Fumarase a from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme.
It has been shown previously that Escherichia coli contains three fumarase genes designated fumA, fumB, and fumC. The gene products fumarases A, B, and C have been divided into two classes. Class I contains fumarases A and B, which have amino acid sequences that are 90% identical to each other, but have almost no similarity to the sequence of porcine fumarase. Class II contains fumarase C and porcine fumarase, which have amino acid sequences 60% identical to each other [Woods, S.A., Schwartzbach, S.D., & Guest, J.R. (1988) Biochim. Biophys. Acta 954, 14-26]. In this work it is shown that purified fumarase A contains a [4Fe-4S] cluster. This conclusion is based on the following observations. Fumarase A contains 4 Fe and 4 S2- per mole of protein monomer. (The mobility of fumarase A in native polyacrylamide gel electrophoresis and the elution volume on a gel permeation column indicate that it is a homodimer.) Its visible and circular dichroism spectra are characteristic of proteins containing an Fe-S cluster. Fumarase A can be reduced to an EPR active-state exhibiting a spectrum consisting of a rhombic spectrum at high fields (g-values = 2.03, 1.94, and 1.88) and a broad peak at g = 5.4. Upon addition of substrate, the high field signal shifts upfield (g-values = 2.035, 1.92, and 1.815) and increases in total spins by 8-fold, while the g = 5.4 signal disappears.(ABSTRACT TRUNCATED AT 250 WORDS)
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E. I. du Pont de Nemours and Company, Central Research and Development, Wilmington, Delaware 19880-0328.
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D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005292:Ferricyanides / Q000494:pharmacology; D005649:Fumarate Hydratase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D005810:Multigene Family; D010084:Oxidation-Reduction; D010957:Plasmids; D011487:Protein Conformation; D013053:Spectrophotometry
D005292:Ferricyanides; D007506:Iron-Sulfur Proteins; D007527:Isoenzymes; D005649:Fumarate Hydratase; C028033:potassium ferricyanide