PHOTOELECTROCHEMICAL VERIFICATION OF PROTON-RELEASING GROUPS IN BACTERIORHODOPSIN

Light-induced transient currents of bacteriorhodopsin (bR) and its mut ants (D96N, D85N, E204Q and E194Q) were measured at the interface of a n electrode and the aqueous solution in an electrochemical cell. The t ransient positive (cathodic) and negative (anodic) photocurrents gener ated upon the onsets of continuous illumination and of turning off lig ht, respectively, were investigated at different electrolyte pH, The w ild type exhibits both positive and negative responses, with the sign of the response inverted at pH < 5, In D85N the response is entirely s uppressed, while D96N lacks the negative response. Laser pulse excitat ion (532 mm) of bR and the mutants showed the response rise time of th e positive transient to be uniformly about 100 mu s, indicating that t he response is linked to the L to M transition in the photocycle. Acco rding to these results the positive and negative signals originate fro m the proton release and uptake reactions, respectively. Photoexcitati on of the mutants E204Q and E194Q that lack protonatable residues at p osition 204 and 194, respectively, produced a negative response at neu tral pH, which indicates that in these proteins proton uptake precedes proton release. This result is consistent with our observations made earlier with pH indicator dyes and demonstrates that Glu-204 and Glu-1 93 constitute the terminal proton release complex in the extracellular region of bR.