Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR
- Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
Author: | Stéphane GrimaldiORCiDGND, Rodrigo Arias-CartinORCiDGND, Pascal Lanciano, Sevdalina LyubenovaGND, Rodolphe Szenes, Burkhard EndewardORCiD, Thomas F. PrisnerORCiD, Bruno GuigliarelliORCiD, Axel MagalonORCiD |
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URN: | urn:nbn:de:hebis:30:3-766700 |
DOI: | https://doi.org/10.1074/jbc.M111.325100 |
ISSN: | 0021-9258 |
Pubmed Id: | https://pubmed.ncbi.nlm.nih.gov/22190684 |
Parent Title (English): | Journal of biological chemistry |
Publisher: | American Society for Biochemistry and Molecular Biology Publications |
Place of publication: | Bethesda, Md |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2021/01/04 |
Year of first Publication: | 2012 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2024/03/05 |
Tag: | Bioenergetics; Electron Paramagnetic Resonance (EPR); Electron Transfer; Metalloenzymes; Quinones |
Volume: | 287.2012 |
Issue: | 7 |
Page Number: | 9 |
First Page: | 4662 |
Last Page: | 4670 |
Institutes: | Biochemie, Chemie und Pharmazie / Biochemie und Chemie |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - CC BY - Namensnennung 4.0 International |