The effect of the substitution of the axial ligand to the primary electron acceptor A0 on the photosynthetic electron transfer performance is investigated in two site-directed mutants of the green alga C. reinhardtii (PsaA-M684H, PsaBM664H). Both mutations affects the stability of the PS I reaction centre, the accumulation of which is about halved compared to the wild-type. In whole cells of the PsaA-M684H and the PsaBM664H mutants the rate of linear electron transfer (ET) is decreased by 10% and 90% compared to the wild-type, respectively, under saturating light conditions. Under limiting light conditions these rates are decreased by 50% and 90%, respectively. These functional differences in the ET reactions involving the PsaA-bound and the PsaB-bound cofactors are not explained at full by previous spectroscopic investigations. We interpret the results in terms of an asymmetric effect of the axial donor substitution to A0 on the maximal photochemical efficiency of PS I. These results also highlight the physiological importance of ET reactions involving PsaB-bound cofactors.

Ammonia and methanol both bind to the water oxidising complex of photosystem II during its turnover, possibly at sites where water binds during the normal water oxidation process. We have investigated the interaction between these two water analogues at the S2 state of the water oxidising cycle using electron magnetic resonance techniques. We find evidence that ammonia displaces methanol from its binding site.

The spin-correlated radical pair [P(700)(+)A(1)(-)] gives rise to a characteristic "out-of-phase" electron spin-echo signal. The electron spin-echo envelope modulation (ESEEM) of these signals has been studied in thylakoids prepared from the wild-type strain of Chlamydomonas reinhardtii and in two site-directed mutants, in which the methionine residue which acts as the axial ligand to the chlorin electron acceptor A(0) has been substituted with a histidine either on the PsaA (PsaA-M684H) or the PsaB (PsaB-M664H) reaction center subunits. The analysis of the time domain ESEEM provides information about the spin-spin interaction in the [P(700)(+)A(1)(-)] radical pair, and the values of the dipolar (D) and the exchange (J) interaction can be extracted. From the distance dependence of the dipolar coupling term, the distance between the unpaired electron spin density clouds of the primary donor P(700)(+) and the phyllosemiquinone A(1)(-) can be determined. The [P(700)(+)A(1)(-)] ESEEM spectrum obtained in wild-type thylakoids can be reconstructed using a linear combination of the spectra measured in the PsaA and PsaB A(0) mutants, demonstrating that electron transfer resulting in charge separation is occurring on both the PsaA and PsaB branches. The [P(700)(+)A(1B)(-)] distance in the point dipole approximation in the PsaA-M684H mutant is 24.27 +/- 0.02 A, and the [P(700)(+)A(1A)(-)] distance in the PsaB-M664H mutant is 25.43 +/- 0.01 A. An intermediate value of 25.01 +/- 0.02 A is obtained in the wild-type membranes which exhibit both spin-polarized pairs.

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 FX and the phylloquinone bound to either the PsaA (A1A) or the PsaB (A1B) subunit of the reaction centre and the equilibrium between the iron–sulfur centres FA and FB. 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 FX. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A1B quinone and slightly endergonic, in the case of the A1A 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 FA by FX.

This mini-review outlines the involvement of the tyrosine electron carriers, Y(D) and Y(Z), in the mechanism of electron transfer from water to P680. We discuss our data and put forward our ideas on the role of Y(D) and Y(Z).

The interaction of water with the water oxidizing Mn complex of photosystem II has been investigated using electron spin-echo envelope modulation spectroscopy in the presence of H(2)(17)O. The spectra show interaction of the (17)O with the preparation in the S(2) state induced by 200 K illumination. The modulation is observed only in the center of the multiline spectrum. The inferred hyperfine coupling terms are compatible with water (not hydroxyl) oxygen bound to a particular quasi-axial Mn(III) center in a coupled Mn cluster.

The characteristic Mn hyperfine ‘multiline’ signal exhibited in the S2 state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S2 state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol.

Chlorin anion radicals have been produced by photoaccumulation of photosystem I and the type I reaction centres of the anoxygenic bacteria Chlorobium limicola and Heliobacterium chlorum. Proton electron nuclear double resonance (ENDOR) spectrometry of the photoaccumulated radicals demonstrates that the photoaccumulation technique is reducing chlorophyll anions rather than bacteriochlorophyll anions, indicating that photoaccumulation specifically reduces the primary electron acceptors (A0) in these anoxygenic reaction centres and is not reducing the antenna bacteriochlorophylls. Detailed analysis of the in vivo proton ENDOR spectra in comparison with in vitro (bacterio)chlorophyll and (bacterio)pheophytin anion radicals points up differences between the environment of the A0 in photosystem I and the anoxygenic type I reaction centres.

Previous work in many laboratories has established that hydroxylamine reduces the S, state of the water oxidizing complex (WOC) in one-electron steps. Significant levels of what can now be defined as the S(-1)* state are achieved by specific (concentration and incubation length) hydroxylamine treatments. This state has already been studied by electron paramagnetic resonance spectrometry (EPR), and unusual EPR signals were noted (for example, see Sivaraja, M., and Dismukes, G. C. (1988) Biochemistry 27, 3467-3475). We have now reinvestigated these initial experiments and confirmed many of the original observations. We then utilized more recent EPR markers for the So and S, states to further explore the S(-1)* state. The broad radical "split" type EPR signal, produced by 200 K illumination of samples prepared to give a high yield of the S(-1)* state, is shown to most likely reflect a trapped intermediate state between S(-1)* and S(0)*, since samples where this signal is present can be warmed in the dark to produce S(0)*. The threshold for advancement from S(-1)* to S(0)* is near 200 K, as the yield of broad radical decreases and S(0)* multiline EPR signal increases with length of 200 K illumination. Advancement of S(0)* to S(1) is limited at 200 K, but S, can be restored by 273 K illumination. Illumination of these hydroxylamine-treated samples at temperatures below 77 K gives a second broad radical EPR signal. The line shape, decay, and other properties of this new radical signal suggest that it may arise from an interaction in the S(-2)* or lower S states, which are probably present in low yield in these samples. Illumination below 20 K of S(0)* state samples containing methanol, and therefore exhibiting the So multiline signal, gives rise to a third broad radical with distinctive line shape. The characteristics of the three broad radicals are similar to those found from interactions between Y(Z)(.) and other S states. The evidence is presented that they do represent intermediate states in S state turnover. Further work is now needed to identify these radicals.

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.

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700+. Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700+, and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.

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. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science BN. All rights reserved.

The electron transfer chain of PSI comprises a primary electron donor (P700; chlorophyll a dimer), and five electron acceptors: A0 (chlorophyll a monomer); A1 (phylloquinone); FX; FA and FB (all 4Fe4S2- centres). P700 and FX exist on the apparent C2 axis of symmetry of the PsaA/PsaB heterodimeric core, and there are two symmetrical branches of the redox cofactors A0 and A1 between them. It has recently been suggested that both branches of electron transfer may be functional, with a fast branch (PsaB branch) and a slow branch (PsaA branch) [1]. We have been using a combination of site-directed mutagenesis and spectroscopy to probe the protein environment surrounding the cofactor, A0. The suggested ligand to the central magnesium of the chlorophyll A0 is residue M684 on the PsaA polypeptide of PSI. The presence of a methionine residue as a ligand to a chlorophyll is highly unusual. We have been studying the effect of mutating this residue in the unicellular green alga, Chlamydomonas reinhardtii, on the dynamics of electron transfer in PSI and the properties of A0. The analysis of the mutants (M684A, M684H and M684V) has been carried out using paramagnetic spectroscopic techniques. These techniques have enabled us to investigate whether residue PsaA:M684 is a factor controlling the redox potential of A0. Inactivation of electron transfer on the PsaA branch in some of these mutants has enabled study of the significance of bidirectional electron transfer in PSI. 1. Guergova-Kuras, M., Boudreaux, B., Joliot, A., Joliot, P., Redding, K. (2001) PNAS, in press.

We have been using site-directed mutagenesis in conjunction with spectroscopic techniques to probe structure-function relationships in the Photosytem I reaction centre of the green alga Chlamydomonas reinhardtii. We recently reported [1] that substitution of the conserved tryptophan W693/H/L on the PsaA polypeptide slowed down or abolished forward electron transfer from the phylloquinone to the FeS centre Fx, as monitored by the decay of the Electron Spin Polarised signal (ESP) arising from the P700+ /A1- radical pair at 260K. We concluded that the photoaccumulated phyllosemiquinone EPR signal, and the ESP signal, monitored the phylloquinone on the PsaA branch of electron transfer. We have recently used Electron Nuclear Double Resonance spectroscopy (ENDOR) to demonstrate that we can photoaccumulate under certain conditions the phyllosemiquinone on the PsaB branch of electron transfer, and that there are differences in the electronic structure of the two phyllosemiquinones. By monitoring the decay of the ESP signal at 100K where it reflects the properties of the P700.+/A1.- radical pair we have obtained evidence for forward electron transfer from P700 to A1 on both the PsaA and PsaB sides of the reaction centre. [1] S. Purton, D. Stevens, I. P. Muhiuddin, M. C. W. Evans, S. Carter, S. E. J. Rigby, and P. Heathcote (2001) Biochemistry 40, 2167-2175.

This mini-review outlines the current theories on the mechanism of electron transfer from water to P680, the location and structure of the water oxidising complex and the role of the manganese cluster. We discuss how our data fit in with current theories and put forward our ideas on the location and mechanism of water oxidation.