Photocurrents Generated by Bacteriorhodopsin Adsorbed on Thiol/Lipid Bilayers Supported by Mercury

Abstract

The kinetics of light-driven proton transport by bacteriorhodopsin (bR) were investigated over a broad pH range upon adsorbing purple membrane (PM) fragments on a mercury-supported mixed alkanethiol/phospholipid bilayer. The light-on and light-off capacitive photocurrents were measured under short-circuit conditions in the absence of photoartifacts. Using dioleoylphosphatidylcholine as the lipid monolayer, a bell-shaped curve of the peak current versus pH, with a maximum in the proximity of 6, was obtained. The analysis of the biphasic decay kinetics of the light-on and light-off currents allows an estimate of the pKa values for the steps releasing protons to, and taking up protons from, the bathing solution. In particular, the pKa values obtained from the light-off current (pK1 = 3.5, pK2 = 5.3, pK3 = 7.5, and pK4 = 9.0) suggest a mechanism similar to that proposed by Balashov et al. for dark adaptation, albeit in the opposite direction (Balashov, S. P.; Imasheva, E. S.; Govindjee, R.; Sheves, M.; Ebrey, T. G. Biophys. J. 1996, 70, 473). The time dependence of the light-on and light-off currents in the proximity of pH 6 is interpreted on the basis of both a simple equivalent circuit and a kinetic model making use of spectroscopic data available in the literature. When using dioleoylphosphatidylserine (DOPS) as the lipid monolayer, an inversion in the sign of both light-on and light-off currents, as well as a change in their shape and magnitude, was observed by increasing the pH above 9 and then, at all pH values from 9 to 1, by subsequently decreasing the pH on the same mercury-supported mixed alkanethiol/DOPS bilayer. The normal situation was restored only by adding sodium azide. This inversion in current and the notable hysteresis observed under these conditions are critically discussed.