Abstract

The efficiency of oxidative phosphorylation in mitochondria is fundamentally linked to the proton-to-oxygen (H+/O) stoichiometry of the electron transport chain (ETC) complexes. This study aimed to precisely determine the H+/O ratios for Complex III and Complex IV of the liver mitochondrial respiratory chain under conditions of "free respiration," which mimics the physiological state of uncoupled electron flow. We employed a novel methodological approach utilizing N,N,N',N'-Tetramethyl-p-Phenylenediamine (TMPD) as an artificial electron donor to Complex IV, and α,ω-Hexadecanedioic Acid (HDDA) to selectively modulate Complex III activity. Isolated rat liver mitochondria were used to measure oxygen consumption and proton translocation simultaneously. The results demonstrate that the H+/O ratio for Complex IV, when supplied with electrons via TMPD, is approximately 2.0, while the ratio for Complex III, under the influence of HDDA, is determined to be 2.0. These findings suggest that the total H+/O ratio for the segment spanning Complexes III and IV is 4.0, challenging some previously reported values and providing a more accurate assessment of proton pumping efficiency in the uncoupled state. This refined understanding of ETC stoichiometry is crucial for modeling mitochondrial energy metabolism and understanding the bioenergetic defects associated with various pathologies.