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 lambda(max) of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color-controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.