Back photoreaction from intermediate M of bacteriorhodopsin photocycle

The back photoreaction from intermediate M of the photocycle has been studi ed on wild-type and several mutant bacteriorhodopsins. Data are collected b y spectroscopic, absorption kinetic and electric signal measurements. To dr ive the photocycle in intermediate M, different techniques are applied. For wild-type bacteriorhodopsin double-flash experiments are carried out. The mutant D96N is driven in intermediate M with background light on the sample at pH 9. The mutant D85N produces an M-like intermediate at pH 8 in the da rk. The accumulated M intermediate is excited by a 420 nm laser flash. The electric signals of the wild-type protein and mutant D96N, belonging to the back reaction from intermediate M, have only negative components. The M-li ke intermediate of the D85N mutant has only a positive component. A photocy cle model for the light-driven back reaction of the M intermediate is sugge sted. The spectra of intermediates and the sign and time constant of the el ectric signals suggest that the deprotonated Schiff base in the M state aft er light excitation is reprotonated fast, but the protein reaches its groun d state only in several seconds. The proton taken up after photoexcitation comes from the same side at which it was released in the previous process. (C) 1999 Elsevier Science S.A. All rights reserved.