Regeneration of the high-affinity manganese-binding site in the reaction center of an oxygen-evolution deficient mutant of Scenedesmus by protease action.

The O2-evolution deficient mutant (LF-1) of Scenedesmus obliquus inserts an unprocessed D1 protein into the thylakoid membrane and binds less than half the wild type (WT) level of Mn. LF-1 photosystem II (PS II) membrane fragments lack that part of the high-affinity Mn(2+)-binding site found in WT membranes which may be associated with histidine residues on the D1 protein (Seibert et al. 1989 Biochim Biophys Acta 974: 185-191). Hsu et al. (1987 Biochim Biophys Acta 890: 89-96) purport that the high-affinity site (characterized by competitive inhibition of DPC-supported DCIP photoreduction by μM concentrations of Mn(2+)) in Mn-extracted PS II membranes is also the binding site for Mn functional in O2 evolution. Proteases (papain, subtilisin, and carboxypeptidase A) can be used to regenerate the high-affinity Mn(2+)-binding site in LF-1 PS II membranes but not in thylakoids. Experiments with the histidine modifier, DEPC, suggest that the regenerated high-affinity Mn(2+)-binding sites produced by either subtilisin or carboxypeptidase A treatments were the same sites observed in WT membranes. However, none of the protease treatments produced LF-1 PS II membranes that could be photoactivated. Reassessment of the processing studies of Taylor et al. (1988 FEBS Lett 237: 229-233) lead us to believe that their procedure also does not result in substantial photoactivation of LF-1 PS II membranes. We conclude that (1) the unprocessed carboxyl end of the D1 protein in LF-1 is located on the lumenal side of the PS II membrane, (2) the unprocessed fragment physically obstructs or perturbs that part of the high-affinity Mn(2+)-binding site undetectable in LF-1, and (3) the D1 protein must be processed at the time of insertion into the membrane for normal O2-evolution function to result.