EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF A RECONSTITUTED RECOMBINANT 7FE FERREDOXIN FROM DESULFOVIBRIO-AFRICANUS

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecul ar mass of 6585 Da) that contains one [3Fe-4S](1+/0) and one [4Fe-4S]( 2+/1+) cluster when isolated aerobically. The amino acid sequence cons ists 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 D. africanus ferredoxin III, we have overexpressed it i n Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was e xpressed in E. coli as an apoprotein. We have reconstituted the holopr otein by incubating the apoprotein with excess iron and sulphide in th e presence of a, reducing agent. The reconstituted recombinant ferredo xin appeared to have a lower stability than that of wildtype D. africa nus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contai ns a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) cluster similar to those fou nd in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant fer redoxin.