Time-resolved resonance Raman spectroscopy of bacteriorhodopsin

Abstract

BACTERIORHODOPSIN is found in purple patches on the cell membrane of halophilic bacteria¹. Similar in structure to rhodopsin (but differing in function), it consists of a retinal–protein complex. Bacteriorhodopsin is a photosynthetic system which, when it absorbs light, passes through a cycle of several intermediates (Fig. 1) approximately 200 times per second, pumping protons from the inside of the bacterial cell to the outside. The resultant electrochemical gradient drives the phosphorylation of ADP to ATP (ref. 1). The intermediates have been identified by measuring their relatively broad structureless optical absorption spectra^2–4 as a function of time after a photolytic flash. The most precise structural information has come from resonance Raman spectra^5–8 taken with continuous wave (cw) optical excitation. These studies have provided information about the bR₅₇₀ and bM₄₁₂ intermediates only, since they have appreciable steady state concentrations at room temperature under constant illumination. Resonance Raman spectra of these species have also been obtained at liquid N₂ temperatures and in ether saturated solutions. These conditions, however, may not be representative of actual physiological conditions. We report here a time-resolved resonance Raman spectrum of an intermediate with a risetime of < 6 ns. Our data show a decrease in the C = C stretching frequency by 10–20 cm⁻¹. From the known inverse correlation between the C = C stretching frequency and the position of the absorption maxima⁷ for a series of model retinal Schiff bases in various solvents⁹, the observed C = C Raman band was assigned to the bK₅₉₀ intermediate, which is known to have a risetime of 10 ps⁴.