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Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2 H and 15 N Labeling, HYSCORE Spectroscopy, and DFT Modeling
Author(s) -
Seif Eddine Maryam,
Biaso Frédéric,
AriasCartin Rodrigo,
Pilet Eric,
Rendon Julia,
Lyubenova Sevdalina,
Seduk Farida,
Guigliarelli Bruno,
Magalon Axel,
Grimaldi Stéphane
Publication year - 2017
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201700571
Subject(s) - chemistry , substituent , imidazole , hydrogen bond , hyperfine structure , stereochemistry , crystallography , spectroscopy , computational chemistry , molecule , organic chemistry , physics , quantum mechanics
In vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli . 15 N selective labeling of His 15 Nδ or Lys 15 Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His 15 Nδ as the direct hydrogen‐bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys 15 Nζ consistent with a through‐space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2 H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2 H—and hence of the 1 H—methyl isotropic hyperfine coupling by 2 H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen‐bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one‐sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode.