On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin

The widespread occurrence of Pro residues adjacent to Cys ligandsin the sequences of [4Fe-4S] electron transfer proteins hasnot yet found a functional basis. The two such Pro of Clostridiumpasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directedmutagenesis. Any one of them, but not both simultaneously, canbe substituted without impairing the proper folding of the protein.The reduction potentials of the ferredoxin variants fall ina narrow range of <20 mV above the potential of the nativeprotein. The biological activities with C.pasteurianum hydrogenaseand pyruvate-ferredoxin oxidoreductase do not change significantly,except when Lys replaces Pro. In these cases, the data suggestthat the two clusters of 2[4Fe-4S] ferredoxin may not alwaysbe equivalent in the interaction with the redox partners. Destabilizationof the structure has been observed as the consequence of theProl9 or Pro48 substitutions. Using 2-D NMR, this effect hasbeen associated with perturbations of both the hydrogen bondnetwork 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 butdoes not play an essential role in the mechanism of electrontransfer.