiquinones in photosynthetic proteins.

[QA- - QA] Fourier transform infrared difference spectra have previously been obtained using purple bacterial reaction centers from Rhodobacter sphaeroides with unlabeled, 18O and 13C isotope labeled phylloquinone (PhQ, also known as vitamin K1) incorporated into the QA protein binding site (Breton, (1997), Proc. Natl. Acad. Sci. USA94 11318-11323). The nature of the bands in these spectra and the isotope induced band shifts are poorly understood, especially for the phyllosemiquinone anion (PhQ-) state. To aid in the interpretation of the bands in these experimental spectra, ONIOM type QM/MM vibrational frequency calculations were undertaken. Calculations were also undertaken for PhQ- in solution. Surprisingly, both sets of calculated spectra are similar and agree well with the experimental spectra. This similarity suggests pigment-protein interactions do not perturb the electronic structure of the semiquinone in the QA binding site. This is not found to be the case for the neutral PhQ species in the same protein binding site. PhQ also occupies the A1 protein binding site in photosystem I, and the vibrational properties of PhQ- in the QA and A1 binding sites are compared and shown to exhibit considerable differences. These differences probably arise because of changes in the degree of asymmetry of hydrogen bonding of PhQ- in the A1 and QA binding sites.

Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site.

Time-resolved step-scan Fourier transform infrared difference spectroscopy has been used to study cyanobacterial photosystem I photosynthetic reaction centers from Synechocystis sp. PCC 6803 (S6803) with four high-potential, 1,4-naphthoquinones incorporated into the A1 binding site. The high-potential naphthoquinones are 2-chloro-, 2-bromo-, 2,3-dichloro- and 2,3-dibromo-1,4-naphthoquinone. "Foreign minus native" double difference spectra (DDS) were constructed by subtracting difference spectra for native photosystem I (with phylloquinone in the A1 binding site) from corresponding spectra obtained using photosystem I with the different quinones incorporated. To help assess and assign bands in the difference and double difference spectra, density functional theory based vibrational frequency calculations for the different quinones in solvent, or in the presence of a single asymmetric H- bond to either a water molecule or a peptide backbone NH group, were undertaken. Calculated and experimental spectra agree best for the peptide backbone asymmetrically H- bonded system. By comparing multiple sets of double difference spectra, several new bands for the native quinone (phylloquinone) are identified. By comparing calculated and experimental spectra we conclude that the mono-substituted halogenated NQs can occupy the binding site in either of two different orientations, with the chlorine or bromine atom being either ortho or meta to the H- bonded CO group.

Experimental and calculated infrared spectra of disubstituted naphthoquinones.

In recent years there has been interest in incorporating substituted 1,4-naphthoquinones (NQs) into the A1 binding site in photosystem I (PSI) photosynthetic protein complexes. This interest in part stems from the considerably altered bioenergetics of electron transfer that occur in PSI with such substitutions. Time resolved FTIR studies of PSI complexes with disubstituted NQs incorporated have and currently are being undertaken, and with this in mind it is worth considering FTIR absorption spectra of these disubstituted NQs in solution. Here we present FTIR absorbance spectra for 2-bromo-3-methyl-1,4-naphthoquinone (BrMeNQ), 2-chloromethyl-3-methyl-1,4-naphthoquinone (CMMeNQ) and 2-ethylthio-3-methyl-1,4-naphthoquinone (ETMeNQ) in tetrahydrofuran (THF). The FTIR spectra of these di-substituted naphthoquinones (NQs) were compared to FTIR spectra of 2-methyl-3-phytyl-1,4-naphthoquinone [phylloquinone (PhQ)], 2,3-dimethyl-1,4-naphthoquinone (DMNQ), and 2-methyl-1,4-naphthoquinone (2MNQ). To aid in the assignment of bands in the experimental spectra, density functional theory (DFT) based vibrational frequency calculations for all the substituted NQs in solution were undertaken. The calculated and experimental spectra agree well. By calculating normal mode potential energy distributions, unambiguous quantitative band assignments were made. The calculated and experimental spectra together make predictions about what may be observable in time resolved FTIR difference spectra obtained using PSI with the different NQs incorporated. Time resolved FTIR difference spectra are presented that support these predictions.

The effect of hydrogen-bonding on flavin's infrared absorption spectrum.

Cluster and continuum solvation computational models are employed to model the effect of hydrogen bonding interactions on the vibrational modes of lumiflavin. Calculated spectra were compared to experimental Fourier-transform infrared (FTIR) spectra in the diagnostic 1450-1800 cm-1 range, where intense νC=C, νC=N, [Formula: see text] , and [Formula: see text] stretching modes of flavin's isoalloxazine ring are found. Local mode analysis is used to describe the strength of hydrogen-bonding in cluster models. The computations indicate that νC=C and νC=N mode frequencies are relatively insensitive to intermolecular interactions while the [Formula: see text] and [Formula: see text] modes are sensitive to direct (and also indirect for [Formula: see text] ) hydrogen-bonding interactions. Although flavin is neutral, basis sets without the diffuse functions provide incorrect relative frequencies and intensities. The 6-31+G* basis set is found to be adequate for this system, and there is limited benefit to considering larger basis sets. Calculated vibrational mode frequencies agree with experimentally determined frequencies in solution when cluster models with multiple water molecules are used. Accurate simulation of relative FTIR band intensities, on the other hand, requires a continuum (or possibly quantum mechanical/molecular mechanical) model that accounts for long-range electrostatic effects. Finally, an experimental peak at ca. 1624 cm-1 that is typically assigned to the [Formula: see text] vibrational stretching mode has a complicated shape that suggests multiple underlying contributions. Our calculations show that this band has contributions from both the C6-C7 and C2 = O stretching vibrations.

Assessment of the orientation and conformation of pigments in protein binding sites from infrared difference spectra.

Time resolved FTIR difference spectroscopy (DS) has been used to study photosystem I (PSI) with the disubstituted 1,4-naphthoquinones acequinocyl (AcQ) and lapachol (Lpc) incorporated into the A1 binding site. AcQ is a 2-acetoxy-3-dodecyl-1,4-naphthoquinone, Lpc is a 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone. To assess whether the experimental spectra are specific to different orientations of the quinone and their substitutions ONIOM-type QM/MM vibrational frequency calculations were undertaken for various orientations of the pigments and side-chain conformations in the A1 binding site. Comparison of calculated and experimental spectra for the reduced species (semiquinone anion) suggests that the orientation for the naphthoquinone ring in the binding site and specific side-chain conformations can be identified based on the spectra. In native PSI phylloquinone (PhQ) in the A1 binding site binds with its phytyl chain ortho to the hydrogen bonded carbonyl group. This is not found to be the case for the hydrocarbon tail of AcQ, which is meta to the H-bonded carbonyl group. In contrast, Lpc in PSI binds with its hydrocarbon tail also ortho to the H-bonded carbonyl group. Furthermore, comparison of calculated and experimental spectra indicates which conformations the acetoxy group of AcQ and the hydroxy group of Lpc adopt in the A1 binding site.

Reversible inhibition and reactivation of electron transfer in photosystem I.

In photosystem I (PSI) complexes at room temperature electron transfer from A1- to FX is an order of magnitude faster on the B-branch compared to the A-branch. One factor that might contribute to this branch asymmetry in time constants is TrpB673 (Thermosynechococcus elongatus numbering), which is located between A1B and FX. The corresponding residue on the A-branch, between A1A and FX, is GlyA693. Here, microsecond time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study isolated PSI complexes from wild type and TrpB673Phe mutant (WB673F mutant) cells from Synechocystis sp. PCC 6803. WB673F mutant cells require glucose for growth and are light sensitive. Photoaccumulated FTIR difference spectra indicate changes in amide I and II protein vibrations upon mutation of TrpB673 to Phe, indicating the protein environment near FX is altered upon mutation. In the WB673F mutant PSI samples, but not in WT PSI samples, the phylloquinone molecule that occupies the A1 binding site is likely doubly protonated following long periods of repetitive flash illumination at room temperature. PSI with (doubly) protonated quinone in the A1 binding site are not functional in electron transfer. However, electron transfer functionality can be restored by incubating the light-treated mutant PSI samples in the presence of added phylloquinone.

Time-resolved FTIR difference spectroscopy for the study of quinones in the A1 binding site in photosystem I: Identification of neutral state quinone bands.

Infrared absorption bands associated with the neutral state of quinones in the A1 binding site in photosystem I (PSI) have been difficult to identify in the past. This problem is addressed here, where time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study PSI with six different quinones incorporated into the A1 binding site. (P700+A1- - P700A1) and (A1- - A1) FTIR difference spectra (DS) were obtained for PSI with the different quinones incorporated, and several double-difference spectra (DDS) were constructed from the DS. From analysis of the DS and DDS, in combination with density functional theory based vibrational frequency calculations of the quinones, the neutral state bands of the incorporated quinones are identified and assigned. For neutral PhQ in the A1 binding site, infrared absorption bands were identified near 1665 and 1635 cm-1, that are due to the C1O and C4O stretching vibrations of the incorporated PhQ, respectively. These assignments indicate a 30 cm-1 separation between the C1O and C4O modes, considerably less than the ~80 cm-1 found for similar modes of PhQ-. The C4O mode downshifts due to hydrogen bonding, so the suggestion is that hydrogen bonding is weaker for the neutral state compared to the anion state, indicating radical-induced proton dynamics associated with the quinone in the A1 binding site in PSI.

Calculated vibrational properties of semiquinones in the A1 binding site in photosystem I.

Time-resolved (P700+A1- - P700A1) FTIR difference spectra have been obtained using photosystem I (PSI) particles with several different quinones incorporated into the A1 protein binding site. Difference spectra were obtained for PSI with unlabeled and 18O labeled phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) and 2-methyl-1,4-naphthaquinone (2MNQ) incorporated, and for PSI with unlabeled 2,3-dimethyl-1,4-naphthoquinone (DMNQ) incorporated. (18O - 16O), (2MNQ - PhQ) and (DMNQ - PhQ) FTIR double difference spectra were constructed from the difference spectra. These double difference spectra allow one to more easily distinguish protein and pigment bands in convoluted difference spectra. To further aid in the interpretation of the difference spectra, particularly the spectra associated with the semiquinones, we have used two-layer ONIOM methods to calculate corresponding difference and double difference spectra. In all cases, the experimental and calculated double difference spectra are in excellent agreement. In previous two and three-layer ONIOM calculations it was not possible to adequately simulate multiple difference and double difference spectra. So, the computational approach outlined here is an improvement over previous calculations. It is shown that the calculated spectra can vary depending on the details of the molecular model that is used. Specifically, a molecular model that includes several water molecules that are near the incorporated semiquinones is required.

Fourier transform visible and infrared difference spectroscopy for the study of P700 in photosystem I from Fischerella thermalis PCC 7521 cells grown under white light and far-red light: Evidence that the A-1 cofactor is chlorophyll f.

(P700+ - P700) Fourier transform visible and infrared difference spectra (DS) have been obtained using photosystem I (PSI) complexes isolated from cells of Fischerella thermalis PCC 7521 grown under white light (WL) or far-red light (FRL). PSI from cells grown under FRL (FRL-PSI) contain ~8 chlorophyll f (Chl f) molecules (Shen et al., Photosynth. Res. Jan. 2019). Both the visible and infrared DS indicate that neither the PA or PB pigments of P700 are Chl f molecules, but do support the conclusion that at least one of the A-1 cofactors is a Chl f molecule. The FTIR DS indicate that the hydrogen bond to the 131-keto CO group of the PA pigment of P700 is weakened in FRL-PSI, as might be expected given that the proteins that bind the P700 pigments are substantially different in FRL-PSI (Gan et al., Science 345, 1312-1317, 2014). The FTIR DS obtained using FRL-PSI display a band at 1664 cm-1 that is assigned (based on density functional theory calculations) to the 21-formyl CO group of Chl f, that upshifts 5 cm-1 upon P700+ formation. This is much less than expected for a cation-induced upshift, indicating that the Chl f molecule is not one of the pigments of P700. In WL-PSI the A-1 cofactor is a Chl a molecule with 131-keto and 133-methylester CO mode vibrations at 1696 and 1750 cm-1, respectively. In FRL-PSI the A-1 cofactor is a Chl f molecule with 131-keto and 133-methylester CO mode vibrations at 1702 and 1754 cm-1, respectively.

Modelling electron transfer in photosystem I: limits and perspectives.

Uncovering the parameters underlying the electron transfer (ET) in photosynthetic reaction centres is of importance for understanding the molecular mechanisms underpinning their functionality. The reductive nature of most cofactors involved in photosynthetic ET makes the direct estimation of their properties difficult. Photosystem I (PSI) operates in a highly reducing regime, making the assessment of cofactor properties even more difficult. Kinetic modelling coupled to a non-adiabatic description of ET is a useful approach in overcoming this hindrance. Here we review the theory and modelling approaches that have been used in assessing parameters associated with ET reactions in PSI, with particular attention to ET reactions involving the phylloquinones and the iron-sulphur clusters. In most modelling studies, the goal is to estimate the driving force of ET, which is usually associated with the cofactor midpoint potentials. The driving force is sensitive to many factors, which define the ET rate, i.e. the reorganisation energy, the coupling with nuclear modes and the electronic matrix elements, which are explored and discussed here. The importance of an inclusive modelling of both forward and reverse ET processes is discussed and highlighted. It is shown that although estimates are indeed sensitive to the exact parameter sets employed in the modelling, a general consensus is still attained, pointing to a scenario where Δ G A 1 A → F X 0 / Δ G A 1 B → F X 0 is weakly endergonic/exergonic, respectively. It is emphasised that to further refine those estimates, it will require a joint effort between computational modelling and more wide-ranging experimental studies.

Time-resolved step-scan FTIR difference spectroscopy for the study of photosystem I with different benzoquinones incorporated into the A1 binding site.

Time-resolved step-scan FTIR difference spectroscopy has been used to study photosystem I (PSI) with plastoquinone-9 (PQ) and two other benzoquinones (2,6-dimethyl-1,4-benzoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone) incorporated into the A1 binding site. By subtracting a (P700+A1- - P700A1) FTIR difference spectrum for PSI with the native phylloquinone (PhQ) incorporated from corresponding spectra for PSI with different benzoquinones (BQs) incorporated, FTIR double difference spectra are produced, that display bands associated with vibrational modes of the quinones, without interference from features associated with protein vibrational modes. Molecular models for BQs involved in asymmetric hydrogen bonding were constructed and used in vibrational mode frequency calculations. The calculated data were used to aid in the interpretation and assignment of bands in the experimental spectra. We show that the calculations capture the general trends found in the experimental spectra. By comparing four different FTIR double difference spectra we are able to verify unambiguously bands associated with phyllosemiquinone in PSI at 1495 and 1415 cm-1. We also resolve a previously unrecognized band of phyllosemiquinone at 1476 cm-1 that calculations suggest is due in part to a C4-⃛O stretching mode. For PSI with PQ incorporated, calculations and experiment taken together indicate that the C1-⃛O and C4-⃛O vibrational modes of the semiquinone give rise to bands at 1487 and 1444 cm-1, respectively. This is very distinct compared to PSI with PhQ incorporated. From the calculated and experimental spectra, we show that it is possible to distinguish between two possible orientations of PQ in the A1 protein binding site.

Probing structural changes in single enveloped virus particles using nano-infrared spectroscopic imaging.

Enveloped viruses, such as HIV, Ebola and Influenza, are among the most deadly known viruses. Cellular membrane penetration of enveloped viruses is a critical step in the cascade of events that lead to entry into the host cell. Conventional ensemble fusion assays rely on collective responses to membrane fusion events, and do not allow direct and quantitative studies of the subtle and intricate fusion details. Such details are accessible via single particle investigation techniques, however. Here, we implement nano-infrared spectroscopic imaging to investigate the chemical and structural modifications that occur prior to membrane fusion in the single archetypal enveloped virus, influenza X31. We traced in real-space structural and spectroscopic alterations that occur during environmental pH variations in single virus particles. In addition, using nanospectroscopic imaging we quantified the effectiveness of an antiviral compound in stopping viral membrane disruption (a novel mechanism for inhibiting viral entry into cells) during environmental pH variations.

Photosystem I with benzoquinone analogues incorporated into the A1 binding site.

Time-resolved FTIR difference spectroscopy has been used to study photosystem I (PSI) particles with three different benzoquinones [plastoquinone-9 (PQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (Cl4BQ)] incorporated into the A1 binding site. If PSI samples are cooled in the dark to 77 K, the incorporated benzoquinones are shown to be functional, allowing the production of time-resolved (P700+A1--P700A1) FTIR difference spectra. If samples are subjected to repetitive flash illumination at room temperature prior to cooling, however, the time-resolved FTIR difference spectra at 77 K display contributions typical of the P700 triplet state (3P700), indicating a loss of functionality of the incorporated benzoquinones, that occurs because of double protonation of the incorporated benzoquinones. The benzoquinone protonation mechanism likely involves nearby water molecules but does not involve the terminal iron-sulfur clusters FA and FB. These results and conclusions resolve discrepancies between results from previous low-temperature FTIR and EPR studies on similar PSI samples with PQ incorporated.

Inverted-region electron transfer as a mechanism for enhancing photosynthetic solar energy conversion efficiency.

In all photosynthetic organisms, light energy is used to drive electrons from a donor chlorophyll species via a series of acceptors across a biological membrane. These light-induced electron-transfer processes display a remarkably high quantum efficiency, indicating a near-complete inhibition of unproductive charge recombination reactions. It has been suggested that unproductive charge recombination could be inhibited if the reaction occurs in the so-called inverted region. However, inverted-region electron transfer has never been demonstrated in any native photosynthetic system. Here we demonstrate that the unproductive charge recombination in native photosystem I photosynthetic reaction centers does occur in the inverted region, at both room and cryogenic temperatures. Computational modeling of light-induced electron-transfer processes in photosystem I demonstrate a marked decrease in photosynthetic quantum efficiency, from 98% to below 72%, if the unproductive charge recombination process does not occur in the inverted region. Inverted-region electron transfer is therefore demonstrated to be an important mechanism contributing to efficient solar energy conversion in photosystem I. Inverted-region electron transfer does not appear to be an important mechanism in other photosystems; it is likely because of the highly reducing nature of photosystem I, and the energetic requirements placed on the pigments to operate in such a regime, that the inverted-region electron transfer mechanism becomes important.

Quinones in the A1 binding site in photosystem I studied using time-resolved FTIR difference spectroscopy.

Time-resolved step-scan FTIR difference spectroscopy at low temperature (77 K) has been used to study photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthaquinone) and menadione (2-methyl-1,4-naphthaquinone) incorporated into the A1 binding site. By subtracting spectra for PSI with phylloquinone incorporated from spectra for PSI with menadione incorporated a (menadione - phylloquinone) double difference spectrum was constructed. In the double difference spectrum bands associated with protein vibrational modes effectively cancel, and the bands in the spectrum are primarily associated with the neutral and reduced states of the two quinones in the A1 binding site. To aid in the assignment of bands in the experimental double difference spectrum, a double difference spectrum was calculated using three-layer ONIOM methods. The calculated and experimental spectra agree well, allowing unambiguous band assignments to be made. The ONIOM calculations show that both quinones in the A1 binding site are similarly oriented, with only a single hydrogen bond between the C4=O quinone carbonyl group and the backbone NH group of a leucine residue. For the semi-quinone species, but not for the neutral species, this hydrogen bond appears to be very strong. Finally, we have for the first time been able to unmask and identify infrared difference bands associated with neutral naphthoquinone species occupying the A1 binding site in PSI.

Time-resolved visible and infrared absorption spectroscopy data obtained using photosystem I particles with non-native quinones incorporated into the A1 binding site.

Time-resolved visible and infrared absorption difference spectroscopy data at both 298 and 77 K were obtained using cyanobacterial menB (-) mutant photosystem I particles with several non-native quinones incorporated into the A1 binding site. Data was obtained for photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone), 2-bromo-1,4-naphthoquinone, 2-chloro-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 2,3-dibromo-1,4-naphthoquinone, 2,3-dichloro-1,4-naphthoquinone, and 9,10-anthraquinone incorporated. Transient absorption data were obtained at 487 and 703 nm in the visible spectral range, and 1950-1100 cm(-1) in the infrared region. Time constants obtained from fitting the time-resolved infrared and visible data are in good agreement. The measured time constants are crucial for the development of appropriate kinetic models that can describe electron transfer processes in photosystem I, "Modeling Electron Transfer in Photosystem I" Makita and Hastings (2016) [1].