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.

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.

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.

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 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.

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.