Exploring the ubiquinone binding cavity of respiratory complex I.
Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.
Johann Wolfgang Goethe-Universität, Fachbereich Medizin, Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Frankfurt Macromolecular Complexes, D-60590 Frankfurt am Main, Germany.
Maja A
MA
Tocilescu
Uta
U
Fendel
Klaus
K
Zwicker
Stefan
S
Kerscher
Ulrich
U
Brandt
D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D011522:Protons; D016963:Thermus thermophilus / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry; D014451:Ubiquinone / Q000737:chemistry*; D025062:Yarrowia / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D011522:Protons; D014451:Ubiquinone; D013912:Threonine; D042967:Electron Transport Complex I