Nearest neighbor relationships among constituent proteins of oxygen-evolving Photosystem II membranes: binding and function of the extrinsic 33 kDa protein

Nearest neighbor relationships among constituent proteins of oxygen-evolving Photosystem II (PS II) membrane preparations from spinach were investigated by means of crosslinking with a cleavable bifunctional crosslinker, dithiobis(succinimidylpropionate) (DSP). (1) Diagonal gel electrophoresis revealed crosslinking between two extrinsic proteins of 17 and 23 kDa, between the extrinsic 33 kDa protein and the 47 kDa chlorophyll-carrying protein and between the 26 and 27 kDa apoproteins of light-harvesting chlorophyll a/b protein. In addition, a product which involved a protein of 29 kDa was detected. (2) Amounts of the extrinsic proteins crosslinked were determined by washing DSP-treated membranes with high concentrations of urea and NaCl, or CaCl₂. Neither of the two extrinsic proteins of 17 and 23 kDa was crosslinked with intrinsic membrane proteins, whereas 15 to 20% of the 33 kDa protein was immobilized by treatment with 0.1% DSP. Oxygen evolution became resistant to the urea/NaCl-wash proportionally to the amount of the 33 kDa protein crosslinked, indicating that the 33 kDa protein covalently bound to the 47 kDa protein is still fully functional. (3) The crosslinking of the 33 kDa protein accompanied by parallel increases in the amount of Mn remained unextracted, and the rate of oxygen evolution survived after 30-min treatment of PS IImembranes at pH 9.0. Thus the 33 kDa protein has a protective effect on the Mn cluster at the alkaline pH. In contrast, Mn was mostly extracted with a high concentration of Tris, irrespective of the crosslinking of the 33 kDa protein.