Azide as a Probe of Proton Transfer Reactions in Photosynthetic Oxygen Evolution

In oxygenic photosynthesis, photosystem II (PSII) is the multisubunit membrane protein responsible for the oxidation of water to O2 and the reduction of plastoquinone to plastoquinol. One electron charge separation in the PSII reaction center is coupled to sequential oxidation reactions at the oxygen-evolving complex (OEC), which is composed of four manganese ions and one calcium ion. The sequentially oxidized forms of the OEC are referred to as the Sn states. S1 is the dark-adapted state of the OEC. Flash-induced oxygen production oscillates with period four and occurs during the S3 to S0 transition. Chloride plays an important, but poorly understood role in photosynthetic water oxidation. Chloride removal is known to block manganese oxidation during the S2 to S3 transition. In this work, we have used azide as a probe of proton transfer reactions in PSII. PSII was sulfate-treated to deplete chloride and then treated with azide. Steady state oxygen evolution measurements demonstrate that azide inhibits oxygen evolution in a chloride-dependent manner and that azide is a mixed or noncompetitive inhibitor. This result is consistent with two azide binding sites, one at which azide competes with chloride and one at which azide and chloride do not compete. At pH 7.5, the Ki for the competing site was estimated as 1 mM, and the Ki′ for the uncompetitive site was estimated as 8 mM. Vibrational spectroscopy was then used to monitor perturbations in the frequency and amplitude of the azide antisymmetric stretching band. These changes were induced by laser-induced charge separation in the PSII reaction center. The results suggest that azide is involved in proton transfer reactions, which occur before manganese oxidation, on the donor side of chloride-depleted PSII.