Generation of high delta psi in respiring submitochondrial particles by steady-state accumulation of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine.

Abstract

Oxidation of N,N,N′,N′‐tetramethyl‐p‐phenylenediame [Ph(NMe2)2] plus ascorbate by inside out submitochondrial particles is associated with accumulation of the oxidized form [Ph(NMe2)2+] in the inner space of the vesicles. The concentration of [Ph(NMe2)2+]in, can exceed by 5–6‐fold the concentration of Ph(NMe2)2 added to the reaction mixture.The steady‐state concentration of [Ph(NMe2)2+]in, is a function of the rate of Ph(NMe2)2 influx driven by concentration gradient, the rate of Ph(NMe2)2+ oxidation, determined by its concentration, and Ph(NMe2)2+ efflux. The last rate is determined by Ph(NMe2)2+ concentration gradient and the membrane potential component (δɛ) of the proton‐motive force. Collapse of δɛ by permeant anions, ionophores or carbonylcyanide p‐trifluoromethoxyphenylhydrazone, builds up the internal concentration of [Ph(NMe2)2+]in till the concentration gradient can support an efflux identical to the rate of Ph(NMe2)2+ formation (equivalent to the rate of respiration). This relationship is analyzed according to Nernst‐Planck equation and δɛ calculated by this method amounts to 100–120 mV (positive inside). This value is corroborated by equilibrium dialysis. The measured membrane potential is built by the positive charge of Ph(NMe2)2+itself plus that of accumulated H+. Still, the contribution of Ph(NMe2)2+ is about half of the total electric force.At high Ph(NMe2)2 concentrations (0.5–5 mM), substantial electron flux through cytochrome oxidase bypasses cytochrome c and is carried by direct interaction of Ph(NMe2)2 with the terminal oxidase.