Time-resolved detection of transient movement of helices F and G in doublyspin-labeled bacteriorhodopsin

Photo-excited structural changes of the light-driven proton pump bacteriorh odopsin were monitored using double-site-directed spin labeling combined wi th electron paramagnetic resonance (EPR) spectroscopy. The inter-spin dista nces between nitroxides attached at residue positions 100 and 226, 101 and 160, and 101 and 168 were determined for the BR initial state and the trapp ed M photo-intermediate. Distance changes that occur during the photocycle were followed with millisecond time resolution under physiological conditio ns at 293 K. The kinetic analysis of the EPR data and comparison with the a bsorbance changes in the visible spectrum reveal an outward movement of hel ix F during the late M intermediate and a subsequent approach of helix G to ward the proton channel. The displacements of the cytoplasmic moieties of t hese helices amount to 0.1-0.2 nm. We propose that the resulting opening of the proton channel decreases the pK of the proton donor D96 and facilitate s proton transfer to the Schiff base during the M-to-N transition.