540 Chemie und zugeordnete Wissenschaften
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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 (β1-β2S2 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.
Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.