Determination of the primary charge separation rate in isolated photosystem II reaction centers with 500-fs time resolution

We have measured directly the rate of formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a^- (Pheoa^-) following excitation of isolated spinach photosystem II reaction centers at 4°C. The reaction-center complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure that stabilizes the protein complex. Transient absorption difference spectra were measured from 440 to 850 nm as a function of time with 500-fs resolution following 610-nm laser excitation. The formation of P680⁺-Pheoa^- is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 505 and 540 nm due to formation of Pheoa^-. The appearance of the 820-nm band is monoexponential with τ = 3.0 ± 0.6 ps. The time constant for decay of ^1*P680, the lowest excited singlet state of P680, monitored at 650 nm, is τ = 2.6 ± 0.6 ps and agrees with that of the appearance of P680⁺ within experimental error. Treatment of the photosystem II reaction centers with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of Pheoa^-, results in formation of a transient absorption spectrum due to ^1*P680. We find no evidence for an electron acceptor that precedes the formation of Pheoa^-.