Two bathointermediates of the bacteriorhodopsin photocycle, from time-resolved nanosecond spectra in the visible.

Time-resolved measurements were performed on wild-type bacteriorhodopsin with an optical multichannel analyzer in the spectral range 350-735 nm, from 100 ns to the photocycle completion, at four temperatures in the 5-30 degrees C range. The intent was to examine the possibility of two K-like bathochromic intermediates and to obtain their spectra and kinetics in the visible. The existence of a second K-like intermediate, termed KL, had been postulated (Shichida et al., Biochim. Biophys. Acta 1983, 723, 240-246) to reconcile inconsistencies in data in the pico- and microsecond time domains. However, introduction of KL led to a controversy, since neither its visible spectrum nor its kinetics could be confirmed. Infrared data (Dioumaev and Braiman, J. Phys. Chem. B 1997, 101, 1655-1662) revealed a state which might have been considered a homologue to KL, but it had a kinetic pattern different from that of the earlier proposed KL. Here, we characterize two distinct K-like intermediates, K(E) ("early") and K(L) ("late"), by their spectra and kinetics in the visible as revealed by global kinetic analysis. The K(E)-to-K(L) transition has a time constant of approximately 250 ns at 20 degrees C, and describes a shift from K(E) with lambda(max) at approximately 600 nm and extinction of approximately 56,000 M(-1) x cm(-1) to K(L) with lambda(max) at approximately 590 nm and extinction of approximately 50,000 M(-1) x cm(-1). The temperature dependence of this transition is characterized by an enthalpy of activation of DeltaH(++) approximately 40 kJ/mol and a positive entropy of activation of DeltaS(++)/R approximately 4. The consequences of multiple K-like states for interpreting the spectral evolution in the early stages of the photocycle are discussed.