The effect of azide on the photocycle of bacteriorhodopsin

Abstract

Azide has previously been shown to have an effect on the photocycle of bacteriorhodopsin mutants. In appropriate mutants, both the proton release from the Schiff base and reprotonation are accelerated. However, no effect on native bacteriorhodopsin has been found. In this work, we show that protonated azide influences the photocycle of native bacteriorhodopsin, although to a lesser extent than in certain mutants. Several transitions are influenced by protonated azide: however, the overall effect is dominated by the modification of a single step. The M to N transition, i.e. reprotonation of the Schiff base, is accelerated by azide. As a consequence of this kinetic effect, the concentrations of several intermediates are influenced. The interpretation of the kinetic effect of azide involves an alternative parallel reaction path from the M form to the ground state in native bacteriorhodopsin. It is concluded that there are two routes of Schiff base reprotonation: via mediation by Asp-96 (in this case, the molecule returns to the ground state through the N intermediate; this is the generally accepted scenario): via Schiff base reprotonation directly from the external medium (in this case, the molecule returns from another M form to the ground state in one step).