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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
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
MacMillan, Fraser; Kacprzak, Sylwia; Hellwig, Petra; Grimaldi, Stephane; Michel, Hartmut; Kaupp, Martin
Inst. Chemie
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.
- Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
- This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.
