Photochemistry of K-590 in the room-temperature bacteriorhodopsin photocycle

The photochemistry of the K-590 intermediate in the room-temperature (RT) b acteriorhodopsin (BR) photocycle (i.e., K-590 back reaction) is examined wi th picosecond time resolution over the 50 ps to 4.5 ns period of its lifeti me. 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 (p ump 2 at 650-660 nm), and (iii) monitor the changes in sample absorbance af ter BR-570 or BR-570 and K-590 excitation (robe at 570-620 nm). The wavelen gths of these laser pulses are selected to optimize their respective functi ons in terms of photolysis or monitoring changes in absorbance. The timing relationships between the pump i, pump 2, and probe pulses, all with indepe ndently controlled pulse widths, energies, and wavelengths, are selected to obtain two different types of pulse sequences: (i) a two-pulse timing sequ ence designed to monitor intermediate concentrations in the forward, uninte rrupted BR photocycle and (ii) two different, three-pulse timing sequences designed to characterize the optically induced, picosecond RT/K-590 photoch emistry (back reaction). The results show that (i) the species formed by th e 650-660-nm excitation of K-590 can be identified via its absorption spect rum as BR-570, (ii) BR-570 is formed from K-590 within the 5-ps cross-corre lation time defined by the pump 2 and probe pulses, (iii) the K-590 to BR-5 70 mechanism does not appear to involve an intermediate analogous to J-625 found in the forward BR photocycle, and (iv) the spectroscopic characterist ics 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 quantu m efficiency and photoproduct) remains constant over this time interval. Th ese results are discussed with respect to previous studies of the K-590 bac k reaction (i) at low temperatures and (ii) at RT using high-power, nanosec ond pulsed excitation both of which create photostationary mixtures of inte rmediates. The mechanistic interpretation of these picosecond, RT results, including the relationship(s) to the forward BR photocycle, derives from st ructural changes in the retinal chromophore and its protein binding pocket, as well as their respective interactions.