We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (ρ° cells) by transforming them with the alternative oxidase Aox of Emericella nidulans . Cotransforming ρ° cells with the NADH dehydrogenase of Saccharomyces cerevisiae , Ndi1 and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of ρ° cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD + . Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes.