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

ABSTRACT: 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 (?1-?2^S2 loop), ND1 (TMH5-6^ND1 loop) and ND3 (TMH1-2^ND3 loop) were suggested to be critical for its proton pumping mechanism. Here, we show that stabilization of the TMH1-2^ND3 loop by accessory subunit LYRM6 (NDUFA6) is pivotal for energy conversion by mitochondrial complex I. We determined the high-resolution structure of inactive mutant F89A^LYRM6 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.

SUBMITTER: Galemou Yoga E

PROVIDER: S-EPMC7693276 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Galemou Yoga Etienne E Parey Kristian K Djurabekova Amina A Haapanen Outi O Siegmund Karin K Zwicker Klaus K Sharma Vivek V Zickermann Volker V Angerer Heike H

Nature communications 20201126 1

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 ...[more]

PMID: 33243981

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Project description:RATIONALE:Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. OBJECTIVES:To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. METHODS AND RESULTS:Isolated ventilated and perfused lungs from Cox4i2-/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2-/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2-/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2-/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2-/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. CONCLUSIONS:Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

Project description:RATIONALE:Hypoxic pulmonary vasoconstriction (HPV) optimizes systemic oxygen delivery by matching ventilation to perfusion. HPV is intrinsic to pulmonary artery smooth muscle cells (PASMCs). Hypoxia dilates systemic arteries, including renal arteries. Hypoxia is sensed by changes in mitochondrial-derived reactive oxygen species, notably hydrogen peroxide (H2O2) ([H2O2]mito). Decreases in [H2O2]mito elevate pulmonary vascular tone by increasing intracellular calcium ([Ca2+]i) through reduction-oxidation regulation of ion channels. Although HPV is mimicked by the Complex I inhibitor, rotenone, the molecular identity of the O2 sensor is unknown. OBJECTIVE:To determine the role of Ndufs2 (NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 2), Complex I's rotenone binding site, in pulmonary vascular oxygen-sensing. METHODS AND RESULTS:Mitochondria-conditioned media from pulmonary and renal mitochondria isolated from normoxic and chronically hypoxic rats were infused into an isolated lung bioassay. Mitochondria-conditioned media from normoxic lungs contained more H2O2 than mitochondria-conditioned media from chronic hypoxic lungs or kidneys and uniquely attenuated HPV via a catalase-dependent mechanism. In PASMC, acute hypoxia decreased H2O2 within 112±7 seconds, followed, within 205±34 seconds, by increased intracellular calcium concentration, [Ca2+]i. Hypoxia had no effects on [Ca2+]i in renal artery SMC. Hypoxia decreases both cytosolic and mitochondrial H2O2 in PASMC while increasing cytosolic H2O2 in renal artery SMC. Ndufs2 expression was greater in PASMC versus renal artery SMC. Lung Ndufs2 cysteine residues became reduced during acute hypoxia and both hypoxia and reducing agents caused functional inhibition of Complex I. In PASMC, siNdufs2 (cells/tissue treated with Ndufs2 siRNA) decreased normoxic H2O2, prevented hypoxic increases in [Ca2+]i, and mimicked aspects of chronic hypoxia, including decreasing Complex I activity, elevating the nicotinamide adenine dinucleotide (NADH/NAD+) ratio and decreasing expression of the O2-sensitive ion channel, Kv1.5. Knocking down another Fe-S center within Complex I (Ndufs1, NADH [nicotinamide adenine dinucleotide] dehydrogenase [ubiquinone] iron-sulfur protein 1) or other mitochondrial subunits proposed as putative oxygen sensors (Complex III's Rieske Fe-S center and COX4i2 [cytochrome c oxidase subunit 4 isoform 2] in Complex IV) had no effect on hypoxic increases in [Ca2+]i. In vivo, siNdufs2 significantly decreased hypoxia- and rotenone-induced constriction while enhancing phenylephrine-induced constriction. CONCLUSIONS:Ndufs2 is essential for oxygen-sensing and HPV.

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

Publications

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

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