Resonance Raman spectroscopy of the retinylidene chromophore in bacteriorhodopsin (bR570), bR560, M412, and other intermediates: structural conclusions based on kinetics, analogs, models, and isotopically labeled membranes

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Abstract
Resonance Raman spectra of various intermediates in the bacteriorhodopsin proton pumping cycle were obtained at physiological and low temperatures [from Halobacterium halobium]. To interpret these data, spectra of model compounds, bacteriorhodopsin analogs and isotopically labeled membranes were measured. A protein group apparently interacts with the Schiff base proton and, thus, the chromophore in protonated bacteriorhodopsin species is not a simple protonated Schiff base. This accounts for the abnormally low frequency of the C .dbd. N+H vibrational mode in bacteriorhodopsin and other failures to model the chromophore in bR570 with a simple butylamine protonated Schiff base of all-trans-retinal. To obtain the resonance Raman spectrum of M412 at physiological pH and temperatures, a dual beam kinetic technique was developed. In the fingerprint region of the resonance Raman spectrum M412 is modeled accurately by a simple unprotonated butylamine Schiff base of all-trans-retinal. Spectral resolution and the solution environment of the membrane suspensions play important roles in this conclusion. Kinetic resonance Raman techniques are also used to monitor the time evolution of the M412 species and the intermediates which precede it. Spectral features in the kinetic data which can be assigned to L550 are found, and evidence are presented for a new unprotonated species (X) which occurs before M412. Single pass flow resonance Raman spectra of bR560 also were obtained, and, although bR570 and M412 appear to have all-trans chromophores, there are 13-cis-like features in the spectra of bR560, L550 and X.