P700: the primary electron donor of photosystem I.

The primary electron donor of photosystem I, P700, is a chlorophyll species that in its excited state has a potential of approximately -1.2 V. The precise chemical composition and electronic structure of P700 is still unknown. Recent evidence indicates that P700 is a dimer of one chlorophyll (Chl) a and one Chl a'. The Chl a' and Chl a are axially coordinated by His residues provided by protein subunits PsaA and PsaB, respectively. The Chl a', but not the Chl a, is also H-bonded to the protein. The H-bonding is likely responsible for selective insertion of Chl a' into the reaction center. EPR studies of P700(+*) in frozen solution and single crystals indicate a large asymmetry in the electron spin and charge distribution towards one Chl of the dimer. Molecular orbital calculations indicate that H-bonding will specifically stabilize the Chl a'-side of the dimer, suggesting that the unpaired electron would predominantly reside on the Chl a. This is supported by results of specific mutagenesis of the PsaA and PsaB axial His residues, which show that only mutations of the PsaB subunit significantly alter the hyperfine coupling constants associated with a single Chl molecule. The PsaB mutants also alter the microwave induced triplet-minus-singlet spectrum indicating that the triplet state is localized on the same Chl. Excitonic coupling between the two Chl a of P700 is weak due to the distance and overlap of the porphyrin planes. Evidence of excitonic coupling is found in PsaB mutants which show a new bleaching band at 665 nm that likely represents an increased intensity of the upper exciton band of P700. Additional properties of P700 that may give rise to its unusually low potential are discussed.     

Electrogenic reduction of the primary electron donor P700 by plastocyanin in photosystem I complexes

An electrometric technique was used to investigate electron transfer between spinach plastocyanin (Pc) and photooxidized primary electron donor P700 in photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of Pc, the fast unresolvable kinetic phase of membrane potential generation related to electron transfer between P700 and the terminal iron–sulfur acceptor F_B was followed by additional electrogenic phases in the microsecond and millisecond time scales, which contribute approximately 20% to the overall electrogenicity. These phases are attributed to the vectorial electron transfer from Pc to the protein-embedded chlorophyll dimer P700⁺ within the PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic phase exhibited a saturation profile at increasing Pc concentration, suggesting the formation of a transient complex between Pc and PS I with the dissociation constant K_d of about 80 μM. A small but detectable fast electrogenic phase was observed at high Pc concentration. The rate constant of this phase was independent of Pc concentration, indicating that it is related to a first-order process.     

Species-dependence of the redox potential of the primary quinone electron acceptor QA in photosystem II verified by spectroelectrochemistry

The redox potentials E_m(Q_A/) of the primary quinone electron acceptor Q_A in oxygen-evolving photosystem II complexes of three species were determined by spectroelectrochemistry. The E_m(Q_A/) values were experimentally found to be −162 ± 3 mV for a higher plant spinach, −171 ± 3 mV for a green alga Chlamydomonas reinhardtii and −104 ± 4 mV vs. SHE for a red alga Cyanidioschyzonmerolae. On the basis of possible deviations for the experimental values, as estimated to differ by 9-29 mV from each true value, plausible causes for such remarkable species-dependence of E_m(Q_A/) are discussed, mainly by invoking the effects of extrinsic subunits on the delicate structural environment around Q_A.     

Stark effect in P700, the primary electron donor of Photosystem I

Quadratic Stark effect in CP1 pigment-protein complex was examined at low temperatures in the red spectral region. The Stark spectra of samples containing P700 in reduced form exhibit a strong negative band at 704 nm, which disappears on chemical oxidation of P700. The change in permanent dipole moment, delta mu, of P700 on electronic excitation estimated from these spectra was found to be between 4.7 and 7.7 Debye units. It is suggested to reflect the charge-transfer contribution to the excited state of P700. For antenna chlorophyll, delta mu approximately equal to 1 D was obtained in accordance with the data for monomeric chlorophyll.     

The two histidine axial ligands of the primary electron donor chlorophylls (P700) in photosystem I are similarly perturbed upon P700+ formation

The extent of delocalization of the positive charge in the oxidized dimer of chlorophyll (Chl) constituting P700, the primary electron donor of photosystem I (PSI), has been investigated by analyzing the perturbation upon P700(+) formation of infrared (IR) vibrational modes of the two His axial ligands of the two P700 Chl molecules. Fourier transform IR (FTIR) difference spectra of the photooxidation of P700 in PSI core complexes isolated from Synechocystis sp. PCC 6803 isotopically labeled either globally with (15)N or more specifically with (13)C on all the His residues reveal isotopic shifts of a differential signal at 1102/1108 cm(-)(1). This signal is assigned to a downshift upon P700(+) formation of the predominantly C(5)-Ntau imidazole stretching mode of His residue(s). The amplitude of this signal is reduced by approximately half in FTIR spectra of Synechocystis mutants in which His PsaB 651, the axial ligand to one of the two Chl molecules in P700, is replaced by Cys, Gln, or Leu. These observations provide further evidence that the positive charge in P700(+) is essentially delocalized over the two Chl molecules, in agreement with a previous FTIR study in which the frequency of the vibrational modes of the 9-keto and 10a-ester C=O groups of the two Chl's in P700, P700(+), and (3)P700 were firmly established for the first time [Breton, J., et al. (1999) Biochemistry 38, 11585-11592]. Only limited perturbations of the amplitude and frequency of the 9-keto and 10a-ester C=O bands of the P700 Chl are elicited by the mutations. On the basis of comparable mutational studies of the primary electron donor in purple bacteria, these perturbations are attributed to small molecular rearrangements of the Chl macrocycle and substituents caused by the repositioning of the P700 dimer in the new protein cavity generated by the mutations. It is proposed that the perturbation of the FTIR spectra upon mutation of a His axial ligand of the P700 Chl recently reported in Chlamydomonas reinhardtii [Hastings, G., et al. (2001) Biochemistry 40, 12943-12949] can be explained by the same effect without the need for a new assignment of the C=O bands of P700. The distribution of charge/spin in P700(+) and (3)P700 determined by FTIR spectroscopy is discussed in relation with the contrasting interpretations derived from recent magnetic resonance experiments.     

Effect of trypsin treatment of photosystem I particles on the electron donation to p700

Proteolysis of photosystem I particles had no effect on P700 oxidation but did inhibit the rate of P700⁺ reduction. The V_max values were decreased for both dichlorophenol and plastocyanin, but the K_m values were unaffected indicating that trypsin treatment altered electron transfer rather than the binding of the donor to the photosystem I complex. The salt dependence of P700⁺ reduction was unaffected. The effects of P700⁺ reduction were the same for the preparations of different workers (Shiozawa, Alberte, Thornber 1974 Arch Biochem Biophys 165: 388; and Bengis, Nelson 1975 J Biol Chem 250: 2783).

In both cases, the 70-kilodalton, chlorophyll-containing polypeptide was digested confirming its role in transferring electrons from plastocyanin to P700. The fact that the preparation of Shiozawa et al. lacks subunit (III) but still used plastocyanin as the electron donor rules out a role for this subunit as “the plastocyanin binding protein.” Subunit III was also digested in the Bengis and Nelson preparation.

     

Structure of the primary electron donor in photosystem I: a resonance Raman study

Low-temperature resonance Raman (RR) spectra have been obtained at resonance with the Soret transition of chlorophyll a in photosystem I particles containing large amounts either of the triplet state of P700 or of its radical cation state. Subtracting these spectra from those of resting reaction centers yielded RR spectra of P700 in its neutral, ground state. These spectra arise from two distinct chlorophyll a molecules differing by the strengths of the bonding interactions assumed by their keto carbonyl groups, the stretching frequencies of which are found at 1655 and 1675 cm-1. The present results rule out previous hypotheses that P700 might have consisted of a single, chemically modified chlorophyll a molecule. Neither of the bonding interactions assumed by the keto carbonyls of the P700 chlorophylls most probably involves chlorophyll-chlorophyll bridging through water molecules, as surmised in the so-called special pair models, but likely consists of H bonds with distinct protein sites. The magnesium atoms of the two P700 chlorophylls are 5-coordinated. Hence, the structural model of P700 provided by the present data is qualitatively the same, in terms of bonding interactions, as that currently accepted for the bacterial primary donor.     

Multiple pathways of charge recombination revealed by the temperature dependence of electron transfer kinetics in cyanobacterial photosystem I

The kinetics of charge recombination in Photosystem I P₇₀₀-F_A/F_B complexes and P₇₀₀-F_X cores lacking the terminal iron?sulfur clusters were studied over a temperatures range of 310 K to 4.2 K. Analysis of the charge recombination kinetics in this temperature range allowed the assignment of backward electron transfer from the different electron acceptors to P₇₀₀⁺. The kinetic and thermodynamic parameters of these recombination reactions were determined. The kinetics of all electron transfer reactions were activation-less below 170 K, the glass transition temperature of the water-glycerol solution. Above this temperature, recombination from [F_A/F_B]^? in P₇₀₀-F_A/F_B complexes was found to proceed along two pathways with different activation energies (E_a). The charge recombination via A_1A has an E_a of ~290 meV and is dominant at temperatures above ~280 K, whereas the direct recombination from F_X^? has an E_a of 22 meV and is prevalent in the 200 K to 270 K temperature range. Charge recombination from the F_X cluster becomes highly heterogeneous at temperatures below 200 K. The conformational mobility of Photosystem I was studied by molecular dynamics simulations. The F_X cluster was found to ‘swing’ by ~30° along the axis between the two sulfur atoms proximal to F_A/F_B. The partial rotation of F_X is accompanied by significant changes of electric potential within the iron?sulfur cluster, which may induce preferential electron localization at different atoms of the F_X cluster. These effects may account for the partial arrest of forward electron transfer and for the heterogeneity of charge recombination observed at the glass transition temperature.     

Redox properties and regulatory mechanism of the iron-quinone electron acceptor in photosystem II as revealed by FTIR spectroelectrochemistry

Photosynthesis Research - Photosystem II (PSII) performs oxidation of water and reduction of plastoquinone through light-induced electron transfer. Electron transfer reactions at individual redox...     

Influence of the redox potential of the primary quinone electron acceptor on photoinhibition in photosystem II

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, Q(A), in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of Q(A) by approximately -60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of Q(A). Thus the influence of the redox potential of Q(A) on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving Q(A) was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of Q(A) causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of Q(A). The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.     

Mutation of the putative hydrogen-bond donor to P700 of photosystem I

The primary electron donor of photosystem I (PS1), called P(700), is a heterodimer of chlorophyll (Chl) a and a'. The crystal structure of photosystem I reveals that the chlorophyll a' (P(A)) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (P(B)) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P(700), PsaA-Thr739 was converted to alanine to remove the H-bond to the 13(1)-keto group of the chlorophyll a' in Chlamydomonas reinhardtii. The PsaA-T739A mutant was capable of assembling active PS1. Furthermore the mutant PS1 contained approximately one chlorophyll a' molecule per reaction center, indicating that P(700) was still a Chl a/a' heterodimer in the mutant. However, the mutation induced several band shifts in the visible P(700)(+) - P(700) absorbance difference spectrum. Redox titration of P(700) revealed a 60 mV decrease in the P(700)/P(700)(+) midpoint potential of the mutant, consistent with loss of a H-bond. Fourier transform infrared (FTIR) spectroscopy indicates that the ground state of P(700) is somewhat modified by mutation of ThrA739 to alanine. Comparison of FTIR difference band shifts upon P(700)(+) formation in WT and mutant PS1 suggests that the mutation modifies the charge distribution over the pigments in the P(700)(+) state, with approximately 14-18% of the positive charge on P(B) in WT being relocated onto P(A) in the mutant. (1)H-electron-nuclear double resonance (ENDOR) analysis of the P(700)(+) cation radical was also consistent with a slight redistribution of spin from the P(B) chlorophyll to P(A), as well as some redistribution of spin within the P(B) chlorophyll. High-field electron paramagnetic resonance (EPR) spectroscopy at 330-GHz was used to resolve the g-tensor of P(700)(+), but no significant differences from wild-type were observed, except for a slight decrease of anisotropy. The mutation did, however, provoke changes in the zero-field splitting parameters of the triplet state of P(700) ((3)P(700)), as determined by EPR. Interestingly, the mutation-induced change in asymmetry of P(700) did not cause an observable change in the directionality of electron transfer within PS1.     

Photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina

Fourier transform infrared spectroscopy (FTIR) difference spectroscopy in combination with deuterium exchange experiments has been used to study the photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina. Comparison of (P740(+)-P740) and (P700(+)-P700) FTIR difference spectra show that P700 and P740 share many structural similarities. However, there are several distinct differences also: 1), The (P740(+)-P740) FTIR difference spectrum is significantly altered upon proton exchange, considerably more so than the (P700(+)-P700) FTIR difference spectrum. The P740 binding pocket is therefore more accessible than the P700 binding pocket. 2), Broad, "dimer" absorption bands are observed for both P700(+) and P740(+). These bands differ significantly in substructure, however, suggesting differences in the electronic organization of P700(+) and P740(+). 3), Bands are observed at 2727(-) and 2715(-) cm(-1) in the (P740(+)-P740) FTIR difference spectrum, but are absent in the (P700(+)-P700) FTIR difference spectrum. These bands are due to formyl CH modes of chlorophyll d. Therefore, P740 consists of two chlorophyll d molecules. Deuterium-induced modification of the (P740(+)-P740) FTIR difference spectrum indicates that only the highest frequency 13(3) ester carbonyl mode of P740 downshifts, indicating that this ester mode is weakly H-bonded. In contrast, the highest frequency ester carbonyl mode of P700 is free from H-bonding. Deuterium-induced changes in (P740(+)-P740) FTIR difference spectrum could also indicate that one of the chlorophyll d 3(1) carbonyls of P740 is hydrogen bonded.     

Significant species-dependence of P700 redox potential as verified by spectroelectrochemistry: comparison of spinach and Theromosynechococcus elongatus

The redox potentials of P700, the primary electron donor of photosystem (PS) I, of spinach and Thermosynechococcus elongatus were determined by means of spectroelectrochemistry with an error range of +/-2-3 mV, to find that the redox potential of P700 in T. elongatus is lower by ca. 50 mV as compared with spinach. The shift in the P700 redox potential of PS I core particles prepared by harsh detergent treatments remained to within 10 mV for both organisms. These results show that the 50 mV difference in the P700 redox potential between the two organisms is not a detergent-induced artifact but reflects an intrinsic property of each PS I.     

FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700(+) and localization of the triplet character in (3)P700.

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.     

Tuning the redox potential of the primary electron donor in bacterial reaction centers by manganese binding and light-induced structural changes

The influence of transition metal binding on the charge storage ability of native bacterial reaction centers (BRCs) was investigated. Binding of manganous ions uniquely prevented the light-induced conformational changes that would yield to long lifetimes of the charge separated state and the drop of the redox potential of the primary electron donor (P). The lifetimes of the stable charge pair in the terminal conformations were shortened by 50-fold and 7-fold upon manganous and cupric ion binding, respectively. Nickel and zinc binding had only marginal effects. Binding of manganese not only prevented the drop of the potential of P/P⁺ but also elevated it by up to 117 mV depending on where the metal was binding. With variable conditions, facilitating either manganese binding or light-induced structural changes a controlled tuning of the potential of P/P⁺ in multiple steps was demonstrated in a range of ~200 mV without the need of a mutation or synthesis. Under the selected conditions, manganese binding was achieved without its photochemical oxidation thus, the energized but still native BRCs can be utilized in photochemistry that is not reachable with regular BRCs. A 42 Å long hydrophobic tunnel was identified that became obstructed upon manganese binding and its likely role is to deliver protons from the hydrophobic core to the surface during conformational changes.     

PRoperties of the low-temperature photosystem I primary reaction in the P-700-chlorophyll alpha-protein.

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15 degrees K in P-700-chlorophyll alpha-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300 degrees K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15 degrees K as well as at 300 degrees K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.     

Inhibition of the photosynthetic electron transport of isolated thylakoids by hemolyzed rabbit sera. Evidence for the potential involvement of parallel electron transport in photosystem I Mehler reactions

The inhibition patterns of rabbit sera (RS1 & RS2) from two different rabbits on the photosynthetic electron transport of isolated spinach thylakoids were studied. Fifty l of RSI were required for 100% inhibition of a H₂O MV/O₂ reaction, while only 10 l of a 1:10 dilution of RS2 were needed for 100% inhibition. The RS2 serum was greatly hemolyzed. The -globulin fraction from purified rabbit serum (RS1) did not inhibit photosynthetic electron transport, indicating that the antibody fraction of the rabbit serum does not contain the inhibitor. It appears that the inhibitor is from the hemolyzed red blood cells. Rabbit sera added prior to chloroplast illumination caused no inhibition, while addition of rabbit sera during illumination inhibited a H₂O MV/O₂ reaction within 1–3s. Aminotriazole, a catalase inhibitor, did not affect the efficacy of the rabbit sera indicating that the unknown rabbit serum inhibitor is not catalase. Various Hill reactions were employed to determine the site of inhibition. Rabbit sera inhibited the following reactions: DHQ/DCMU MV/O₂, DAD/Asc/DBMIB MV/O₂, and DCIP/Asc/DBMIB MV/O₂. Rabbit sera did not inhibit a H₂O DAD_ox reaction indicating that inhibition is on the reducing side of PSI. However, a H₂O Fd/NADP⁺ reaction was not inhibited by rabbit sera. NADP did not interfere with the ability of RS2 to inhibit a MV-mediated Mehler reaction. In simultaneously measured assays of Fd-mediated O₂ and NADP⁺ reductions, RS2 serum inhibited the reduction of O₂ by ferredoxin without inhibiting the reduction of NADP⁺. These results indicate the potential involvement of parallel (branched) electron transport of the reducing side of PSI in the reduction of oxygen.Abbreviations RS1 and RS2 Rabbit serum 1 and 2 - MV methylviologen - DCMU 3,4-dichlorophenyl-N,N-dimethylurea - KFeCN potassium ferricyanide - DCIP dichlorophenolindolphenol - DAD 2,3,5,6-tetramethyl-p-phenylenediamine - DHQ tetramethyl-p-hydroquinone (durohydroquinone) - MES [2-(N-morpholino)-esthanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid] - DBMIB dibromothymoquinone - PSI and PSII photosystem I and II - Fd ferredoxin - Chl chlorophyll - Asc ascorbate - SOD superoxide dismutase     

Polyribosome loading of spinach mRNAs for photosystem I subunits is controlled by photosynthetic electron transport

In light-, but not in dark-grown spinach seedlings, the mRNAs for the nuclear-encoded photosystem I subunits D, F and L are associated with polyribosomes and this association is prevented by the application of 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea (DCMU), an inhibitor of the photosynthetic electron transport. To identify the cis-elements which are responsible for this regulation, we generated a series of chimeric PsaD constructs and tested them in transgenic tobacco. The spinach PsaD 5'-untranslated region is sufficient to confer light- and photosynthesis-dependent polyribosome association onto the uidA reporter gene, while the tobacco PsaD 5'-untranslated region directs constitutive polyribosome association. These results are discussed with regard to signals from photosynthetic electron flow which control processes in the cytoplasm.     

Analysis of donors of electrons to photosystem I and cyclic electron flow by redox kinetics of P700 in chloroplasts of isolated bundle sheath strands of maize

Bundle sheath chloroplasts of NADP-malic enzyme (NADP-ME) type C₄ species have a high demand for ATP, while being deficient in linear electron flow and oxidation of water by photosystem II (PSII). To evaluate electron donors to photosystem I (PSI) and possible pathways of cyclic electron flow (CEF1) in isolated bundle sheath strands of maize (Zea mays L.), an NADP-ME species, light-induced redox kinetics of the reaction center chlorophyll of PSI (P700) were followed under aerobic conditions. Donors of electrons to CEF1 are needed to compensate for electrons lost from the cycle. When stromal electron donors to CEF1 are generated during pre-illumination with actinic light (AL), they retard the subsequent rate of oxidation of P700 by far-red light. Ascorbate was more effective than malate in generating stromal electron donors by AL. The generation of stromal donors by ascorbate was inhibited by DCMU, showing ascorbate donates electrons to the oxidizing side of PSII. The inhibitors of NADPH dehydrogenase (NDH), amytal and rotenone, accelerated the oxidation rate of P700 by far-red light after AL, indicating donation of electrons to the intersystem from stromal donors via NDH. These inhibitors, however, did not affect the steady-state level of P700⁺ under AL, which represents a balance of input and output of electrons in P700. In contrast, antimycin A, the inhibitor of the ferredoxin-plastoquinone reductase-dependent CEF1, substantially lowered the level of P700⁺ under AL. Thus, the primary pathway of ATP generation by CEF1 may be through ferredoxin-plastoquinone, while function of CEF1 via NDH may be restricted by low levels of ferredoxin-NADP reductase. NDH may contribute to redox poising of CEF1, or function to generate ATP in linear electron flow to O₂ via PSI, utilizing NADPH generated from malate by chloroplastic NADP-ME.     

Fluorescence of the various red antenna states in photosystem I complexes from cyanobacteria is affected differently by the redox state of P700

Photosystem I of cyanobacteria contains different spectral pools of chlorophylls called red or long-wavelength chlorophylls that absorb at longer wavelengths than the primary electron donor P700. We measured the fluorescence spectra at the ensemble and the single-molecule level at low temperatures in the presence of oxidized and reduced P700. In accordance with the literature, it was observed that the fluorescence is quenched by P700(+). However, the efficiency of the fluorescence quenching by oxidized P700(+) was found to be extremely different for the various red states in PS I from different cyanobacteria. The emission of the longest-wavelength absorbing antenna state in PS I trimers from Thermosynechococcus elongatus (absorption maximum at 5K: ≈ 719nm; emission maximum at 5K: ≈ 740nm) was found to be strongly quenched by P700(+) similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760nm at 5K. The fluorescence of these red states is diminished by more than a factor of 10 in the presence of oxidized P700. For the first time, the emission of the reddest states in A. platensis and T. elongatus has been monitored using single-molecule fluorescence techniques.