title: Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism.
abstract: A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family.
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, LCB UMR7283, Marseille, France.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Arias-Cartin
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, LCB UMR7283, Marseille, France.|Aix-Marseille Univ, CNRS, IMM, BIP UMR7281, Marseille, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Ceccaldi
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, BIP UMR7281, Marseille, France.
authors.forename: Barbara
authors.initials: B
authors.lastname: Schoepp-Cothenet
authors.affiliation: Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany.
authors.forename: Klaudia
authors.initials: K
authors.lastname: Frick
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, LCB UMR7283, Marseille, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Blanc
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, BIP UMR7281, Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, LCB UMR7283, Marseille, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Walburger
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, BIP UMR7281, Marseille, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Grimaldi
authors.affiliation: Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Friedrich
authors.affiliation: Aix-Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM UMR7258, Marseille, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Receveur-Brechot
authors.affiliation: Aix-Marseille Univ, CNRS, IMM, LCB UMR7283, Marseille, France.
authors.forename: Axel
authors.initials: A
authors.lastname: Magalon
mesh_terms: D000595:Amino Acid Sequence; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008982:Molybdenum / Q000378:metabolism*; D050901:Nitrate Reductase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
chemical_list: D008667:Metalloproteins; D008982:Molybdenum; D050901:Nitrate Reductase