The obligate aerobic yeast Yarrowia lipolytica has been established as a powerful model system for the analysis of mitochondrial complex I. Using a combination of genomic and proteomic approaches, a total of 37 subunits was identified. Several of the accessory subunits are predicted to be STMD (single transmembrane domain) proteins. Site-directed mutagenesis of Y. lipolytica complex I has provided strong evidence that a significant part of the ubiquinone reducing catalytic core resides in the 49 kDa and PSST subunits and can be modelled using X-ray structures of distantly related enzymes, i.e. water-soluble [NiFe] hydrogenases from Desulfovibrio spp. Iron–sulphur cluster N2, which is related to the hydrogenase proximal cluster, is directly involved in quinone reduction. Mutagenesis of His²²⁶ and Arg¹⁴¹ of the 49 kDa subunit provided detailed insight into the structure–function relationships around cluster N2. Overall, our findings suggest that proton pumping by complex I employs long-range conformational interactions and ubiquinone intermediates play a critical role in this mechanism.