pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome b(559) (Cyt b(559)). At pH 6.5 it was found that Cyt b(559) was reversibly photoreduced by red actinic light when Mn2+ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b(559) was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942-10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b(559) is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b(559) increases, with increasing pH, the rate of irreversible photo-oxidation of beta-carotene decreases, a result expected if the HP form protects against donor side photoinhibition.