Independent Photocycles of the Spectrally Distinct Forms of Bacteriorhodopsin

Time-resolved, flash-induced difference absorbance spectra (300-700 nm) at pH 10.5 and 5 degrees C for the bacteriorhodopsin photocycle fast and slow decaying forms of the M intermediate (Mf and Ms, respectively) and R intermediate are reported. The main distinguishing features are as follows: For Mf, Δ Amax = 412 nm a shoulder at 436 nm, no absorbance change at 350 nm; Δ Amin = 565 nm; Δ A412/Δ A565= 0.85. For Ms, Δ Amax = 412 nm, a shoulder at 386 nm; Δ Amin = 575 nm; Δ A412/Δ A575 = 0.6. For R, Δ Amax = 336 and 350nm (double peak), smaller peaks at 386 and 412 nm; Δ Amin = 585 nm; Δ A350/Δ A585 = 0.2. The different difference spectra for Mf and Ms provide direct evidence that these species, initially identified by their kinetics, are physically distinct. With fast transient absorption spectroscopy, it was shown that R may form very fast, perhaps faster than the L intermediate decays. On the basis of the different bleaching peaks for Mf and Ms, we propose that Mf and Ms are in independent photocycles formed from slightly different forms of bacteriorhodopsin. R may also be in a different photocycle. The different forms of bacteriorhodopsin are probably in dynamic equilibrium with their ratios, controlled by pH and temperature.