Multiple Orientations in a Physiological Complex: The Pyruvate-Ferredoxin Oxidoreductase-Ferredoxin System ‡

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 × 105 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 wit...