Q_H^•- Ubisemiquinone Radical in the bo₃-Type Ubiquinol Oxidase Studied by Pulsed Electron Paramagnetic Resonance and Hyperfine Sublevel Correlation Spectroscopy

Abstract

The high-affinity QH ubiquinone-binding site in the bo(3) ubiquinol oxidase from Escherichia coli has been characterized by an investigation of the native ubiquinone radical anion QH(*-) by pulsed electron paramagnetic resonance (EPR) spectroscopy. One- and two-dimensional electron spin-echo envelope modulation (ESEEM) spectra reveal strong interactions of the unpaired electron of QH(*-) with a nitrogen nucleus from the surrounding protein matrix. From analysis of the experimental data, the (14)N nuclear quadrupolar parameters have been determined: kappa = e(2)qQ/4h = 0.93 MHz and eta = 0.50. This assignment is confirmed by hyperfine sublevel correlation (HYSCORE) spectroscopy. On the basis of a comparison of these data with those obtained previously for other membrane-protein bound semiquinone radicals and model systems, this nucleus is assigned to a protein backbone nitrogen. This result is discussed with regard to the location and potential function of QH in the enzyme.

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

“…The above theoretical analysis was strongly indicative for the interpretation of the data and it was further supported using HYSCORE experiments 16 ; see later for a detailed discussion.…”

“…16,21 ). In all cases except D75H the observed EPR lineshape was partially resolved due to 1 H CH3 HFC's as has been observed and analysed previously.…”

“…16 Although it was not possible to identify the origin of the nitrogen signal solely by means of the ESEEM data, useful indications were obtained by comparison of the 14 N nuclear quadrupolar parameters (k, h) determined from the ESEEM data, with nuclear quadrupole resonance (NQR) data available from the literature (see Fig. 6).…”

“…The validity of such NQR parameters has been further substantiated N(3). 16,23 Recently First 14 N NQR quantum chemical calculations (DFT) have also been performed on quinone-histidine model compounds and in proteins. 73,106 using…”

“…[1][2][3] It catalyses both the two-electron oxidation of ubiquinol-8 (UQ 8 ) and the four-electron reduction of dioxygen. The presence of up to demonstrated that this semiquinone radical in QOX forms a hydrogen bond with a nitrogen atom either from the polypeptide backbone of the protein 16 or from an arginine side chain 17 and very recently studies of Lin at al. have identified N 3 of Arg71 as a H-bond donor.…”