Temperature dependence of light-induced proton movement in reconstituted purple membrane

Abstract

Bacteriorhodopsin (BR) was incorporated into phosphatidylcholine (PC) vesicles containing different amounts of other lipids. Under the conditions of nullified membrane potential, light-induced proton movement seemed to follow a kinetic scheme which assumed the existence of a proton-pumping inhibition process characterized by a rate constant, k I . The temperature dependence of both k I and the membrane proton leak rate constant ( k D ) obeyed a simple Arrhenius equation. The presence of cholesterol in the membrane significantly increased the activation energy ( E a ) of both the inhibition and leak process. However, further addition of phosphatidic acid (PA) suppressed the increase of E a associated with k I . The initial proton pumping rate ( R 0) of vesicles reconstituted with PC showed a bell-shaped temperature dependence with a maximum at ~20 °C. The addition of cholesterol abolished this dependence. These results suggest that the molecular origin of the inhibition process characterized by k I is different from that of R 0 or k D . The temperature dependence of the steady-state fluorescence polarization of dansylated bacteriorhodopsin in vesicles was also investigated. The polarization of the labels in the vesicles without cholesterol showed a bell-shaped temperature dependence with a maximum at ~20 °C. However, in the presence of cholesterol, the polarization increased linearly as temperature decreased. A comparison of these results with the observed proton movement in similarly reconstituted systems with unmodified protein indicates that membranes with a low fluidity and negatively charged surfaces enhance proton pumping efficiency of bacteriorhodopsin.