Abstract
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 language | English |
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Pages (from-to) | 315-344 |
Number of pages | 30 |
Journal | Faraday Discussions |
Volume | 148 |
Early online date | 1 Dec 2010 |
DOIs | |
Publication status | Published - 2011 |