Structural and kinetic studies of the pyruvate-ferredoxin oxidoreductase/ferredoxin complex from Desulfovibrio africanus

The pyruvate-ferredoxin oxidoreductase (PFOR)/ferredoxin (Fd) system of Des ulfovibrio africanus has been investigated with the aim of understanding mo re fully protein-protein interaction and the kinetic characteristics of ele ctron transfer between the two redox partners. D. africanus contains three Fds (Fd I, Fd II and Fd III) able to function as electron accepters for PFO R. The complete amino acid sequence of Fd II was determined by automatic Ed man degradation. It revealed a striking similarity to that of Fd I. The pro tein consists of 63 residues and its amino acid sequence is in agreement wi th a molecular mass of 6822.5 Da as measured by electrospray MS. Fd II cont ains five cysteine residues of which the first four (Cys11, Cys14, Cys17 an d Cys54) are likely ligands for the single [4Fe-4S] cluster. A covalently c ross-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 f erredoxin for one PFOR subunit and is dependent on the ionic strength. The second-older rate constants for electron transfer between PFOR and Fds dete rmined electrochemically using cyclic voltammetry are 7 x 10(7) M-(1).s(-1) for Fd I and 2 x 10(7) M-1.s(-1) for Fd II and Fd III. The K-m values of P FOR for Fd I and Fd II measured both by the electrochemical and the spectro photometric method have been found to be 3 mu M and 5 mu M, respectively. T he 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 bet ween aspartic acid/glutamic acid invariant residues of Fds and lysine resid ues surrounding the distal [4Fe-4S] cluster of PFOR. All of these studies a re indicative of the involvement of electrostatic interactions between the two redox partners.