Identification of Amino Acids of the 33 kDa Mn-Stabilizing Protein that are Involved in Binding to PSII Complexes

Abstract

Photosynthetic oxygen evolution takes place in the photosystem II (PSII) reaction center complex that spans the thylakoid membrane (1). The extrinsic 33 kDa Mn-stabilizing protein (MSP) that is associated with the lumenal surface of the complex is essential for optimal operation of the oxygen-evolving reaction (2–5) and for stabilization of the Mn cluster that plays a central role in oxidation of water (4). Recently, we have shown that proteolytic cleavage of the MSP from the thermophilic cyanobacterium Synechococcus elongatus at a site between Phe156 and Gly163 or between Arg184 and Ser191 results in total loss of the binding capacity of the protein to PSI’ complexes (6, 7). Mutagenetic experiments further showed that the ability of the MSP to restore oxygen evolution was abolished by introduction of a nick or a single amino acid (Met) between Phe156 and Leu157, a chymotrypsin-cleavage site (7). We suggested, therefore, that the two eight-amino acid sequences are regions for interaction with the functional binding site of the PSII complex. In this communication, site-directed mutagenesis was performed to identify amino acids that are involved in the molecular link between the MSP and PSII complex. There are several lines of evidence suggesting involvement of electrostatic interactions in the binding of MSP to the PSII complex (2, 3, 8–10). We constructed, therefore, mutant MSPs with a basic amino acid replaced by Gln and examined the oxygen-evolving activity of PSII complexes that were reconstituted with each mutant MSP. Substituted were three basic amino acids, Lys160, Arg162 and Lys188, that are located in the two protease-sensitive sequences and five other basic amino acids that are well conserved from cyanobacteria to higher plants.