Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.

Tunable laser resonance raman spectroscopy of bacteriorhodopsin.

Bacteriorhodopsin is a rhodopsin-like protein found in the cell membrane of Halobacterium halobium. It shows an absorption maximum at 570 nm and, in the light, undergoes cyclic spectral changes which include a relatively long-lived complex absorbing maximally at 412 nm. Excitation profiles have been obtained with several laser frequencies for two vibrations in the resonance Raman spectrum of bacteriorhodopsin. The results show that the Schiff base retinylidene lysine linkage is protonated in the 570 nm complex and that in the 412 nm complex it is unprotonated. The 412 nm complex must be present at appreciable concentrations when bacteriorhodopsin is exposed to high-energy argon ion laser light of the Raman spectrophotometer at room temperature. We conclude that the observed C=N stretch at 1622 cm(-1) in the room temperature spectra, which in an earlier study by Mendelsohn was interpreted as evidence for an unprotonated linkage in bacteriorhodopsin, results from the presence of the 412 nm complex.