The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase.
Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000. The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a [2Fe-2S]2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution, the reduced [4Fe-4S]+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the [4Fe-4S]cluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear [3Fe-4S] cluster or a [2Fe-2S] cluster. Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.
Flint
D H
DH
Central Research and Development Department, E.I. du Pont de Nemours & Company, Wilmington, Delaware 19880.
Emptage
M H
MH
Finnegan
M G
MG
Fu
W
W
Johnson
M K
MK
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
D007506:Iron-Sulfur Proteins; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase