Volume 148, 2011
From the journal:

Faraday Discussions

Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

Fraser MacMillan,*a   Sylwia Kacprzak,b   Petra Hellwig,c   Stephane Grimaldi,d   Hartmut Michele  and  Martin Kaupp*f  

* Corresponding authors

a Henry Wellcome Unit of Biological EPR, School of Chemistry, University of East Anglia, Norwich, UK
E-mail: fraser.macmillan@uea.ac.uk

b Institut für Physikalische Chemie, Universität Freiburg, Freiburg, Germany

c Institut de Chimie, UMR 7177, Université de Strasbourg, Strasbourg, France

d Université d'Aix-Marseille/CNRS, BIP UPR 9036, Marseille, France

e Max-Planck Institut für Biophysik, Frankfurt am Main, Germany

f Technische Universität Berlin, Institut für Chemie, Sekr. C7, Strasse des 17. Juni 135, D-10623 Berlin, Germany

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.

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Article information

DOI
https://doi.org/10.1039/C005149G
Article type
Paper
Submitted
20 Apr 2010
Accepted
18 May 2010
First published
01 Dec 2010

Faraday Discuss., 2011,148, 315-344

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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

F. MacMillan, S. Kacprzak, P. Hellwig, S. Grimaldi, H. Michel and M. Kaupp, Faraday Discuss., 2011, 148, 315 DOI: 10.1039/C005149G

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