Characterization of the proton-transporting photocycle of pharaonis halorhodopsin

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN3 and 1 M Na2SO4. Recent observations established that the rep lacement of the chloride ion with azide transforms the photocycle from a ch loride-transporting one into a proton-transporting one. Kinetic analysis pr oves that the photocycle is very similar to that of bacteriorhodopsin. Afte r K and L, intermediate M appears, which is missing from the chloride-trans porting photocycle. In this intermediate the retinal Schiff base deprotonat es. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multipha sic. Intermediate N cannot be detected, but a large amount of O accumulates . The multiphasic character of the last step of the photocycle could be exp lained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes i ts sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride ph otocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchan ged by exchanging chloride with azide.