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. This is consistent with the [4Fe-4S]+ cluster existing in a mixture of S = 1/2 and S = 3/2 ground states, while the cluster-substrate complex has only S = 1/2 ground state. The redox potential of the Fe-S cluster in the presence of substrate is -480 mV. The activity of enzyme is dramatically effected (10-50-fold decrease) by photoreduction, which lowers the V(max) and increases the K(m) for malate. Fumarase A can also be oxidized with O2 and K3Fe(CN)6 to an inactive form which has an axial EPR signal at g = 2.02, consistent with the presence of a [3Fe-4S]+ cluster. The facile oxidation of the cluster by O2 is the basis of the instability of the activity of fumarase A in air. The activity of partially inactivated fumarase A can be restored by incubation with iron and reductant. These results provide strong evidence that, like aconitase, fumarase A contains a catalytically active [4Fe-4S] cluster that can be readily oxidized to an inactive [3Fe-4S] cluster.