No abstract

The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.

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.