title: Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site.
abstract: Quinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E'm,7.5 (DMK/DMKH2) ~-70mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI.
authors.affiliation: Aix-Marseille Université, CNRS, Unité de Bioénergétique et Ingénierie des Protéines (UMR7281), F-13402 Marseille, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Rendon
authors.affiliation: Aix-Marseille Université, CNRS, Unité de Bioénergétique et Ingénierie des Protéines (UMR7281), F-13402 Marseille, France; Université Pierre et Marie Curie, Faculté de Biologie (UFR927), F-75005 Paris, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Pilet
authors.affiliation: Aix-Marseille Université, CNRS, Unité de Bioénergétique et Ingénierie des Protéines (UMR7281), F-13402 Marseille, France; Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), F-13402 Marseille, France.
authors.forename: Zeinab
authors.initials: Z
authors.lastname: Fahs
authors.affiliation: Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), F-13402 Marseille, France.
authors.forename: Farida
authors.initials: F
authors.lastname: Seduk
authors.affiliation: Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), F-13402 Marseille, France.
authors.forename: Léa
authors.initials: L
authors.lastname: Sylvi
authors.affiliation: Université Grenoble Alpes, Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), F-38000 Grenoble, France; Centre National de la Recherche Scientifique (CNRS), LAPM, F-38000 Grenoble, France.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Hajj Chehade
authors.affiliation: Université Grenoble Alpes, Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), F-38000 Grenoble, France; Centre National de la Recherche Scientifique (CNRS), LAPM, F-38000 Grenoble, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Pierrel
authors.affiliation: Aix-Marseille Université, CNRS, Unité de Bioénergétique et Ingénierie des Protéines (UMR7281), F-13402 Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.affiliation: Aix-Marseille Université, CNRS, Laboratoire de Chimie Bactérienne (UMR7283), F-13402 Marseille, France. Electronic address: magalon@imm.cnrs.fr.
authors.forename: Axel
authors.initials: A
authors.lastname: Magalon
authors.affiliation: Aix-Marseille Université, CNRS, Unité de Bioénergétique et Ingénierie des Protéines (UMR7281), F-13402 Marseille, France. Electronic address: grimaldi@imm.cnrs.fr.
authors.forename: Stephane
authors.initials: S
authors.lastname: Grimaldi
mesh_terms: D016227:Benzoquinones / Q000378:metabolism; D019069:Cell Respiration; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D006873:Hydroquinones / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009284:Naphthols / Q000737:chemistry; D050901:Nitrate Reductase / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D024482:Vitamin K 2 / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D006873:Hydroquinones; D009284:Naphthols; D009566:Nitrates; D024482:Vitamin K 2; C013917:demethylmenaquinone; D050901:Nitrate Reductase; C031927:hydroquinone
keywords: EPR spectroscopy; Electron transfer; Nitrate reductase; Protein cofactor interactions; Semiquinone