Essential role of accessory subunit LYRM6 in the mechanism of mitochondrial complex I.

Respiratory complex I catalyzes electron transfer from NADH to ubiquinone (Q) coupled to vectorial proton translocation across the inner mitochondrial membrane. Despite recent progress in structure determination of this very large membrane protein complex, the coupling mechanism is a matter of ongoing debate and the function of accessory subunits surrounding the canonical core subunits is essentially unknown. Concerted rearrangements within a cluster of conserved loops of central subunits NDUFS2 (ß1-ß2S2 loop), ND1 (TMH5-6ND1 loop) and ND3 (TMH1-2ND3 loop) were suggested to be critical for its proton pumping mechanism. Here, we show that stabilization of the TMH1-2ND3 loop by accessory subunit LYRM6 (NDUFA6) is pivotal for energy conversion by mitochondrial complex I. We determined the high-resolution structure of inactive mutant F89ALYRM6 of eukaryotic complex I from the yeast Yarrowia lipolytica and found long-range structural changes affecting the entire loop cluster. In atomistic molecular dynamics simulations of the mutant, we observed conformational transitions in the loop cluster that disrupted a putative pathway for delivery of substrate protons required in Q redox chemistry. Our results elucidate in detail the essential role of accessory subunit LYRM6 for the function of eukaryotic complex I and offer clues on its redox-linked proton pumping mechanism.

Concise Synthesis of 1,4-Benzoquinone-Based Natural Products as Mitochondrial Complex I Substrates and Substrate-Based Inhibitors.

A short, efficient one-step synthesis of 2-methyl-5-(3-methyl-2-butenyl)-1,4-benzoquinone, a natural product from Pyrola media is described. The synthesis is based on a direct late C-H functionalization of the quinone scaffold. The formation of the natural product was confirmed by means of 2D-NMR spectroscopy. Additional derivatives were synthesized and tested alongside the natural product as potential substrate and substrate-based inhibitors of mitochondrial complex I (MCI). The structure-activity relationship study led to the discovery of 3-methylbuteneoxide-1,4-anthraquinone (1 i), an inhibitor with an IC50 of 5 µM against MCI. The identified molecule showed high selectivity for MCI when tested against other quinone-converting enzymes, including succinate dehydrogenase, and the Na (+)-translocating NADH:quinone oxidoreductase. Moreover, the identified inhibitor was also active in cell-based proliferation assays. Therefore, 1 i can be considered as a novel chemical probe for MCI.

Respiratory complex I - Mechanistic insights and advances in structure determination.

Complex I is the largest and most intricate redox-driven proton pump of the respiratory chain. The structure of bacterial and mitochondrial complex I has been determined by X-ray crystallography and cryo-EM at increasing resolution. The recent cryo-EM structures of the complex I-like NDH complex and membrane bound hydrogenase open a new and more comprehensive perspective on the complex I superfamily. Functional studies and molecular modeling approaches have greatly advanced our understanding of the catalytic cycle of complex I. However, the molecular mechanism by which energy is extracted from the redox reaction and utilized to drive proton translocation is unresolved and a matter of ongoing debate. Here, we review progress in structure determination and functional characterization of complex I and discuss current mechanistic models.

Acyl modification and binding of mitochondrial ACP to multiprotein complexes.

The mitochondrial acyl carrier protein (ACPM/NDUFAB1) is a central element of the mitochondrial fatty acid synthesis type II machinery. Originally ACPM was detected as a subunit of respiratory complex I but the reason for the association with the large enzyme complex remained elusive. Complex I from the aerobic yeast Yarrowia lipolytica comprises two different ACPMs, ACPM1 and ACPM2. They are anchored to the protein complex by LYR (leucine-tyrosine-arginine) motif containing protein (LYRM) subunits LYRM3 (NDUFB9) and LYRM6 (NDUFA6). The ACPM1-LYRM6 and ACPM2-LYRM3 modules are essential for complex I activity and assembly/stability, respectively. We show that in addition to the complex I bound fraction, ACPM1 is present as a free matrix protein and in complex with the soluble LYRM4(ISD11)/NFS1 complex implicated in Fe-S cluster biogenesis. We show that the presence of a long acyl chain bound to the phosphopantetheine cofactor is important for docking ACPMs to protein complexes and we propose that association of ACPMs and LYRMs is universally based on a new protein-protein interaction motif.

Supercomplex-associated Cox26 protein binds to cytochrome c oxidase.

Here we identified a hydrophobic 6.4kDa protein, Cox26, as a novel component of yeast mitochondrial supercomplex comprising respiratory complexes III and IV. Multi-dimensional native and denaturing electrophoretic techniques were used to identify proteins interacting with Cox26. The majority of the Cox26 protein was found non-covalently bound to the complex IV moiety of the III-IV supercomplexes. A population of Cox26 was observed to exist in a disulfide bond partnership with the Cox2 subunit of complex IV. No pronounced growth phenotype for Cox26 deficiency was observed, indicating that Cox26 may not play a critical role in the COX enzymology, and we speculate that Cox26 may serve to regulate or support the Cox2 protein. Respiratory supercomplexes are assembled in the absence of the Cox26 protein, however their pattern slightly differs to the wild type III-IV supercomplex appearance. The catalytic activities of complexes III and IV were observed to be normal and respiration was comparable to wild type as long as cells were cultivated under normal growth conditions. Stress conditions, such as elevated temperatures resulted in mild decrease of respiration in non-fermentative media when the Cox26 protein was absent.

Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

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.

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

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.

The superfamily of mitochondrial Complex1_LYR motif-containing (LYRM) proteins.

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'.

Tracing the tail of ubiquinone in mitochondrial complex I.

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron-sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. This redox-driven proton pump catalyzes the four-electron reduction of molecular oxygen to water, one of the most fundamental processes in biology. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding both the mechanism of oxygen reduction and its coupling to proton pumping. Using CcO from Paracoccus denitrificans, we demonstrate that the artificial F state, classically generated by reaction with an excess of hydrogen peroxide, can be converted into a new P state (in contradiction to the conventional direction of the catalytic cycle) by addition of ammonia at pH 9. We suggest that ammonia coordinates directly to Cu(B) in the binuclear active center in this P state and discuss the chemical structures of both oxoferryl intermediates F and P. Our results are compatible with a superoxide bound to Cu(B) in the F state.

Small single transmembrane domain (STMD) proteins organize the hydrophobic subunits of large membrane protein complexes.

The large membrane protein complexes of mitochondrial oxidative phosphorylation are composed of central subunits that are essential for their bioenergetic core function and accessory subunits that may assist in regulation, assembly or stabilization. Although sequence conservation is low, a significant proportion of the accessory subunits is characterized by a common single transmembrane (STMD) topology. The STMD signature is also found in subunits of other membrane protein complexes. We hypothesize that the general function of STMD subunits is to organize the hydrophobic subunits of large membrane protein complexes in specialized environments like the inner mitochondrial membrane.

High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: new insights into the active site and the proton transfer pathways.

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.

A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate.

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.

The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase.

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P(M)-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P(M) and F* intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P(M) and F* intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.