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. M itochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase, EC 1.6.5.3) is a 1-MDa membrane protein complex with a central function in cellular energy conversion (1). Redox-linked proton translocation by complex I contributes to the electrochemical proton gradient across the inner mitochondrial membrane that drives ATP synthesis by ATP synthase. Complex I dysfunction caused by mutations or toxins is associated with a number of neuromuscular and neurodegenerative human disorders, such as Parkinson's disease. Fourteen central subunits are conserved from bacteria to humans that harbor the core function of energy conversion. In eukaryotes, a substantial fraction of the mass of the holoenzyme is contributed by so-called accessory subunits (2). A broad range of functions from structural reinforcement to regulation of the enzyme complex has been suggested for the accessory subunits, but in most cases, their specific role remains unresolved. Electron microscopy (EM) (3) and X-ray crystallographic analysis of mitochondrial complex I at 6.3 Å resolution (4) revealed the arrangement of functional modules within the L-shaped complex of a highly hydrophobic membrane arm consisting of the proximal and distal pump-modules (P P and P D modules) and a hydrophilic peripheral arm extruding into the mitochondrial matrix that comprises the NADH oxidation and ubiquinone reduction modules (N and Q modules). However, information on the structure and position of individual accessory subunits is still very limited.In this study, we focused on accessory complex I subunit NB4M (alternative designations NDUFA6 and B14). NB4M/ NDUFA6 belongs to the Complex1_LYR family of LYRM proteins (5-7) that is characterized by a motif comprising an N-terminal leucine-tyrosine-arginine sequence located upstream of several conserved arginines and an invariant phenylalanine. The human genome contains at least 11 proteins of the LYRM superfamily, and the mammalian complex I subunit NDUFA6 is identical to LYR motif containing protein 6 (LYRM6). Several other LYRM proteins were shown ...

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

“…Previous studies demonstrate that ACP, in addition to its central role in mtFAS, forms a complex with LYRM3 and LYRM6 of Respiratory Complex I (17,18,(21)(22)(23). Similar to the SDA ec complex, the LYR motif and invariant Phe residue are required to anchor ACP to LYRM6 and to produce a functional complex (20). Overlay of ISD11-ACP with previously determined LYR-ACP structures reveals that all three LYR proteins have the same relative orientation to ACP (Fig.…”

“…Although the incorporation of ACP is a recently discovered component for the eukaryotic Fe-S cluster assembly machinery, its inclusion follows an emerging paradigm for interactions between mitochondrial ACP and LYR proteins (20). Previous studies demonstrate that ACP, in addition to its central role in mtFAS, forms a complex with LYRM3 and LYRM6 of Respiratory Complex I (17,18,(21)(22)(23).…”

“…One of the best known functions of the mtFAS pathway is to generate octanoyl-ACP, which is required for lipoic acid biosynthesis. However, mitochondrial ACP also functions as a required subunit for respiratory complex I and is predominately associated with the long-chain fatty acid 3-hydroxytetradecanoate (16)(17)(18)(19)(20)(21)(22)(23). More recently, ACP has been identified as an essential functional component of the eukaryotic Fe-S cluster biosynthetic complex (1).…”

Modulation of the conformational state of mitochondrial complex I as a target for therapeutic intervention

“…Therefore, it seems likely that this transition involves at least one of the accessory subunits, e.g. 39 kDa (NDUFA9) [32], B14 (NDUFA6) and SDAP-a (NDUFAB1) [34] or B13 (NDUFA5) [35] which are located in close proximity to the hydrophilic loop of ND3 [23,24,28]. At least two different conformational states of complex I were observed in recent cryo-electron microscopy studies [23,24].…”

Iron–sulfur cluster biogenesis and trafficking in mitochondria

“…ates (41)(42)(43). Although S. cerevisiae has no complex I, that of the yeast Yarrowia lipolytica contains the LYRM6 protein, which is essential for the catalytic activity of this respiratory complex.…”