The two substrate-water molecules are already bound to the oxygen-evolving complex in the S2 state of photosystem II

The first direct evidence which shows that both substrate-water molecules are bound to the O-2-evolving catalytic site in the S-2 state of photosystem II (PSII) is presented. Rapid O-18 isotope exchange measurements between (H2O)-O-18 incubated in the S-2 state of PSII-enriched membrane samples and the photogenerated O-2 reveal a fast and a slow phase of exchange at mle 34 (which measures the level of the (OO)-O-16-O-18 product). The rate constant for the slow phase of exchange ((34)k(1)) equals 1.9 +/- 0.3 s(-1) at 10degreesC, while the fast phase of exchange is unresolved by our current experimental setup ((34)k(2) greater than or equal to 175 s-1). The unresolvable fast phase has left open the possibility that the second substrate-water molecule binds to the catalytic site only after the formation of the S-3 state [Hillier, W., and Wydrzynski, T. (2000) Biochemistry, 39, 4399-4405]. However, for PSII samples depleted of the 17 and 23 kDa extrinsic proteins (Ex-depleted PSII), two completely resolvable phases of O-18 exchange are observed in the S-2 state of the residual activity, with the following rate constants: (34)k(1) = 2.6 +/-0.3.s(-1) and (34)k(2) = 120 +/- 14 s(-1) at 10degreesC. Upon addition of 15 mM CaCl2 to Ex-depleted PSII, the O-2 evolution activity increases to similar to80% of the control level, while the two resolvable phases of exchange remain the same. In measurements of Ex-depleted PSII at mle 36 (which measures the level of the (OO)-O-18-O-18 product), only a single phase of exchange is observed in the S-2 state, with a rate constant ((36)k(1) = 2.5 +/- 0.2 s(-1)) that is identical to the slow rate of exchange in the We 34 data. Taken together, these results show that the fast phase of O-18 exchange is specifically slowed by the removal of the 17 and 23 kDa extrinsic proteins and that the two substrate-water molecules must be bound to independent sites already in the S-2 state. In contrast, the O-18 exchange behavior in the S-1 state of Ex-depleted PSII is no different from what is observed for the control, with or without the addition of CaCl2. Since the fast phase of exchange in the S-1 state is unresolved (i.e., (34)k(2) > 100 s(-1)), the possibility remains that the second substrate-water molecule binds to the catalytic site only after the formation of the S-2 state. The role of the 17 and 23 kDa extrinsic proteins in establishing an asymmetric dielectric environment around the substrate binding sites is discussed.