Assignment of a kinetic component to electron transfer between iron-sulfur clusters F(X) and F(A/B) of Photosystem I

We studied the kinetics of reoxidation of the phylloquinones in Chlamydomonas reinhardtii Photosystem I using site-directed mutations in the PhQ(A)-binding site and of the residues serving as the axial ligand to ec3(A) and ec3(B) chlorophylls. In wild type PS I, these kinetics are biphasic, and mutations in the binding region of PhQ(A) induced a specific slowing down of the slow component. This slowing allowed detection of a previously unobserved 180-ns phase having spectral characteristics that differ from electron transfer between phylloquinones and F(X). The new kinetic phase thus reflects a different reaction that we ascribe to oxidation of F(X)(-) by the F(A/B) FeS clusters. These absorption changes partly account for the differences between the spectra associated with the two kinetic components assigned to phylloquinone reoxidation. In the mutant in which the axial ligand to ec3(A) (PsaA-Met688) was targeted, about 25% of charge separations ended in P(700)(+)A(0)(-) charge recombination; no such recombination was detected in the B-side symmetric mutant. Despite significant changes in the amplitude of the components ascribed to phylloquinone reoxidation in the two mutants, the overall nanosecond absorption changes were similar to the wild type. This suggests that these absorption changes are similar for the two different phylloquinones and that part of the differences between the decay-associated spectra of the two components reflect a contribution from different electron acceptors, i.e. from an inter-FeS cluster electron transfer.     

A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts

Photosystem I activity of Tris-washed chloroplasts was measured at room temperature as the rate of photoreduction of NADP and as the rate of oxygen uptake mediated by methyl viologen in both cases using dichlorophenolindophenol plus ascorbate as the source of electrons for Photosystem I. With both assay systems the rate of electron transport by Photosystem I was stimulated approx. 20 % by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which caused the Photosystem II reaction centers to close. Photosystem I activity of chloroplasts was measured at low temperature as the rate of photooxidation of P-700. Chloroplasts suspended in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea were frozen to ?196 °C after adaptation to darkness or after a preillumination at room temperature. The Photosystem II reaction centers of the frozen dark-adapted sample were all open; those of the preilluminated sample were all closed. The rate of photooxidation of P-700 at ?196 °C with the preilluminated sample was approx. 25 % faster than with the dark-adapted sample. We conclude from both the room temperature and the low temperature experiments that there is greater energy transfer from Photosystem II to Photosystem I when the Photosystem II reaction centers are closed and that these results are a direct demonstration of spillover.     

Light-induced alteration of low-temperature interprotein electron transfer between photosystem I and flavodoxin

Electron paramagnetic resonance (EPR) was used to study light-induced electron transfer in Photosystem I-flavodoxin complexes. Deuteration of flavodoxin enables the signals of the reduced flavin acceptor and oxidized primary donor, P(700)(+), to be well-resolved at X- and D-band EPR. In dark-adapted samples, photoinitiated interprotein electron transfer does not occur at 5 K. However, for samples prepared in dim light, significant interprotein electron transfer occurs at 5 K and a concomitant loss of the spin-correlated radical pair P(+)A(1A)(-) signal is observed. These results indicate a light-induced reorientation of flavodoxin in the PSI docking site that allows a high quantum yield efficiency for the interprotein electron transfer reaction.     

Kinetics of electron transfer from Q(a) to Q(b) in photosystem II

The oxidation kinetics of the reduced photosystem II electron acceptor Q(A)(-) was investigated by measurement of the chlorophyll fluorescence yield transients on illumination of dark-adapted spinach chloroplasts by a series of saturating flashes. Q(A)(-) oxidation depends on the occupancy of the "Q(B) binding site", where this reaction reduces plastoquinone to plastoquinol in two successive photoreactions. The intermediate, one-electron-reduced plastosemiquinone anion Q(B)(-) remains tightly bound, and its reduction by Q(A)(-) may proceed with simple first-order kinetics. The next photoreaction, in contrast, may find the Q(B) binding site occupied by a plastoquinone, a plastoquinol, or neither of the two, resulting in heterogeneous Q(A)(-) oxidation kinetics. The assumption of monophasic Q(B)(-) reduction kinetics is shown to allow unambiguous decomposition of the observed multiphasic Q(A)(-) oxidation. At pH 6.5 the time constant for Q(A)(-) oxidation was found to be 0.2-0.4 ms with Q(B) in the site, 0.6-0.8 ms with Q(B)(-) in the site, 2-3 ms when the site is empty and Q(B) has to bind first, and of the order of 0.1 s if the site is temporarily blocked by the presence of Q(B)H(2) or other low-affinity inhibitors such as carbonyl cyanide m-chlorophenylhydrazone (CCCP). Effects of pH and H(2)O/D(2)O exchange were found to be remarkably nonspecific. No influence of the S-states could be demonstrated.     

Reversible inhibition and reactivation of electron transfer in photosystem I

Photosynthesis Research - 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...     

Flash-photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I.

Plastocyanin (Pc) has been modified by site-directed mutagenesis at two separate electron-transfer (ET) sites: Leu-12-Glu at a hydrophobic patch, and Tyr-83-His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu-12-Glu) and increased by 35 mV in Pc(Tyr-83-His). The latter mutant shows a 2-fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu-12-Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.     

Photo-induced electron transfer from photosystem I to NADP(+): Characterization and tentative simulation of the in vivo environment

The photoproduction of NADPH in photosynthetic organisms requires the successive or concomitant interaction of at least three proteins: photosystem I (PSI), ferredoxin (Fd) and ferredoxin:NADP(+) oxidoreductase (FNR). These proteins and their surrounding medium have been carefully analysed in the cyanobacterium Synechocystis sp. PCC 6803. A high value of 550mg/ml was determined for the overall solute content of the cell soluble compartment. PSI and Fd are present at similar concentrations, around 500μM, whereas the FNR associated to phycobilisome is about 4 fold less concentrated. Membrane densities of FNR and trimeric PSI have been estimated to 2000 and 2550 per μm(2), respectively. An artificial confinement of Fd to PSI was designed using fused constructs between Fd and PsaE, a peripheral and stroma located PSI subunit. The best covalent system in terms of photocatalysed NADPH synthesis can be equivalent to the free system in a dilute medium. In a macrosolute crowded medium (375mg/ml), this optimized PSI/Fd covalent complex exhibited a huge superiority compared to the free system. This is a likely consequence of restrained diffusion constraints due to the vicinity of two out of the three protein partners. In vivo, Fd is the free partner, but the constant proximity between PSI and the phycobilisome associated FNR creates a similar situation, with two closely associated partners. This organization seems well adapted for an efficient in vivo production of the stable and fast diffusing NADPH.     

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.     

Temperature dependence of biphasic forward electron transfer from the phylloquinone(s) A1 in photosystem I: only the slower phase is activated

The temperature dependence of the biphasic electron transfer (ET) from the secondary acceptor A1 (phylloquinone) to iron-sulfur cluster F(X) was investigated by flash absorption spectroscopy in photosystem I (PS I) isolated from Synechocystis sp. PCC 6803. While the slower phase (tau=340 ns at 295 K) slowed upon cooling according to an activation energy of 110 meV, the time constant of the faster phase (tau=11 ns at 295 K) was virtually independent of temperature. Following a suggestion in the literature that the two phases arise from bidirectional ET involving two symmetrically arranged phylloquinones, Q(K)-A and Q(K)-B, it is concluded that energetic parameters (most likely the driving forces) rather than the electronic couplings are different for ET from Q(K)-A to F(X) and from Q(K)-B to F(X). Two alternative schemes of ET in PS I are presented and discussed.     

Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.     

Electron transfer in cyanobacterial photosystem I: I. Physiological and spectroscopic characterization of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are pi-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1- radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1- and the CW EPR spectra of photoaccumulated A1-. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR.     

Flavodoxin: A compromise between efficiency and versatility in the electron transfer from Photosystem I to Ferredoxin-NADP reductase

Under iron-deficient conditions Flavodoxin (Fld) replaces Ferredoxin in Anabaena as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). Several residues modulate the Fld interaction with FNR and PSI, but no one appears as specifically critical for efficient electron transfer (ET). Fld shows a strong dipole moment, with its negative end directed towards the flavin ring. The role of this dipole moment in the processes of interaction and ET with positively charged surfaces exhibited by PSI and FNR has been analysed by introducing single and multiple charge reversal mutations on the Fld surface. Our data confirm that in this system interactions do not rely on a precise complementary surface of the reacting molecules. In fact, they indicate that the initial orientation driven by the alignment of dipole moment of the Fld molecule with that of the partner contributes to the formation of a bunch of alternative binding modes competent for the efficient ET reaction. Additionally, the fact that Fld uses different interaction surfaces to dock to PSI and to FNR is confirmed.     

Insertional inactivation of the menG gene, encoding 2-phytyl-1,4-naphthoquinone methyltransferase of Synechocystis sp. PCC 6803, results in the incorporation of 2-phytyl-1,4-naphthoquinone into the A(1) site and alteration of the equilibrium constant between A(1) and F(X) in photosystem I.

A gene encoding a methyltransferase (menG) was identified in Synechocystis sp. PCC 6803 as responsible for transferring the methyl group to 2-phytyl-1,4-naphthoquinone in the biosynthetic pathway of phylloquinone, the secondary electron acceptor in photosystem I (PS I). Mass spectrometric measurements showed that targeted inactivation of the menG gene prevented the methylation step in the synthesis of phylloquinone and led to the accumulation of 2-phytyl-1,4-naphthoquinone in PS I. Growth rates of the wild-type and the menG mutant strains under photoautotrophic and photomixotrophic conditions were virtually identical. The chlorophyll a content of the menG mutant strain was similar to that of wild type when the cells were grown at a light intensity of 50 microE m(-2) s(-1) but was slightly lower when grown at 300 microE m(-2) s(-1). Chlorophyll fluorescence emission measurements at 77 K showed a larger increase in the ratio of PS II to PS I in the menG mutant strain relative to the wild type as the light intensity was elevated from 50 to 300 microE m(-2) s(-1). CW EPR studies at 34 GHz and transient EPR studies at multiple frequencies showed that the quinone radical in the menG mutant has a similar overall line width as that for the wild type, but consistent with the presence of an aromatic proton at ring position 2, the pattern of hyperfine splittings showed two lines in the low-field region. The spin polarization pattern indicated that 2-phytyl-1,4-naphthoquinone is in the same orientation as phylloquinone, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to Q(-) center-to-center distance as in wild-type PS I. Transient EPR studies indicated that the lifetime for forward electron transfer from Q(-) to F(X) is slowed from 290 ns in the wild type to 600 ns in the menG mutant. The redox potential of 2-phytyl-1,4-naphthoquinone is estimated to be 50 to 60 mV more oxidizing than phylloquinone in the A(1) site, which translates to a lowering of the equilibrium constant between Q(-)/Q and F(X)(-)/F(X) by a factor of ca. 10. The lifetime of the P700(+) [F(A)/F(B)](-) backreaction decreased from 80 ms in the wild type to 20 ms in the menG mutant strain and is evidence for a thermally activated, uphill electron transfer through the quinone rather than a direct charge recombination between [F(A)/F(B)](-) and P700(+).     

Bidirectional electron transfer in photosystem I: replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone A1B with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures

A conserved tryptophan residue located between the A(1B) and F(X) redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A(1A) and the A(1B) phyllo(semi)quinones. The kinetics of A(1)(-) reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F(X) is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F(X) leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F(A) and F(B) by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A(1B).     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X)

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).     

Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.     

Interactions between thylakoid electron transfer complexes. I. In vitro kinetic studies with isolated photosystem II and cytochrome b6-f complexes

The cytochrome b6-f complex from spinach thylakoids has been reconstituted with an oxygen-evolving Photosystem II (PSII) preparation isolated from the same source to give oxygenic plastocyanin reductase activity. We observe that (i) mixing of the two complexes in concentrated form prior to dilution with the assay medium is necessary for optimal reconstitution of activity; (ii) incubation for longer times after dilution can also give substantial reconstitution if the two complexes are added separately to the assay mixture; (iii) either monovalent or divalent cations are required for optimum activity in the reconstituted system; (iv) titration of the cytochrome complex with varying amounts of the PSII complex gave a saturation of the plastocyanin reduction activity at a cytochrome complex/PSII ratio of 3-4; (v) kinetic analysis of plastocyanin photoreduction by Photosystem II shows nonlinearity, while first-order reduction kinetics are observed with duroquinol as electron donor; and (vi) as the concentration of plastocyanin is increased, the half-time of the reduction increases. These observations are considered in terms of a functional association between PSII and the cytochrome b6-f complex in this reconstituted system, and the relevance of these observations to the situation in vivo is discussed.     

Evidence for asymmetric electron transfer in cyanobacterial photosystem I: analysis of a methionine-to-leucine mutation of the ligand to the primary electron acceptor A0

The X-ray crystal structure of photosystem I (PS I) depicts six chlorophyll a molecules (in three pairs), two phylloquinones, and a [4Fe-4S] cluster arranged in two pseudo C2-symmetric branches that diverge at the P700 special pair and reconverge at the interpolypeptide FX cluster. At present, there is agreement that light-induced electron transfer proceeds via the PsaA branch, but there is conflicting evidence whether, and to what extent, the PsaB branch is active. This problem is addressed in cyanobacterial PS I by changing Met688(PsaA) and Met668(PsaB), which provide the axial ligands to the Mg2+ of the eC-A3 and eC-B3-chlorophylls, to Leu. The premise of the experiment is that alteration or removal of the ligand should alter the midpoint potential of the A0-/A0 redox pair and thereby result in a change in the forward electron-transfer kinetics from A0- to A1. In comparison with the wild type, the PsaA-branch mutant shows: (i) slower growth rates, higher light sensitivity, and reduced amounts of PS I; (ii) a reduced yield of electron transfer from P700 to the FA/FB iron-sulfur clusters at room temperature; (iii) an increased formation of the 3P700 triplet state due to P700(+)A0- recombination; and (iv) a change in the intensity and shape of the polarization patterns of the consecutive radical pair states P700(+)A1- and P700(+)FX-. The latter changes are temperature dependent and most pronounced at 298 K. These results are interpreted as being due to disorder in the A0 binding site, which leads to a distribution of lifetimes for A0- in the PsaA branch of cofactors. This allows a greater degree of singlet-triplet mixing during the lifetime of the radical pair P700(+)A0-, which changes the polarization patterns of P700(+)A1- and P700(+)FX-. The lower quantum yield of electron transfer is also the likely cause of the physiological changes in this mutant. In contrast, the PsaB-branch mutant showed only minor changes in its physiological and spectroscopic properties. Because the environments of eC-A3 and eC-B3 are nearly identical, these results provide evidence for asymmetric electron-transfer activity primarily along the PsaA branch in cyanobacterial PS I.     

Electrometrical study of electron transfer from the terminal FA/FB iron-sulfur clusters to external acceptors in photosystem I

An electrometrical technique was used to investigate electron transfer between the terminal iron-sulfur centers F(A)/F(B) and external electron acceptors in photosystem I (PS I) complexes from the cyanobacterium Synechococcus sp. PCC 6301 and from spinach. The increase of the relative contribution of the slow components of the membrane potential decay kinetics in the presence of both native (ferredoxin, flavodoxin) and artificial (methyl viologen) electron acceptors indicate the effective interaction between the terminal 14Fe-4S] cluster and acceptors. The finding that FA fails to donate electrons to flavodoxin in F(B)-less (HgCl2-treated) PS I complexes suggests that F(B) is the direct electron donor to flavodoxin. The lack of additional electrogenicity under conditions of effective electron transfer from the F(B) redox center to soluble acceptors indicates that this reaction is electrically silent.