Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

Abstract

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 .+-. 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 .+-. 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13.dbd.C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode of 1618 cm-1 in D2O indicates that the schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C.dbd.N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base. These results, together with the recent confirmation that L550 contains a 14-s trans chromophore [Fodor, S P. A., Pollard, W. T., Gebhard, R., van den Berg, E. M. M., Lugtenburg, J., and Mathies, R. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 2156-2160], argue against models for the bacteriorhodopsin photocycle that employ C14-C15 bond rotation or inversion of the Schiff base nitrogen as a "reprotonation switch". An alternative "C-T model" for the photocycle is proposed that invokes isomerization-driven protein conformational changes as the reprotonation switch.