The Affinities for the Two Substrate Water Binding Sites in the O2 Evolving Complex of Photosystem II Vary Independently during S-State Turnover

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Abstract
The first determinations of substrate water binding to the O2 evolving complex in photosystem II as a complete function of the S states have been made. H218O was rapidly injected into spinach thylakoid samples preset in either the S0, S1, S2, or S3 states, and the rate of 18O incorporation into the O2 produced was determined by time-resolved mass spectrometry. For measurements at m/e = 34 (i.e., for the 16O18O product), the rate of 18O incorporation in all S states shows biphasic kinetics, reflecting the binding of the two substrate water molecules to the catalytic site. The slow phase kinetics yield rate constants at 10 °C of 8 ± 2, 0.021 ± 0.002, 2.2 ± 0.3, and 1.9 ± 0.2 s-1 for the S0, S1, S2, and S3 states, respectively, while the fast phase kinetics yield a rate constant of 36.8 ± 1.9 s-1 for the S3 state but remain unresolvable (>100 s-1) for the S0, S1, and S2 states. Comparisons of the 18O exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S0 to S1 transition, then decreases during the S1 to S2 transition, but stays the same during the S2 to S3 transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S2 to S3 transition. These findings are discussed in terms of two independent MnIII substrate binding sites within the O2 evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the MnIII sites may only first bind a substrate water molecule during the S2 to S3 transition.