Elucidating mechanisms in haem copper oxidases: The high-affinity Q_H binding site in quinol oxidase as studied by DONUTHYSCORE spectroscopy and density functional theory

摘要

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 Q_H binding site in detail to resolve these issues. Use is made of site-directed variants as well as globally ~(15)N/~(14)N-exchanged protein. Comparison of computed and experimental ~(13)C 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.