The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction.
Iron-sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only approximately 7A in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q1 and Q2 that were coupled to proton pumping. Apparent Km values for Q1 and Q2 were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron-sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.
Tocilescu
Maja A
MA
Molecular Bioenergetics Group, Medical School, Cluster of Excellence Frankfurt Macromolecular Complexes, Center for Membrane Proteomics, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Fendel
Uta
U
Zwicker
Klaus
K
Dröse
Stefan
S
Kerscher
Stefan
S
Brandt
Ulrich
U
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry; D014451:Ubiquinone / Q000378:metabolism*; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014451:Ubiquinone; D014443:Tyrosine; D042967:Electron Transport Complex I