Characterization and photochemistry of 13-desmethyl bacteriorhodopsin.

The photochemistry of the 13-desmethyl (DM) analogue of bacteriorhodopsin (BR) is examined by using spectroscopy, 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, P 1 D M (λ m a x = 525 nm), which can be generated efficiently by exciting the resting state, bR D M with yellow or red light (A > 590 nm). Chromophore extraction analysis reveals that the retinal configuration in P 1 D M is 9-cis, identical to that of the retinal configuration in the native BR P 1 state. Fourier transform infrared and Raman experiments on P 1 D M indicate an anti configuration around the C 1 5 =N bond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient, time-resolved studies indicate that the P 1 D M state forms primarily via thermal relaxation from the L D D M state. Theoretical studies on the BR binding site show that 13-dm retinal is capable of isomerizing into a 9-cis configuration with minimal steric hindrance from surrounding residues, in contrast to the native chromophore in which surrounding residues significantly obstruct the corresponding motion. Analysis of the photokinetic experiments indicates that the Arrhenius activation energy of the bR D M → P 1 D M transition in 13-dm-BR is less than 0.6 kcal/mol (vs 22 ′5 kcal/mol measured for the bR → P (Pi and P 2 ) reaction in 85:15 glycerol: water suspensions of wild type). Consequently, the P 1 D M state in 13-dm-BR can form directly from all-trans, 15-anti intermediates (bR D M and O D M ) or all-trans, 15-syn (K D D M /L D D M ) intermediates. This study demonstrates that the 13-methyl group, and its interactions with nearby binding site residues, is primarily responsible for channeling one-photon photochemical and thermal reactions and is limited to the all-trans and 13-cis species interconversions in the native protein.