[3FE-4S][--][4FE-4S] CLUSTER INTERCONVERSION IN DESULFOVIBRIO-AFRICANUS FERREDOXIN-III - PROPERTIES OF AN ASP(14)-]CYS MUTANT

The 8Fe ferredoxin III from Desulfovibrio africanus is a monomeric pro tein which contains two [4Fe-4S](2+/1+) clusters, one of which is labi le and can readily and reversibly lose one Fe under oxidative conditio ns to yield a [3Fe-4S](1+/0) cluster. This 4Fe cluster has an S = 3/2 ground spin state instead of S = 1/2 in the reduced + 1 state [George, Armstrong, Hatchikian and Thomson (1989) Biochem. J. 264, 275-284]. T he co-ordination to this cluster is unusual in that an aspartate (Asp( 14), D14) is found where a cysteine residue normally occurs. Using a m utant protein obtained from the overexpression in Escherichia coli of a synthetic gene in which Asp(14), the putative ligand to the removabl e Fe, has been changed to Cys, we have studied the cluster interconver sion properties of the labile cluster. Analysis by EPR and magnetic-ci rcular-dichroism spectroscopies showed that the Asp(14) --> Cys (D14C) mutant contains two [4Fe-4S](2+/1+) clusters, both with S=1/2 in the reduced state. Also, unlike in native 8Fe D. africanus ferredoxin III, the 4Fe <-> 3Fe cluster interconversion reaction was found to be slug gish and did not go to completion. It is inferred that the reversibili ty of the reaction in the native protein is due to the presence of the aspartate residue at position 14 and that this residue might protect the [3Fe-4S] cluster from further degradation.