[3Fe-4S] <--> [4Fe-4S] cluster interconversion in Desulfovibrio africanus ferredoxin III: properties of an Asp14 --> Cys mutant.
The 8Fe ferredoxin III from Desulfovibrio africanus is a monomeric protein which contains two [4Fe-4S]2+/1+ clusters, one of which is labile and can readily and reversibly lose one Fe under oxidative conditions to yield a [3Fe-4S]1+/0 cluster. This 4Fe cluster has an S = 3/2 ground sping state insteaed of S = 1/2 in the reduced +1 state [George, Armstrong, Hatchikian and Thomson (1989) Biochem. J. 264, 275-284]. The co-ordination to this cluster is unusual in that an aspartate (Asp14, D14, is found where a cysteine residue normally occurs. Using a mutant protein obtained from the overexpression in Escherichia coli of a synthetic gene in which Asp14, the putative ligand to the removable Fe, has been changed to Cys, we have studied the cluster interconversion properties of the labile cluster. Analysis by EPR and magnetic-circular-dichroism spectroscopies showed that the Asp14 --> 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 sluggish and did not go to completion. It is inferred that the reversibility 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.
Busch
J L
JL
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Breton
J L
JL
Bartlett
B M
BM
Armstrong
F A
FA
James
R
R
Thomson
A J
AJ
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D003545:Cysteine; D003901:Desulfovibrio / Q000737:chemistry*; D004227:Dithionite / Q000378:metabolism; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D007501:Iron; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015183:Restriction Mapping; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur
D005288:Ferredoxins; D004227:Dithionite; D001224:Aspartic Acid; D013455:Sulfur; D007501:Iron; D003545:Cysteine