The mode of binding of three extrinsic proteins of 33 kDa, 23 kDa and 18 kDa in the Photosystem II complex of spinach

Binding of three extrinsic proteins of 33 kDa, 23 kDa and 18 kDa to the oxygen-evolving Photosystem II (PS II) complex was investigated under equilibrium conditions using spinach PS II membranes. Dissociation constants for the functional and stoichiometric binding of the extrinsic proteins were determined to be 12, 2.7 and 3.8 nM for the 33 kDa, 23 kDa and 18 kDa proteins, respectively. These values indicate the binding energy between the proteins and the PS II complex on the order of 10-11 kcal.mol-1. Effect of removal of the extrinsic proteins and Mn atoms from the PS II complex on the dissociation constant and the number of binding sites for the proteins was also examined. The results indicate that the binding site for the 33 kDa protein is present in the PS II complex devoid of the functional Mn and that the Mn stabilizes the binding of the 33 kDa protein. The change in the binding energy of the 33 kDa protein afforded by the Mn was estimated to be about 1 kcal.mol-1, indicating that the interaction between the 33 kDa protein and the functional Mn is, if any, very weak. It is also indicated that the 33 kDa protein bound to the PS II complex provides the high-affinity binding site for the 23 kDa protein in the complex and that the 23 kDa protein together with the 33 kDa protein does so for the 18 kDa protein. The measurement of the sedimentation coefficients of the extrinsic proteins and protein mixtures in an aqueous solution showed that the extrinsic proteins did not interact with each other when they were apart from the PS II complex. These observations suggest that the binding of the extrinsic proteins to the PS II complex alters the conformation of the extrinsic proteins themselves and/or that of the intrinsic part of the complex so as to create the binding sites for the other extrinsic proteins.