Functional analysis of site-directed mutants on the C-terminal Leu-343 and Ala-344 of D1 protein

Abstract

Previous studies suggested that mutagenesis around the C-terminal processing site on D1 protein could have major effects on the formation and/or conformation of the oxygen-evolving complex. Some of the mutants had a very slow processing rate (Hatano-Iwasaki et al. PMB (2000) 42, 353-363) and the other mutant carried two functionally distinct Mn complexes (Hatano-Iwasaki et al. BBA (2001) 1504, 299-310). In this study, we further mutagenized Leu343 and Ala344 of D1 protein in C. reinhardtii and performed biophysical characterizations. Thermoluminescence measurements revealed that 1) the L343P(LP) and A344P(AP) mutants have modified PSII centers which have higher redox potential of the Mn complex, and 2) only the LP mutant evolves oxygen at a lower rate. Measurements of fluorescence kinetics demonstrated that 1) the LP mutant has a lower charge recombination rate which may account for the lower oxygen evolving activity, and 2) AP mutant has a very slow re-reduction rate of the oxidized P680, inhibiting the normal electron transfer reaction around the donor-side. These mutants are further being analyzed by EPR to gain detailed information about the modification(s) on the Mn complex.