Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680(+)) and the reduced electron acceptor pheophytin a (-) (Pheo a (-)) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D1, D2, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450-840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680(+)-Pheo a (-) is indicated by the appearance of a band due to P680(+) at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a (-). The appearance of the 490 nm and 820 nm bands is monoexponenital with τ=1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a (-). Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to (1*)P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Qy absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.