Mutations D1-D59N and D1-D61E in the putative Ca
2+-binding lumenal interhelical a-bloop of the photosystem II (PSII) D1 protein [Chu, H. A., Nguyen, A. P., and Debus (1995),
Biochemistry34, 5839-5858] were further characterized in terms of S-state cycling and photoactivation. Bare platinumelectrode measurements of centrifugally deposited O
2-evolving membranes isolated from the a-b loopmutants demonstrated a retarded appearance of O
2 following single turnover flashes, although not to theextent of retardation seen in the
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psb0 mutant, which lacks the extrinsic manganese-stabilizing protein(MSP). Double flash measurements indicate that retarded O
2 release in mutants coincides with a decreasein overall PSII turnover during the S
3-[S
4]-S
0 transition. S
2 and S
3 decay measurements in the isolatedmembranes indicate that D1-D59N and D1-D61E have faster decays of these higher S-states in contrastto slowed decays in the
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psb0 mutant. Measurements of the flash interval dependence of photoactivationindicate that intermediates of photoactivation [light-dependent assembly of the (Mn)
4 complex] are highlydestabilized in the a-b loop mutants compared to both
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psbO and the wild-type: flash intervals of greaterthan 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
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psbOand wild-type samples. These results are consistent with a role for the residues D1-D59 and D1-D61in modulating the redox properties of the higher S-states and, also, possibly in the binding the calciumion involved in photoactivation.