Elucidating mechanisms in haem copper oxidases: The high-affinity Q(H) binding site in quinol oxidase as studied by DONUT-HYSCORE spectroscopy and density functional theory

Fraser Macmillan, Sylwia Kacprzak, Petra Hellwig, Stéphane Grimaldi, Hartmut Michel, Martin Kaupp

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15 Citations (Scopus)


The Cytochrome bo(3) ubiquinol oxidase (QOX) from Escherichia coli (E. coli) contains a redox-active quinone, the so-called "high-affinity'' Q(H) 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 N-15/(14) N-exchanged protein. Comparison of computed and experimental C-13 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.
Original languageEnglish
Pages (from-to)315-344
Number of pages30
JournalFaraday Discussions
Early online date1 Dec 2010
Publication statusPublished - 2011

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