title: Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR,14,15N HYSCORE, and DFT Study.
abstract: Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in98Mo and15N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively15N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.
authors.affiliation: Aix Marseille Univ, CNRS, BIP , Marseille, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Rendon
authors.affiliation: Aix Marseille Univ, CNRS, BIP , Marseille, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Biaso
authors.affiliation: Aix Marseille Univ, CNRS, BIP , Marseille, France.|Aix Marseille Univ, CNRS, LCB , Marseille, France.
authors.forename: Pierre
authors.identifier: http://orcid.org/0000-0001-8156-5035
authors.initials: P
authors.lastname: Ceccaldi
authors.affiliation: Aix Marseille Univ, CNRS, LCB , Marseille, France.
authors.forename: René
authors.initials: R
authors.lastname: Toci
authors.affiliation: Aix Marseille Univ, CNRS, LCB , Marseille, France.
authors.forename: Farida
authors.initials: F
authors.lastname: Seduk
authors.affiliation: Aix Marseille Univ, CNRS, LCB , Marseille, France.
authors.forename: Axel
authors.initials: A
authors.lastname: Magalon
authors.affiliation: Aix Marseille Univ, CNRS, BIP , Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.affiliation: Aix Marseille Univ, CNRS, BIP , Marseille, France.
authors.forename: Stéphane
authors.identifier: http://orcid.org/0000-0002-9559-6112
authors.initials: S
authors.lastname: Grimaldi
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D009565:Nitrate Reductases / Q000737:chemistry* / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D011789:Quantum Theory
chemical_list: D009942:Organometallic Compounds; D008982:Molybdenum; D009565:Nitrate Reductases