Role of Atypical D1 Proteins in Photosystem II

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In photosynthesis, light is used to remove the hydrogen from water to give oxygen. The hydrogen equivalents are then used to chemically reduce carbon dioxide to organic molecules. The water oxidation reaction is catalysed by an enzyme known as photosystem II (PSII). It is hard to catalyse this reaction with light, so far the only known effective system is the natural one. The reaction takes place at a metallocluster containing 4 manganese ions and a calcium ion. This cluster is bound and stabilised by a highly conserved protein known as the "D1 protein" in PSII. The PSII complex contains more than 20 other protein subunits. However, in an active leaf, the D1 protein is degraded extremely quickly, every 30 minutes or so. This is thought to be because of the generation of reactive oxygen side products from the water-splitting reaction which damage the protein. A sophisticated system of repair exists to regenerate PSII with fresh D1 protein. Cyanobacteria have several different D1 genes suited to different situations. Some are synthesized in response to high light, others in low oxygen environments. Some photosynthetic cyanobacteria can also fix nitrogen from the air. This can be a problem as the nitrogenase enzyme is irreversibly inhibited by oxygen. These organisms either physically separate the nitrogenase from photosynthesis, or only fix nitrogen at night when PSII is inactive.There is a recently discovered a class of D1 genes that are mutated relative to the "canonical D1" at the sites binding the manganese cluster. The mutations are such that these "rogue" D1 are not thought to be capable of oxygen evolution, however, there are sufficient functional groups for metal binding to be a possibility. We believe that the atypical D1 sequences might be a mechanism to inactivate PSII with a non-catalytic D1, which is then replaced when activity is required again. There is a further class of even more atypical D1 ("super-rogue D1") that is associated with cyanobacteria that adapt to far-red light by making a red-shifted chlorophyll, chlorophyll f. This chlorophyll has a peak absorption in the infra-red, yet seems to still be capable of using these lower energy photons to drive water oxidation. The super-rogue D1 is 1000-fold up-regulated in far-red light conditions, so probably has a role in adaptation to far-red light.The atypical D1 sequences are phylogenetically early, so are reminiscent of an ancestral D1, so could provide information on the evolution of oxygenic photosynthesis. If functional in substrate oxidation, the variant PSII may use a substrate other than water. Of interest in itself and again providing insight into the evolution of photosynthesis. Such a reaction centre would be a novel finding.We will investigate the function of the atypical D1 proteins in PSII both in vivo and in vitro. For in vivo studies we will culture cyanobacteria with atypical D1 under a variety of conditions, including circadian light-dark rhythms. We will investigate the expression pattern of the atypical D1 in comparison to normal D1, in relation to other factors, such as light-dark, nitrogen fixation, and external carbon sources. For in vitro studies we will purify PSII with the rogue and super-rogue D1. We will investigate their function, such as metal content, ability to oxidise substrates and transfer electrons. We will assess the presence of all of the PSII subunits in the modified reaction centres. With a combination of the in vivo and in vitro approaches we will learn what the biological function of the atypical D1 sequences is, and how, at a biochemical and biophysical level, it is accomplished.