Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of watersoluble [NiFe] hydrogenases.Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the first of five complexes of oxidative phosphorylation in the mitochondrial inner membrane. In complex I, two electrons are transported from NADH to ubiquinone via FMN and eight ironsulfur clusters (1). In this process four protons are translocated across the membrane (2). The mechanism of this process and the location and nature of the ubiquinone binding sites are still unclear (3). Evidence obtained with bovine heart submitochondrial particles strongly suggests that iron-sulfur cluster N2 is directly involved in ubiquinone reduction: based on paramagnetic interaction, the distance between an ubisemiquinone radical and cluster N2 was estimated to be 8-11 Å (4). Subunit PSST is the most likely carrier of iron-sulfur cluster N2. Site-directed mutagenesis of the homologue subunits in Escherichia coli (5) and Neurospora crassa (6) indicated that three cysteines of subunit PSST are ligands for cluster N2. These cysteines correspond to positions 86, 150, and 180 in the PSST homologous NUKM subunit of Yarrowia lipolytica. A glutamate residue (Glu-89 in Y. lipolytica) had been proposed as the fourth ligand (7), but this could be excluded by site-directed mutagenesis (8). Thus, the fourth ligand of cluster N2 is still not known. Albracht and co-workers (9) have proposed that complex I contains two iron-sulfur clusters N2 that have identical electron paramagnetic resonance (EPR) spectra and are bound by the two ferredoxin-like Fe 4 S 4 binding motifs in the TYKY subunit. However, these two clusters in the TYKY subunit have been shown to be EPR-silent and designated N6a and N6b by Friedrich and coworkers (10). Ubiquinone-analogous...