Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.

Dysfunctions in mitochondrial complex I (NADH:ubiquinone oxidoreductase) are both genetically and clinically highly diverse and a major cause of human mitochondrial diseases. The genetic determinants of individual clinical cases are increasingly being described, but how these genetic defects affect complex I on the molecular and cellular level, and have different clinical consequences in different individuals, is little understood. Furthermore, without molecular-level information innocent genetic variants may be misassigned as pathogenic. Here, we have used a yeast model system (Yarrowia lipolytica) to study the molecular consequences of 16 single amino acid substitutions, classified as pathogenic, in the NDUFV1 subunit of complex I. NDUFV1 binds the flavin cofactor that oxidizes NADH and is the site of complex I-mediated reactive oxygen species production. Seven mutations caused loss of complex I expression, suggesting they are detrimental but precluding further study. In two variants complex I was fully assembled but did not contain any flavin, and four mutations led to functionally compromised enzymes. Our study provides a molecular rationale for assignment of all these variants as pathogenic. However, three variants provided complex I that was functionally equivalent to the wild-type enzyme, challenging their assignment as pathogenic. By combining structural, bioinformatic and functional data, a simple scoring system for the initial evaluation of future NDUFV1 variants is proposed. Overall, our results broaden understanding of how mutations in this centrally important core subunit of complex I affect its function and provide a basis for understanding the role of NDUFV1 mutations in mitochondrial dysfunction.