title: Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR.
abstract: 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).
authors.affiliation: Unité de Bioénergétique et Ingénierie des Protéines (UPR9036), Institut de Microbiologie de la Méditerranée, CNRS and Aix-Marseille University, 13009 Marseille, France. grimaldi@ifr88.cnrs-mrs.fr
authors.forename: Stéphane
authors.initials: S
authors.lastname: Grimaldi
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Arias-Cartin
authors.forename: Pascal
authors.initials: P
authors.lastname: Lanciano
authors.forename: Sevdalina
authors.initials: S
authors.lastname: Lyubenova
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Szenes
authors.forename: Burkhard
authors.initials: B
authors.lastname: Endeward
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Prisner
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.forename: Axel
authors.initials: A
authors.lastname: Magalon
mesh_terms: D041961:Deuterium Exchange Measurement; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D050901:Nitrate Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010971:Plastoquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011522:Protons
chemical_list: D029968:Escherichia coli Proteins; D011522:Protons; C100461:menasemiquinone; D050901:Nitrate Reductase; C494404:napA protein, E coli; D010971:Plastoquinone