H+ production by antimycin-inhibited mitochondria

Area of Science:

• Mitochondrial respiration
• Cellular energy production
• Biochemistry

Background:

• Mitochondria are key organelles for cellular energy production through oxidative phosphorylation.
• Cytochrome c oxidase is the terminal enzyme in the electron transport chain, crucial for oxygen consumption and proton pumping.
• Antimycin A is a known inhibitor of the mitochondrial electron transport chain at Complex III.

Purpose of the Study:

• To investigate the presence and role of endogenous reductants in liver mitochondria.
• To determine the stoichiometry of proton translocation relative to oxygen consumption in the presence of an endogenous reductant.
• To assess the contribution of cytochrome oxidase to proton translocation independent of known substrates.

Main Methods:

• Oxygen-pulse experiments were conducted on liver mitochondria isolated from young rats.
• Mitochondria were treated with antimycin A in the absence of added substrates.
• Competition assays were performed using ferrocyanide and ascorbate/NNN'N'-tetramethyl-p-phenylenediamine as alternative electron donors.

Main Results:

• An endogenous hydrogenated reductant was detected on the oxygen side of the antimycin inhibition site, in a 20-50 fold molar excess over cytochrome aa3.
• Oxidation of this reductant yielded a proton translocated:oxygen atom uptake ratio of 2.1 +/- 0.2.
• The endogenous reductant competed with external electron donors, indicating its role in the respiratory chain.
• Respiration-dependent proton translocation, consistent with cytochrome oxidase activity, was observed even after accounting for endogenous substrate contributions.

Conclusions:

• Liver mitochondria possess an endogenous reductant that participates in electron transfer and proton translocation.
• This endogenous reductant plays a significant role in mitochondrial respiration and energy coupling.
• Cytochrome oxidase exhibits proton pumping activity that is partially independent of the identified endogenous reductant and substrate availability.