Photochemistry of K-590 in the Room-Temperature Bacteriorhodopsin Photocycle

The photochemistry of the K-590 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle (i.e., K-590 back reaction) is examined with picosecond time resolution over the 50 ps to 4.5 ns period of its lifetime. Three separate, 4−5-ps (fwhm) pulses are used at different wavelengths to sequentially (i) initiate the BR photocycle by optical excitation of BR-570 (pump 1 at 578 nm), (ii) photolytically interrupt the RT/BR photocycle at selected time delays between 50 ps and 4.5 ns after BR-570 excitation (pump 2 at 650−660 nm), and (iii) monitor the changes in sample absorbance after BR-570 or BR-570 and K-590 excitation (probe at 570−620 nm). The wavelengths of these laser pulses are selected to optimize their respective functions in terms of photolysis or monitoring changes in absorbance. The timing relationships between the pump 1, pump 2, and probe pulses, all with independently controlled pulse widths, energies, and wavelengths, are selected to obtain two different types of pulse sequences: (i) a two-pulse timing sequence designed to monitor intermediate concentrations in the forward, uninterrupted BR photocycle and (ii) two different, three-pulse timing sequences designed to characterize the optically induced, picosecond RT/K-590 photochemistry (back reaction). The results show that (i) the species formed by the 650−660-nm excitation of K-590 can be identified via its absorption spectrum as BR-570, (ii) BR-570 is formed from K-590 within the 5-ps cross-correlation time defined by the pump 2 and probe pulses, (iii) the K-590 to BR-570 mechanism does not appear to involve an intermediate analogous to J-625 found in the forward BR photocycle, and (iv) the spectroscopic characteristics of the K-590 back reaction remain unchanged for pump 2 delays of 100 ps to 4.5 ns, indicating that the K-590 photochemistry (i.e., relative quantum efficiency and photoproduct) remains constant over this time interval. These results are discussed with respect to previous studies of the K-590 back reaction (i) at low temperatures and (ii) at RT using high-power, nanosecond pulsed excitation both of which create photostationary mixtures of intermediates. The mechanistic interpretation of these picosecond, RT results, including the relationship(s) to the forward BR photocycle, derives from structural changes in the retinal chromophore and its protein binding pocket, as well as their respective interactions.