Application of N,N,N',N'-Tetramethyl-<i>p</i>-Phenylenediamine and α,ω-Hexadecanedioic Acid for Determination of the H<sup>+</sup>/O Ratios of Complexes III and IV of the Liver Mitochondrial Respiratory Chain under Free Respiration Conditions

Stimulation of mitochondrial respiration in state 4 without changes in passive proton leakage is known to be accompanied by a decrease in the H+/O ratio (Luvisetto et al., 1991. J. Biol. Chem. 266, 1034–1042). In the present work, it was found that during the oxidation of succinate by liver mitochondria, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and α,ω-hexadecanedioic acid (HDA) effectively stimulate respiration in state 4, and their action, unlike the protonophore uncoupler DNP, is not caused by an increase in the proton conductivity of the inner membrane. Under these conditions, TMPD and HDA do not significantly affect the efficiency of oxidative ATP synthesis and energy transformation by complex IV (cytochrome c oxidase). The data obtained are considered as evidence that during the oxidation of succinate by liver mitochondria, TMPD and HDA selectively disable ETC complex III from energy transformation. It is theoretically substantiated that, under these conditions, the H+/O ratio can be determined based on the ratio of respiratory rates in the absence and presence of TMPD and HDA. Based on this model, we considered the change in the H+/O ratio depending on the stimulation of mitochondrial respiration in state 4 by TMPD and HDA. It has been established that under the influence of TMPD or HDA, the value of the H+/O ratio decreases during the oxidation of succinate from 6 to the limiting values of 2. We conclude that in liver mitochondria during free respiration, in contrast to the oxidative ATP synthesis, the values of the H+/O ratio are 4 and 2 for complexes III and IV, respectively.