Cysteine labeling studies of beef heart aconitase containing a 4Fe, a cubane 3Fe, or a linear 3Fe cluster.
The reactivity of cysteines following cluster destruction by iron chelation was investigated for [4Fe-4S]2+ and cubane [3Fe-4S]+ beef heart aconitase. When the chelator orthobathophenanthroline disulfonate was used, the formation of sulfur-sulfur bonds and the retention of inorganic sulfur from the cluster was observed. For both the 4Fe and 3Fe forms of aconitase, the two cysteines in peptide 7, the cysteine in peptide 3, and the cysteine in peptide 2 were found as the primary constituents of sulfur-sulfur bonds (the peptide sequences and nomenclature are from Plank, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189). Three of these four cysteines (peptides 3 and 7) correlated with those proposed to be cluster ligands recently determined by x-ray crystallography (Robbins, A. H. and Stout, C. D. (1989) Proteins, in press; Robbins, A. H., and Stout, C. D.,, (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) for pig heart aconitase. A mechanism is proposed whereby the greater affinity of orthobathophenanthroline disulfonate for Fe2+ relative to Fe3+ shifts the equilibrium toward reduction of ferric iron through sulfur-sulfur bond formation at the cluster site. Aconitase which has been oxidized with ferricyanide and from which the cluster iron has been removed by EDTA has been shown to have two di- or polysulfides (Kennedy, M. C., and Beinert, H. (1988) J. Biol. Chem. 263, 8194-8198). The cysteines found in the sulfur-sulfur bonds generated by this treatment also were predominantly those from peptides 3 and 7. In addition, the putative thiol ligands for the linear [3Fe-4S]+ cluster of aconitase are reported. The four cysteines of peptides 7 and 9 (two in each peptide) were found to be protected by the cluster from alkylation when the protein was denatured. The difference in the ligands between the cubane and linear forms indicates that a specific thiol exchange occurs during the conversion.
Plank
D W
DW
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Kennedy
M C
MC
Beinert
H
H
Howard
J B
JB
D000154:Aconitate Hydratase / Q000378:metabolism*; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology; D005292:Ferricyanides / Q000494:pharmacology; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007501:Iron / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010084:Oxidation-Reduction; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D004220:Disulfides; D005292:Ferricyanides; D007461:Iodoacetates; C007931:hexacyanoferrate III; D004492:Edetic Acid; D007501:Iron; D000154:Aconitate Hydratase; D003545:Cysteine; D019807:Iodoacetic Acid