title: Exploring the inhibitor binding pocket of respiratory complex I.
abstract: Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.
authors.affiliation: Johann Wolfgang Goethe-Universität, Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Cluster of Excellence Frankfurt Macromolecular Complexes, Frankfurt am Main, Germany.
authors.forename: Uta
authors.initials: U
authors.lastname: Fendel
authors.forename: Maja A
authors.initials: MA
authors.lastname: Tocilescu
authors.forename: Stefan
authors.initials: S
authors.lastname: Kerscher
authors.forename: Ulrich
authors.initials: U
authors.lastname: Brandt
mesh_terms: D001665:Binding Sites; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D025062:Yarrowia / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D012402:Rotenone; D042967:Electron Transport Complex I