Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

Abstract

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.

Keywords: FeS cluster; NDUFA12; NDUFAF2; assembly; metal protein.

Fig. 1.

Migration profiles of complex I subunits NUMM, N7BM, and assembly factor N7BML. The migration profiles were derived from the complexome profiling analysis of intact mitochondria from the parental strain and the deletion strains for NUMM, N7BM, and N7BML shown in Fig. S2. Each panel shows the relative abundance of the indicated proteins in relation to the migration distance in the BN-gel; mass scale calibrated based on complex I, dimeric complex IV and monomeric and dimeric ATP synthase.

Fig. 2.

Subunit composition of purified complex I from parental, numm∆ and n7bml∆ strains. Complex I was chromatographically purified and resolved by dSDS PAGE; protein spots were stained with silver. The assignment of subunits NUMM, N7BM and complex I assembly factor N7BML labeled in violet was confirmed by LC-ESI MS/MS; assignment of all other subunits labeled in green is based on (35).

Fig. 3.

Position of the Zn-binding site in complex I. Structure of Yarrowia lipolytica complex I showing the position of the C-terminal NUMM domain and its bound Zn. Complex I subunits are represented in gray except for subunits TYKY (green) 49-kDa (blue), 75-kDa (yellow), and the fitted model of the C-terminal domain of NUMM (red). The Zn-specific anomalous Fourier map is contoured at 8σ and is represented in red. (Upper Left Inset) The position of the Zn is represented in context of the iron–sulfur cluster chain involved in electron transfer. Distances are measured in Å. (Lower Right Inset) Detailed view of the Zn-binding site is shown; NUMM is adjacent to elements from TYKY, 49-kDa and 75-kDa subunits.

Fig. 4.

EPR spectra of complex I isolated from the numm∆ and n7bml∆ strains. EPR spectra of purified complexes were recorded at different temperatures and microwave power settings after reduction with 1.8 mM NADH. At 12 K, 1 mW microwave power clusters N1–N4 are detectable in the control. Although n7bmlΔ CI displayed a spectrum identical to the parental one, in complex I of nummΔ EPR signal N4 is missing and intensities of signals N2 and N1 are diminished by 50% and 25%, respectively. At 8 K, 10 mW, conditions more suitable for detection of N4, the lack of N4 EPR signal in nummΔ is evident. Under these conditions N2 is hardly detectable due to distinct power saturation. Spectra were normalized to an equal signal amplitude of N3. Characteristic contributions of individual iron–sulfur clusters to the EPR spectrum are indicated.