NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (B20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc 1 complex. We have examined the interaction of atovaquone with the bc 1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc 1 complex competitively with apparent K i ‫؍‬ 9 nM, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc 1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc 1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc 1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc 1 complex (K i ‫؍‬ 80 nM) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (K i ‫؍‬ 100 nM) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc 1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.

We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit of [NiFe] hydrogenases. None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of ironsulfur cluster N2, a prosthetic group not located in the 49-kDa subunit. In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster. We propose that the fold around the hydrogen reactive site of [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region. We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.Oxidative phosphorylation is a universal process that converts most of the energy provided by foodstuffs into the general energy source ATP (1). During this process electrons pass through a series of membrane-bound multiprotein complexes that translocate protons across the membrane. The resulting proton motive force is used by ATP synthase to make ATP (2). Complex I (NADH:ubiquinone oxidoreductase) is the first of these electron transfer complexes and accounts for up to 40% of the pumped protons. In human mitochondria, complex I is regarded as a major source of deleterious reactive oxygen species. Hereditary and acquired defects affecting this multiprotein complex have been implicated in numerous degenerative diseases and seem to promote aging (3). The physiological and medical importance of complex I contrasts sharply with our still rather limited knowledge about the molecular structure and the catalytic mechanism of this multisubunit membrane protein with a total mass close to 1000 kDa (4).The core of complex I is formed by 14 central subunits of which in animals and most fungi seven are encoded by the mitochondrial genome (5). The mitochondrial enzyme contains up to 29 additional "supernumerary" subunits. As bacterial enzymes comprise only the 14 central subunits, these must contain all functional modules required for electron and proton transfer. Except for the electron entry point that is formed by FMN and iron-sulfur center N3 in the 51-kDa subunit, the organization of these functional modules, the location and number of prosthetic groups, and the location of the ubiquinone substrate binding site are largely unknown. In particular, the function of nine of the 14 central subunits that carry none of the known prosthetic g...