Time and pH Dependence of the L-to-M Transition in the Photocycle of Bacteriorhodopsin and Its Correlation with Proton Release

Abstract

The pH dependence of the L-to-M transition in the photocycle of bacteriorhodopsin was studied in pump-probe resonance Raman (RR) flow experiments in the range pH 3.5-7.8 on a time scale of 0-700 micros. For pH < 5, following the initial decay of L to M, the two intermediates approach nearly constant levels. From a specially designed perturbation-relaxation experiment at pH 4.6, in which the composition of L and M is perturbed by photoreversal of M, it could be concluded that the incomplete decay of L is due to an intermediate equilibration between L and M. It was found, by both RR and optical transient spectroscopy, that the maximum level of M (approximately 500 micros) increases with pH according to a pKa of 5. 6 (150 mM Na+). Since the proton release from an internal group XH to the extracellular surface is determined by nearly the same pKa of 5.7 [Zimanyi, L., Varo, G., Chang, M., Ni, B., Needleman, R., and Lanyi, J. K. (1992) Biochemistry 31, 8535-8543], it is concluded that this increase is controlled by the dissociation of XH. From the analysis of the perturbation-relaxation experiments and the multiexponential rise of M, a kinetic scheme with two sequential L-M equilibria is proposed for the L-M transition. By comparison with the time behavior of proton release [Heberle, J., and Dencher, N. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5996-6000], it is suggested that it is the second equilibrium which is further shifted toward the M state by the dissociation of XH. From the magnitude of this shift, it is concluded that the L-M transition and proton release are not as strongly coupled as is generally assumed. Instead, it is proposed that structural changes during the photocycle are the dominating factors which reduce the pKa of XH to approximately 5.7 so that proton release becomes possible under normal conditions.

Crystal Structure of the M Intermediate of Bacteriorhodopsin: Allosteric Structural Changes Mediated by Sliding Movement of a Transmembrane Helix

“…55 This partial deprotonation is indicative of a transient state in which the Schiff base and Asp85 share a proton. This transient state seems to correspond to the L/M state that is generated when the P622 crystal is illuminated at 180 K. 11 For clarity, the M intermediate in this mixture state is designated as M cold .…”

Crystal Structures of Acid Blue and Alkaline Purple Forms of Bacteriorhodopsin

“…It has been reported that the fast rising component of M becomes significant when the pH increases above pH 10. 30 This phenomenon can be explained readily because the energy barrier for re-arrangement of the hydrogen-bonding network in the proton release channel becomes smaller when the paired structure of Glu194 and Glu204 is already broken in the initial unexcited state.…”

“…30 This phenomenon has been suggested to correlate with a remarkable decrease in the rate constant of the dark adaptation at high pH. 31 To explain this correlation, Balashov et al have proposed that the pK a of Asp85 is affected by the protonation state of another residue locating near the extracellular surface.…”

Kinetic Optimization of Bacteriorhodopsin Films for Holographic Interferometry

“…An equilibration between M I and L at reduced humidities is described in the literature. [36][37][38] The L concentration in dried samples was found to be low. 32 An increase in the L population reduces the net forward reaction rate k 1A -k 1B (see Figure 10) because L is photochemically converted back to B.…”