Bacteriorhodopsin's M412 intermediate contains a 13-cis,14-s-trans,15-anti-retinal Schiff base chromophore

Abstract

The structure of the retinal chromophore about the C.dbd.N and C14.sbd.C15 bonds in bacteriorhodopsin''s M412 intermediate has been determined by analyzing resonance Raman spectra of 2H and 13C isotopic derivatives. Normal model calculations on 13-cis-retinal Schiff bases demonstrate that the C15-D rock and N-CLys stretch are strongly coupled for C.dbd.N-syn chromophores and weakly coupled for C.dbd.N-anti chromophores. When the Schiff base geometry is anti, the C15-D rock appears as a localized resonance Raman active mode at .apprx. 980 cm-1, which is moderately sensitive to 13C substitution at positions 14 and 15 (.apprx. 7 cm-1) and insensitive to 13C substitution at the .epsilon. position of lysine. When the Schiff base geometry is syn, in-phase and out-of-phase combinations of the C15-D rock and N-CLys stretch are predicted at .apprx. 1060 and .apprx. 910 cm-1, respectively. The in-phase mode is more sensitive to 13C substitution at positions 14 and 15 (.apprx. 15 cm-1) and at the .epsilon. position of lysine (.apprx. 4 cm-1). Calculations and comparison with experimental data on dark-adapted bacteriorhodopsin indicate that the in-phase mode at .apprx. 1060 cm-1 carries the majority of the resonance Raman intensity. M412 exhibits a C15-D rock at 968 cm-1 that shifts 8 cm-1 when 13C is added at positions 14 and 15 and is insensitive to 13C substitution at the .epsilon.-position of lysine. This demonstrates that M412 contains a C.dbd.N-anti Schiff base. Time-resolved Raman experiments on M412 under a variety of conditions show that the various rise and decay components of M412 in the light-adapted photocycle all contain C.dbd.N-anti chromophores. Finally, M412 exhibits a 14,15-D2 coupled rock at 958 cm-1 which shows that the chromophore has a 14-s-trans conformation. We conclude that M412 contains a 13-cis,14-s-trans,15-anti chromophore. This result supports the recently proposed C-T model for the mechanism of the proton pump in bacteriorhodopsin [Fodor, S.P.A., Ames, J.B., Gebhard, R., van den Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., and Mathies, R. A. (1988) Biochemistry 27, 7097-7101].