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 temper ature in aqueous suspensions of purple membranes using time-resolved r esonance Raman (RR) and optical transient spectroscopy (OTS). The samp les were photolyzed, and the fractional concentrations of the retinyli dene chromophore in its parent state, BR570, and in the intermediate s tates 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 compo nents could be identified. At pH 7 one fraction of L (approximately 65 %) decays in 80 mus to M (deprotonation of the Schiff base), whereas t he residual part is converted in approximately 0.5 ms to N. The RR spe ctra 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) Biochem istry 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 valu e 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 subcomponen ts which can be distinguished by their different reaction pathways (bu t 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 sequen ce, 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 diffe rent reaction sequences of the form L --> M(s) --> BR. The quantitativ e analysis reveals that each ''titration effect'' can be related to a certain fraction of bR. It is proposed that each fraction can be ident ified with a ''subspecies'' of bR which undergoes an independent and i ndividual cyclic reaction. A complete reaction scheme is set up which represents the manifold of observed phenomena. It is concluded from th e 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 pro ton 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 catal yze the reisomerization of retinal is the one which is pumped through the membrane during the photocycle of bR.