Michael C.W. Evans

The role of trace metals in photosynthetic electron transport in O2-evolving organisms

Iron is the quantitatively most important trace metal involved in thylakoid reactions of all oxygenic organisms since linear (= non-cyclic) electron flow from H2O to NADP+ involves PS II (2–3 Fe), cytochrome b6-f (5 Fe), PS I (12 Fe), and ferredoxin (2 Fe); (replaceable by metal-free flavodoxin in certain cyanobacteria and algae under iron deficiency). Cytochrome c6 (1 Fe) is the only redox catalyst linking the cytochrome b6-f complex to PS I in most algae; in many cyanobacteria and Chlorophyta cytochrome c6 and the copper-containing plastocyanin are alternatives, with the availability of iron and copper regulating their relative expression, while higher plants only have plastocyanin. Iron, copper and zinc occur in enzymes that remove active oxygen species and that are in part bound to the thylakoid membrane. These enzymes are ascorbate peroxidase (Fe) and iron-(cyanobacteria, and most al gae) and copper-zinc- (some algae; higher plants) superoxide dismutase. Iron-containing NAD(P)H-PQ oxidoreductase in thylakoids of cyanobacteria and many eukaryotes may be involved in cyclic electron transport around PS I and in chlororespiration. Manganese is second to iron in its quantitative role in the thylakoids, with four Mn (and 1 Ca) per PS II involved in O2 evolution. The roles of the transition metals in redox catalysts can in broad terms be related to their redox chemistry and to their availability to organisms at the time when the pathways evolved. The quantitative roles of these trace metals varies genotypically (e.g. the greater need for iron in thylakoid reactions of cyanobacteria and rhodophytes than in other O2-evolvers as a result of their lower PS II:PS I ratio) and phenotypically (e.g. as a result of variations in PS II:PS I ratio with the spectral quality of incident radiation).

Isolation of a photosystem 2 preparation from higher plants with highly enriched oxygen evolution activity

Detergent‐treatment of higher plant thylakoids with Triton X‐100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl−1.h−1). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680+ is particularly well preserved in the preparation, as judged by P680+ reduction kinetics, the detection by EPR of Signal IILT and the presence of the high potential form of cytochrome b‐559 (at a ratio of 1:1 with the reaction centre).

Determination of the oxidation—reduction potential of the bound iron-sulphur proteins of the primary electron acceptor complex of photosystem I in spinach chloroplasts

Malkin and Bearden [l] showed that spinach chloroplasts contain an EPR-detectable component, which can be photoreduced at low temperatures. Further experiments showed that this component is concentrated in photosystem I particles and is photoreduced by far red light [2,3]. Using varying conditions for photoreduction of the acceptor we were able to show [3] that two EPR detectable components could be detected, both of which had the characteristics of iron-sulphur centres. We further suggested that the two components might be two active centres of a single protein. More recently, Ke et al. [4] have measured the intensities of the EPR signals of these components as a function of redox potential in digitonin prepared photosystem I particles. They found that these components have extremely low redox potentials. Their interpretation of the data was that there are at least three species of iron-sulphur centres present; and that each of these reductions involved a two-electron change. This result implies that the groups must represent a novel type of redox carrier, since all known ironsulphur centres accept electrons one at a time. Even if

Direct detection of the electron acceptor of photosystem II: Evidence that Q is an iron—quinone complex

The primary pboto~h~~st~ of the photosystem II reaction centre involves the transfer of an electron from the primary chlorophyll donor (P680) to the primary acceptor, Q. The properties of Q, a specialised plastoquinone [ 1,2] have been studied in detail using indirect fluorescence measurements [3-61 and absorbance changes [7-l 21 but no electron paramagnetic resonance (EPR) signal was observed to confirm the suggestion [ 10,l l] that as in bacterial reaction centres, a transition metal ion (probably Fe’+) was complexed to Q. When Q is chemic~y reduced, the photoredu~tion of a pheophytin intermediate electron carrier (I) has been observed [ 131. Recent EPR investigations have identified a signal near g = 2.00 corresponding to I[14-171 which, when present in samples containing Q-, gives rise to an EPR doublet [ 14,151 with similar properties to those found in purple photosynthetic bacterial reaction centres (reviewed in [IS]). This and further work [ 191 strongly suggested that the interaction producing the doublet involved a semiquinoneiron complex of the type exhibiting characteristic EPR signals near g = 1.82 in purple photosynthetic bacteria [20-231. Using broken c~oroplasts from a mutant of barley (Hordeurn vulgare viridis zb 63)lacking photosystem I [16,17], the photoreduction of Iwas also observed at 5-77 K indicating the presence of a fast donor to P680 under these conditions. In 1241 a highly active photosystem II particle was prepared from a mutant of the green alga ~~~ydornonas reinhardii, originally isolated by P. Bennoun. Fluorescence measurements [24] suggest that the particles lack the secondary quinone acceptor B. Using

Identification of multiple components in the intermediary electron carrier complex of photosystem I

Illumination at 230 K of dithionite‐reduced particles results in the appearance of an EPR detectable radical 13 G wide with g = 2.0033. This radical is formed in a ratio of 2.28 (±0.5)/P700. Investigation of the time course of formation shows two components are present. One (A 1) has g = 2.0051 and the other (A o g= 2.0024. Reduction of A 1 results in an increase in reaction centre triplet formation, subsequent reduction of A o results in a decrease of triplet formation to the base level. We propose that these components function sequentially in the transfer of electrons from P700 to the iron—sulphur acceptors.

Direct measurement of the redox potential of the primary and secondary quinone electron acceptors in Rhodopseudomonas sphaeroides (wild-type) by EPR Spectrometry

The secondary acceptor in bacterial photosynthesis is a specialized quinone molecule, Qz, which is bound to the reaction centre (reviewed [ 11). By carrying out flash experiments upon reaction centres of Rhodopseudomonas sphaeroides it was demonstrated that QZ can accept two electrons (e-) consecutively, in a process which involves the formation of the stable semiquinone on odd-numbered flashes and the donation of a pair of electrons (and protons) to exog&ous ubiquinone on even-numbered flashes [2,3]. Subsequently binary oscillations of the semiquinone were reported in chromatophores of Rps. sphaeroides [4,5], Rhodopseudomonas capsulata [6] and in whole cells of Rhodospirillum rubrum [5]. Out of phase oscillations of the slow phase of the carotenoid bandshift [5] and of cytochrome b reductions [6] have also been reported in chromatophore preparations. Since the presence of the ubiquinone pool is not necessary for cyclic electron transport to occur [7] and cytochrome bsO, a leacceptor, is thought to be the oxidant of Qz (e.g. [S]), mechanisms of electron transfer from QZ have had to be invoked for chromatophores [5,6] which are more complicated than that indicated from the reaction centre data [2,3]. These models were formulated in the absence ofEm values for the redox changes of Q?. We have reported the presence of a semiquinoneiron signal attributable to Q2--Fe in Rhodopseudomonas viridis [9,10]. Redox titrations were carried out measuring this signal and EmSD values for the Q?/Q;H and Q;H/Q,H, couples were estimated to be t67 mV and -15 mV, respectively. Little work on the electron transport chain of this species has been done, so application of these values to the formulation of an electron-transport model is purely speculative. Here we report semiquinone-iron EPR signals in chromatophores of Rps. sphaeroides which are attributable to the primary and secondary acceptors. Directly measured Em values have been obtained for the QI/Q;H, Q2/Q;H and QiH/Q& couples.

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.

Photosynthetic water oxidation: towards a mechanism

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.

The role of the membrane-bound iron-sulphur centres A and B in the photosystem I reaction centre of spinach chloroplasts.

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I (P-700) and X. Oxidation-reduction potential titrations confirmed that Centre A had Em congruent to -550 mV, Centre B had Em congruent to -585 mV. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, P-700 photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible P-700 photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with Em congruent to -585 mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.

Optical difference spectrum of the electron acceptor Ao in photosystem I

Photosystem I Primary acceptor Optical difference spectrum Chlorophyll a monomer