Back photoreaction from intermediate M of bacteriorhodopsin photocycle

Abstract

The back photoreaction from intermediate M of the photocycle has been studied on wild-type and several mutant bacteriorhodopsins. Data are collected by spectroscopic, absorption kinetic and electric signal measurements. To drive 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 dark. 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-like intermediate of the D85N mutant has only a positive component. A photocycle model for the light-driven back reaction of the M intermediate is suggested. The spectra of intermediates and the sign and time constant of the electric signals suggest that the deprotonated Schiff base in the M state after light excitation is reprotonated fast, but the protein reaches its ground 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.