Pathway of proton uptake in the bacteriorhodopsin photocycle

Abstract

The time courses of chromophore reactions and proton uptake in the second half of the photocycle of the proton pump bacteriorhodopsin (BR) were examined. At pH > 8.5, the kinetics are simplified by the fact that only the M and N intermediates accumulate. The relaxation kinetics after perturbation of M with a second, blue flash confirm that M<==>N equilibration is the only significant cause of the biphasic M decay. With this feature, the analysis of time-resolved difference spectra yields a scheme which contains two sequential N states connected by a nearly unidirectional reaction. The proton uptake from the bulk, as measured with the pH-indicator dye pyranine, occurs during the decay of the first N rather than the recovery of BR. The results thus suggest the model M2(-1)<==>N(-1) + H+ (from the bulk)<==>N(0)-->BR, where the superscripts indicate the protonation state of the protein relative to BR. M2(-1)-->N(-1) is reprotonation of the Schiff base from D96, N(-1) + H+ (from the bulk)-->N(0) is uptake of proton from the cytoplasmic side, and N(0)-->BR represents 13-cis to all-trans reisomerization of the retinal and other relaxations which regenerate the initial state. R227, a residue near D96, affects the deprotonation of D96 more than the subsequent proton uptake. According to the changed [M2(-1)]/[N(-1)] equilibrium in the R227Q protein, interaction between R227 and D96 is responsible for about 1 pH unit of the decrease in the pKa of D96 during the reprotonation of the Schiff base. According to the pH dependencies of the rate constants in the N(-1)<==>N(0) equilibrium in wild-type and R227Q, interaction with R227 lowers the pKa for proton uptake from the bulk by 0.5 pH unit, to 11. We conclude from the proton uptake kinetics that at physiological pH free energy is converted to proton electrochemical potential in this pump not only as protons are released on the extracellular side [Zimányi, L., Váró. G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] but also as protons are taken up on the cytoplasmic side.

Breaking the Carboxyl Rule

“…In its hydrophobic environment, Asp-96 in the initial state of bacteriorhodopsin has a pK a of Ͼ11.5 (28,46), elevated from its value of ϳ 4 in water. This extremely high pK a is from the environment of Asp-96, the hydrophobic "barrel" that surrounds it (47).…”

“…This would explain the key differences in the sequence of events after photoexcitation. In bacteriorhodopsin, protonation of the Schiff base by Asp-96 during the M to N transition is distinctively more rapid than the proton uptake (39,42,46,55,56) leading to reprotonation of Asp-96. Asp-96 appears to be inaccessible to the bulk at the time of the proton exchange between Asp-96 and the Schiff base (33), because it does not lose the proton to the bulk up to very high pH values.…”

Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

“…The results suggest that rapid protein motions, allowing thermal averaging of substates on the time scale of the photocycle reactions, do become possible at ambient temperature, and one would predict therefore conventional kinetics. Attempts have been made to account for experimental discrepancies from simple models with distributed kinetics (45,46); however, although they gave better fits, the validity of this approach (vs. more complex conventional kinetic schemes) remains an open question.…”

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

“…Two arginines, Arg-225 and Arg-227, are located at the cytoplasmic end of helix G, one helix turn below Gly-231. Arg-227 mutations affect the kinetics of Mdecay (47) and proton uptake (48), indicating the interaction of the positively charged guanidinium group with a hydrogenbonded network, which is proposed to be part of a proton uptake cluster (48). Exchange of the small unpolar and highly flexible glycine with the polar and ionizable cysteine in position 231 may lead to an alteration in the hydrogen-bonded network and/or water-filled cavities and to changes in the peptide backbone.…”