The high-affinity QH 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

doi:10.1039/c005149g

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The high-affinity Q_H binding site in quinol oxidase as studied by DONUT-HYSCORE spectroscopy and density functional theory

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Michel, Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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MacMillan, F., Kacprzak, S., Hellwig, P., Grimaldi, S., Michel, H., & Kaupp, M. (2011). The high-affinity Q_H binding site in quinol oxidase as studied by DONUT-HYSCORE spectroscopy and density functional theory. Faraday Discussions, 148, 315-344. doi:10.1039/c005149g.

Cite as: https://hdl.handle.net/11858/00-001M-0000-0024-D625-E

Abstract

The Cytochrome bo₃ 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 Q_H 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 Q_H in the overall mechanism of function of this family of haem copper oxidases.