Mutations of arginine 64 within the putative Ca(2+)-binding lumenal interhelical a-b loop of the photosystem II D1 protein disrupt binding of the manganese stabilizing protein and cytochrome c(550) in Synechocystis sp. PCC6803.

Mutations D1-R64E, D1-R64Q, and D1-R64V in the putative calcium-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein were characterized in terms of impact on growth, extrinsic protein binding, photoactivation, and properties of the H(2)O-oxidation complex. The D1-R64E charge reversal mutation greatly weakened the binding of the extrinsic manganese-stabilizing protein (MSP) and, to a considerably lesser extent, weakened the binding of cytochrome c(550) (c550). Both D1-R64Q and D1-R64E exhibited an increased requirement for Ca(2+) in the cell growth medium. Bare platinum electrode measurements of O(2)-evolving membranes showed a retarded appearance of O(2) following single turn-over flashes, especially in the case of the D1-R64E mutant. The D1-R64E mutant also had a pronounced tendency to lose O(2) evolution activity in the dark and exhibited an increased relative quantum yield of photoactivation, which are characteristics shared by mutants that lack extrinsic proteins. S(2) and S(3) decay measurements in the isolated membranes indicate that D1-R64E and D1-R64Q have faster decays of these higher S-states as compared to the wild-type. However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination with PSII donors, showed somewhat slower decays. Taken together, the fluorescence and S-state decay indicate that the midpoint of either Q(B)(-) has been modified to be more negative in the mutants or that a recombination path presumably involving either Q(B)(-) or Y(D) has become kinetically more accessible.