Resonance Raman spectroscopy is used to examine the possibility that C14-C15 single bond isomerizations of the retinal prosthetic group are involved in the photochemical reactions of bacteriorhodopsin. Normal mode calculations show that the vibration that contains predominantly C14`C15 stretch character is -70 cm-' lower in frequency in the 14-s-cis conformer than in the s-trans case. This geometric effect is insensitive to out-of-plane twists and should be observed in the sterically hindered 13-cis, 14-s-cis retinal protonated Schiff base, which has been proposed as the chromophore in the K and L intermediates of bacterio- has a localized C14-C15 stretch at 1172 cm-', consistent with a 14-s-trans chromophore. These results argue that the primary step in bacteriorhodopsin is a C13=C14 trans -* cis photoisomerization that does not involve C14-C15 s-cis structures.There is a great deal of interest in the mechanism by which changes in retinal chromophore structure in bacteriorhodopsin (BR) of this model is that thermal 14-s-cis --14-s-trans isomerization could occur after deprotonation of the Schiff base and function as a "proton switch." An analogous model has been presented recently by Liu and coworkers (6). These suggestions have remained untested due to the lack of a method for determining C-C single bond conformation in retinal pigments.Resonance Raman spectroscopy can be used to probe the in situ structure of the retinal chromophore in BR (7,8 In this paper we show that an s-trans --s-cis isomerization produces a characteristic "100 cm-' lowering of the C-C stretching frequency of the isomerized bond. To establish the conformation of the C,4-C15 bond in BR, the contribution of C14-C15 stretching character to the fingerprint normal modes must first be determined by selective 13C-or 2H-labeling. Comparison of the C14-C15 frequency in the pigment with that of the s-trans retinal PSB model compounds can then be used to determine the C14-C15 conformation in BR568, K625, and L550. EXPERIMENTALThe synthesis of the 10-13C, 11-13C, 14-13C, and 15-13C retinal isotopic derivatives has been described by Lugtenburg (10). Incorporation of deuterium at C15 was accomplished by reducing the retinoic ester or acid with lithium aluminum deuteride prior to oxidation. The isotopic purity was -98% Abbreviations: BR, bacteriorhodopsin; PSB, protonated Schiffbase.