EPR kinetic studies of oxygen release in thylakoids and PSII membranes: a kinetic intermediate in the S3 to S0 transition

Time-resolved EPR oximetry has been used to determine the oxygen release kinetics in spinach thylakoids and PSII membranes. We observe release kinetics with half-times of approximately 0.85 and approximately 1.45 ms for thylakoids and PSII membranes, respectively, which are in close agreement with the EPR determined Yz decay kinetics for the S3 --> --> S0 transition in these systems. The results show conclusively that water-oxygen chemistry is not a rate-limiting step in the donor side of PSII under normal turnover conditions. By analyzing the oxygen release kinetics in thylakoids under nonphysiological, but still functionally competent conditions (low pH or high salt), we observed an initial delay in the O2 release of up to 200 microseconds following flash turnover from the S3 state. This is the first direct indication of a probable quasi-stable intermediate in the S3 --> --> S0 turnover of PSII, possibly representing the putative S4 state. Under conditions more closely approaching physiological, no such delay was resolved, indicating that the S4 --> O2 transition occurs within 50 microseconds under such circumstances. Two possible reaction sequences for O2 formation consistent with these and other data are discussed. It is suggested that the more probable form of "S4" is in fact the S3 + Yz* combination, which must undergo some molecular rearrangement on the tens to hundreds of microseconds time scale before O2 formation chemistry occurs.