Triphenyltetrazolium and its derivatives are anisotropic inhibitors of energy transduction in oxidative phosphorylation in rat liver mitochondria.

Triphenyltetrazolium and its derivatives inhibited energy transduction in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. Triphenyltetrazolium incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. Triphenyltetrazolium also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced sites on the membranes for triphenyltetrazolium and its derivatives. The maximum amounts of energy-dependent binding sites for triphenyltetrazolium on membranes energized with succinate and ATP, respectively, were 14 and 4 nmol/mg protein. Triphenyltetrazolium also induced H+ ejection from the energized membranes. The maximum amounts of H+ ejection from membranes energized with succinate and ATP, respectively, were 4 and 2.4 nmol/mg protein. Triphenyltetrazolium also decreased the membrane potential up to about half the control value and caused shrinkage of mitochondria in an energy-dependent fashion. Comparison of the Hammett's sigma constants of triphenyltetrazolium derivatives with various substituents on the 3-benzene ring showed that lower concentrations of triphenyltetrazolium derivatives with a stronger positive charge were required for inhibition of energy transduction. The present findings show that triphenyltetrazolium and its derivatives act as anisotropic inhibitors of energy transduction by binding to negative charges created on the outer side (C-side) of energized mitochondria, and that the positive charge of these inhibitors is one of important factors for their inhibitory activity. These negative charges may be an essential part of the H+ pump.