FLUORESCENCE FROM THE PRIMARY PRODUCTS OF THE BACTERIORHODOPSIN PHOTOCYCLE - PICOSECOND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

The fluorescence spectrum of the K-590 intermediate, formed several picoseconds after the optical initiation of the bacteriorhodopsin (BR) photocycle, is observed at room temperature using a two laser, pump-probe technique. The BR photocycle is initiated with a 565 nm pump laser pulse (6 ps) while the observed fluorescence is generated by the optically delayed 590 nm probe laser pulse (4 ps). The distinct emission from K-590, while unstructured and occurring in the same general spectral region as the emission from BR-570 itself, is distinguished by its approximately twofold greater emission quantum yield and a shift of the position of maximum emission intensity to higher energy (corresponding to a blue shift of 17 nm). The assignment of this emission to the K-590 is based on its time dependence. The K-590 fluorescence spectrum described here is obtained using a 40 ps pump-probe delay. At this time in the photocycle, the only transient species which can be excited by the picosecond probe laser is K-590. The changes in the ground and excited-state properties of the retinal chromophore of BR during the initial 100 ps of the photocycle can be quantitatively described by a rate-equation model in which the instantaneous populations of each of the photocycle species is calculated. These results are used to fit the picosecond transient absorption (PTA) and time resolved fluorescence (PTRF) data. Differences in the time-dependence of the PTA and PTRF data require the inclusion into the rate-equation model of vibrationally-excited, ground state species, BR' and K', which are formed during the optical pumping and by decay of J-625, respectively.