NDLI: Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A*

Content Provider	PubMed Central
Author	Grimaldi, Stéphane Rodrigo, Arias-cartin Lanciano, Pascal Lyubenova, Sevdalina Burkhard, Endeward Prisner, Thomas F. Magalon, Axel Bruno, Guigliarelli
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 b D. 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 b D 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.
Related Links	http://dx.doi.org/10.1074/jbc.m109.060251
Ending Page	187
Page Count	9
Starting Page	179
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	1
Volume Number	285
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2010-01-01
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

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

Characterization by electron paramagnetic resonance of the role of the Escherichia coli nitrate reductase (NarGHI) iron-sulfur clusters in electron transfer to nitrate and identification of a semiquinone radical intermediate.

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

Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site

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Proton translocation and the respiratory nitrate reductase of Escherichia coli

Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation*

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A*

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Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

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