Advertisement

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR*

  • Stéphane Grimaldi
    Correspondence
    To whom correspondence may be addressed: Unité de Bioénergétique et Ingénierie des Protéines (BIP – CNRS UPR9036), Institut de Microbiologie de la Méditerranée, CNRS and Aix-Marseille Université, 31, chemin Joseph Aiguier 13402 Marseille cedex 20, France. Tel.: 33-491-164-557; Fax: 33-491-164-097; E-mail: .
    Affiliations
    Unité de Bioénergétique et Ingénierie des Protéines (UPR9036), CNRS and Aix-Marseille University, 13009 Marseille, France
    Search for articles by this author
  • Rodrigo Arias-Cartin
    Footnotes
    Affiliations
    Laboratoire de Chimie Bactérienne (UPR9043), Institut de Microbiologie de la Méditerranée, CNRS and Aix-Marseille University, 13009 Marseille, France and
    Search for articles by this author
  • Pascal Lanciano
    Footnotes
    Affiliations
    Unité de Bioénergétique et Ingénierie des Protéines (UPR9036), CNRS and Aix-Marseille University, 13009 Marseille, France
    Search for articles by this author
  • Sevdalina Lyubenova
    Footnotes
    Affiliations
    Institut für Physikalische und Theoretische Chemie, University of Frankfurt, 60438 Frankfurt, Germany
    Search for articles by this author
  • Rodolphe Szenes
    Affiliations
    Unité de Bioénergétique et Ingénierie des Protéines (UPR9036), CNRS and Aix-Marseille University, 13009 Marseille, France
    Search for articles by this author
  • Burkhard Endeward
    Affiliations
    Institut für Physikalische und Theoretische Chemie, University of Frankfurt, 60438 Frankfurt, Germany
    Search for articles by this author
  • Thomas F. Prisner
    Affiliations
    Institut für Physikalische und Theoretische Chemie, University of Frankfurt, 60438 Frankfurt, Germany
    Search for articles by this author
  • Bruno Guigliarelli
    Affiliations
    Unité de Bioénergétique et Ingénierie des Protéines (UPR9036), CNRS and Aix-Marseille University, 13009 Marseille, France
    Search for articles by this author
  • Axel Magalon
    Affiliations
    Laboratoire de Chimie Bactérienne (UPR9043), Institut de Microbiologie de la Méditerranée, CNRS and Aix-Marseille University, 13009 Marseille, France and
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the CNRS, the Agence Nationale de la Recherche, and Aix-Marseille Université. This work was also supported by Research Infrastructures Activity in the 6th Framework Program of the European Community (Contract RII3-026145, EU-NMR) for financial support (to S. G.), as well as the European Cooperation in Science and Technology Action (COST P15) “Advanced Paramagnetic Resonance Methods in Molecular Biophysics” for Short-Term Scientific Mission funding (to S. G.).
    This article contains supplemental Figs. S1 and S2 and Tables S1 and S2.
    2 Present address: Dept. of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520.
    3 Present address: Dept. of Biology, University of Pennsylvania, Philadelphia, PA 19104.
    4 Present address: EPR Division, Bruker BioSpin GmbH, 76287 Rheinstetten, Germany.
      Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (QD) 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 QD site (MSQD) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with 1H2O/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 MSQD 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 bD. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the QD site are discussed, in light of the unusually high thermodynamic stability of MSQD.

      Introduction

      Quinones are small lipophilic organic molecules found in energy-transducing membranes of all living organisms except methanogens (
      • Nicholls D.G.
      • Ferguson S.J.
      ). Due to their ability to transfer up to two electrons and two protons, they are widely used in photosynthetic and respiratory electron transfer chains. Quinones can freely diffuse in the hydrophobic core of lipid membranes. They can therefore bind into specific quinone-reactive sites (Q-sites)
      The abbreviations used are: Q-site
      quinone-reactive site
      Q
      quinone
      SQ
      semiquinone
      QH2
      quinol
      ENDOR
      electron nuclear double resonance
      ESE
      electron spin echo
      ESEEM
      electron spin echo envelope modulation
      HYSCORE
      hyperfine sublevel correlation
      IMV
      inner membrane vesicle
      MSQD
      menasemiquinone stabilized at the QD site of NarGHI
      USQD
      ubisemiquinone stabilized at the QD site of NarGHI
      NarGHI
      membrane-bound form of the native enzyme complex
      RC
      photosynthetic reaction center
      mT
      milliteslas.
      of membrane proteins in which they function as two-electron and proton carriers and are responsible for exchange of reducing equivalents between different electron transport complexes. In this case, the quinones leave the protein after completion of the redox cycle. Typical examples are the QB site of bacterial reaction center (RC) or photosystem II and the Q-sites (Qo and Qi) of bc1 complex. In contrast, non-dissociable quinones can be tightly bound at specific quinone-reactive sites of proteins in which they can be involved in electron transfer processes as prosthetic groups. Well known representatives of this type include quinones in the QA site in RCs of purple bacteria and in photosystem II or the A1 sites in photosystem I (
      • Lubitz W.
      • Feher G.
      The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals QA•− and QB•− by EPR and ENDOR.
      ,
      • Srinivasan N.
      • Golbeck J.H.
      Protein-cofactor interactions in bioenergetic complexes: the role of the A1A and A1B phylloquinones in photosystem I.
      ).
      The different redox states of quinones may also adopt different conformations in the quinone-binding pockets, as evidenced for ubiquinone and ubisemiquinone at the QB site of bacterial RC (
      • Stowell M.H.
      • McPhillips T.M.
      • Rees D.C.
      • Soltis S.M.
      • Abresch E.
      • Feher G.
      Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer.
      ). The functional diversity of Q-sites arises from a particular tuning of the protein environment. Despite the fact that high-resolution structural data are available for several Q-sites, how protein-cofactor interactions relate to and control the functional properties of the bound quinone is largely unknown. In particular, understanding the molecular mechanism underlying the coupling between electron transfer and proton translocation that occurs at dissociable Q-sites requires obtaining structural information on all three forms, quinone (Q), semiquinone (SQ), and quinol (QH2). For this purpose, high-resolution EPR methods such as ENDOR (electron nuclear double resonance) and ESEEM (electron spin echo envelope modulation) spectroscopies were proven to be valuable by giving detailed structural information on protein-bound semiquinone intermediates, provided that this paramagnetic state can be trapped for spectroscopic studies.
      Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound heterotrimeric enzyme induced by anaerobiosis and the presence of nitrate. Involved in the nitrate respiratory pathway, a major alternative to the bacterial oxidative phosphorylation, it couples the oxidation of menaquinols or ubiquinols at a periplasmically oriented Q-site (named QD) to the cytoplasmic reduction of nitrate. Thus, both substrate turnovers contribute to the generation of a proton motive force across the cytoplasmic membrane. NarGHI contains eight redox-active metal centers (
      • Lanciano P.
      • Savoyant A.
      • Grimaldi S.
      • Magalon A.
      • Guigliarelli B.
      • Bertrand P.
      New method for the spin quantitation of [4Fe-4S]+ clusters with S = 3/2: application to the FS0 center of the NarGHI nitrate reductase from Escherichia coli.
      ,
      • Guigliarelli B.
      • Magalon A.
      • Asso M.
      • Bertrand P.
      • Frixon C.
      • Giordano G.
      • Blasco F.
      Complete coordination of the four Fe-S centers of the β subunit from Escherichia coli nitrate reductase: physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters.
      ,
      • Blasco F.
      • Guigliarelli B.
      • Magalon A.
      • Asso M.
      • Giordano G.
      • Rothery R.A.
      The coordination and function of the redox centers of the membrane-bound nitrate reductases.
      ,
      • Rothery R.A.
      • Blasco F.
      • Magalon A.
      • Weiner J.H.
      The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols.
      ,
      • Bertero M.G.
      • Rothery R.A.
      • Palak M.
      • Hou C.
      • Lim D.
      • Blasco F.
      • Weiner J.H.
      • Strynadka N.C.
      Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A.
      ): a molybdenum cofactor and an Fe4S4 cluster (FS0) in the nitrate-reducing subunit NarG; one Fe3S4 cluster (FS4) and three Fe4S4 clusters (FS1–3) in the electron transfer subunit NarH; and two low spin hemes b in the membrane-anchor subunit NarI, termed bD and bP to indicate their distal and proximal position to the catalytic site. Importantly, NarI stabilizes an EPR-detectable semiquinone intermediate of both natural substrates at its quinol oxidation site QD close to heme bD (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ,
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      ,
      • Arias-Cartin R.
      • Lyubenova S.
      • Ceccaldi P.
      • Prisner T.
      • Magalon A.
      • Guigliarelli B.
      • Grimaldi S.
      HYSCORE evidence that endogenous mena- and ubisemiquinone bind at the same Q site (QD) of Escherichia coli nitrate reductase A.
      ). Remarkably, the resulting menasemiquinone species herein referred to as MSQD has the largest thermodynamic stability measured so far in respiratory complexes stabilizing semiquinone intermediates. These peculiar properties render NarGHI ideally suited for investigating the molecular factors responsible for the reactivity of respiratory enzymes toward quinols.
      Although no high-resolution structural data revealing the binding mode of the natural quinol/quinone substrate are available, we recently utilized high-resolution EPR techniques on endogenous MSQD and ubisemiquinone radical (USQD) stabilized in NarGHI-enriched inner membrane vesicles (IMVs) of E. coli to explore their environment using the unpaired electron as a probe. The use of ESEEM and HYSCORE (hyperfine sublevel correlation) spectroscopies on either the wild-type enzyme or the enzyme uniformly enriched with 15N nuclei provided direct evidence for nitrogen ligation to MSQD and USQD. On the basis of the direct determination of the quadrupolar parameters of the corresponding interacting 14N by S-band (∼3 GHz) HYSCORE experiments, we assigned the latter to an Nδ imidazole nitrogen and proposed it to arise from the heme bD axial ligand His-66 (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). The non-zero isotropic hyperfine coupling of this nitrogen suggests that the interaction occurs via a hydrogen bond, allowing electron spin density to be transferred from the radical to the interacting nucleus. Interestingly, these experiments did not support a direct H-bond between MSQD (or USQD) and Lys-86, a residue in the QD site that was previously shown to be essential for quinol oxidation and menasemiquinone detection (
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      ,
      • Bertero M.G.
      • Rothery R.A.
      • Boroumand N.
      • Palak M.
      • Blasco F.
      • Ginet N.
      • Weiner J.H.
      • Strynadka N.C.
      Structural and biochemical characterization of a quinol-binding site of Escherichia coli nitrate reductase A.
      ). Indeed, no evidence for the transfer of a measurable spin density on any other nuclei than that mentioned above was found. Thus, we tentatively proposed that a water-mediated interaction is formed between MSQD (or USQD) and Lys-86, consistent with the latter being involved in reactivity toward quinols (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). Moreover, we have recently shown that a cardiolipin molecule specifically bound to the complex is necessary for quinol substrate fixation at the QD site, probably through the action of one of its acyl chains located in the vicinity of His-66 (
      • Arias-Cartin R.
      • Grimaldi S.
      • Pommier J.
      • Lanciano P.
      • Schaefer C.
      • Arnoux P.
      • Giordano G.
      • Guigliarelli B.
      • Magalon A.
      Cardiolipin-based respiratory complex activation in bacteria.
      ). Clearly, additional information is required to improve our understanding of the semiquinone binding mode in the QD site and of its functional tuning by the protein environment.
      In this work, high-resolution EPR techniques have been used to map the environment and the binding mode of MSQD via the detection of proton hyperfine couplings to the radical. Using a combination of X-band (∼9 GHz) ESEEM/HYSCORE and Q-band (∼34 GHz) Mims ENDOR experiments on MSQD prepared in either a protonated or a deuterated solvent, one exchangeable and two non-exchangeable protons magnetically coupled to the radical were detected. Their detailed characterization allows their assignment to specific protons in the vicinity of the radical. Implications of these results for deciphering the semiquinone binding mode and the catalytic mechanism at the QD site are discussed.

      EXPERIMENTAL PROCEDURES

       Sample Preparation

      NarGHI was expressed in an E. coli nitrate reductase-deficient strain LCB3063 (RK4353, ΔnapA-B, narG::ery, ΔnarZ::Ω, SpcR) (
      • Potter L.C.
      • Millington P.
      • Griffiths L.
      • Thomas G.H.
      • Cole J.A.
      Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth?.
      ) using pVA700 plasmid (AmpR) (
      • Guigliarelli B.
      • Magalon A.
      • Asso M.
      • Bertrand P.
      • Frixon C.
      • Giordano G.
      • Blasco F.
      Complete coordination of the four Fe-S centers of the β subunit from Escherichia coli nitrate reductase: physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters.
      ), which encodes for the narGHJI operon under control of the tac promoter. Cells were grown in Terrific Broth under semi-anaerobic conditions at 37 °C as described in Ref.
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      with ampicillin (100 μg ml−1) and spectinomycin (50 μg ml−1) included in the growth medium.
      Purified E. coli NarGHI-enriched IMVs were used for this study, allowing us to maintain an unmodified lipid environment and to study the interaction of NarGHI with its endogenous menaquinol substrate. For this purpose, purified E. coli NarGHI-enriched IMVs were isolated by differential centrifugation and sucrose gradient step as described in Ref.
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      using a buffer containing 100 mm MOPS and 5 mm EDTA at pH 7.5. Deuterium-exchanged samples were prepared using the same membrane extraction protocol with a buffer containing 2H2O (99.9% atom 2H) instead of 1H2O. The functionality of NarGHI in our samples was confirmed spectrophotometrically by measuring the quinol:nitrate oxidoreductase activity. Stabilization of the semiquinone at the QD site was achieved through redox titrations under the same conditions as those used in our previous works (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ,
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      ,
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). Redox potentials are given in the text with respect to the standard hydrogen electrode. The semiquinone concentration in our samples estimated from the double integration of their corresponding EPR spectra and by comparison with a standard (1 mm CuSO4) was estimated in the range of 10–12 μM.

       Pulsed EPR/ENDOR Experiments

      X-band (∼9 GHz) and Q-band (∼34 GHz) pulsed EPR/ENDOR experiments were performed using a Bruker EleXsys E580-Q spectrometer equipped with an Oxford Instruments CF 935 cryostat. Spectra were measured at 90 K to avoid contamination from fast relaxing metal centers such as FeS centers in NarGHI (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). For the two-pulse experiments (π/2-τ-π), the echo intensity was measured as a function of magnetic field at fixed time interval τ between the two microwave pulses for field sweep ESE or as a function of τ at a fixed magnetic field value for two-pulse ESEEM.
      Two-pulse and four-pulse (π/2-τ-π/2-T/2-π-T/2-π/2) ESEEM and HYSCORE (π/2-τ-π/2-t1-π-t2-π/2) experiments were performed at a magnetic field corresponding to the maximum intensity of the MSQD two-pulse field sweep ESE spectrum where all orientations of the semiquinone with respect to the external magnetic field contribute, giving rise to powder ESEEM/HYSCORE spectra (see the supplemental material). Spectra were processed using the Bruker Xepr software. Relaxation decays were subtracted (fitting by polynomial functions) followed by zero-filling and tapering with a Hamming window, before Fourier transformation, which finally gives the spectrum in frequency domain. All spectra are shown in absolute value mode. HYSCORE spectra are presented as contour plots.
      Q-band pulsed 2H ENDOR spectra were obtained using the Mims (π/2-τ-π/2-t-π/2-τ-echo) sequence (
      • Mims W.B.
      Pulsed ENDOR experiments.
      ). A radio frequency π pulse was applied during the time interval t. The radio frequency power was delivered by a 2-kilowatt Dressler solid state radio frequency amplifier. It was optimized for radio frequency π pulse lengths of 40 μs for deuterium Mims ENDOR experiments. Pulsed ENDOR spectra were recorded at a magnetic field corresponding to the g position of the nearly axial Q-band EPR signal of MSQD (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ). Pulsed EPR/ENDOR spectra were simulated in the MATLAB environment using the Easy-spin software package (release 3.1.0) (
      • Stoll S.
      • Schweiger A.
      EasySpin, a comprehensive software package for spectral simulation and analysis in EPR.
      ).

       Hyperfine and Quadrupole Interactions

      A hyperfine coupling between an S = ½ radical and a nucleus with nuclear spin value I consists in general of (i) the isotropic contribution Aiso = 2μ0gegnβeβn0(0)|2/3h, where |ψ0(0)|2 is the electron spin density at the nucleus, ge and gn are electron and nuclear g-factors, respectively, βe and βn are Bohr and nuclear magnetons, respectively, h is Planck's constant, and (ii) the anisotropic contribution described by the traceless dipolar coupling tensor T̃. In most cases, T̃ can be assumed to be axial, with principal values (−T, −T, 2T).
      The hyperfine couplings of different isotopes of the same element are proportional to a very good approximation to the corresponding gn values. In this study, the direct and simultaneous determination of Aiso and T of the protons interacting with MSQD were derived from the analysis of HYSCORE cross-peak contours as detailed in the supplemental material (
      • Dikanov S.A.
      • Bowman M.K.
      Cross-peak lineshape of two-dimensional ESEEM spectra in disordered S = ½, I = ½ spin systems.
      ).
      A 2H nucleus has a quadrupole moment that interacts with the electric field gradient at the nucleus. The components of the electric field gradient tensor are defined in its principal axis system and ordered according to |qZZ| ≥ |qYY| ≥ |qXX|. This traceless tensor can then be fully described by only two parameters: (i) the 2H nuclear quadrupole coupling constant κ = |e2qZZQ/h|, where e is the charge of electron, Q is the 2H nuclear electric quadrupole moment; and (ii) the asymmetry parameter η = |qYY − qXX/qZZ|. κ is a measure of the strength of the interaction between the nuclear quadrupole moment and the electric field gradient at the 2H nucleus site due to anisotropic charge distribution around the nucleus, whereas η is a measure of the deviation of this distribution from axial symmetry. Thus, the electric field gradient is related to the specific binding geometry. Its components can, therefore, be used to obtain detailed information on hydrogen bonds (
      • Flores M.
      • Isaacson R.
      • Abresch E.
      • Calvo R.
      • Lubitz W.
      • Feher G.
      Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II: geometry of the hydrogen bonds to the primary quinone formula by 1H and 2H ENDOR spectroscopy.
      ,
      • Sinnecker S.
      • Flores M.
      • Lubitz W.
      Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: effect of hydrogen bonding on the electronic and geometric structure of the primary quinone. A density functional theory study.
      ,
      • Sinnecker S.
      • Reijerse E.
      • Neese F.
      • Lubitz W.
      Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions.
      ,
      • Epel B.
      • Niklas J.
      • Sinnecker S.
      • Zimmermann H.
      • Lubitz W.
      Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
      ,
      • Flores M.
      • Isaacson R.A.
      • Calvo R.
      • Feher G.
      • Lubitz W.
      Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR Spectroscopy at 35 GHz.
      ,
      • Soda G.
      • Chiba T.
      Deuteron magnetic resonance study of cupric sulfate pentahydrate.
      ,
      • Hunt M.J.
      • Mackay A.L.
      Deuterium and nitrogen pure quadrupole resonance in deuterated amino acids.
      ). In this study the parameters κ and η of the 2H interacting with MSQD were estimated by simulation of the Q-band 2H Mims ENDOR spectrum.

      RESULTS

       X-band Pulsed EPR (Field Sweep, Two-pulse ESEEM)

      X-band field sweep ESE spectra of NarGHI-enriched IMVs were recorded at 90 K in samples redox-poised at ∼−100 mV prepared in either 1H2O or 2H2O. They show a single line from the MSQ stabilized at the QD site of NarGHI with g ∼2.0045 and the width ∼0.8 mT in 1H2O (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). Replacement of 1H2O by 2H2O decreases the line width by less than 0.1 mT (Fig. 1A). The weakness of this effect is due to the primary contribution to the line shape of the g-tensor anisotropy, which was previously resolved using numerical simulation of the MSQD Q-band EPR spectrum (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ). The two-pulse spin echo decay of the radical measured in 1H2O at 90 K is depicted in Fig. 1B. It mainly shows the modulation associated with weakly coupled protons in the immediate environment, with Zeeman frequencies νI(1H) ∼14.7 MHz. A characteristic deep additional modulation of the echo intensity appears in the sample prepared in 2H2O (Fig. 1B). Fourier transformation of this echo envelope reveals that the major contribution to the deep variations occurs at the frequency ∼2.3 MHz, corresponding to the Zeeman frequency of deuterium (not shown). These results give a first indication of solvent accessibility and 1H/2H exchange around MSQD. To increase spectral resolution and thus provide more detailed information about the proton environment of MSQD, HYSCORE experiments were carried out and are shown below.
      Figure thumbnail gr1
      FIGURE 1Two-pulse experiments of MSQD. A, field sweep ESE spectra in redox-poised samples prepared in 1H2O (−107 mV, solid line) and 2H2O (−106 mV, dotted lines). B, ESEEM patterns of the corresponding samples in 1H2O (top) and 2H2O (bottom). For the sample in 1H2O, the microwave frequency was 9.6912 GHz, and the magnetic field was 345.2 mT. For the sample in 2H2O, these were 9.6899 GHz and 345.3 mT, respectively.

       X-band 1H HYSCORE

      The low frequency parts of the X-band HYSCORE spectra of MSQD were previously shown and analyzed in detail. They revealed cross-peaks arising from a single 14N hyperfine coupling assigned to the heme bD ligand His-66 residue (
      • Arias-Cartin R.
      • Lyubenova S.
      • Ceccaldi P.
      • Prisner T.
      • Magalon A.
      • Guigliarelli B.
      • Grimaldi S.
      HYSCORE evidence that endogenous mena- and ubisemiquinone bind at the same Q site (QD) of Escherichia coli nitrate reductase A.
      ,
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). In addition to these 14N signals, several cross-features from protons symmetrically positioned with respect to the 1H Zeeman frequency (νI(1H) ∼14.7 MHz) are clearly resolved in the 10–20-MHz frequency range in the (+,+) quadrant of these spectra (Fig. 2A). This indicates that several protons are magnetically coupled to the radical. The appearance of these correlations in the (+,+) quadrant indicates that the corresponding hyperfine couplings for a given proton satisfy the relationships |T+2Aiso| ≪ 4νI(1H) (
      • Schweiger A.
      • Jeschke G.
      ). To further analyze the spectrum and discriminate between exchangeable and non-exchangeable features, HYSCORE experiments were also performed under the same conditions in the sample prepared in 2H2O. Fig. 2 shows the proton region of the corresponding HYSCORE spectra recorded with τ = 204 ns in 1H2O (Fig. 2A) or 2H2O (Fig. 2B). In addition to the diagonal peak at νI(1H) ∼14.7 MHz, four pairs of cross-features located symmetrically relative to the diagonal are well resolved in the spectrum shown in Fig. 2A. They are designated 1, 1′, 2, 2′, 3, 3′, 4, and 4′. The ridges 2-2′ exhibit the smallest resolved hyperfine splitting, of the order of ∼2 MHz, whereas the largest one is observed for cross-peaks 1-1′. Cross-ridges 3-3′ possess the most extended anisotropic contour, with the largest deviation from the diagonal, whereas cross-peaks 4-4′ deviate significantly from the normal to the diagonal. These two features indicate a significant anisotropic hyperfine component. Contours 1-1′ and 2-2′ are approximately normal to the diagonal, suggesting a smaller anisotropy. Cross-peaks 1 and 4 partially overlap.
      Figure thumbnail gr2
      FIGURE 2Proton part of HYSCORE spectra of MSQD in 1H2O (A) or in 2H2O (B) with time τ = 204 ns. The microwave frequency was 9.6944 GHz (A) and 9.6934 GHz (B), and the magnetic field was 345.2 mT. For both spectra, the durations of the π/2 and π pulses were 12 and 24 ns, respectively, with equal amplitude. 256 points were recorded in each dimension. t1 and t2 were incremented in steps of 16 ns from their initial value.
      Cross-peaks 3-3′ and 4-4′ completely disappear in the proton HYSCORE spectrum measured in 2H2O, demonstrating that they are produced by at least one exchangeable proton (Fig. 2B). In contrast, cross-peaks 1-1′ and 2-2′ still appear in the spectrum measured in 2H2O, showing that they arise from non-exchangeable (i.e. covalently bound) protons.
      Quantitative analysis of the cross-peak contour line shapes indicates that cross-peaks 3, 3′, 4, and 4′ are produced by a single exchangeable proton (supplemental Fig. S2 and supplemental Table S1). Hence, HYSCORE signals derive from three protons coupled to MSQD: H1 (1-1′), H2 (2-2′), and H3 (3-3′-4-4′). Among them, H3 is exchangeable. The isotropic (Aiso) and anisotropic (T) components of the three 1H hyperfine tensors are given in Table 1. The magnitude of the hyperfine couplings deduced from the analysis of HYSCORE spectra are consistent with our previous preliminary observations of 1H continuous wave ENDOR resonances with corresponding estimated hyperfine couplings A1 ∼5.7 MHz (H3) and A2 ∼3.3 MHz (H2) (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ,
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      ).
      TABLE 1Hyperfine tensors derived from contour line shape analysis of HYSCORE spectra
      Table thumbnail tbl1

       X-band 1H Four-pulse ESEEM

      Additional information about the interacting protons was obtained from one-dimensional four-pulse ESEEM spectra, which are particularly useful for the observation of proton sum combination lines with improved resolution (
      • Schweiger A.
      • Jeschke G.
      ,
      • Reijerse E.J.
      • Dikanov S.A.
      Electron spin echo envelope modulation spectroscopy on orientationally disordered systems: Line shape singularities in S = ½, I = ½ spin systems.
      ). The four-pulse ESEEM spectrum of MSQD in 1H2O buffer contains two well resolved lines in the region of the proton around 2νI(1H) as shown in Fig. 3A. The most intense line appears exactly at the 2νI(1H) frequency and represents the contribution of weakly coupled protons from the protein environment. In addition, the spectrum exhibits a peak of lower intensity shifted from 2νI(1H) to higher frequencies by ∼1.2 MHz. This shifted peak completely disappears in the spectra of the sample prepared in 2H2O (Fig. 3B). This indicates that the line shifted from 2νI(1H) arises from an exchangeable proton. The shift observed in the four-pulse ESEEM is well described by
      Δ=9T2/16ν11H
      (Eq. 1)


      from which the anisotropic component T of the hyperfine coupling can be estimated (see supplemental material). The shift of ∼1.2 MHz corresponds to T = 5.7 MHz, which is in very good agreement with the corresponding value determined for H3 from the analysis of the HYSCORE spectra. The expected shifts from H1 and H2 (0.06 and 0.05 MHz, respectively) are too small to be resolved in a four-pulse ESEEM spectrum (supplemental Table S2). The proton sum combination peak therefore confirms the assignment made in the HYSCORE spectra for the exchangeable proton and the hyperfine coupling determined from these spectra.
      Figure thumbnail gr3
      FIGURE 3Stacked presentations of the two-dimensional set of the four-pulse ESEEM spectra of MSQD in 1H2O (A) and 2H2O (B). The spectra show modulus Fourier transforms along the time T/2 axis (1024 points with a 4 ns step) at 30 different times τ. The initial τ is 96 ns and was increased by 8 ns in successive traces. The microwave frequency was 9.6912 GHz (A) and 9.6898 GHz, and the magnetic field was 345.2 mT (A) and 345.4 mT (B).

       Q-band 2H Mims ENDOR

      Further details concerning exchangeable protons coupled to MSQD were obtained through the use of pulsed 2H ENDOR spectroscopy. Fig. 4 shows the Q-band 2H Mims ENDOR spectrum of MSQD in NarGHI-enriched IMVs prepared in 2H2O. It has been recorded at a magnetic field value corresponding to the maximum intensity of the nearly axially symmetric Q-band EPR spectrum of MSQD with g-tensor principal values gx = 2.0061, gy = 2.0051, gz = 2.0023 ± 0.0001 (Fig. 4, top) (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ). The ENDOR spectrum exhibits two pairs of well resolved intense lines located symmetrically with respect to the 2H nuclear Larmor frequency (νI(2H) ∼7.84 MHz) and subject to the nuclear quadrupole interaction. The splitting of their center away from the Larmor frequency (∼0.8 MHz) is determined by the hyperfine coupling constant, and the splitting within the pair (∼0.13 MHz) is given by the quadrupole interaction. The same holds for the two pairs of less intense lines resolved in the spectrum shown in Fig. 4, separated by ∼1.5 MHz and split each by ∼0.26 MHz. The hyperfine coupling values of ∼0.8 and ∼1.5 MHz are first estimates of the A and A components of an almost purely dipolar hyperfine tensor. They are very close to those found for H3 and scaled to the 2H nucleus, i.e. A = 0.87 and A = 1.77 MHz. Similarly, a nuclear quadrupole coupling constant κ ∼
      43


      × 0.13 ∼0.173 MHz can be estimated from the 0.13-MHz splitting of the two most intense doublets. A numerical simulation of the spectrum is shown in Fig. 4. It was obtained using the proton hyperfine coupling values of H3 deduced from the analysis of the HYSCORE spectra and rescaled by the factor gn(1H)/gn(2H) ∼6.5. This best simulation was obtained assuming that the g-, A-, and Q-tensors are collinear, with quadrupole parameters κ = (0.176 ± 0.004) MHz and η = 0.20 ± 0.05. Finally, this procedure allowed us to select unambiguously the right set (|Aiso|, |T|) for H3 from the two alternatives (Table 1). Overall, the data show that a single exchangeable proton is coupled to the radical, in agreement with the analysis above.
      Figure thumbnail gr4
      FIGURE 4Q-band 2H Mims ENDOR spectrum of MSQD in 2H2O. Experimental conditions were as follows: microwave pulse length, 24 ns; microwave frequency, 33.684926 GHz; magnetic field value, 1.1999 T; measurement time, 20 h. The simulated spectrum (dotted lines) was generated from the following parameters: a single hyperfine tensor with components (Aiso = 0.06/6.5, ∼0.009 MHz, T = 5.73/6.5, ∼0.88 MHz) and with nuclear quadrupole coupling parameters (κ = 0.18 MHz, η = 0.2), with g-, A-, and Q- collinear to each other. The Q-band field-swept ESE spectrum of MSQD is shown on top. Its simulation, shown as dotted lines, has been performed using the g-tensor principal values given under “Results,” an isotropic convolutional Gaussian line width with full width at half-maximum of 0.76 mT. Experimental conditions were as follows: microwave pulse lengths, 24 and 48 ns for π/2 and π pulses, respectively; microwave frequency, 33.68504 GHz.

      DISCUSSION

       Single Exchangeable Proton Coupling with Peculiar Hyperfine Coupling Characteristics

      Our data clearly show the presence of a single exchangeable proton in the vicinity of MSQD characterized by |Aiso| = 0.06 MHz and |T| = 5.73 MHz. Such hyperfine coupling constants are in the range of those measured for exchangeable protons coupled to protein-bound semiquinones and assigned to protons hydrogen-bonded to the quinone carbonyl oxygens. Typical examples include exchangeable proton couplings to the menasemiquinone stabilized at the QH site of the aa3 menaquinol oxidase from Bacillus subtilis (|T| = 5.6 MHz and |Aiso| = 5.4 MHz) or to the photoaccumulated phyllosemiquinone A1•− in Thermococcus elongatus photosystem I (|T| ∼3.7 MHz and |Aiso| ∼0.1 MHz) (
      • Yi S.M.
      • Narasimhulu K.V.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
      ,
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ). Thus, we assign H3 to a proton involved in H-bonding to one of the MSQD carbonyl oxygens. However, the hyperfine coupling tensor to H3 has atypical properties as it combines both an almost zero isotropic hyperfine coupling constant Aiso and a large anisotropic part T. The magnitude of the H-bond tensor is determined by the geometry of the H-bond. A small Aiso is expected when the H-bond lies in the molecular plane due to the small overlap between the hydrogen 1s orbital and the oxygen 2pz orbital forming part of the semiquinone singly occupied molecular orbital. Almost purely anisotropic hyperfine tensors have thus been observed for in-plane hydrogen-bonded protons to unsubstituted quinones measured in alcoholic solvent, whereas the corresponding |T| values do not exceed 3 MHz (
      • Epel B.
      • Niklas J.
      • Sinnecker S.
      • Zimmermann H.
      • Lubitz W.
      Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
      ,
      • Flores M.
      • Isaacson R.A.
      • Calvo R.
      • Feher G.
      • Lubitz W.
      Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR Spectroscopy at 35 GHz.
      ,
      • O'Malley P.J.
      • Babcock G.T.
      Powder ENDOR spectra of p-Benzoquinone anion radical: Principal hyperfine tensor components for ring protons and hydrogen-bonded protons.
      ,
      • MacMillan F.
      • Lendzian F.
      • Lubitz W.
      EPR and ENDOR characterization of semiquinone anion radicals related to photosynthesis.
      ). The T value measured for H3 is one of the largest measured so far for a proton hydrogen-bonded to a semiquinone. According to density functional theory calculations, the large T value of H3 is predicted to account for a short hydrogen-bond length, typically in the range of 1.3–1.4 Å (
      • Sinnecker S.
      • Reijerse E.
      • Neese F.
      • Lubitz W.
      Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions.
      ). In this case, Sinnecker et al. (
      • Sinnecker S.
      • Flores M.
      • Lubitz W.
      Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: effect of hydrogen bonding on the electronic and geometric structure of the primary quinone. A density functional theory study.
      ,
      • Sinnecker S.
      • Reijerse E.
      • Neese F.
      • Lubitz W.
      Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions.
      ,
      • Flores M.
      • Isaacson R.A.
      • Calvo R.
      • Feher G.
      • Lubitz W.
      Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR Spectroscopy at 35 GHz.
      ) have shown that the point dipole model does not work due to the increased covalent character of the H-bond that is not covered in the point dipole approximation. A more reliable alternative approach is to evaluate H-bond distances from the nuclear quadrupole coupling constant of 2H. As empirically proposed by Soda and Chiba (
      • Soda G.
      • Chiba T.
      Deuteron magnetic resonance study of cupric sulfate pentahydrate.
      ) and Hunt and Mackay (
      • Hunt M.J.
      • Mackay A.L.
      Deuterium and nitrogen pure quadrupole resonance in deuterated amino acids.
      ), it has been shown that the nuclear quadrupole coupling constant of 2H nuclei H-bonded to semiquinones follows a r−3(O–2H) dependence of the form
      κ=abr3O2H[kHz]
      (Eq. 2)


      where a and b are empirical parameters (
      • Sinnecker S.
      • Reijerse E.
      • Neese F.
      • Lubitz W.
      Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions.
      ). Using a = 319 kHz and b = 607 kHz Å3 (
      • Flores M.
      • Isaacson R.
      • Abresch E.
      • Calvo R.
      • Lubitz W.
      • Feher G.
      Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II: geometry of the hydrogen bonds to the primary quinone formula by 1H and 2H ENDOR spectroscopy.
      ) and the value of κ = 176 ± 4 kHz deduced from our work, we obtain from Equation 2 a bond length of r(O–2H) = 1.62 ± 0.02 Å. This value is in the range of short hydrogen bonds for biological systems. For instance, it is similar to that formed from the carbonyl oxygen O4 of the ubisemiquinone QA•− in the RC from Rhodobacter sphaeroides R-26 to the imidazole nitrogen Nδ of His M219 (r(O–2H) = 1.60 ± 0.04 Å) (
      • Flores M.
      • Isaacson R.
      • Abresch E.
      • Calvo R.
      • Lubitz W.
      • Feher G.
      Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II: geometry of the hydrogen bonds to the primary quinone formula by 1H and 2H ENDOR spectroscopy.
      ).

       Assignment of Non-exchangeable Proton Couplings

      In addition to H3, two non-exchangeable proton couplings H1 and H2 are clearly resolved in the HYSCORE spectra of MSQD measured in 2H2O. For their assignment, we rely on previous experimental and theoretical studies on phylloquinone (also called vitamin K1) and menaquinone (vitamin K2) radicals examined in liquid and solid organic solvents or in proteins (
      • Epel B.
      • Niklas J.
      • Sinnecker S.
      • Zimmermann H.
      • Lubitz W.
      Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
      ,
      • Yi S.M.
      • Narasimhulu K.V.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
      ,
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ,
      • Das M.R.
      • Connor H.D.
      • Leniart D.S.
      • Freed J.H.
      An electron nuclear double resonance and electron spin resonance study of semiquinones related to vitamins K and E.
      ,
      • Teutloff C.
      • Bittl R.
      • Lubitz W.
      Pulse ENDOR studies on the radical pair P700·+ A1·− and the photoaccumulated quinone acceptor A1·− of photosystem I.
      ,
      • O'Malley P.J.
      Density functional calculated spin densities and hyperfine couplings for hydrogen-bonded 1,4-naphthosemiquinone and phyllosemiquinone anion radicals: a model for the A1 free radical formed in photosystem I.
      ,
      • Hastings S.F.
      • Heathcote P.
      • Ingledew W.J.
      • Rigby S.E.
      ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.
      ,
      • Rigby S.E.
      • Evans M.C.
      • Heathcote P.
      Electron nuclear double resonance (ENDOR) spectroscopy of radicals in photosystem I and related type 1 photosynthetic reaction centers.
      ). Indeed, these quinones share the same naphthoquinone ring structure methylated at the second position but differ in their aliphatic side chain attached at the 3-position (see supplemental Fig. S1). It has been shown both experimentally and theoretically that the aliphatic side chain properties have only a weak influence on the proton hyperfine coupling tensors measured in organic solvents (
      • Epel B.
      • Niklas J.
      • Sinnecker S.
      • Zimmermann H.
      • Lubitz W.
      Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
      ,
      • Das M.R.
      • Connor H.D.
      • Leniart D.S.
      • Freed J.H.
      An electron nuclear double resonance and electron spin resonance study of semiquinones related to vitamins K and E.
      ,
      • Teutloff C.
      • Bittl R.
      • Lubitz W.
      Pulse ENDOR studies on the radical pair P700·+ A1·− and the photoaccumulated quinone acceptor A1·− of photosystem I.
      ,
      • Rigby S.E.
      • Evans M.C.
      • Heathcote P.
      ENDOR and special triple resonance spectroscopy of A1•− of photosystem 1.
      ,
      • Gardiner A.T.
      • Zech S.G.
      • MacMillan F.
      • Käss H.
      • Bittl R.
      • Schlodder E.
      • Lendzian F.
      • Lubitz W.
      Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.
      ). From these previous studies, it is evident that the non-exchangeable proton couplings from H1 and H2 > 2 MHz originate from the ring methyl protons, from the β-methylene isoprenyl protons, or from α-protons directly attached to the quinone ring. Due to rapid rotation of the methyl group even at low temperature, methyl protons of vitamin K molecules have equal hyperfine tensors and give prominent ENDOR/ESEEM signals. They are characterized by an almost axial hyperfine tensor, a predominant isotropic hyperfine coupling value in the range of 6.8–12.3 MHz, and a characteristic relative hyperfine anisotropy (A −A)/Aiso in the range of 0.26–0.45 with A > A > 0. The latter value increases up to 0.76 for the methyl protons of the asymmetrically bound ubisemiquinone QA•− in the RC from R. sphaeroides (
      • Lubitz W.
      • Feher G.
      The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals QA•− and QB•− by EPR and ENDOR.
      ). Based on these results, we assign H1 to the methyl protons of MSQD, with Aiso = +5.53 MHz and T = +1.25 MHz leading to a (A − A)/Aiso value of ∼0.68 (Fig. 5).
      Figure thumbnail gr5
      FIGURE 5Working model of MSQD binding mode in E. coli NarGHI based on our spectroscopic work. Strongly asymmetric binding of MSQD occurs via a short in-plane H-bond to the Nδ of His-66, whereas Lys-86 does not appear to be a direct H-bond donor to the radical in the semiquinone state (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). The MSQD O4 oxygen is deprotonated. The protons H1, H2, and H3 discussed under “Results” are indicated by arrows. r = NHCCO(CH2)3.
      Hyperfine data for β-methylene protons and ring α protons in semiquinones are less abundant. Unlike the methyl protons, the methylene protons are not expected to rotate freely (
      • Zheng M.
      • Dismukes G.C.
      The conformation of the isoprenyl chain relative to the semiquinone head in the primary electron acceptor (QA) of higher plant PSII (plastosemiquinone) differs from that in bacterial reaction centers (ubisemiquinone or menasemiquinone) by ∼90 degrees.
      ). They are also characterized by a nearly axial hyperfine tensor with A > A > 0 (
      • Zheng M.
      • Dismukes G.C.
      The conformation of the isoprenyl chain relative to the semiquinone head in the primary electron acceptor (QA) of higher plant PSII (plastosemiquinone) differs from that in bacterial reaction centers (ubisemiquinone or menasemiquinone) by ∼90 degrees.
      ,
      • Kevan L.
      • Kispert L.D.
      ). In contrast, a high degree of anisotropy is expected for a ring α proton hyperfine tensor due to the short distance of the proton and the spin density (
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ,
      • Zheng M.
      • Dismukes G.C.
      The conformation of the isoprenyl chain relative to the semiquinone head in the primary electron acceptor (QA) of higher plant PSII (plastosemiquinone) differs from that in bacterial reaction centers (ubisemiquinone or menasemiquinone) by ∼90 degrees.
      ). Because of the axial symmetry of its hyperfine coupling tensor, we tentatively assign H2 to one of the β-methylene isoprenyl protons with Aiso = +0.96 MHz and T = +1.18 MHz (Fig. 5). Overall, the 1H hyperfine coupling constants determined in this work are consistent with those estimated from our previous continuous wave X-band 1H ENDOR studies of MSQD (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ,
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      ).

       Strongly Asymmetric Spin Distribution in MSQD

      The 1H hyperfine coupling constants are an excellent probe of the asymmetry of the spin density distribution in the quinone. In particular, the predominant isotropic component, Aiso, of hyperfine coupling to the methyl protons is directly proportional to the unpaired spin density in the π orbital on the adjacent α-carbon (ρC), as described by the McConnell relation, Aiso = ρCB2/2, where B2 has been taken in the range from 120 to 212 MHz (
      • McConnell H.M.
      Indirect hyperfine interactions in the paramagnetic resonance spectra of aromatic free radicals.
      ). The corresponding value for the methyl protons of MSQD (Aiso = 5.53 MHz) is the smallest one ever reported for these protons for vitamin K molecules bound to proteins or in protic solvents (
      • Epel B.
      • Niklas J.
      • Sinnecker S.
      • Zimmermann H.
      • Lubitz W.
      Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
      ,
      • Yi S.M.
      • Narasimhulu K.V.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
      ,
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ,
      • Das M.R.
      • Connor H.D.
      • Leniart D.S.
      • Freed J.H.
      An electron nuclear double resonance and electron spin resonance study of semiquinones related to vitamins K and E.
      ,
      • Teutloff C.
      • Bittl R.
      • Lubitz W.
      Pulse ENDOR studies on the radical pair P700·+ A1·− and the photoaccumulated quinone acceptor A1·− of photosystem I.
      ,
      • Hastings S.F.
      • Heathcote P.
      • Ingledew W.J.
      • Rigby S.E.
      ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.
      ,
      • Rigby S.E.
      • Evans M.C.
      • Heathcote P.
      ENDOR and special triple resonance spectroscopy of A1•− of photosystem 1.
      ,
      • Gardiner A.T.
      • Zech S.G.
      • MacMillan F.
      • Käss H.
      • Bittl R.
      • Schlodder E.
      • Lendzian F.
      • Lubitz W.
      Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.
      ). In particular, the spin density on the Cα at the 2-position, which is sensed by the methyl protons, is reduced by ∼30% in MSQD as compared with the symmetrically hydrogen bonded MSQ-4 prepared in 2-propanol (
      • Gardiner A.T.
      • Zech S.G.
      • MacMillan F.
      • Käss H.
      • Bittl R.
      • Schlodder E.
      • Lendzian F.
      • Lubitz W.
      Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.
      ). This decrease can be explained by a strong asymmetry of hydrogen bonding to the carbonyl oxygens of MSQD in NarGHI, which leads to a redistribution of both the spin density and charges within the quinone ring (
      • Lubitz W.
      • Feher G.
      The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals QA•− and QB•− by EPR and ENDOR.
      ,
      • Lubitz W.
      • Abresch E.C.
      • Debus R.J.
      • Isaacson R.A.
      • Okamura M.Y.
      • Feher G.
      Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides.
      ,
      • Feher G.
      • Isaacson R.A.
      • Okamura M.Y.
      • Lubitz W.
      ). Indeed, a stronger hydrogen bond to the carbonyl oxygen O1, as compared with oxygen O4, is expected to lead to an increase of spin density on carbon 3 but a decrease of the spin density on carbon 2 as observed here for MSQD. A similar but less pronounced spin density shift has been proposed for the menasemiquinone-9 in the QA site of the RC from Rhodopseudomonas viridis for which the methyl protons isotropic hyperfine coupling constant is about 6.8 MHz (
      • Gardiner A.T.
      • Zech S.G.
      • MacMillan F.
      • Käss H.
      • Bittl R.
      • Schlodder E.
      • Lendzian F.
      • Lubitz W.
      Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.
      ). In contrast, the large isotropic constants for methyl protons of the MSQ-7 in the aa3 menaquinol oxidase from B. subtilis (Aiso ∼11.0 MHz) (
      • Yi S.M.
      • Narasimhulu K.V.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
      ) or of the radical anion of phylloquinone in the A1 site of photosystem I from Thermosynechococcus elongatus (Aiso = 9.8 MHz) (
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ) suggest that a stronger hydrogen bond to oxygen O4 is formed as compared with oxygen O1, a strongly asymmetric binding mode reverse to that observed in NarGHI.

       Model for MSQD Binding to NarGHI and Mechanistic Implications

      Altogether, the data inferred from the present work allow us to refine the MSQD binding model previously proposed (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). We conclude that the asymmetrical spin density distribution in MSQD is primarily due to the strong hydrogen bond formed to the O1 oxygen of MSQD involving the exchangeable H3 proton coupling (Fig. 5). Hence, the latter appears mostly responsible for the transfer of spin density from the radical to the interacting nitrogen nucleus that was deduced from the measurement of a small 14N isotropic hyperfine coupling of Aiso ∼0.8 MHz to MSQD using HYSCORE spectroscopy. Based on the measurement of its nuclear quadrupole parameters by S-band HYSCORE spectroscopy (κ = 0.49, η = 0.50), this nucleus was assigned to the Nδ imidazole nitrogen from the heme bD axial ligand His-66 (
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ). In addition, this model is consistent with the relatively small η value found for this imidazole nitrogen that is close to that predicted by density functional theory calculations on imidazoles forming a strong in-plane hydrogen bond to one of the benzosemiquinone oxygen atoms (
      • Fritscher J.
      Influence of hydrogen bond geometry on quadrupole coupling parameters: a theoretical study of imidazole–water and imidazole–semiquinone complexes.
      ). Finally, the presence of the positive charges on the nearby heme Fe2+ ion may also contribute to the observed asymmetrical spin distribution in MSQD.
      Interestingly, a single strong and highly ordered H-bond to MSQD was detected in the present work by using both 1H HYSCORE and 2H Q-band ENDOR spectroscopies. In the most recent studies of protein-bound semiquinones, the radicals appear to be coupled to at least two exchangeable protons assigned to those involved in H-bonds (
      • Flores M.
      • Isaacson R.
      • Abresch E.
      • Calvo R.
      • Lubitz W.
      • Feher G.
      Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II: geometry of the hydrogen bonds to the primary quinone formula by 1H and 2H ENDOR spectroscopy.
      ,
      • Yi S.M.
      • Narasimhulu K.V.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
      ,
      • Yap L.L.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli.
      ,
      • Dikanov S.A.
      • Samoilova R.I.
      • Kolling D.R.
      • Holland J.T.
      • Crofts A.R.
      Hydrogen bonds involved in binding the Qi site semiquinone in the bc1 complex, identified through deuterium exchange using pulsed EPR.
      ,
      • Martin E.
      • Samoilova R.I.
      • Narasimhulu K.V.
      • Lin T.J.
      • O'Malley P.J.
      • Wraight C.A.
      • Dikanov S.A.
      Hydrogen bonding and spin density distribution in the QB semiquinone of bacterial reaction centers and comparison with the QA site.
      ,
      • Chatterjee R.
      • Milikisiyants S.
      • Coates C.S.
      • Lakshmi K.V.
      High-resolution two-dimensional 1H and 14N hyperfine sublevel correlation spectroscopy of the primary quinone of photosystem II.
      ). One-sided H-bond was resolved only for the photoaccumulated phyllosemiquinone A1•− in photosystem I (
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ,
      • Srinivasan N.
      • Chatterjee R.
      • Milikisiyants S.
      • Golbeck J.H.
      • Lakshmi K.V.
      Effect of hydrogen bond strength on the redox properties of phylloquinones: a two-dimensional hyperfine sublevel correlation spectroscopy study of photosystem I.
      ). Based on their experimental and theoretical results, Niklas et al. (
      • Niklas J.
      • Epel B.
      • Antonkine M.L.
      • Sinnecker S.
      • Pandelia M.E.
      • Lubitz W.
      Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
      ) have shown that the single detected short ∼1.64 Å H-bond can fully account for the observed asymmetry in the spin density distribution of the SQ in the A1 site. Remarkably, both the H-bond length and the ∼30% variation of the spin density on the Cα at the 2-position of the phyllosemiquinone in the A1 site with respect to the symmetrically hydrogen-bonded radical are comparable with the corresponding values measured for MSQD. Thus, our results indicate that MSQD most likely binds to the protein via a one-sided H-bond.
      Consequently, our present work indicates that the O4 carbonyl oxygen of MSQD is not protonated in this intermediate state, showing that at least one proton has to be released to the periplasm consecutively to the first electron transfer step to heme bD. This step likely involves Lys-86, a residue that is located at the protein surface, at the entrance of the QD cavity, and that is required for correct binding of quinol analogues and for semiquinone detection at the QD site of NarGHI (
      • Lanciano P.
      • Magalon A.
      • Bertrand P.
      • Guigliarelli B.
      • Grimaldi S.
      High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
      ,
      • Grimaldi S.
      • Arias-Cartin R.
      • Lanciano P.
      • Lyubenova S.
      • Endeward B.
      • Prisner T.F.
      • Magalon A.
      • Guigliarelli B.
      Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
      ,
      • Bertero M.G.
      • Rothery R.A.
      • Boroumand N.
      • Palak M.
      • Blasco F.
      • Ginet N.
      • Weiner J.H.
      • Strynadka N.C.
      Structural and biochemical characterization of a quinol-binding site of Escherichia coli nitrate reductase A.
      ). Thus, we speculate that Lys-86 could be a direct hydrogen bond donor to the quinol molecule, facilitating proton abstraction at the quinol O4 oxygen and, concomitantly, the first electron transfer step. This would be accompanied by a movement of Lys-86 away from the substrate, leading to an asymmetric binding mode of the semiquinone intermediate and allowing for proton release toward the periplasm (Fig. 5). Whether quinol deprotonation at the O1 oxygen is coupled to the first or the second electron transfer step remains unclear. Additional studies aimed at measuring the pH dependence of the redox reactions occurring at the QD site will be useful to further understanding how quinol deprotonation and electron transfer are synchronized at the QD site. Such studies are currently being performed in our laboratories.

       Role of Protein Environment in Quinol Utilization and Semiquinone Stabilization

      The high stabilization of MSQD in NarGHI is directly related to the redox potentials of the redox transitions MQH2/MSQ (Em,7.5 = −150 mV) and MSQ/MQ (Em,7.5 = −40 mV), which are both thermodynamically favorable for electron transfer to the bD heme (
      • Grimaldi S.
      • Lanciano P.
      • Bertrand P.
      • Blasco F.
      • Guigliarelli B.
      Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
      ). We questioned whether the unusually high stabilization of the semiquinone state at the NarGHI QD site can be related to the strongly asymmetric binding mode of MSQD mainly due to the short in-plane H-bond formed to the radical. Remarkably, the two other protein-bound semiquinones with redox properties most strongly affected by the protein environment as compared with the corresponding species in alcoholic solvents are the very low potential (Em ∼−750 mV) phyllosemiquinone anion A1•− in photosystem I and the high affinity ubisemiquinone at the QH site of cytochrome bo3, which has a high stability, albeit 10 times smaller than that measured for MSQD. In both cases, the semiquinones interact with the protein environment in a very asymmetric manner (
      • Yap L.L.
      • Samoilova R.I.
      • Gennis R.B.
      • Dikanov S.A.
      Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli.
      ,
      • Grimaldi S.
      • Ostermann T.
      • Weiden N.
      • Mogi T.
      • Miyoshi H.
      • Ludwig B.
      • Michel H.
      • Prisner T.F.
      • MacMillan F.
      Asymmetric binding of the high-affinity QH•− ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy.
      ,
      • Kacprzak S.
      • Kaupp M.
      • MacMillan F.
      Protein-cofactor interactions and EPR parameters for the QH quinone-binding site of quinol oxidase: a density functional study.
      ,
      • MacMillan F.
      • Kacprzak S.
      • Hellwig P.
      • Grimaldi S.
      • Michel H.
      • Kaupp M.
      Elucidating mechanisms in heme copper oxidases: the high-affinity QH-binding site in quinol oxidase as studied by DONUT-HYSCORE spectroscopy and density functional theory.
      ). This contrasts to the much lower stability of the more symmetrically bound semiquinones at the QB site of photosynthetic bacterial RC (
      • Martin E.
      • Samoilova R.I.
      • Narasimhulu K.V.
      • Lin T.J.
      • O'Malley P.J.
      • Wraight C.A.
      • Dikanov S.A.
      Hydrogen bonding and spin density distribution in the QB semiquinone of bacterial reaction centers and comparison with the QA site.
      ,
      • Rutherford A.W.
      • Evans M.C.
      Direct measurement of the redox potential of the primary and secondary quinone electron acceptors in Rhodopseudomonas sphaeroides (wild-type) by EPR spectrometry.
      ) or at the Qi site in R. sphaeroides bc1 complex (
      • Dikanov S.A.
      • Samoilova R.I.
      • Kolling D.R.
      • Holland J.T.
      • Crofts A.R.
      Hydrogen bonds involved in binding the Qi site semiquinone in the bc1 complex, identified through deuterium exchange using pulsed EPR.
      ,
      • Robertson D.E.
      • Prince R.C.
      • Bowyer J.R.
      • Matsuura K.
      • Dutton P.L.
      • Ohnishi T.
      Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems.
      ). In addition, the strong H-bond to MSQD is expected to withdraw electron density and stabilize the semiquinone form, thereby raising the redox potential of the second oxidation step from semiquinone to quinone, as experimentally measured. Consequently, we hypothesize that the atypical binding mode of MSQD could strongly contribute to its unusual redox properties. In addition, other effects such as the electrostatic environment of the nearby protein should also be taken into account. Evaluating their respective contribution to the MSQD redox properties requires further work. Finally, the functional implications of this high stabilization remain to be established.
      It has been shown both experimentally and theoretically that the presence of bulky substituents on the quinone ring force hydrogen-bond formation out-of-plane, thereby increasing simultaneously the isotropic and anisotropic coupling of the hydrogen-bonded protons (
      • MacMillan F.
      • Lendzian F.
      • Lubitz W.
      EPR and ENDOR characterization of semiquinone anion radicals related to photosynthesis.
      ,
      • O'Malley P.J.
      A density functional study of the effect of orientation of hydrogen bond donation on the hyperfine couplings of benzosemiquinones: relevance to semiquinone-protein hydrogen bonding interactions in vivo.
      ). Remarkably, measurement of a small Aiso and a simultaneous large T value for the proton hydrogen-bonded to MSQD indicates that the protein environment around the radical strongly constrains the geometry of the hydrogen bond by maintaining a short in-plane H-bond to the radical, thus leading to the observed peculiar hyperfine coupling characteristics. We have recently shown that an endogenous USQD can also be stabilized at the NarGHI QD site (
      • Arias-Cartin R.
      • Lyubenova S.
      • Ceccaldi P.
      • Prisner T.
      • Magalon A.
      • Guigliarelli B.
      • Grimaldi S.
      HYSCORE evidence that endogenous mena- and ubisemiquinone bind at the same Q site (QD) of Escherichia coli nitrate reductase A.
      ). It binds to the protein via an H-bond to the same nitrogen as menasemiquinone does, i.e. most likely His-66 Nδ. The similar 14N HYSCORE pattern observed for both radicals suggests that the H-bond involved in binding USQD has similar characteristics to that detected in the present study. This provides further support for its involvement in ubisemiquinone stabilization at the QD site. Finally, our work indicates that the protein environment counteracts the effect of the presence of bulky substituents to impose an atypical binding mode. Further work is in progress in our laboratories to evaluate the importance of the quinone substituents to accommodate and utilize various substrates at this Q-site and to stabilize semiquinone intermediates.

       Concluding Remarks

      From the experiments reported in this work, we conclude that MSQD is involved in a single strong in-plane and highly ordered H-bond with a solvent exchangeable proton. This strongly asymmetric binding causes a shift of the electron spin density over the quinone ring consistent with the formation of a strong hydrogen bond to the quinone carbonyl oxygen O1. This peculiar binding mode could strongly contribute to the unusual redox properties of MSQD.

      Acknowledgments

      We thank Guillaume Gerbaud and Emilien Etienne for maintenance of the Aix-Marseille EPR facility and Patrick Bertrand and Frédéric Biaso for helpful discussions.

      Supplementary Material

      REFERENCES

        • Nicholls D.G.
        • Ferguson S.J.
        Bioenergetics. Academic Press, London2002
        • Lubitz W.
        • Feher G.
        The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals QA•− and QB•− by EPR and ENDOR.
        Appl. Magn. Reson. 1999; 17: 1-48
        • Srinivasan N.
        • Golbeck J.H.
        Protein-cofactor interactions in bioenergetic complexes: the role of the A1A and A1B phylloquinones in photosystem I.
        Biochim. Biophys. Acta. 2009; 1787: 1057-1088
        • Stowell M.H.
        • McPhillips T.M.
        • Rees D.C.
        • Soltis S.M.
        • Abresch E.
        • Feher G.
        Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer.
        Science. 1997; 276: 812-816
        • Lanciano P.
        • Savoyant A.
        • Grimaldi S.
        • Magalon A.
        • Guigliarelli B.
        • Bertrand P.
        New method for the spin quantitation of [4Fe-4S]+ clusters with S = 3/2: application to the FS0 center of the NarGHI nitrate reductase from Escherichia coli.
        J. Phys. Chem. B. 2007; 111: 13632-13637
        • Guigliarelli B.
        • Magalon A.
        • Asso M.
        • Bertrand P.
        • Frixon C.
        • Giordano G.
        • Blasco F.
        Complete coordination of the four Fe-S centers of the β subunit from Escherichia coli nitrate reductase: physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters.
        Biochemistry. 1996; 35: 4828-4836
        • Blasco F.
        • Guigliarelli B.
        • Magalon A.
        • Asso M.
        • Giordano G.
        • Rothery R.A.
        The coordination and function of the redox centers of the membrane-bound nitrate reductases.
        Cell Mol. Life Sci. 2001; 58: 179-193
        • Rothery R.A.
        • Blasco F.
        • Magalon A.
        • Weiner J.H.
        The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols.
        J. Mol. Microbiol. Biotechnol. 2001; 3: 273-283
        • Bertero M.G.
        • Rothery R.A.
        • Palak M.
        • Hou C.
        • Lim D.
        • Blasco F.
        • Weiner J.H.
        • Strynadka N.C.
        Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A.
        Nat. Struct. Biol. 2003; 10: 681-687
        • Grimaldi S.
        • Lanciano P.
        • Bertrand P.
        • Blasco F.
        • Guigliarelli B.
        Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
        Biochemistry. 2005; 44: 1300-1308
        • Lanciano P.
        • Magalon A.
        • Bertrand P.
        • Guigliarelli B.
        • Grimaldi S.
        High stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.
        Biochemistry. 2007; 46: 5323-5329
        • Arias-Cartin R.
        • Lyubenova S.
        • Ceccaldi P.
        • Prisner T.
        • Magalon A.
        • Guigliarelli B.
        • Grimaldi S.
        HYSCORE evidence that endogenous mena- and ubisemiquinone bind at the same Q site (QD) of Escherichia coli nitrate reductase A.
        J. Am. Chem. Soc. 2010; 132: 5942-5943
        • Grimaldi S.
        • Arias-Cartin R.
        • Lanciano P.
        • Lyubenova S.
        • Endeward B.
        • Prisner T.F.
        • Magalon A.
        • Guigliarelli B.
        Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.
        J. Biol. Chem. 2010; 285: 179-187
        • Bertero M.G.
        • Rothery R.A.
        • Boroumand N.
        • Palak M.
        • Blasco F.
        • Ginet N.
        • Weiner J.H.
        • Strynadka N.C.
        Structural and biochemical characterization of a quinol-binding site of Escherichia coli nitrate reductase A.
        J. Biol. Chem. 2005; 280: 14836-14843
        • Arias-Cartin R.
        • Grimaldi S.
        • Pommier J.
        • Lanciano P.
        • Schaefer C.
        • Arnoux P.
        • Giordano G.
        • Guigliarelli B.
        • Magalon A.
        Cardiolipin-based respiratory complex activation in bacteria.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 7781-7786
        • Potter L.C.
        • Millington P.
        • Griffiths L.
        • Thomas G.H.
        • Cole J.A.
        Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth?.
        Biochem. J. 1999; 344: 77-84
        • Mims W.B.
        Pulsed ENDOR experiments.
        Proc. R. Soc. Lond. Ser. A. 1965; 283: 452-457
        • Stoll S.
        • Schweiger A.
        EasySpin, a comprehensive software package for spectral simulation and analysis in EPR.
        J. Magn. Reson. 2006; 178: 42-55
        • Dikanov S.A.
        • Bowman M.K.
        Cross-peak lineshape of two-dimensional ESEEM spectra in disordered S = ½, I = ½ spin systems.
        J. Magn. Reson. 1995; 116: 125-128
        • Flores M.
        • Isaacson R.
        • Abresch E.
        • Calvo R.
        • Lubitz W.
        • Feher G.
        Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: II: geometry of the hydrogen bonds to the primary quinone formula by 1H and 2H ENDOR spectroscopy.
        Biophys. J. 2007; 92: 671-682
        • Sinnecker S.
        • Flores M.
        • Lubitz W.
        Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: effect of hydrogen bonding on the electronic and geometric structure of the primary quinone. A density functional theory study.
        Phys. Chem. Chem. Phys. 2006; 8: 5659-5670
        • Sinnecker S.
        • Reijerse E.
        • Neese F.
        • Lubitz W.
        Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions.
        J. Am. Chem. Soc. 2004; 126: 3280-3290
        • Epel B.
        • Niklas J.
        • Sinnecker S.
        • Zimmermann H.
        • Lubitz W.
        Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
        J. Phys. Chem. B. 2006; 110: 11549-11560
        • Flores M.
        • Isaacson R.A.
        • Calvo R.
        • Feher G.
        • Lubitz W.
        Probing hydrogen bonding to quinone anion radicals by 1H and 2H ENDOR Spectroscopy at 35 GHz.
        Chem. Phys. 2003; 294: 401-413
        • Soda G.
        • Chiba T.
        Deuteron magnetic resonance study of cupric sulfate pentahydrate.
        J. Chem. Phys. 1969; 50: 439-455
        • Hunt M.J.
        • Mackay A.L.
        Deuterium and nitrogen pure quadrupole resonance in deuterated amino acids.
        J. Magn. Reson. 1974; 15: 402-414
        • Schweiger A.
        • Jeschke G.
        Principles of Pulse Electron Paramagnetic Resonance. Oxford University Press, New York2001
        • Reijerse E.J.
        • Dikanov S.A.
        Electron spin echo envelope modulation spectroscopy on orientationally disordered systems: Line shape singularities in S = ½, I = ½ spin systems.
        J. Chem. Phys. 1991; 95: 836-845
        • Yi S.M.
        • Narasimhulu K.V.
        • Samoilova R.I.
        • Gennis R.B.
        • Dikanov S.A.
        Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
        J. Biol. Chem. 2010; 285: 18241-18251
        • Niklas J.
        • Epel B.
        • Antonkine M.L.
        • Sinnecker S.
        • Pandelia M.E.
        • Lubitz W.
        Electronic structure of the quinone radical anion A1•− of photosystem I investigated by advanced pulse EPR and ENDOR techniques.
        J. Phys. Chem. B. 2009; 113: 10367-10379
        • O'Malley P.J.
        • Babcock G.T.
        Powder ENDOR spectra of p-Benzoquinone anion radical: Principal hyperfine tensor components for ring protons and hydrogen-bonded protons.
        J. Am. Chem. Soc. 1986; 108: 3995-4001
        • MacMillan F.
        • Lendzian F.
        • Lubitz W.
        EPR and ENDOR characterization of semiquinone anion radicals related to photosynthesis.
        Magn. Reson. Chem. 1995; 33: S81-S93
        • Das M.R.
        • Connor H.D.
        • Leniart D.S.
        • Freed J.H.
        An electron nuclear double resonance and electron spin resonance study of semiquinones related to vitamins K and E.
        J. Am. Chem. Soc. 1970; 92: 2258-2268
        • Teutloff C.
        • Bittl R.
        • Lubitz W.
        Pulse ENDOR studies on the radical pair P700·+ A1·− and the photoaccumulated quinone acceptor A1·− of photosystem I.
        Appl. Magn. Reson. 2004; 26: 5-21
        • O'Malley P.J.
        Density functional calculated spin densities and hyperfine couplings for hydrogen-bonded 1,4-naphthosemiquinone and phyllosemiquinone anion radicals: a model for the A1 free radical formed in photosystem I.
        Biochim. Biophys. Acta. 1999; 1411: 101-113
        • Hastings S.F.
        • Heathcote P.
        • Ingledew W.J.
        • Rigby S.E.
        ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.
        Eur. J. Biochem. 2000; 267: 5638-5645
        • Rigby S.E.
        • Evans M.C.
        • Heathcote P.
        Electron nuclear double resonance (ENDOR) spectroscopy of radicals in photosystem I and related type 1 photosynthetic reaction centers.
        Biochim. Biophys. Acta. 2001; 1507: 247-259
        • Rigby S.E.
        • Evans M.C.
        • Heathcote P.
        ENDOR and special triple resonance spectroscopy of A1•− of photosystem 1.
        Biochemistry. 1996; 35: 6651-6656
        • Gardiner A.T.
        • Zech S.G.
        • MacMillan F.
        • Käss H.
        • Bittl R.
        • Schlodder E.
        • Lendzian F.
        • Lubitz W.
        Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.
        Biochemistry. 1999; 38: 11773-11787
        • Zheng M.
        • Dismukes G.C.
        The conformation of the isoprenyl chain relative to the semiquinone head in the primary electron acceptor (QA) of higher plant PSII (plastosemiquinone) differs from that in bacterial reaction centers (ubisemiquinone or menasemiquinone) by ∼90 degrees.
        Biochemistry. 1996; 35: 8955-8963
        • Kevan L.
        • Kispert L.D.
        Electron Spin Double Resonance Spectroscopy. John Wiley & Sons, Inc., New York1976
        • McConnell H.M.
        Indirect hyperfine interactions in the paramagnetic resonance spectra of aromatic free radicals.
        J. Chem. Phys. 1956; 24: 764-766
        • Lubitz W.
        • Abresch E.C.
        • Debus R.J.
        • Isaacson R.A.
        • Okamura M.Y.
        • Feher G.
        Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides.
        Biochim. Biophys. Acta. 1985; 808: 464-469
        • Feher G.
        • Isaacson R.A.
        • Okamura M.Y.
        • Lubitz W.
        Michel-Beyerle M.E. Antennas and Reaction Centers of Photosynthetic Bacteria. Vol. 42. Springer-Verlag, Berlin1985: 174-189
        • Fritscher J.
        Influence of hydrogen bond geometry on quadrupole coupling parameters: a theoretical study of imidazole–water and imidazole–semiquinone complexes.
        Phys. Chem. Chem. Phys. 2004; 6: 4950-4956
        • Yap L.L.
        • Samoilova R.I.
        • Gennis R.B.
        • Dikanov S.A.
        Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli.
        J. Biol. Chem. 2006; 281: 16879-16887
        • Dikanov S.A.
        • Samoilova R.I.
        • Kolling D.R.
        • Holland J.T.
        • Crofts A.R.
        Hydrogen bonds involved in binding the Qi site semiquinone in the bc1 complex, identified through deuterium exchange using pulsed EPR.
        J. Biol. Chem. 2004; 279: 15814-15823
        • Martin E.
        • Samoilova R.I.
        • Narasimhulu K.V.
        • Lin T.J.
        • O'Malley P.J.
        • Wraight C.A.
        • Dikanov S.A.
        Hydrogen bonding and spin density distribution in the QB semiquinone of bacterial reaction centers and comparison with the QA site.
        J. Am. Chem. Soc. 2011; 133: 5525-5537
        • Chatterjee R.
        • Milikisiyants S.
        • Coates C.S.
        • Lakshmi K.V.
        High-resolution two-dimensional 1H and 14N hyperfine sublevel correlation spectroscopy of the primary quinone of photosystem II.
        Biochemistry. 2011; 50: 491-501
        • Srinivasan N.
        • Chatterjee R.
        • Milikisiyants S.
        • Golbeck J.H.
        • Lakshmi K.V.
        Effect of hydrogen bond strength on the redox properties of phylloquinones: a two-dimensional hyperfine sublevel correlation spectroscopy study of photosystem I.
        Biochemistry. 2011; 50: 3495-3501
        • Grimaldi S.
        • Ostermann T.
        • Weiden N.
        • Mogi T.
        • Miyoshi H.
        • Ludwig B.
        • Michel H.
        • Prisner T.F.
        • MacMillan F.
        Asymmetric binding of the high-affinity QH•− ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy.
        Biochemistry. 2003; 42: 5632-5639
        • Kacprzak S.
        • Kaupp M.
        • MacMillan F.
        Protein-cofactor interactions and EPR parameters for the QH quinone-binding site of quinol oxidase: a density functional study.
        J. Am. Chem. Soc. 2006; 128: 5659-5671
        • MacMillan F.
        • Kacprzak S.
        • Hellwig P.
        • Grimaldi S.
        • Michel H.
        • Kaupp M.
        Elucidating mechanisms in heme copper oxidases: the high-affinity QH-binding site in quinol oxidase as studied by DONUT-HYSCORE spectroscopy and density functional theory.
        Faraday Discuss. 2011; 148: 315-344
        • Rutherford A.W.
        • Evans M.C.
        Direct measurement of the redox potential of the primary and secondary quinone electron acceptors in Rhodopseudomonas sphaeroides (wild-type) by EPR spectrometry.
        FEBS Lett. 1980; 110: 257-261
        • Robertson D.E.
        • Prince R.C.
        • Bowyer J.R.
        • Matsuura K.
        • Dutton P.L.
        • Ohnishi T.
        Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems.
        J. Biol. Chem. 1984; 259: 1758-1763
        • O'Malley P.J.
        A density functional study of the effect of orientation of hydrogen bond donation on the hyperfine couplings of benzosemiquinones: relevance to semiquinone-protein hydrogen bonding interactions in vivo.
        Chem. Phys. Lett. 1998; 291: 367-374