Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a la mbda(max) of about 500 nm in water at neutral pH and a great influence of p H and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of h ydroxylamine in the dark as well as in light. Both the proton release kinet ics and the photocycle are altered, as indicated by a delayed proton releas e after proton uptake and changed M kinetics. Moreover, affinity of the col or-controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of B W, (C) 1999 Federation of European Biochemical Societies.