Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the H-1/H-2 kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp(85), Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of similar to 100 ns and similar to 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 mu s, is absent at low pH, and increases in amplitude with a pK(a) of similar to 6. Because the proton release group deprotonates after 70-100 mu s and has a transient pK(a) of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp(85) elevated, and is probably due to the restoration of the pK of Asp(85) to it, initial low value, This provides further evidence for coupling between the pK's of Asp(85) and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg(82) from a position near the release group on the surface to Asp(85) makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (similar to 8), Arg(82) stays near the surface, leading to a corresponding signal reduction.