Our interest in the function of photosystem II dates back to early studies of the mechanism of delayed fluorescence, and our subsequent investigation of the interaction of protons with the oxygen evolving apparatus. We suggested a mechanism for the enhancement of delayed fluorescence which occurs when the lumenal pH is lowered by proton uptake, which involved the interaction of protons with the reactions of oxygen-evolution. We went on to show by direct measurement of H+-release using indicators that when chloroplasts are illuminated from the dark adapted state, protons are released to the inner thylakoid space in synchrony with the transitions of the oxygen evolving complex, giving a quartenary pattern (starting in the S0-state) of approximately 1, 0, 1, 2 . We interpreted this pattern as indicating that the elements of water were incorporated into the enzymic cycle at transitions earlier than those leading to release of oxygen.
On the acceptor side of photosystem II, we have shown that the two steps by which electrons reduce the secondary quinone occur with different kinetics. We showed that the electron transfer rates are differentially sensitive to a variety of inhibitors and herbicides, and are modified in plants which develop a resistance to herbicides. We have characterized the kinetics and thermodynamics of the PS II two-electron gate, and the role of protons in stabilizing semiquinones.
More recently, these interests have diversified as detailed below.
Click here for a brief review of photosystem II
We have used the coordinates of the reaction center from Rps. viridis as a template on which to build structural models of the D1 and D2 proteins,and the chromophores of photosystem II. The models have been useful in deciding sites for molecular engineering, and for trying to understand the relation between structure and function. More recently, we have been using a more complete photosystem II model by Dr. Jonathan Nugent of the University of London, which incorporates interhelical loops, bicarbonate, and a Mn-cluster.
We have made an extensive study of the mechanism of the two-electon gate by which photosystem II reduces the plastoquinone pool. Using fluorescence techniques, we were able to explore the kinetics and thermodynamics of electron transfer in the two reactions at the QB-site, and to provide a detailed kinetic model for the reduction of QB to QB.-(H+). We have also explored the mechanism of inhibition by herbicides which bind at this site, and the structural basis of herbicide resistance in mutant strains.
Mechanism of the two-electron gate, and herbicide resistance
We have characterized a number of new inhibitors, and studied the mechanism of action of these and some other established inhibitors. In chloroplasts, UHDBT inhibits the oxidation of the primary quinone acceptor of photosystem II. We have shown that DBMIB, benzoquinone, and 3-undecyl-2-hydroxy-l,4-naphthoquinone have a similar inhibitory effect, and that the inhibition can be accounted for by competition of the quinone analogue with the endogenous quinone or quinol for the binding site at which the quinone is reduced to a stable bound semiquinone during the operation of the two-electron gate. We are studying a number of other quinone analogues and other inhibitory sites to see if a similar mechanism can be shown to operate elsewhere.
We have built a regenerative stopped flow apparatus linked to a fluorescence photometer to assay the kinetics of binding of inhibitors at the two-electron gate. Binding can be assayed under conditions in which the quinone at the QB-site is oxidized, or when the site is occupied by a semiquinone. The results are consistent with a detailed model for the site previously developed, and allow us to describe many of the properties of inhibitor binding at the two-electron gate in terms of a relatively simple model.
We made the first computer models of the D1 and D2 proteins which form the core of photosystem II, by using the X-ray crystallographic structure of Rps. viridis as a template. We have used these to predict sites for modification of the catalytic sites by site-directed mutagenesis. We have developed a new protocol for molecular engineering in the D1 protein of Chlamydamonas reinhardtii by construction of a plasmid containing an intron-free psbA gene in tandem with a aadA spectinomycin resistance cassette. We have used this to construct specific mutations in residues thought to contribute to the QB-site, and on the oxygen-evolving apparatus on the donor side.
Studies of the two-electron gate in mutant strains.
In collaboration with Dr. Sue Golden (Texas A & M), we have studied the kinetic consequences of specific mutations in the secondary quinone binding site of photosystem II of Anacystis, and are now extending this work to C. reinhardtii. Some mutations occur naturally, giving rise to herbicide resistant strains, and we are constructing others using PCR-based techniques for site directed mutagenesis. We have developed a set of PCR-based tools for amplification and sequencing of the psbA gene which codes for the D1 protein of Photosystem II. We have sequenced wild-type and herbicide resistant strains of Amaranthus hybridus, and shown the codon change leading to modification of the QB-site. We are using our new protocols to explore mutations in C. reinhardtii. At present we are investigating the modified function in D1 mutants H252Q and S264G..
Studies of the donor-side
The D1 protein is thought to contain the primary ligands for the Mn-center which catalyses the oxidation of water. We are investigating the structure-function relationships in these spans, by using computer modelling of structure, and biophysical analysis of the kinetic and thermodynamic parameters to assay the consequences of mutation, in order to identify contributions of specific residues to the mechanism of catalysis. We have used model structures to decide on sites for molecular engineering. We have generated a large number of mutations to test potential ligands, and the role of tyrosine 161 as secondary donor.
We have pursued our interest in the involvement of protons by using other techniques for studying these reactions, including a detailed study of the relation between the proton gradient and the partial reactions of the donor-side, through studies of delayed fluorescence. We have investigated inhibitory treatments to characterize the role of the different subunits of the oxygen evolving complex, the role of cyt b-559, the mechanism of the ADRY effect, and the role of Cl-.
We have shown that inhibition of photosynthesis by exposure to UV-irradiation leads to a loss of components on the donor side of photosystem II, including Mn in oxygen evolving preparations, and the EPR signals due to the Tyr+ radicals of Z and D. Kinetics of electron transfer on the acceptor side of PS II were unaffected in centers which retained a normal fluorescence yield. However, a loss of fluorescence accompanied loss of activity and damage to the donor side. We are further investigating the mechanism of UV photoinhibition, and comparing it with photoinhibition by visible light of high intensity, or in systems with restricted donation to photosystem II. We have explored the role of the donor side reactions in the down-regulation of photosynthesis at high light intensity, associated with loss of fluorescence yield, and demonstrated that qE-quenching does not require an active photosystem II, or the presence of the donor-side Mn-complex. We found that although the donor-side reactions were not needed for qE-quenching, an impaired donor side led to photoinhibition at relatively low light intensity, and suggested that this might indicate the importance of the oxidized donor-side in generation of damage to photosystem II.