PROTEASE TREATMENTS OF PHOTOSYSTEM-II MEMBRANE-FRAGMENTS REVEAL THAT THERE ARE 4 SEPARATE HIGH-AFFINITY MN-BINDING SITES

The "high-affinity Mn-binding site" in Mn-depleted photosystem II (PS II) membrane fragments isolated from Scenedesmus obliquus was examined by using the diphenylcarbazide (DPC)/Mn2+ non-competitive inhibition assay [Preston, C., & Seibert, M. (1991) Biochemistry (preceding paper in this issue)]. Different proteases were used to degrade lumenal surface protein segments from these PS II membranes, and a total of four independent high-affinity Mn-binding sites (ligands) were identified. Carboxypeptidase A, subtilisin, and Staphylococcus aureus V8 protease each degrade one of two high-affinity Mn-binding sites sensitive to the histidine chemical modifier diethyl pyrocarbonate (DEPC). However, sequential treatment experiments indicate that subtilisin degrades a DEPC-sensitive Mn-binding site that is different from the one degraded by the other two proteases. Trypsin also was found to degrade one of the DEPC-sensitive Mn-binding sites (that degraded by carboxypeptidase A and V8 protease). In addition, trypsin degrades one of two 1-ethyl-3-[(3-dimethylamino)propyl]carbodiimide (EDC) sensitive Mn-binding sites, but only in the absence of the 30-kDa extrinsic protein. Thus, the 30-kDa extrinsic protein associated with O2 evolution appears to protect the EDC-sensitive binding site from trypsin degradation. No protease has yet been identified that will degrade the trypsin-insensitive EDC-sensitive Mn-binding site. Under the conditions of the assay (high DPC concentration), more than three Mn per reaction center were found bound to the membrane with a K(M) of about 0.4-mu-M, as determined by direct metal analysis. This is consistent with the idea that each of the four high-affinity sites binds (or provides a ligand for) one of four Mn. These four high-affinity Mn-binding sites may correspond to the four sites formed, albeit sequentially, in the normal photoactivation process. We provide evidence that a histidine residue (His 337) on D1 is a prime candidate for one of the high-affinity Mn-binding sites and suggest that it also provides a nitrogen ligand for binding functional Mn. Finally, an alternative explanation of our data might be that the four high-affinity sites represent four ligands binding a single Mn, and ligands to other membrane bound Mn are not detectable by the DPC/Mn2+ assay.