Resonance Raman and optical transient studies on the light-induced proton pump of bacteriorhodopsin reveal parallel photocycles

Abstract

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 microseconds-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 microseconds 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 pKa approximately 6.2. The decay of M412 monitored by OTS can be fitted for each pH value by two different amplitudes and time constants (Mf, tau f; Ms, tau s; f = fast, s = slow). Both Mf and Ms consist of subcomponents which can be distinguished by their different reaction pathways (but not by OTS). Mf occurs in the reaction sequences L-->Mf-->N-->BR and L-->Mf-->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. Ms is also found in two different reaction sequences of the form L-->Ms-->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 Ms and N that the reconstitution of BR570 in the reaction steps Ms-->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.

Evidence for the Rate of the Final Step in the Bacteriorhodopsin Photocycle Being Controlled by the Proton Release Group: R134H Mutant†

“…A decrease of absorbance is due to proton release into the suspension; an increase of absorbance is due to proton uptake. Traces 1-5 were taken at pH 6.0, 6.3, 6.5, 6.8, and 7.0. with a pK a of 7.2 seen in the WT (27,(35)(36)(37)(38) and the E204Q and E194Q mutants (27) was tentatively assigned to D96 in the late N or O states (27). The reason for the decreased pK a (and decreased amount of O intermediate at neutral pH) in R134H as well as in E194D (27) should be investigated further.…”

Chemical and functional studies on the importance of purple membrane lipids in bacteriorhodopsin photocycle behavior

“…More recently, it has been shown that Mf decays to the ground state through the O intermediate whereas Ms does not [4,5]. A recent study showed that very mild treatment of purple membranes (PM) with Triton X-100 destroyed the ability of actinic light to influence the photocycle and altered the decay pathway of Mf without disrupting the trimer BR structure [1].…”

In situ determination of transient pK a changes of internal amino acids of bacteriorhodopsin by using time-resolved attenuated total reflection Fourier-transform infrared spectroscopy

“…For example, the L-M transition is accelerated at alkaline pH (15,29). At low pH the O intermediate is accumulated, whereas the lifetime of the N intermediate is prolonged at high pH (15,(30)(31)(32). The sequence of proton release and uptake is reversed at a pH lower than 6 [late proton release (33,34)].…”