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).     

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.     

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).     

Asymmetric electron transfer in cyanobacterial Photosystem I: charge separation and secondary electron transfer dynamics of mutations near the primary electron acceptor A0

Point mutations were introduced near the primary electron acceptor sites assigned to A0 in both the PsaA and PsaB branches of Photosystem I in the cyanobacterium Synechocystis sp. PCC 6803. The residues Met688PsaA and Met668PsaB, which provide the axial ligands to the Mg2+ of the eC-A3 and eC-B3 chlorophylls, were changed to leucine and asparagine (chlorophyll notation follows Jordan et al., 2001). The removal of the ligand is expected to alter the midpoint potential of the A0/A0- redox pair and result in a change in the intrinsic charge separation rate and secondary electron transfer kinetics from A0- to A1. The dynamics of primary charge separation and secondary electron transfer were studied at 690 nm and 390 nm in these mutants by ultrafast optical pump-probe spectroscopy. The data reveal that mutations in the PsaB branch do not alter electron transfer dynamics, whereas mutations in the PsaA branch have a distinct effect on electron transfer, slowing down both the primary charge separation and the secondary electron transfer step (the latter by a factor of 3-10). These results suggest that electron transfer in cyanobacterial Photosystem I is asymmetric and occurs primarily along the PsaA branch of cofactors.     

Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A₀, A₁ and Fe-S_X. Fe-S_X is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-S_X is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-S_A/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.Dedicated to the memory of Daniel I. Arnon.     

Iron-sulfur clusters in type I reaction centers.

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.     

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.     

The role of Na2S in anoxygenic photosynthesis and H2 production in the cyanobacterium Nostoc muscorum

Na2S is known to support anoxygenic photosynthesis in some strains of cyanobacteria and to stimulate H2 production in N2 fixing filaments of Nostoc muscorum. We have shown electron transfer between Na2S and Photosystem I to be dependent on cytochrome b559 which was detected only in vegetative cells. An electron mediator was required to support Na2S driven nitrogenase activity in isolated heterocysts. Na2S was also found to deplete the ATP pool, probably by inhibiting electron transfer from Photosystem I.     

Control of electron transport in photosystem I by the iron-sulfur cluster FX in response to intra- and intersubunit interactions

Photosystem I (PS I) is a transmembranal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an approximately 10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I.     

Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors F(A) and F(B) in Heliobacterium modesticaldum

The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates FA- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichia coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/FB]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798+ and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.     

Bidirectional electron transfer in photosystem I: accumulation of A0- in A-side or B-side mutants of the axial ligand to chlorophyll A0

Photosystem I contains two potential electron transfer pathways between P(700) and F(X). These branches are made up of the electron transfer chain components A, A(0), and A(1). The primary electron acceptor A(0) is a chlorophyll a monomer that could be one or both of the two chlorophyll molecules, eC-A(3)/eC-B(3), identified in the 2.5 A resolution structure. The eC-A(3)/eC-B(3) chlorophylls are both coordinated by the sulfur atom of a methionine. This coordination is highly unusual, as interactions between the acid Mg(2+) and the soft base sulfur are weak. The eC-A(3)/eC-B(3) chlorophylls also are located close to one of the connecting chlorophylls that may link the antenna and the electron transfer chain chlorophylls. Due to their location in the structure, the eC-A(3)/eC-B(3) chlorophylls may play a role in both excitation energy transfer and electron transfer. To test the role of the eC-A(3)/eC-B(3) chlorophylls in electron transfer, Met-684 of PsaA and Met-664 of PsaB have been changed to His, Ser, and Leu. Replacement of either M(A684) or M(B664) results in a significant alteration in growth phenotype. The His and Leu mutants are very light sensitive in the presence of oxygen. Growth is impaired to a greater extent in the B-side mutants. However, all of the mutants are able to grow anaerobically at comparable rates. The His and Ser mutants all accumulate PSI at a level similar to that of wild type, whereas the Leu mutants have reduced amounts of PSI. Ultrafast transient absorbance measurements show that the (A(0)(-) - A(0)) difference signal accumulates in the MH(A684) and MH(B664) mutants under neutral conditions, demonstrating that electron transfer between A(0)(-) and A(1) is blocked or significantly slowed. The results show that both the A-branch and the B-branch of the ETC are active in PSI from Chlamydomonas reinhardtii.     

Protein sequences and redox titrations indicate that the electron acceptors in reaction centers from heliobacteria are similar to Photosystem I

Photosynthetic reaction centers isolated from Heliobacillus mobilis exhibit a single major protein on SDS-PAGE of 47 000 M_r. Attempts to sequence the reaction center polypeptide indicated that the N-terminus is blocked. After enzymatic and chemical cleavage, four peptide fragments were sequenced from the Heliobacillus mobilis apoprotein. Only one of these sequences showed significant specific similarity to any of the protein and deduced protein sequences in the GenBank data base. This fragment is identical with 56% of the residues, including both cysteines, found in the highly conserved region that is proposed to bind iron-sulfur center F_X in the Photosystem I reaction center peptide that is the psaB gene product. The similarity to the psaA gene product in this region is 48%.Redox titrations of laser-flash-induced photobleaching with millisecond decay kinetics on isolated reaction centers from Heliobacterium gestii indicate a midpoint potential of –414 mV with n=2 titration behavior. In membranes, the behavior is intermediate between n=1 and n=2, and the apparent midpoint potential is –444 mV. This is compared to the behavior in Photosystem I, where the intermediate electron acceptor A₁, thought to be a phylloquinone molecule, has been proposed to undergo a double reduction at low redox potentials in the presence of viologen redox mediators.These results strongly suggest that the acceptor side electron transfer system in reaction centers from heliobacteria is indeed analogous to that found in Photosystem I. The sequence similarities indicate that the divergence of the heliobacteria from the Photosystem I line occurred before the gene duplication and subsequent divergence that lead to the heterodimeric protein core of the Photosystem I reaction center.Abbreviations BChl bacteriochlorophyll - %C percent bisacrylamide as a percentage of total acrylamide - DTT dithiothreitol - EPR electron paramagnetic resonance - Fe-S iron-sulfur center - H. Heliobacterium - Hb. Heliobacillus - k one thousand - M_r molecular retention - PS I Photosystem I - PS II Photosystem II - RCs reaction centers - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide electrophoresis - %T percent total acrylamide - Tris tris(hydroxymethyl)aminomethane     

Evidence from time resolved studies of the P700(.+)/A1(.-) radical pair for photosynthetic electron transfer on both the PsaA and PsaB branches of the photosystem I reaction centre

Kinetic analysis using pulsed electron paramagnetic resonance (EPR) of photosynthetic electron transfer in the photosystem I reaction centres of Synechocystis 6803, in wild-type Chlamydomonas reinhardtii, and in site directed mutants of the phylloquinone binding sites in C. reinhardtii, indicates that electron transfer from the reaction centre primary electron donor, P700, to the iron-sulphur centres, Fe-S(X/A/B), can occur through either the PsaA or PsaB side phylloquinone. At low temperature reaction centres are frozen in states which allow electron transfer on one side of the reaction centre only. A fraction always donates electrons to the PsaA side quinone, the remainder to the PsaB side.     

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 A₁⁻ 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.     

The mechanism of the degradation of psaB gene product,one of the photosynthetic reaction center subunits of Photosystem I,upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala⁵⁰⁰ and Val⁵⁰¹ on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0- reoxidation dynamics in photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P(700), and a sequence of electron acceptors, A, A(0) and A(1), bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A(0), are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A(0)(-) (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A(0)(-) on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A(0)(-) is transient, and that reoxidation of A(0)(-) occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A(1). This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A(0) in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.