Jonathan H. A. Nugent

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

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.

Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochrome b6f complex.

The random integration of transforming DNA into the nuclear genome of Chlamydomonas has been employed as an insertional mutagen to generate a collection of photosynthetic mutants that display abnormal steady-state fluorescence levels and an acetate-requiring phenotype. Electron paramagnetic resonance spectroscopy was then used to identify those mutants that specifically lack a functional cytochrome b6f complex. Our analysis of RNA and protein synthesis in five of these mutants reveals four separate phenotypes. One mutant fails to accumulate transcript for cytochrome f, whilst a second displays a severely reduced accumulation of the cytochrome b6 transcript. Two other mutants appear to be affected in the insertion of the haem co-factor into cytochrome b6. The fifth mutant displays no detectable defect in the synthesis of any of the known subunits of the complex. Genetic analysis of the mutants demonstrates that in three cases, the mutant phenotype co-segregates with the introduced DNA. For the mutant affected in the accumulation of the cytochrome f transcript, we have used the introduced DNA as a tag to isolate the wild-type version of the affected gene.

The 9-kDa phosphoprotein of photosystem II. Generation and characterisation of Chlamydomonas mutants lacking PSII-H and a site-directed mutant lacking the phosphorylation site.

The chloroplast gene psbH encodes a 9-10 kDa thylakoid membrane protein (PSII-H) that is associated with photosystem II and is subject to light-dependent phosphorylation at a threonine residue located on the stromal side of the membrane. The function of PSII-H is not known, neither is it clear what regulatory role phosphorylation may play in the control of PSII activity. Using particle gun-mediated transformation, we have created chloroplast transformants of Chlamydomonas reinhardtii in which the synthesis of PSII-H is prevented by the disruption of psbH, or in which the phosphorylatable threonine is replaced by alanine through site-directed mutagenesis of the gene. The mutants lacking PSII-H have a photosystem II-deficient phenotype, with no detectable functioning PSII complex present in whole cells or isolated thylakoid membranes. In contrast, the alanine mutant (T3A) grows photoautotrophically, and PSII activity is comparable to wild-type cells as determined by various biochemical and biophysical assays.

EPR signals of cytochromes in subchloroplast particles

~~0~~ cytochromes are important components in photosynthetic electron transport, their study in plants by electron paramagneti~ resonance (EPR) has been confined to only a few reports. A brief report [I] assigned signals in the g = 6.0 region to high-spin forms of cytochrome b6 and low potential cytochrome b-559. A study of low-spin cytochrome signals in the g = 3.0 region [Z] revealed signals attributed to cytochrome f and low potential cytochrome b-559. Photooxidation of the cytochrome b-559 was observed at cryogenic temperatures in samples reduced by ascorbate prior to freezing. Information on cytochrome b-S59 using EPR techniques would be valuable in understanding the role of this cytochrome and its various potential forms in photosystem II and the electron-transpo~ chain of ~~oroplasts. Here we investigated both the highand low-spin cytochrome regions of the EPR spectrum at cryogenic temperatures. This study confirms some of the previous work but also reports new high spin signals, some of which respond to illumination at cryogenic temperatures.

Heat stress attenuates free radical release in the isolated perfused rat heart.

Prior heat stress leads to an enhancement of postischemic mechanical function and improvement in biochemical indices of injury in the rat heart, associated with an elevation in endogenous catalase activity. We have examined the effect of heat stress on free radical release during reperfusion in the isolated rat heart using electron spin resonance (ESR). Twenty four hours after heat stress or sham treatment, hearts were perfused in the Langendorff mode and subjected to 10 min of no-flow global ischemia followed by 10 min of reperfusion. Coronary effluent was collected at specific time points in PBN for ESR measurement. A PBN adduct was identified with characteristics consistent with an alkoxyl radical: PBN-LO. In sham hearts there was a rapid rise in adduct release to a maximum (228 +/- 15% of stabilization values, p < .05) occurring 1 min into reperfusion. In heat stress hearts there was no significant rise in adduct release during the reperfusion period. Pretreatment of hearts with 3-amino triazole, an inhibitor of catalase, failed to clarify whether the protection seen in heat stress hearts was a result of the elevation in catalase activity. These results suggest that heat stress protects the myocardium against the oxidative stress of ischemia-reperfusion.

Electron transfer in the isolated photosystem II reaction centre complex

We have characterised the electron‐transfer properties of the D1/D2/cytochrome b‐559 complex using EPR spectrometry. The complex can transfer electrons to silicomolybdate and ferricyanide at cryogenic temperatures. In the presence of silicomolybdate or ferricyanide, two chlorophyll cation radicals were observed from P680+ (0.8 mT) and monomeric Chl (1.0 mT). Reduction of silicomolybdate was detected as a 2.7 mT signal at g = 1.942. A radical attributed to a tyrosine cation radical (D+/Z+) was also observed in a small percentage of centres.

Comparison of the EPR properties of Photosytem I iron-sulphur centres A and B in spinach and barley

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.

Electron donation in photosystem II

Abstract The electron transfer resulting from illumination and dark storage of PS II has been studied using EPR signals from several electron carriers. The recombination of D + (Signal II) and Q − A formed by illumination occurred during dark storage at 77 K and was used to deplete reaction centres of D + . The donor D was then shown to be oxidized in the dark by the S 2 state of the oxygen-evolving complex. A slow change which occurred during dark storage of PS II samples was detected using the power saturation characteristics of D. We interpret this effect on D to be an indirect result of a rearrangement of the manganese complex during long-term dark adaptation. A role for D in the stability, protection and perhaps initial manganese binding of the oxygen-evolving complex is suggested.

Bicarbonate binding and the properties of photosystem II electron acceptors

We have investigated the quinone‐binding region of PS II using exchange reactions to replace key components. Bicarbonate was replaced by formate and QB by exogenous quinones or herbicide. The effects of these changes were monitored using the ESR signals from the QA iron‐semiquinone and Fe3+ non‐haem iron components. We observed that QB binding caused characteristic changes in the Q− A ESR signal and confirmed that the characteristics of Q− A depend on bicarbonate binding. The non‐haem iron was oxidised only under conditions where bicarbonate was bound. The characteristics of bicarbonate binding were observed in Phormidium laminosum PS II, showing that the bicarbonate effect occurs in cyanobacteria. The results support a hypothesis which gives a central role to bicarbonate in providing the conditions for electron transfer in both cyanobacteria and higher plant PS II. It is suggested that bicarbonate binds at or close to the non‐haem iron and influences the characteristics of QA, QB and the non‐haem iron.