The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity

Abstract

Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I.

Fig. 1.

Proteomic analysis of complex I from the parental strain and strain nb4mΔ. (A) Coomassie-stained 4–16% blue native (BN)-PAGE of dodecylmaltoside-solubilized mitochondrial membranes and affinity-purified complex I from parental strain and strain nb4mΔ. Roman numerals indicate complexes of oxidative phosphorylation in monomeric or dimeric form. (B) Silver-stained dSDS-PAGE of affinity-purified complex I from parental strain and strain nb4mΔ; blue captions, central subunits; black captions, accessory subunits; NB4M and ACPM1 are highlighted in green. NB4M and the acyl carrier protein ACPM1 are absent in complex I from strain nb4mΔ (red circles). Please note that the assignment of subunits NUNM and NUMM was corrected and differs from that in ref. .

Fig. 2.

EPR spectrum of purified complex I from strain nb4mΔ. EPR spectra of complex I purified from parental and strain nb4mΔ were recorded using the following parameters: microwave frequency, 9.47 GHz; microwave power, 1 mW; modulation amplitude, 0.64 mT; and modulation frequency, 100 kHz. Samples were reduced with NADH (2 mM), and the spectra were recorded at 12 K. Characteristic signal contributions of the four EPR visible iron–sulfur clusters of complex I (N1, N2, N3, N4) detectable under these conditions are indicated above the spectra. For comparison, spectra were normalized on signal intensities of clusters N3 and N4. The difference spectrum largely shows the pure signature of the terminal iron–sulfur cluster N2.

Fig. 3.

Ubiquinone reductase activities and complex I content of mitochondrial membranes from parental strain, strain nb4mΔ, and site-directed mutants. Complex I content (dark gray bars) assessed as nonphysiological NADH:hexaammineruthenium(III) oxidoreductase activity of mitochondrial membranes (100% = 1.38 µmol⋅min⁻¹⋅mg⁻¹). Relative inhibitor sensitive deamino-NADH:decylubiquinone oxidoreductase activities (light gray bars) normalized to complex I content (100% = 0.61 µmol⋅min⁻¹⋅mg⁻¹).

Fig. 4.

Single-particle 2D electron microscopic analysis of complex I purified from parental strain and strain nb4mΔ. Average images of the subcomplex (I) and the holocomplex (II), followed by the difference image (II-I). The circled numbers indicate the previously described subdomains 1, 2, 5, and 6 of the peripheral arm (3). (Scale bar, 10 nm.)

Fig. 5.

Structural modeling of subunits NB4M and ACPM1. (A) Overlay of the X-ray structure of T. thermophilus complex I (ribbon representation, PDB file 4HEA) (16) and the envelope of Y. lipolytica complex I determined by single-particle EM (3). NADH oxidation module (N, orange), ubiquinone reduction module (Q, blue), proton pump modules (green, P_P and P_D), and iron-sulfur cluster N2 (yellow circle). The blue frame indicates the zoom window for B and C. (B) Overlay of X-ray electron density map (4) (gray surface, contour level 1.9) with an envelope of Y. lipolytica complex I (3) (gray mesh), and X-ray structure of T. thermophilus complex I; 49-kDa subunit (NQO4, blue, N-terminal β-sheet highlighted in yellow), PSST subunit (NQO6, cyan), TYKY subunit (NQO9, orange), 30-kDa subunit (NQO5, hot pink), loop connecting helices 1 and 2 of subunit ND3 (NQO7, red); structure of ACP from E. coli [PDB file 2FAE) (18), pink; decanoyl phosphopantetheine, yellow stick representation] fitted to electron density in the distal part of subdomain 6 (magenta), electron density features tentatively assigned to subunit NB4M are highlighted in green (cf. Fig. S8). (C) Alternative view of B rotated by about 180°.