Electron paramagnetic resonance kinetic studies of the S states in spinach thylakoids

The Tyr(z)⁺ decay kinetics have been analyzed by using time-resolved EPR to determine the half-time of each S(i) → S((i+1)) transition in the O₂-evolving complex of spinach thylakoids under physiological conditions. Using dark-adapted thylakoids and appropriate single-turnover flash sequences, we were able to detect the signal II(vf) kinetics of the Tyr(z)⁺ S₀ → Tyr(z) S₁, Tyr(z)⁺ S₁ → Tyr(z) S₂, Tyr(z)⁺ S₂ → Tyr(z) S₃, and Tyr(z)⁺ S₃ → (S₄) → Tyr(z) S₀ transitions. To correct for damping of the S state synchronization during the flash sequence, the Kok parameters were estimated by measuring the oxygen flash pattern in situ using nitroxide- based EPR oximetry. Following deconvolution of the individual S state contributions, the signal II(vf) decay kinetics yield the following half- times for the S state transitions: S₀ → S₁ in 40-60 μs, S₁ → S₂ in 85 μs, S₂ → S₃ in 140 μs, and S₃ → (S₄) → S₀ in 750 μs. Preliminary results with detergent-solubilized PSII membranes suggest that the S₃ → S₀ transition at least is slowed by a factor of ~2 in this system. Ramifications of these half-times in terms of electron transfer events on the donor site of PSII are discussed.