title: Structural and kinetic studies of the pyruvate-ferredoxin oxidoreductase/ferredoxin complex from Desulfovibrio africanus.
abstract: The pyruvate-ferredoxin oxidoreductase (PFOR)/ferredoxin (Fd) system of Desulfovibrio africanus has been investigated with the aim of understanding more fully protein-protein interaction and the kinetic characteristics of electron transfer between the two redox partners. D. africanus contains three Fds (Fd I, Fd II and Fd III) able to function as electron acceptors for PFOR. The complete amino acid sequence of Fd II was determined by automatic Edman degradation. It revealed a striking similarity to that of Fd I. The protein consists of 64 residues and its amino acid sequence is in agreement with a molecular mass of 6822.5 Da as measured by electrospray MS. Fd II contains five cysteine residues of which the first four (Cys11, Cys14, Cys17 and Cys54) are likely ligands for the single [4Fe-4S] cluster. A covalently cross-linked complex between PFOR and Fd I or Fd II was obtained by using a water soluble carbodiimide. This complex exhibited a stoichiometry of one ferredoxin for one PFOR subunit and is dependent on the ionic strength. The second-order rate constants for electron transfer between PFOR and Fds determined electrochemically using cyclic voltammetry are 7 x 107 M-1.s-1 for Fd I and 2 x 107 M-1.s-1 for Fd II and Fd III. The Km values of PFOR for Fd I and Fd II measured both by the electrochemical and the spectrophotometric method have been found to be 3 microM and 5 microM, respectively. The three-dimensional modelling of Fd II and surface analysis of Fd I, Fd II and PFOR suggest that a protein-protein complex is likely to be formed between aspartic acid/glutamic acid invariant residues of Fds and lysine residues surrounding the distal [4Fe-4S] cluster of PFOR. All of these studies are indicative of the involvement of electrostatic interactions between the two redox partners.
authors.affiliation: Unité de Bioénergétique et Ingéniérie des Protéines, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
authors.forename: L
authors.initials: L
authors.lastname: Pieulle
authors.forename: M H
authors.initials: MH
authors.lastname: Charon
authors.forename: P
authors.initials: P
authors.lastname: Bianco
authors.forename: J
authors.initials: J
authors.lastname: Bonicel
authors.forename: Y
authors.initials: Y
authors.lastname: Pétillot
authors.forename: E C
authors.initials: EC
authors.lastname: Hatchikian
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003901:Desulfovibrio / Q000201:enzymology*; D004563:Electrochemistry; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry*; D007658:Ketone Oxidoreductases / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D050743:Pyruvate Synthase; D016415:Sequence Alignment; D017421:Sequence Analysis; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D005288:Ferredoxins; D020033:Protein Isoforms; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase