Fourier transform infrared double-flash experiments resolve bacteriorhodopsin's M-1 to M-2 transition

The orientation of the central proton-binding site, the protonated Schiff base, away from the proton release side to the proton uptake side is crucial for the directionality of the proton pump bacteriorhodopsin. It has been proposed that this movement, called the reprotonation switch, takes place in the M-1 to M-2 transition. To resolve the molecular events in this M-1 to M-2 transition, we performed double-flash experiments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermediate back into the BR ground state. For short delay times between initiating and resetting pulses, most of the M molecules being reset are in the M-2 intermediate, and for longer delay times most of the reset M molecules are in the M-2 intermediate. The BR-M-1 and BR-M-2 difference spectra are monitored with nanosecond step-scan Fourier transform infrared spectroscopy. Because the Schiff base reprotonation rate is k(M1) = 0.8 x 10(7) s(-1) in the light-induced M-1 back-reaction and k(M2) = 0.36 x 10(7) s(-1) in the M-2 back-reaction, the two different M intermediates represent two different proton accessibility configurations of the Schiff base. The results show only a minute movement of one or two peptide bonds in the M-1 to M-2 transition that changes the interaction of the Schiff base with Y185. This backbone movement is distinct from the larger one in the subsequent M to N transition. No evidence of a chromophore isomerization is seen in the M-1 to M-2 transition. Furthermore, the results show time-resolved reprotonation of the Schiff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.