Charge motions during the photocycle of pharaonis halorhodopsin

Oriented gel samples were prepared from halorhodopsin-containing membranes from Nathronobacterium pharaonis, and their photoelectric responses to lase r flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but hig h-sulfate, concentration. The chloride concentration-dependent transition b etween the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that repor ted by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A, D. Kaul en. 1998. FEES Lett. 427:59-63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the vol tage signal. Although no negative electric signal was observed at high chlo ride concentration, the calculated electrogenicity of the K intermediate wa s negative, and very similar to that of bacteriorhodopsin. The late photocy cle intermediates (O, HR', and HR) had almost equal electrogenicities, expl aining why no chloride-dependent time constant was identified earlier by Ka laidzidis et al. The calculated electrogenicities, and the spectroscopic in formation for the chloride release and uptake steps of the photocycle, sugg est a mechanism for the chloride-translocation process in this pump.