ABSTRACT
The rotational spectra of benzyl alcohol and of its OD isotopologue have been assigned and measured in a supersonic expansion, either with pulsed-jet Fourier transform microwave or free jet absorption millimeter wave spectroscopy. The spectrum is consistent with a gauche conformation of the oxygen atom, characterized by a theta (OC(7)-C(1)C(2)) dihedral angle of approximately 55 degrees. Such a configuration is 4-fold degenerate, corresponding to minima with theta approximately +/-60 degrees, +/-120 degrees. The four equivalent minima are separated by two kinds of barrier, corresponding to theta = +/-90 degrees, and 0 or 180 degrees. Only the theta = +/-90 degrees barriers are low enough to generate a tunneling splitting, which has been measured in a spectrum strongly perturbed by tunneling interactions. The observed splittings diminish considerably upon deuterium substitution. The tunneling splittings are consistent with a barrier about 280 cm(-1) and high level ab initio calculations predicting a 320 cm(-1) barrier.
ABSTRACT
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, P1(DM) (lambda(max) = 525 nm), which can be generated efficiently by exciting the resting state, bR(DM) with yellow or red light (lambda > 590 nm). Chromophore extraction analysis reveals that the retinal configuration in P1(DM) is 9-cis, identical to that of the retinal configuration in the native BR P1 state. Fourier transform infrared and Raman experiments on P1(DM) indicate an anti configuration around the C15=N bond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient, time-resolved studies indicate that the P1(DM) state forms primarily via thermal relaxation from the L(D)(DM) 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(DM) --> P1(DM) transition in 13-dm-BR is less 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 P1(DM) state in 13-dm-BR can form directly from all-trans, 15-anti intermediates (bR(DM) and O(DM)) or all-trans, 15-syn (K(D)(DM)/L(D)(DM)) 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.
