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 FX

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 A₁, 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 (A₁) 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 A₀ on both electron transfer branches and the reduction of F_A by F_X.     

Stabilization of iron-sulfur cluster F(X) by intra-subunit interactions unraveled by suppressor and second site-directed mutations in PsaB of Photosystem I

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.     

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.     

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.     

The bound electron acceptors in green sulfur bacteria: resolution of the g-tensor for the F(X) iron-sulfur cluster in Chlorobium tepidum

The photosynthetic reaction center (RC) of green sulfur bacteria contains two [4Fe-4S] clusters named F(A) and F(B), by analogy with photosystem I (PS I). PS I also contains an interpolypeptide [4Fe-4S] cluster named F(X); however, spectroscopic evidence for an analogous iron-sulfur cluster in green sulfur bacteria remains equivocal. To minimize oxidative damage to the iron-sulfur clusters, we studied the sensitivity of F(A) and F(B) to molecular oxygen in whole cells of Chlorobium vibrioforme and Chlorobium tepidum and obtained highly photoactive membranes and RCs from Cb. tepidum by adjusting isolation conditions to maximize the amplitude of the F(A)(-)/F(B)(-) electron paramagnetic resonance signal at g = 1.89 (measured at 126 mW of microwave power and 14 K) relative to the P840(+) signal at g = 2.0028 (measured at 800 microW of microwave power and 14 K). In these optimized preparations we were able to differentiate F(X)(-) from F(A)(-)/F(B)(-) by their different relaxation properties. At temperatures between 4 and 9 K, isolated membranes and RCs of Cb. tepidum show a broad peak at g = 2.12 and a prominent high-field trough at g = 1.76 (measured at 126 mW of microwave power). The complete g-tensor of F(X)(-), extracted by numerical simulation, yields principal values of 2.17, 1.92, and 1. 77 and is similar to F(X) in PS I. An important difference from PS I is that because the bound cytochrome is available as a fast electron donor in Chlorobium, it is not necessary to prereduce F(A) and F(B) to photoaccumulate F(X)(-).     

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     

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.     

The roles of coenzyme A in the pyruvate:ferredoxin oxidoreductase reaction mechanism: rate enhancement of electron transfer from a radical intermediate to an iron-sulfur cluster

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the coenzyme A (CoA)-dependent oxidative decarboxylation of pyruvate. In many autotrophic anaerobes, PFOR links the Wood-Ljungdahl pathway to glycolysis and to cell carbon synthesis. Herein, we cloned and sequenced the M. thermoacetica PFOR, demonstrating strong structural homology with the structurally characterized D. africanus PFOR, including the presence of three [4Fe-4S] clusters per monomeric unit. The PFOR reaction includes a hydroxyethyl-thiamin pyrophosphate (HE-TPP) radical intermediate, which forms rapidly after PFOR reacts with pyruvate. This step precedes electron transfer from the HE-TPP radical intermediate to an intramolecular [4Fe-4S] cluster. We show that CoA increases the rate of this redox reaction by 10(5)-fold. Analysis by Marcus theory indicates that, in the absence of CoA, this is a true electron-transfer reaction; however, in its presence, electron transfer is gated by an adiabatic event. Analysis by the Eyring equation indicates that entropic effects dominate this rate enhancement. Our results indicate that the energy of binding CoA contributes minimally to the rate increase since the thiol group of CoA lends over 40 kJ/mol to the reaction, whereas components of CoA that afford most of the cofactor's binding energy contribute minimally. Major conformational changes also do not appear to explain the rate enhancement. We propose several ways that CoA can accomplish this rate increase, including formation of a highly reducing adduct with the HE-TPP radical to increase the driving force for electron transfer. We also consider the possibility that CoA itself forms part of the electron-transfer pathway.     

A kinetic assessment of the sequence of electron transfer from F(X) to F(A) and further to F(B) in photosystem I: the value of the equilibrium constant between F(X) and F(A)

The x-ray structure analysis of photosystem I (PS I) crystals at 4-A resolution (Schubert et al., 1997, J. Mol. Biol. 272:741-769) has revealed the distances between the three iron-sulfur clusters, labeled F(X), F(1), and F(2), which function on the acceptor side of PS I. There is a general consensus concerning the assignment of the F(X) cluster, which is bound to the PsaA and PsaB polypeptides that constitute the PS I core heterodimer. However, the correspondence between the acceptors labeled F(1) and F(2) on the electron density map and the F(A) and F(B) clusters defined by electron paramagnetic resonance (EPR) spectroscopy remains controversial. Two recent studies (Diaz-Quintana et al., 1998, Biochemistry. 37:3429-3439;, Vassiliev et al., 1998, Biophys. J. 74:2029-2035) provided evidence that F(A) is the cluster proximal to F(X), and F(B) is the cluster that donates electrons to ferredoxin. In this work, we provide a kinetic argument to support this assignment by estimating the rates of electron transfer between the iron-sulfur clusters F(X), F(A), and F(B). The experimentally determined kinetics of P700(+) dark relaxation in PS I complexes (both F(A) and F(B) are present), HgCl(2)-treated PS I complexes (devoid of F(B)), and P700-F(X) cores (devoid of both F(A) and F(B)) from Synechococcus sp. PCC 6301 are compared with the expected dependencies on the rate of electron transfer, based on the x-ray distances between the cofactors. The analysis, which takes into consideration the asymmetrical position of iron-sulfur clusters F(1) and F(2) relative to F(X), supports the F(X) --> F(A) --> F(B) --> Fd sequence of electron transfer on the acceptor side of PS I. Based on this sequence of electron transfer and on the observed kinetics of P700(+) reduction and F(X)(-) oxidation, we estimate the equilibrium constant of electron transfer between F(X) and F(A) at room temperature to be approximately 47. The value of this equilibrium constant is discussed in the context of the midpoint potentials of F(X) and F(A), as determined by low-temperature EPR spectroscopy.     

A mixed-ligand iron-sulfur cluster (C556SPaB or C565SPsaB) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I.

The proposed structure of Photosystem I depicts two cysteines on the PsaA polypeptide and two cysteines on the PsaB polypeptide in a symmetrical environment, each providing ligands for the interpolypeptide Fx cluster. We studied the role of Fx in electron transfer by substituting serine for cysteine (C565SPsaB and C556SPsaB), thereby introducing the first example of a genetically engineered, mixed-ligand [4Fe-4S] cluster into a protein. Optical kinetic spectroscopy shows that after a single-turnover flash at 298 K, the contribution of A1- (lifetime of 10 microseconds, 40% of total and lifetime of 100 microseconds, 20% of total) and Fx- (lifetime of 500-800 microseconds, 10-15% of total) to the overall P700+ back reaction have increased in C565SPsaB and C556SPsaB at the expense of the back reaction from [FA/FB]-. The electron paramagnetic resonance spectrum of Fx shows g-values of 2.04, 1.94, and 1.81 in both mutants and a similarly decreased amount of FA and FB reduced at 15 K after a single-turnover flash. These results indicate that the mixed-ligand (3 cysteines, 1 serine) Fx cluster is an inefficient electron carrier, but that a small leak through Fx still permits FA and FB to be reduced quantitatively when the samples are frozen during continuous illumination. The data confirm that Fx is a necessary intermediate in the electron transfer pathway from A1 to FA and FB in Photosystem I.     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

Analysis of the spin-polarized electron spin echo of the [P700+ A1-] radical pair of photosystem I indicates that both reaction center subunits are competent in electron transfer in cyanobacteria, green algae, and higher plants

The decay of the light-induced spin-correlated radical pair [P700+ A1-] and the associated electron spin echo envelope modulation (ESEEM) have been studied in either thylakoid membranes, cellular membranes, or purified photosystem I prepared from the wild-type strains of Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, and Spinaceae oleracea. The decay of the spin-correlated radical pair is described in the wild-type membrane by two exponential components with lifetimes of 2-4 and 16-25 micros. The proportions of the two components can be altered by preillumination of the membranes in the presence of reductant at temperatures lower than 220 K, which leads to the complete reduction of the iron-sulfur electron acceptors F(A), F(B), and F(X) and partial photoaccumulation of the reduced quinone electron acceptor A1A-. The "out-of-phase" (OOP) ESEEM attributed to the [P700+ A1-] radical pair has been investigated in the three species as a function of the preillumination treatment. Values of the dipolar (D) and the exchange (J) interactions were extracted by time-domain fitting of the OOP-ESEEM. The results obtained in the wild-type systems are compared with two site-directed mutants of C. reinhardtii [Santabarbara et al. (2005) Biochemistry 44, 2119-2128], in which the spin-polarized signal on either the PsaA- or PsaB-bound electron transfer pathway is suppressed so that the radical pair formed on each electron transfer branch could be monitored selectively. This comparison indicates that when all of the iron-sulfur centers are oxidized, only the echo modulation associated with the A branch [P700+ A1A-] radical pair is observed. The reduction of the iron-sulfur clusters and the quinone A1 by preillumination treatment induces a shift in the ESEEM frequency. In all of the systems investigated this observation can be interpreted in terms of different proportions of the signal associated with the [P700+ A1A-] and [P700+ A1B-] radical pairs, suggesting that bidirectionality of electron transfer in photosystem I is a common feature of all species rather than being confined to green algae.