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. Addition of azide accelerates the M-decay to the value of the wild-type protein and restores proton pump activity. Under this condition the recovery of the initial state is no longer correlated with the decay of M. Spectroscopic analysis of this situation reveals that the intermediate following M, the N-state, accumulates and its decay gets the rate-limiting step in the photocycle. This is proved by photostationary measurements 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 lacking Asp-85, a deprotonated Schiff base (410 nm) is the starting point of a photoreaction initiated by blue light leading to a protonated form absorbing maximally at 610 nm. Azide also accelerates the deprotonation of this product. In all cases, the protonated form of azide is the catalytically active species. This suggests that the protonated azide is part of a hydrogen-bond system required for proton movements within the two half-channels of BR.