Mutagenesis of Basic Residues R15 1 and R161 in Manganese-Stabilizing Protein of Photosystem II Causes Inefficient Binding of Chloride to the Oxygen-Evolving Complex.

Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Argl51 and Argl61 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced. Here, we present evidence that replacement of Arg151 or Arg161 yields proteins that have lower PSII binding affinity, and are functionally deficient even though about 2 tool of mutant MSP/mol PSII can be rebound to MSP depleted PSII membranes. R161G reconstitutes O2 evolution activity to 40% of the control, while R151G and R151D reconstitute only 20% of the control activity. Spectroscopic and biochemical techniques fail to detect significant changes in solution structure. More extensive 02 evolution assays revealed that the Mn cluster is stable in samples reconstituted with each mutated MSP, and that all three Arg mutants have the same ability to retain Ca2+ as the wild-type protein. Activity assays exploring the effect of these mutations on retention of CI-, however, showed that the R151 G, R I 51 D, and R 161G MSPs are defective in Cl- binding to the OEC. The mutants have Cl- KM values that are about four (R161G) or six times (R151G and R151D) higher than the value for the wild-type protein. The results reported here suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and, consequently, in retention of Cl- by the O2-evolving complex. [ABSTRACT FROM AUTHOR]