Time-resolved absorption and fluorescence from the bacteriorhodopsin photocycle in the nanosecond time regime

Picosecond transient absorption (PTA) in the 568-660-nm region is measured over the initial 80 ns of the bacteriorhodopsin photocycle. After photocycle initiation with 573-nm excitation (7-ps pulsewidth), these PTA data reflect the formation during the initial 40 ps of two long-recognized intermediates with red-shifted (relative to that of BR-570) absorption bands, namely J-625 and K-590. PTA signals at 568, 628, and 652 nm are unchanged for the remainder of the 80-ns photocycle interval measured, demonstrating that no other intermediates, including the proposed KL, are observable by absorption changes. Picosecond time-resolved fluorescence (PTRF), measured at 740 nm, is initiated by 7 ps excitation of the species present at various time delays after the photocycle begins. PTRF signals change rapidly over the initial 40 ps, reflecting, first, the depletion of the ground state BR-570 population and, subsequently, the formation of K-590. The PTRF signal then decreases monotonically with a time constant of 5.5 +/- 0.5 ns from its maximum near a 50-ps delay until it reaches a minimum at a delay of approximately 13 ns. For time delays between 13 and 80 ns, the PTRF signal remains unchanged and slightly higher than that measured from BR-570 alone. The rapid decrease in PTRF signals over the same photocycle interval in which the PTA signals remain unchanged suggests that the retinal-protein interactions involving electronically excited K-590 (K*) are being significantly altered.