The first determinations of substrate water binding to the Oz evolving complex in photosystem II as a complete function of the S states have been made. (H2O)-O-18 Was rapidly injected into spinach thylakoid samples preset in either the S-0, S-1, S-2, or S-3 states, and the rate of O-18 incorporation into the O-2 produced was determined by time-resolved mass spectrometry. For measurements at mle = 34 (i.e., for the (OO)-O-16-O-18 product), the rate of O-18 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 degrees C of 8 +/- 2, 0.021 +/- 0.002, 2.2 +/- 0.3, and 1.9 +/- 0.2 s(-1) for the S-0, S-1, S-2, and S-3 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 S-0, S-1, and S-2 states. Comparisons of the O-18 exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S-0 to S-1 transition, then decreases during the S-1 to S-2 transition, but stays the same during the S-2 to S-3 transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S-2 to S-3 transition. These findings are discussed in terms of two independent Mn-III substrate binding sites within the O-2 evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the Mn-III sites may only first bind a substrate water molecule during the S-2 to S-3 transition.
