Light-induced delta pH of envelope vesicles containing halorhodopsin measured by use of a spin probe

The spin-labeled amine, 4-amino-2,2,6,6-tetramethyl-piperidino-N-oxyl was used to measure the photoinduced delta pH in the envelope vesicles derived from mutant cells of Halobacterium halobium. The cells contain halorhodopsin (hR) and are spectroscopically free from bacteriorhodopsin (bR). The EPR signals from the extravesicular populations of the probe was quenched by ferricyanide. The membrane bound population of the probe was less than 1% of the amount of the probe entrapped within the vesicle. Hence, we can monitor continuously the concentration of the intravesicular populations of the probe and delta pH can be calculated from the line height of the signal. The kinetic analysis indicated that the time course of the change in the EPR signal represents faithfully delta pH of the vesicle. At pH 7.5, the photoinduced delta pH showed a maximum. The kinetic constant of pH change also displayed a peak at this pH. Addition of uncoupler did not increase either the rate nor magnitude of delta pH at this pH, but at other pH, especially at lower pH, uncoupler showed its effect. A theoretical equation was derived which correlated the photocycle of hR with delta pH. According to the analysis by use of this equation, the time constant of the photocycle was suggested to be constant in pH ranging from 5.5 to 8.25. The maximum in photoinduced delta pH observed at pH 7.5 was concluded to be due to the permeability dependence of ions, especially of H+ on pH. The temperature dependence of delta pH was measured. As the temperature decreased, delta pH was, surprisingly, found to be increased. The rate constant of the photocycle analyzed by the equation, however, decreased with decrease of temperature.