A central functional role for the 49-kDa subunit within the catalytic coreof mitochondrial complex I

We have analyzed a series of eleven mutations in the 49-kDa protein of mito chondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolyti ca to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit o f [NiFe] hydrogenases, None of the mutations affected assembly of complex I , all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of iron-sulfur cluste r N2, a prosthetic group not located in the 49-kDa subunit, In three of the se mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a stra ightforward explanation for the observed, unforeseen effects on this iron-s ulfur cluster. We propose that the fold around the hydrogen reactive site o f [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and ha s become part of the inhibitor and ubiquinone binding region. We discuss th at the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.