FEMTOSECOND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY OF BACTERIORHODOPSIN - DIRECT OBSERVATION OF EXCITED-STATE DYNAMICS IN THE PRIMARY STEP OF THE PROTON PUMP CYCLE

A femtosecond fluorescence upconversion apparatus was used to measure the fluorescence decays in bacteriorhodopsin from Halobacterium halobium. The isotropic fluorescence measurements reveal a weak emission wavelength dependence from 680 nm to 900 nm (which spans most of the steady state fluorescence spectrum). The decays can be well fitted as a sum of three exponential decay components with time constants in the range of 90 fs-240 fs, 0.6 ps-0.9 ps, and 9.0-13.0 ps. We discuss the mechanism for non-exponentiality in terms of the intrinsic characteristics of a barrierless reaction and/or the distribution of parameters affecting the reactive process. We discuss the kinetics by comparing with previous pump-probe transient absorption measurements, and suggest that the first time constant results from either a relaxation process in the first singlet excited state of the retinal molecule or from the reactive process from the excited state to the photoproduct and reactant. We associate the longer components solely with the reactive process from the excited state to the photoproduct and ground state of bacteriorhodopsin. Fluorescence depolarization measurements reveal a wavelength dependent initial anisotropy. The fluorescence data do not seem to be easily explainable with a one-dimensional potential surface model.