ENERGY COUPLING IN AN ION-PUMP - THE REPROTONATION SWITCH OF BACTERIORHODOPSIN

The active site of an ion pump must communicate alternately with the t wo opposite membrane surfaces. In the light-driven proton pump, bacter iorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the accep tor of the proton of D96 (with access to the cytoplasmic side). This ' 'reprotonation switch'' has been associated with a protein conformatio n change observed during the photocycle. When D85 is replaced with asp aragine, the pK(a) value of the Schiff base is lowered from above 13 t o about 9. We determined the direction of the loss or gain of the Schi ff base proton in unphotolyzed and in photoexcited D85N, and the D85N/ D96N and D85N/D96A double mutants, in order to understand the intrinsi c and the induced connectivities of the Schiff base to the two membran e surfaces. The influence of D96 mutations on proton exchange and on a cceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to th e cytoplasmic side. In the wild-type protein (but with the pK(a) of th e Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the e xtracellular side. Raising the pH without illumination of D85N so as t o deprotonate the Schiff base caused the same, or nearly the same, cha nge of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site to the global protein conformation and to the connecti vity of the Schiff base proton to the membrane surfaces. Their relatio nship suggests that the conformation of the unphotolyzed wild-type pro tein is stabilized by coulombic interaction of the Schiff base with it s counter-ion. A proton is translocated across the membrane after ligh t-induced transfer of the Schiff base proton to D85, because the prote in assumes an alternative conformation that separates the donor from t he acceptor and opens new conduction pathways between the active site and the two membrane surfaces.