Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria.
NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, energy transducing, membrane-bound enzyme. It contains 46 different subunits and nine redox cofactors: a noncovalently bound flavin mononucleotide and eight iron-sulfur clusters. The mechanism of complex I is not known. Mechanistic studies using the bovine enzyme, a model for human complex I, have been precluded by the difficulty of preparing complex I which is pure, monodisperse, and fully catalytically active. Here, we describe and characterize a preparation of bovine complex I which fulfills all of these criteria. The catalytic activity is strongly dependent on the phospholipid content of the preparation, and three classes of phospholipid interactions with complex I have been identified. First, complex I contains tightly bound cardiolipin. Cardiolipin may be required for the structural integrity of the complex or play a functional role. Second, the catalytic activity is determined by the amounts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are bound to the complex. They are more weakly bound than cardiolipin, exchange with PC and PE in solution, and can substitute for one another. However, their nontransitory loss leads to irreversible functional impairment. Third, phospholipids are also required in the assay buffer for the purified enzyme to exhibit its full activity. It is likely that they are required for solubilization and presentation of the hydrophobic ubiquinone substrate.
Sharpley
Mark S
MS
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK.
Shannon
Richard J
RJ
Draghi
Federica
F
Hirst
Judy
J
D000818:Animals; D002308:Cardiolipins / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D042967:Electron Transport Complex I / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008928:Mitochondria / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D012995:Solubility; D014451:Ubiquinone / Q000378:metabolism*
D002308:Cardiolipins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D009243:NAD; D014451:Ubiquinone; C483858:phosphatidylethanolamine; D010088:Oxidoreductases; D042967:Electron Transport Complex I