Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2 H and 15 N Labeling, HYSCORE Spectroscopy, and DFT Modeling

• Published in: Chemphyschem Vol. 18; no. 19; pp. 2704 - 2714
• Main Authors: Seif Eddine, Maryam, Biaso, Frédéric, Arias-Cartin, Rodrigo, Pilet, Eric, Rendon, Julia, Lyubenova, Sevdalina, Seduk, Farida, Guigliarelli, Bruno, Magalon, Axel, Grimaldi, Stéphane
• Format: Journal Article
• Language: English
• Published: Germany Wiley-VCH Verlag 06.10.2017
• Subjects: Biochemistry, Molecular Biology; Life Sciences; quinones; bioenergetics; electron transfer; EPR spectroscopy; metalloenzymes
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.201700571

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. N selective labeling of His Nδ or Lys Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His Nδ as the direct hydrogen-bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys 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 H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the H-and hence of the H-methyl isotropic hyperfine coupling by 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.