Protease treatments of photosystem II membrane fragments reveal that there are four 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 KM of about 0.4 microM, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Preston
C
C
Photoconversion Research Branch, Solar Energy Research Institute, Golden, Colorado 80401.
Seibert
M
M
D001665:Binding Sites / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry; D002712:Chlorides; D000460:Chlorophyta / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology*; D008345:Manganese / Q000378:metabolism*; D017895:Manganese Compounds; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000187:drug effects*; D045332:Photosystem II Protein Complex; D010944:Plants / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D002712:Chlorides; D017895:Manganese Compounds; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D008345:Manganese; D010450:Endopeptidases; D014357:Trypsin; D004047:Diethyl Pyrocarbonate; C025340:manganese chloride