A. William Rutherford

Herbicide-induced oxidative stress in photosystem II

Some herbicides act by binding to the exchangeable quinone site in the photosystem II (PSII) reaction centre, thus blocking electron transfer. In this article, it is hypothesized that the plant is killed by light-induced oxidative stress initiated by damage caused by formation of singlet oxygen in the reaction centre itself. This occurs when light-induced charge pairs in herbicide-inhibited PSII decay by a charge recombination route involving the formation of a chlorophyll triplet state that is able to activate oxygen. The binding of phenolic herbicides favours this pathway, thus increasing the efficiency of photodamage in this class of herbicides.

Singlet oxygen production in herbicide‐treated photosystem II

Photo‐generated reactive oxygen species in herbicide‐treated photosystem II were investigated by spin‐trapping. While the production of OH and O2 − was herbicide‐independent, 1O2 with a phenolic was twice that with a urea herbicide. This correlates with the reported influence of these herbicides on the redox properties of the semiquinone QA − and fits with the hypothesis that 1O2 is produced by charge recombination reactions that are stimulated by herbicide binding and modulated by the nature of the herbicide. When phenolic herbicides are bound, charge recombination at the level of P+ Pheo− is thermodynamically favoured forming a chlorophyll triplet and hence 1O2. With urea herbicides this pathway is less favourable.

A change in the midpoint potential of the quinone QA in Photosystem II associated with photoactivation of oxygen evolution

The effect of photoactivation (the assembly of the Mn cluster involved in oxygen evolution) in Photosystem II (PS II), on the redox midpoint potential of the primary quinone electron acceptor, QA, has been investigated. Measurements of the redox state of QA were performed using chlorophyll fluorescence. Cells of Scenedesmus obliquus were grown in the dark to obtain PS II lacking the oxygen-evolving complex. Growth in the light leads to photoactivation. The midpoint potential of QA was shifted, upon photoactivation, from + 110 mV to −80 mV. In cells of a low-fluorescence mutant, LF1, that is unable to assemble the oxygen-evolving complex but that has an otherwise normal PS II, the higher potential form of QA was found. NH2OH treatment of spinach PS II, which releases the Mn and thus inactivates the oxygen-evolving complex, causes an upshift of the redox potential of QA (Krieger and Weis (1992) Photosynthetica 27, 89–98). Oxygen evolution can be reconstituted by incubation in the light in the presence of MnCl2 and CaCl2. Such photoactivation caused the midpoint potential of QA to be shifted back from around +55 mV to lower potentials (−80 mV), typical for active PS II. The above results indicate that the state of the donor side of PS II has a direct influence on the properties of the acceptor side. It is suggested that the change from the high- to the low-potential form of QA may represent a mechanism for protection of PS II during the assembly of the O2-evolving enzyme.

X-ray crystallography identifies two chloride binding sites in the oxygen evolving centre of Photosystem II

Bromide anomalous X-ray diffraction analyses have been used to locate chloride binding sites in the vicinity of the water splitting/oxygen evolving centre (OEC) of Photosystem II. Three-dimensional crystals of PSII from Thermosynechococcus elongatus were grown from (i) isolated PSII crystals infiltrated with bromide or (ii) PSII obtained from cells cultured in a medium in which the chloride content was totally replaced by bromide. In either case, the anomalous diffraction yielded the same result, the existence of two bromide binding sites in the vicinity of the OEC. Neither are in the first coordination sphere of the Mn and Ca ions which form the catalytic centre of the OEC, being about 6 to 7 A from the metal-cluster. Site 1 is located close to the side chain nitrogen of D2-K317 and the backbone nitrogen of D1-Glu333 while Site 2 is adjacent to backbone nitrogens of CP43-Glu354 and D1-Asn338. Their positioning close to postulated hydrophilic channels may suggest a role in proton removal from, or substrate access to, the OEC.

A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site‐directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA‐His676 and PsaB‐His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls.

The origin of 40-50°C thermoluminescence bands in Photosystem II

Abstract We have used thermoluminescence (TL) and EPR measurements of Photosystem II (PS II) from spinach in order to identify charge pairs responsible for TL bands in the region of 40–50°C including the ‘C-band’ (peak V) and the TL band from PS II depleted of calcium. In intact PS II membrane preparations, in the presence of DCMU, a TL band at 50°C is induced following illumination at 77 K. This band decays, at 30°C, with a half-time of 10 min. This decay corresponds to the disappearance of the EPR signal arising from QA− and an accelerated decay of the organic free radical Tyr D+. It is concluded that recombination of this charge pair is probably responsible for the thermoluminescence emission. In PS II preparations that have been depleted of calcium using a salt/EGTA wash followed by rebinding of the extrinsic polypeptides, a TL band is produced at around 45–50°C following 198 K illumination. In such samples a stable S2 state of the water-splitting complex is present, giving rise to a modified form of the EPR multiline signal. During incubation at 30°C in the dark this signal decays with a half-time around 20–25 min. This decay is not accelerated by the presence of QA− induced by low-temperature illumination of the sample. In contrast, low-temperature illumination does result in an acceleration in the decay of Tyr D+, indicating that Tyr D+/Q−A recombination is again the dominant origin of thermoluminescence. In PS II depleted of calcium by incubation at pH 4.0, the possibility that TL emission temperature is determined by a change in the mid-point redox potential of QA (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98) was investigated by comparing TL from equivalent samples of control and Ca2+-depleted PS II. It was shown that the emission temperature of the high temperature TL band induced by illumination at 77 K did not differ significantly between control and treated samples, suggesting that, under the conditions used, the potential of QA does not change significantly.

Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in Ca2+-depleted photosystem II

NaCl/EGTA‐washing of photosystem II (PS‐II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984‐8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained, (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS‐II membranes which are already inhibited by Ca2+‐depletion. (ii) The carboxylic acids act in the S3‐state, are much less effective in S2 and have no effect in the S1‐state. (iii) The extrinsic polypeptides (17‐ and 23‐kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal, (iv) The S3 signal can be observed in Ca2+‐depleted PS‐II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca2+ ‐depleted PS‐II [(1990) Nature 347, 303‐306] also occurs in functional PS‐II.

The electronic structures of the S(2) states of the oxygen evolving complexes of photosystem II in plants and cyanobacteria in the presence and absence of methanol

The electronic properties of the Mn(4)O(x)Ca cluster in the S(2) state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band (55)Mn-ENDOR using photosystem II preparations isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only Mn(III) ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the Mn(III) ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure [Siegbahn, P. E. M. Accounts Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys.2009, 11, 6788-6798]. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the S(T)=½ spin model of the S(2) state. This suggests that the methanol-OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (Mn(A)) and the other three Mn ions that form the trimeric part of the cluster (Mn(B), Mn(C), Mn(D)), by perturbing the linking bis-μ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation.

The heart of photosynthesis in glorious 3D

The input of solar energy into photosynthesis, and thence into the biosphere, occurs via chlorophyll-containing proteins known as reaction centres. There are two kinds of reaction centre in oxygenic photosynthesis: photosystem I (PSI) and photosystem II (PSII). The PSII reaction centre, alias the oxygen-evolving enzyme, the water-oxidizing complex or the water-plastoquinone photo-oxidoreductase, has now been crystallized and its structure solved to a resolution of 3.8 A.

Comparison of chloride-depleted and calcium-depleted PSII: the midpoint potential of QA and susceptibility to photodamage

Abstract Photosystem II has been studied in membranes in which O 2 evolution was inhibited by depletion of either chloride or calcium ions. It has been shown earlier [Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89–98] that depletion of calcium ions results in a 150-mV up-shift of the midpoint redox potential (Em) of Q A /Q A − (the protein-bound plastoquinone which acts as an electron acceptor). Here it is shown that chloride depletion has no effect on the Em of Q A /Q A − . It is also demonstrated that chloride-depleted PSII is more susceptible than Ca 2+ -depleted PSII to damage by light. This extra susceptibility to light in Cl − -depleted PSII is eliminated when the artificial electron acceptor DCPIP is present during illumination. These observations are consistent with the hypothesis that the up-shifted Em of Q A /Q A − in Ca 2+ -depleted PSII results in a protection of the reaction centre from damage by light by changing the dominant charge recombination pathway to one which does not involve formation of the P680 + Ph − radical pair, the P680 triplet and singlet oxygen [Johnson, G.N., Rutherford, A.W. and Krieger, A. (1995) Biochim. Biophys. Acta 1229, 202–207].