The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 A resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a(3) iron and Cu(B) of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH(-) groups do not fit, one water (or OH(-)) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu(B) ligand His326 is present in the imidazolate form.

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 ...

In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.

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Mitochondrial LYRM (leucine/tyrosine/arginine motif) proteins are members of the Complex1_LYR-like superfamily. Individual LYRM proteins have been identified as accessory subunits or assembly factors of mitochondrial OXPHOS (oxidative phosphorylation) complexes I, II, III and V respectively, and they play particular roles in the essential Fe-S cluster biogenesis and in acetate metabolism. LYRM proteins have been implicated in mitochondrial dysfunction, e.g. in the context of insulin resistance. However, the functional significance of the common LYRM is still unknown. Analysis of protein-protein interaction screens suggests that LYRM proteins form protein complexes with phylogenetically ancient proteins of bacterial origin. Interestingly, the mitochondrial FAS (fatty acid synthesis) type II acyl-carrier protein ACPM associates with some of the LYRM protein-containing complexes. Eukaryotic LYRM proteins interfere with mitochondrial homoeostasis and might function as adaptor-like 'accessory factors'.