| Abstract
| - The photochemistry of the 13-desmethyl (DM) analogue of bacteriorhodopsin (BR) is examined by usingspectroscopy, molecular orbital theory, and chromophore extraction followed by conformational analysis.The removal of the 13-methyl group permits the direct photochemical formation of a thermally stable,photochemically reversible state, P1DM (λmax = 525 nm), which can be generated efficiently by exciting theresting state, bRDM with yellow or red light (λ > 590 nm). Chromophore extraction analysis reveals that theretinal configuration in P1DM is 9-cis, identical to that of the retinal configuration in the native BR P1 state.Fourier transform infrared and Raman experiments on P1DM indicate an anti configuration around the C15Nbond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient,time-resolved studies indicate that the P1DM state forms primarily via thermal relaxation from the LDDM state.Theoretical studies on the BR binding site show that 13-dm retinal is capable of isomerizing into a 9-cisconfiguration with minimal steric hindrance from surrounding residues, in contrast to the native chromophorein which surrounding residues significantly obstruct the corresponding motion. Analysis of the photokineticexperiments indicates that the Arrhenius activation energy of the bRDM → P1DM transition in 13-dm-BR isless than 0.6 kcal/mol (vs 22 ±5 kcal/mol measured for the bR → P (P1 and P2) reaction in 85:15 glycerol:water suspensions of wild type). Consequently, the P1DM state in 13-dm-BR can form directly from all-trans,15-anti intermediates (bRDM and ODM) or all-trans, 15-syn (KDDM/LDDM) intermediates. This study demonstratesthat the 13-methyl group, and its interactions with nearby binding site residues, is primarily responsible forchanneling one-photon photochemical and thermal reactions and is limited to the all-trans and 13-cis speciesinterconversions in the native protein.
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