Probing the role of electrostatic forces in the interaction of Clostridium pasteurianum ferredoxin with its redox partners.

The ability of several low-potential redox proteins to mediate electron transfer between Clostridium pasteurianum pyruvate-ferredoxin oxidoreductase and hydrogenase has been evaluated in a coupled enzymatic assay. The active electron mediators, whatever their structure, must have a reduction potential compatible with the two enzymes, but for proteins of similar potentials, a marked specificity is displayed by 2[4Fe-4S] ferredoxins of the clostridial type. Such ferredoxins are small proteins exchanging electrons with many enzymes involved in the metabolism of anaerobic bacteria. The forces underlying the interactions of ferredoxin with hydrogenase and pyruvate-ferredoxin oxidoreductase have been examined with an emphasis on electrostatics: site-directed mutagenesis experiments have been used to individually convert all conserved glutamates and aspartates of C. pasteurianum ferredoxin into either neutral or positively charged amino acids. Also, up to four of these residues have been replaced simultaneously. The biological activities of the resulting variants depend very little on the number and the distribution of the anionic side chains on the surface of the ferredoxin. Only those molecular forms for which the immediate environment of the clusters is perturbed, independently of the charge distribution, display variations in their catalytic properties. It is concluded that electron transfer between C. pasteurianum 2[4Fe-4S] ferredoxin and its partners is far less dependent on electrostatic interactions than in many other well-documented electron transfer systems.