FOURIER-TRANSFORM INFRARED DOUBLE-FLASH EXPERIMENTS RESOLVE BACTERIORHODOPSINS M-1 TO M-2 TRANSITION

The orientation of the central proton-binding site, the protonated Sch iff 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 sw itch, takes place in the M-1 to M-2 transition. To resolve the molecul ar events in this M-1 to M-2 transition, we performed double-flash exp eriments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermedia te back into the BR ground state. For short delay times between initia ting 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 m olecules are in the M-2 intermediate. The BR-M-1 and BR-M-2 difference spectra are monitored with nanosecond step-scan Fourier transform inf rared 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 in termediates represent two different proton accessibility configuration s of the Schiff base. The results show only a minute movement of one o r two peptide bonds in the M-1 to M-2 transition that changes the inte raction of the Schiff base with Y185. This backbone movement is distin ct from the larger one in the subsequent M to N transition. No evidenc e of a chromophore isomerization is seen in the M-1 to M-2 transition. Furthermore, the results show time-resolved reprotonation of the Schi ff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.