Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I Katarzyna Kmitaa, Christophe Wirthb, Judith Warnauc, Sergio Guerrero-Castillod,e, Carola Hunteb, Gerhard Hummerc, Ville R. I. Kailaf, Klaus Zwickerg, Ulrich Brandtd,e, and Volker Zickermanna,e,1 aStructural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe University, 60438 Frankfurt am Main, Germany; bInstitute for Biochemistry and Molecular Biology, ZBMZ, BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg Germany; cDepartment of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany; dNijmegen Center for Mitochondrial Disorders, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands; eCluster of Excellence Frankfurt “Macromolecular Complexes”, Goethe University, 60438 Frankfurt am Main, Germany; fDepartment Chemie, Technische Universität München, 85747 Garching, Germany; and gInstitute of Biochemistry I, Medical School, Goethe University, 60590 Frankfurt am Main, Germany Edited by Hartmut Michel, Max Planck Institute of Biophysics, Frankfurt, Germany, and approved March 11, 2015 (received for review December 19, 2014) Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase harbors a conserved putative Zn-binding motif, comprising three (respiratory complex I) comprises more than 40 polypeptides and cysteines and one histidine in its C-terminal part (11). Zn binding contains eight canonical FeS clusters. The integration of subunits to complex I was previously reported for bovine complex I but its and insertion of cofactors into the nascent complex is a com- functional relevance and the position of the Zn site remained elu- plicated multistep process that is aided by assembly factors. We sive (12, 13). A poly-alanine model of the orthologous 13-kDa show that the accessory NUMM subunit of complex I (human subunit was tentatively fitted into the electron microscopic NDUFS6) harbors a Zn-binding site and resolve its position by structure of bovine complex I but Zn binding was not shown (5). X-ray crystallography. Chromosomal deletion of the NUMM gene In human, mutations leading to the loss of the orthologous or mutation of Zn-binding residues blocked a late step of complex I NDUFS6 subunit or exchange of one of the three conserved assembly. An accumulating assembly intermediate lacked acces- cysteine residues were shown to cause fatal diseases (14, 15). A sory subunit N7BM (NDUFA12), whereas a paralog of this subunit, mouse model to study complex I-linked diseases was generated the assembly factor N7BML (NDUFAF2), was found firmly bound by knock down of the corresponding gene (16). instead. EPR spectroscopic analysis and metal content determina- We analyzed the functional significance of the Zn-binding site tion after chromatographic purification of the assembly interme- of the NUMM subunit in Y. lipolytica complex I and determined diate showed that NUMM is required for insertion or stabilization its position in the X-ray structure of the enzyme complex by Zn of FeS cluster N4. anomalous scattering. We found that deletion of the NUMM gene or site-directed mutagenesis of Zn ligands compromised a assembly | metal protein | FeS cluster | NDUFAF2 | NDUFA12 late step of complex I biogenesis that is required for stable in- sertion of FeS cluster N4. roton-pumping NADH:ubiquinone oxidoreductase (respira- Ptory complex I) is a multisubunit membrane protein complex Results with a central function in aerobic energy metabolism (1, 2). Deletion of the NUMM Subunit Impairs Complex I Biogenesis. To Fourteen central subunits that harbor the bioenergetic core investigate the function of the NUMM subunit and its proposed functions are conserved from bacteria to humans. In addition, Zn-binding site (11) (Fig. S1A) in respiratory complex I we eukaryotic complex I comprises around 30 accessory subunits of largely unknown function. The structures of bacterial and mito- Significance chondrial complex I were analyzed by X-ray crystallography and electron microscopy (3–6). The central subunits can be assigned Respiratory complex I is the largest membrane protein complex to functional modules for NADH oxidation (N-module), ubi- in mitochondria and has a central function in energy metabo- quinone reduction (Q-module), and proton pumping (P-module). lism. Numerous human diseases are linked with complex I The subunits forming the N- and Q- module harbor a chain of dysfunction or assembly defects. The concerted assembly of FeS clusters that connects the NADH oxidation site with the more than 40 subunits and the insertion of cofactors is aided by ubiquinone reduction site where the redox energy is released to specific chaperones. In addition to eight FeS clusters, complex I drive proton translocation. comprises a Zn-binding site of unknown function. Combining The assembly of complex I subunits is a multistep process that X-ray structural analysis of complex I crystals with quantum proceeds via defined intermediates and is aided by a number of chemical modeling and proteomic and spectroscopic analysis assembly factors (7). It also requires the concerted insertion of a purified assembly intermediate, we show that accessory of preformed FeS clusters into several subunits of the N- and subunit NUMM (human ortholog NDUFS6) binds Zn at the in- Q-module (8). Dysfunction of complex I is the most frequent terface of two functional modules of the enzyme complex and cause of mitochondrial disorders (9). Pathogenic mutations were is required for a specific step of complex I biogenesis. BIOCHEMISTRY identified not only in central subunits, encoded by either nuclear or mitochondrial DNA, but also in accessory subunits and as- Author contributions: K.K. and V.Z. designed research; K.K., C.W., J.W., S.G.-C., C.H., G.H., V.R.I.K., K.Z., U.B., and V.Z. performed research; K.K., C.W., J.W., S.G.-C., C.H., G.H., sembly factors. The aerobic yeast Yarrowia lipolytica has been V.R.I.K., K.Z., U.B., and V.Z. analyzed data; and K.K., C.W., J.W., S.G.-C., C.H., G.H., V.R.I.K., established as a yeast genetic model system to study structure K.Z., U.B., and V.Z. wrote the paper. and function of eukaryotic complex I, as well as complex I linked The authors declare no conflict of interest. diseases (10). This article is a PNAS Direct Submission. In this study, we focused on the accessory subunit NUMM of 1To whom correspondence should be addressed. Email: [email protected]. Y. lipolytica complex I. NUMM belongs to a limited subset of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. accessory subunits that is already found in α-proteobacteria and 1073/pnas.1424353112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1424353112 PNAS | May 5, 2015 | vol. 112 | no. 18 | 5685–5690 Downloaded by guest on September 23, 2021 Table 1. Complex I content and ubiquinone reductase activities detect by mass spectrometry because they are highly hydrophobic in mitochondrial membranes and lack suitable cleavage sites (18). Complex I was mainly found † Strain Complex I content*, % Complex I activity ,% in monomeric form but also assembled into higher molecular mass supercomplexes (20); membrane arm subcomplexes, very nummΔ 41 44 likely representing assembly intermediates, were detected at n7bmlΔ 30 83 lower molecular mass (Fig. S2). n7bmΔ 26 35 In strain nummΔ we detected assembly of nearly all expected C97ANUMM 54 63 subunits (Fig. S2). However, we also found a higher fraction of H110ANUMM 60 76 unassembled subunits and subcomplexes of the peripheral arm. C125ANUMM 55 63 Notably, accessory subunit N7BM did not assemble into complex C128ANUMM 54 37 I and was almost exclusively found in free form at the migration *Complex I content was assessed as nonphysiological NADH:hexaamminer- front in the Blue-Native gel (Fig. 1 and Fig. S2). On the other uthenium (III) oxidoreductase activity and compared with parental strain hand, an additional protein was found to be associated with the − − GB20 for chromosomal deletions (100% = 1.41 μmol∙min 1∙mg 1) and with nearly complete complex I subassembly in nummΔ.The27.6-kDa plasmid complemented nummΔ deletion strain for NUMM point mutants protein, mainly detected in free form in the parental strain, was (100% = 1.14 μmol∙min−1∙mg−1). identified as YALI0D27082p in the Y. lipolytica genome (21) and †Inhibitor sensitive dNADH:decylubiquinone oxidoreductase activity was − − has sequence homology to a known complex I assembly factor normalized to complex I content (100% = 0.55 μmol∙min 1 CI 1 for GB20 Homo sapiens Bos taurus −1 −1 called NDUFAF2 in ,B17.2Lin ,and13.4L to compare with chromosomal deletions and 100% = 0.46 μmol∙min CI Neurospora crassa B Δ in (Fig. S1 ) (22, 23). Because its N-terminal for plasmid complemented numm deletion to compare with NUMM Y. lipolytica point mutants). half was also clearly homologous to complex I sub- unit N7BM (Fig. S1D), we named the protein N7BML for N7BM like. Indeed, N7BM is also homologous to subunits NDUFA12, generated a chromosomal deletion of the entire reading frame B17.2, and nuo13.4 from H. sapiens, B. taurus,andN. crassa,re- of the corresponding NUMM gene by homologous recombina- spectively (Fig. S1D) that are paralogs of the respective assembly tion. Complex I content of mitochondrial membranes from the factors indicated above (22–24). deletion strain was decreased to 41% as assessed by NADH: hexammineruthenium (HAR) oxidoreductase activity and nor- A Functional Link Between NUMM, N7BM, and N7BML in Complex I malized dNADH:decylubiquinone (DBQ) oxidoreductase activity Assembly. To investigate the role of N7BML in the complex I dropped to 44% of the parental strain (Table 1). Complementa- assembly process and its functional relation to its paralog, complex tion with variants carrying mutations in the putative Zn ligating I subunit N7BM, we deleted the corresponding genes from the residues C97, H110, C125, C128 did not restore complex I content Y.