Dihydroxy acid dehydratase from spinach contains a [2Fe-2S] cluster.
Dihydroxy acid dehydratase, the third enzyme in the branched-chain amino acid biosynthetic pathway, has been purified to homogeneity (5000-fold) from spinach leaves. The molecular weights of dihydroxy acid dehydratase as determined by sodium dodecyl sulfate and native gel electrophoresis are 63,000 and 110,000, respectively, suggesting the native enzyme is a dimer. 2 moles of iron were found per mol of protein monomer. Chemical analyses of iron and labile sulfide gave an Fe/S2- ratio of 0.95. The EPR spectrum of dithionite-reduced enzyme (gavg = 1.91) is similar to spectra characteristic of Rieske Fe-S proteins and has a spin concentration of 1 spin/1.9 irons. These results strongly suggest that dihydroxy acid dehydratase contains a [2Fe-2S] cluster, a novel finding for enzymes of the hydrolyase class. In contrast to the Rieske Fe-S proteins, the redox potential of the Fe-S cluster is quite low (-470 mV). Upon addition of substrate, the EPR signal of the reduced enzyme changes to one typical of 2Fe ferredoxins (gavg = 1.95), and the visible absorption spectrum of the native enzyme shows substantial changes between 400 and 600 nm. Reduction of the Fe-S cluster decreases the enzyme activity by 6-fold under Vmax conditions. These results suggest the direct involvement of the [2Fe-2S] cluster of dihydroxy acid dehydratase in catalysis. Similar conclusions have been reached for the catalytic involvement of the [4Fe-4S] cluster of the hydrolyase 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).
Flint
D H
DH
E. I. du Pont de Nemours and Co., Central Research and Development Department, Wilmington, Delaware 19898.
Emptage
M H
MH
D004578:Electron Spin Resonance Spectroscopy; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008667:Metalloproteins / Q000302:isolation & purification*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology*; D013455:Sulfur / Q000032:analysis
D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D008667:Metalloproteins; D013455:Sulfur; D007501:Iron; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase