Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

摘要

piDesulfovibrio africanus/i ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]sup1+/0/sup and one [4Fe-4S]sup2+/1+/sup cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of iD. africanus/i ferredoxin III, we have overexpressed it in iEscherichia coli/i by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in iE. coli/i as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type iD. africanus/i ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]sup1+/0/sup and a [4Fe-4S]sup2+/1+/sup cluster similar to those found in native iD. africanus/i ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin./p