Mutations of arginine 64 within the putative Ca2+-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 lum enal interhelical a-b loop of the photosystem II (PSII) D1 protein were cha racterized in terms of impact on growth, extrinsic protein binding, photoac tivation, and properties of the H2O-oxidation complex. The D1-R64E charge r eversal mutation greatly weakened the binding of the extrinsic manganese-st abilizing 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 Ca2+ in the cell growth medium. Bare platinum ele ctrode 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 me mbranes indicate that D1-R64E and D1-R64Q have faster decays of these highe r S-states as compared to the wild-type. However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination wi th PSII donors, showed somewhat slower decays. Taken together, the fluoresc ence and S-state decay indicate that the midpoint of either Q(B)(-) has bee n 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 acce ssible.