Impact of mutations within the putative Ca2+-binding lumenal interhelical a-b loop of the photosystem II D1 protein on the kinetics of photoactivation and H2O-oxidation in Synechocystis sp PCC6803

Mutations D1-D59N and D1-D61E in the putative Ca2+-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein [Chu, H. A., Nguyen, A. P., and Debus (1995), Biochemistry 34, 5839-5858] were further characterized in terms of S-state cycling and photoactivation. Bare platinum electrode measurements of centrifugally deposited O-2-evolving membranes isolated from the a-b loop mutants demonstrated a retarded appearance of O-2 following single turnover flashes, although not to the extent of retardation seen in the Delta psbO mutant, which lacks the extrinsic manganese-stabilizing protein (MSP). Double flash measurements indicate that retarded O-2 release in mutants coincides with a decrease in overall PSII turnover during the S-3-[S-4]-S-0 transition. S-2 and S-3 decay measurements in the isolated membranes indicate that D1-D59N and D1-D61E have faster decays of these higher S-states in contrast to slowed decays in the Delta psbO mutant. Measurements of the flash interval dependence of photoactivation indicate that intermediates of photoactivation [light-dependent assembly of the (Mn)(4) complex] are highly destabilized in the a-b loop mutants compared to both Delta psbO and the wild-type: flash intervals of greater than 2 s result in the nearly complete decay of unstable photointermediate(s) in the D1-D59N and D1-D61E samples, whereas a similar loss does not occur until intervals even greater than 10 s in the Delta psbO and wild-type samples. These results are consistent with a role for the residues D1-D59 and D1-D61 in modulating the redox properties of the higher S-states and, also, possibly in the binding the calcium ion involved in photoactivation.