title: Multiple orientations in a physiological complex: the pyruvate-ferredoxin oxidoreductase-ferredoxin system.
abstract: Ferredoxin I from Desulfovibrio africanus (Da FdI) is a small acidic [4Fe-4S] cluster protein that exchanges electrons with pyruvate-ferredoxin oxidoreductase (PFOR), a key enzyme in the energy metabolism of anaerobes. The thermodynamic properties and the electron transfer between PFOR and either native or mutated FdI have been investigated by microcalorimetry and steady-state kinetics, respectively. The association constant of the PFOR-FdI complex is 3.85 x 10(5) M(-1), and the binding affinity has been found to be highly sensitive to ionic strength, suggesting the involvement of electrostatic forces in formation of the complex. Surprisingly, the punctual or combined neutralizations of carboxylate residues surrounding the [4Fe-4S] cluster slightly affect the PFOR-FdI interaction. Furthermore, hydrophobic residues around the cluster do not seem to be crucial for the PFOR-FdI system activity; however, some of them play an important role in the stability of the FeS cluster. NMR restrained docking associated with site-directed mutagenesis studies suggested the presence of various interacting sites on Da FdI. The modification of additional acidic residues at the interacting interface, generating a FdI pentamutant, evidenced at least two distinct FdI binding sites facing the distal [4Fe-4S] cluster of the PFOR. We also used a set of various small acidic partners to investigate the specificity of PFOR toward redox partners. The remarkable flexibility of the PFOR-FdI system supports the idea that the specificity of the physiological complex has probably been "sacrificed" to improve the turnover rate and thus the efficiency of bacterial electron transfer.
authors.affiliation: Unité de Bioénergétique et Ingéniérie des Protéines, Institut de Biologie Structurale et Microbiologie, CNRS, 31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Pieulle
authors.forename: Matthieu
authors.initials: M
authors.lastname: Nouailler
authors.forename: Xavier
authors.initials: X
authors.lastname: Morelli
authors.forename: Christine
authors.initials: C
authors.lastname: Cavazza
authors.forename: Philippe
authors.initials: P
authors.lastname: Gallice
authors.forename: Stéphane
authors.initials: S
authors.lastname: Blanchet
authors.forename: Pierre
authors.initials: P
authors.lastname: Bianco
authors.forename: Françoise
authors.initials: F
authors.lastname: Guerlesquin
authors.forename: E Claude
authors.initials: EC
authors.lastname: Hatchikian
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D003001:Cloning, Molecular; D045284:Desulfovibrio africanus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D004734:Energy Metabolism / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D057927:Hydrophobic and Hydrophilic Interactions; D007658:Ketone Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D050743:Pyruvate Synthase; D055672:Static Electricity; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D005288:Ferredoxins; D046911:Macromolecular Substances; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase