Functional Implications from an Unexpected Position of the 49-kDa Subunit of NADH:Ubiquinone Oxidoreductase

摘要

Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the “catalytic core” conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer. Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the “catalytic core” conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer. Complex I (NADH:ubiquinone oxidoreductase) 1The abbreviations used are: complex I, NADH:ubiquinone oxidoreductase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TXPBS, Triton X-100 PBS.1The abbreviations used are: complex I, NADH:ubiquinone oxidoreductase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TXPBS, Triton X-100 PBS. is the major entry point for electrons into the respiratory chain. By linking redox chemistry to vectorial proton translocation, complex I converts up to 40% of the energy used in mitochondria to make ATP. The molecular mechanism of catalysis and its structural basis is not at all understood. Complex I from bovine heart mitochondria is composed of more than 40 different subunits adding to a molecular mass of almost 1000 kDa (1Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar). The enzyme from the strictly aerobic yeast Yarrowia lipolytica is of similar size and has been established as a model system to study eucaryotic complex I employing yeast genetics (2Djafarzadeh R. Kerscher S. Zwicker K. Radermacher M. Lindahl M. Schägger H. Brandt U. Biochim. Biophys. Acta. 2000; 1459: 230-238Crossref PubMed Scopus (82) Google Scholar, 3Kerscher S. Dröse S. Zwicker K. Zickermann V. Brandt U. Biochim. Biophys. Acta-Bioenerg. 2002; 1555: 83-91Crossref PubMed Scopus (87) Google Scholar). A major obstacle to progress in understanding this extremely large and complicated enzyme complex has been the lack of detailed structural information. Resolutions in the 20–30 Å range have been obtained by electron microscopy of single particles from different organisms that show an L-shaped structure with a hydrophobic arm residing in the membrane and a peripheral arm protruding into the mitochondrial matrix space (2Djafarzadeh R. Kerscher S. Zwicker K. Radermacher M. Lindahl M. Schägger H. Brandt U. Biochim. Biophys. Acta. 2000; 1459: 230-238Crossref PubMed Scopus (82) Google Scholar, 4Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar, 5Guenebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (204) Google Scholar, 6Grigorieff N. J. Mol. Biol. 1998; 277: 1033-1046Crossref PubMed Scopus (299) Google Scholar). Recently, projection maps