Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1′-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN′N′-Tetramethyl-p-phenylenediamine does not behave as a simple electron carrier. During NNN′N′-tetramethyl-p-phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN′N′-tetramethyl-p-phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN′N′-tetramethyl-p-phenylenediamine is used as a mediator.
AN IMPROVED CATHODE FOR THE MEASUREMENT OF PHOTOSYNTHETIC OXYGEN EVOLUTION BY ISOLATED CHLOROPLASTS
