External electric control of the proton pumping in bacteriorhodopsin

Comparative analysis of the photoelectric response of dried films of purple membranes (PM) depending on their degree of orientation is presented. Time dependence of the photo-induced protein electric response signal (PERS) of oriented and non-oriented films to a single laser pulse in the presence of the external electric field (EEF) was experimentally determined. The signal does not appear in the non-oriented films when the EEF is absent, whereas the PERS of the oriented PM films demonstrates the variable polarity on the microsecond time scale. In the presence of the EEF the PERS of the non-oriented film rises exponentially preserving the same polarization. The polarization of the PERS changes by changing the polarity of the EEF with no influence on the time constant of the PERS kinetics. The EEF effect on the PERS of the oriented films is more complicated. By subtracting the PERS when EEF ≠ 0 from the PERS when EEF = 0 the resulting signal is comparable to that of the non-oriented films. Generalizing the experimental data we conclude that the EEF influence is of the same origin for the films of any orientation. To explain the experimental results the two-state model is suggested. It assumes that the EEF directionally changes the pK_a values of the Schiff base (SB) and of the proton acceptor aspartic acid D85 in bacteriorhodopsin. Because of that the SB→D85 proton transfer might be blocked and consequently the L→M intermediate transition should vanish. Thus, on the characteristic time scale τ _{L → M} ≈ 30 μs; both intermediates, the M intermediate, appearing under normal conditions, and the L intermediate as persisting under the blocked conditions when D85 is protonated, should coexist in the film. The total PERS is a result of the potentials corresponding to the electrogenic products of intermediates L and M that are of the opposite polarity. It is concluded that the ratio of bacteriorhodopsin concentrations corresponding to the L and M intermediates is driven by the EEF and, consequently, it should define the PERS of the non-oriented films. According to this model the orientation degree of the film could be evaluated by describing the PERS.