SPECIFIC ACCELERATION OF DEPROTONATION AND REPROTONATION STEPS BY AZIDE IN MUTATED BACTERIORHODOPSINS

Replacement of Asp-96 by Asn leads to a slowed M-decay (reprotonation of the Schiff base) during the photocycle of bacteriorhodopsin. Additi on of azide accelerates the M-decay to the value of the wild-type prot ein and restores proton pump activity. Under this condition the recove ry of the initial state is no longer correlated with the decay of M. S pectroscopic analysis of this situation reveals that the intermediate following M, the N-state, accumulates and its decay gets the rate-limi ting step in the photocycle. This is proved by photostationary measure ments where a pure N-BR spectrum is measurable. Additional mutation of Leu-93 to Ala leads to decreased decay rates of intermediates N or O, depending on the temperature. Addition of azide has therefore allowed the characterization of the intermediates following M. In mutants lac king Asp-85, a deprotonated Schiff base (410 nm) is the starting point of a photoreaction initiated by blue light leading to a protonated fo rm absorbing maximally at 610 nm. Azide also accelerates the deprotona tion of this product. In all cases, the protonated form of azide is th e catalytically active species. This suggests that the protonated azid e is part of a hydrogen-bond system required for proton movements with in the two half-channels of BR.