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 (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 -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680