title: Elucidating mechanisms in haem copper oxidases: the high-affinity QH binding site in quinol oxidase as studied by DONUT-HYSCORE spectroscopy and density functional theory.
abstract: The Cytochrome bo3 ubiquinol oxidase (QOX) from Escherichia coli (E. coli) contains a redox-active quinone, the so-called "high-affinity" QH quinone. The location of this cofactor and its binding site has yet to be accurately determined by X-ray crystallographic studies. Based on site-directed mutagenesis studies, a putative quinone binding site in the protein has been proposed. The exact binding partner of this cofactor and also whether it is stabilised as an anionic semiquinone or as a neutral radical species is a matter of some speculation. Both Hyperfine Sub-level Correlation (HYSCORE) and Double Nuclear Coherence Transfer Spectroscopy (DONUT-HYSCORE) spectroscopy as well as density functional theory (DFT) have been applied to investigate the QH binding site in detail to resolve these issues. Use is made of site-directed variants as well as globally 15N/14N-exchanged protein. Comparison of computed and experimental 13C hyperfine tensors provides strong support for the binding of the semiquinone radical in an anionic rather than a neutral protonated form. These results are compared with the corresponding information available on other protein binding sites and/or on model systems and are discussed with regard to the location and potential function of QH in the overall mechanism of function of this family of haem copper oxidases.
authors.affiliation: Henry Wellcome Unit of Biological EPR, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK. fraser.macmillan@uea.ac.uk
authors.forename: Fraser
authors.initials: F
authors.lastname: MacMillan
authors.forename: Sylwia
authors.initials: S
authors.lastname: Kacprzak
authors.forename: Petra
authors.initials: P
authors.lastname: Hellwig
authors.forename: Stephane
authors.initials: S
authors.lastname: Grimaldi
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
authors.forename: Martin
authors.initials: M
authors.lastname: Kaupp
mesh_terms: D016227:Benzoquinones / Q000737:chemistry*; D001665:Binding Sites; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011789:Quantum Theory
chemical_list: D016227:Benzoquinones; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; C004532:quinone