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 66,00 0. The combination of UV visible absorption, EPR, magnetic circular di chroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydro xy-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 wi th minor contributions from forms with S = 1/2 and possibly S = 5/2 gr ound states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. c oli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly invol ved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Munck, E. (1983) Proc. Natl. A cad. 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 oxidativ e degradation of the [4Fe-4S] cluster. Compared to aconitase, the oxid ized 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] clus ter. 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.