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High-Stability Semiquinone Intermediate in Nitrate Reductase A (NarGHI) from Escherichia coli Is Located in a Quinol Oxidation Site Close to Heme b D †

Abstract : The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (QD) located in the vicinity of the distal heme bD. Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. 14N and 15N ESEEM and HYSCORE measurements carried out at X-band (∼9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with 15N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant Aiso(14N) ∼0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using 14N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, ∼3.4 GHz), the 14N nuclear quadrupolar parameters of the interacting nitrogen nucleus (κ = 0.49, η = 0.50) were determined and correspond to those of a histidine Nδ, assigned to the heme bD ligand His-66 residue. Moreover S-band 15N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T(15N) = 0.16 MHz). A distance of ∼2.2 Åbetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the QD site of NarGHI.
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https://hal.archives-ouvertes.fr/hal-00336035
Contributor : Laure Azzopardi <>
Submitted on : Friday, October 31, 2008 - 4:14:49 PM
Last modification on : Thursday, January 25, 2018 - 6:26:02 PM

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Pascal Lanciano, Axel Magalon, Patrick Bertrand, Bruno Guigliarelli, Stéphane Grimaldi. High-Stability Semiquinone Intermediate in Nitrate Reductase A (NarGHI) from Escherichia coli Is Located in a Quinol Oxidation Site Close to Heme b D †. Biochemistry, American Chemical Society, 2007, 46 (18), pp.5323 - 5329. ⟨10.1021/bi700074y⟩. ⟨hal-00336035⟩

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