Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.assembly | metal protein | FeS cluster | NDUFAF2 | NDUFA12 P roton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) is a multisubunit membrane protein complex with a central function in aerobic energy metabolism (1, 2). Fourteen central subunits that harbor the bioenergetic core functions are conserved from bacteria to humans. In addition, eukaryotic complex I comprises around 30 accessory subunits of largely unknown function. The structures of bacterial and mitochondrial complex I were analyzed by X-ray crystallography and electron microscopy (3-6). The central subunits can be assigned to functional modules for NADH oxidation (N-module), ubiquinone reduction (Q-module), and proton pumping (P-module). The subunits forming the N-and Q-module harbor a chain of FeS clusters that connects the NADH oxidation site with the ubiquinone reduction site where the redox energy is released to drive proton translocation.The assembly of complex I subunits is a multistep process that proceeds via defined intermediates and is aided by a number of assembly factors (7). It also requires the concerted insertion of preformed FeS clusters into several subunits of the N-and Q-module (8). Dysfunction of complex I is the most frequent cause of mitochondrial disorders (9). Pathogenic mutations were identified not only in central subunits, encoded by either nuclear or mitochondrial DNA, but also in accessory subunits and assembly factors. The aerobic yeast Yarrowia lipolytica has been established as a yeast genetic model system to study structure and function of eukaryotic complex I, as well as complex I linked diseases (10).In this study, we focused on the accessory subunit NUMM of Y. lipolytica complex I. NUMM belongs to a limited subset of accessory subunits that is already found in α-proteobacteria and harbors a conserved putative Zn-binding motif, comprising three cysteines and one histidine in its C-terminal part (11). Zn binding to complex I was previously reported for bovine complex I but its functional relevance and the position of the Zn site remained elusive (12, 13). A poly...

Mitochondrial complex I has a molecular mass of almost 1 MDa and comprises more than 40 polypeptides. Fourteen central subunits harbour the bioenergetic core functions. We are only beginning to understand the significance of the numerous accessory subunits. The present review addresses the role of accessory subunits for assembly, stability and regulation of complex I and for cellular functions not directly associated with redox-linked proton translocation.

Mitochondrial complex I is an intricate 1MDa membrane protein complex with a central role in aerobic energy metabolism. The minimal form of complex I consists of fourteen central subunits that are conserved from bacteria to man. In addition, eukaryotic complex I comprises some 30 accessory subunits of largely unknown function. The gene for the accessory NDUFS4 subunit of human complex I is a hot spot for fatal pathogenic mutations in humans. We have deleted the gene for the orthologous NUYM subunit in the aerobic yeast Yarrowia lipolytica, an established model system to study eukaryotic complex I and complex I linked diseases. We observed assembly of complex I which lacked only subunit NUYM and retained weak interaction with assembly factor N7BML (human NDUFAF2). Absence of NUYM caused distortion of iron sulfur clusters of the electron input domain leading to decreased complex I activity and increased release of reactive oxygen species. We conclude that NUYM has an important stabilizing function for the electron input module of complex I and is essential for proper complex I function.

Complex 1(NADH:ubiquinone oxidoreductase) is the first and largest electron transport chain enzyme in bacteria and in the inner membrane of mitochondria. Complex 1 consists of two arms forming an "L" shape. One arm lies in the inner membrane of the mitochondria, while the other protrudes out into the mitochondrial matrix. After the matrix arm binds NADH, two electrons are transferred via a series of iron-sulfur clusters to ubiquinone with concomitant translocation of four protons across the membrane. The reduced ubiquinone is then transferred to complex III, cytochrome C is reduced and further processed in complex IV. In the process of oxidizing NADH, a total of 10 electrons are translocated across the membrane, creating a membrane potential that powers ATP production by complex V.