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 ~ 0.85 and ~1.45 ms for thylakoids and PSII membranes, respectively, which are in close agreement with the EPR determined Y(z) 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 μs 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 μs 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 + Y(z)· combination, which must undergo some molecular rearrangement on the tens to hundreds of microseconds time scale before O₂ formation chemistry occurs.