RESONANCE RAMAN AND OPTICAL TRANSIENT STUDIES ON THE LIGHT-INDUCED PROTON PUMP OF BACTERIORHODOPSIN REVEAL PARALLEL PHOTOCYCLES

The photocycle of bacteriorhodopsin (bR) was studied at ambient temperature in aqueous suspensions of purple membranes using time-resolved resonance Raman (RR) and optical transient spectroscopy (OTS). The samples were photolyzed, and the fractional concentrations of the retinylidene chromophore in its parent state, BR570, and in the intermediate states L550, M412, N560, and O640 were determined in the time domain 20 mus-1 s and in the pH range 4-10.5. Two kinetically different L components could be identified. At pH 7 one fraction of L (approximately 65%) decays in 80 mus to M (deprotonation of the Schiff base), whereas the residual part is converted in approximately 0.5 ms to N. The RR spectra reveal only minor structural changes of the chromophore in the L --> N transition. These were attributed to a conformational change of the protein backbone [Ormos, P., Chu, K., & Mourant, J. (1992) Biochemistry 31, 6933]. With decreasing pH the L --> N transition is delayed to >2 ms following a titration-like function with pK(a) approximately 6.2. The decay of M412 monitored by OTS can be fitted for each pH value by two different amplitudes and time constants (M(f), tau(f); M(s), tau(s); f = fast, s = slow). Both M(f) and M(s) consist of subcomponents which can be distinguished by their different reaction pathways (but not by OTS). M(f) occurs in the reaction sequences L --> M(f) --> N --> BR and L --> M(f) --> O --> BR. The population of the first sequence, in which N is formed with the time constant tau(f) (approximately 2-4 ms, pH 6-10.5), increases with pH. M(s) is also found in two different reaction sequences of the form L --> M(s) --> BR. The quantitative analysis reveals that each ''titration effect'' can be related to a certain fraction of bR. It is proposed that each fraction can be identified with a ''subspecies'' of bR which undergoes an independent and individual cyclic reaction. A complete reaction scheme is set up which represents the manifold of observed phenomena. It is concluded from the pH dependence of the lifetimes of M(s) and N that the reconstitution of BR570 in the reaction steps M(s) --> BR and N --> BR requires the uptake of a proton from the external phase. It is argued that this proton catalyzes the reisomerization of retinal, whereas the Schiff base is internally reprotonated from Asp-85. A model for proton pumping is proposed in which the proton taken up from the external phase to catalyze the reisomerization of retinal is the one which is pumped through the membrane during the photocycle of bR.