THE INACTIVATION OF FE-S CLUSTER CONTAINING HYDRO-LYASES BY SUPEROXIDE

We report in this paper that highly purified Escherichia coli dihydrox y-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase ar e inactivated by O2- with second order rate constants in the range of 10(6) to 10(7) M-1 s-1. Each of these enzymes belongs to the hydrolyas e class and contains catalytically active [4Fe-4S] clusters. Simultane ous with inactivation by O2- is the release of iron from their cluster s. Our working hypothesis is O2- inactivates these enzymes by oxidizin g their clusterS to an unstable oxidation state, and cluster degradati on follows. Consistent with this hypothesis is our observation that sp inach dihydroxy-acid dehydratase, a member of the hydro-lyase class th at has a catalytically active [2Fe-2S] cluster, is not inactivated and does not lose iron in the presence of O2-. Porcine fumarase, a member of the hydro-lyase class that does not contain an Fe-S cluster, is al so not inactivated by O2-. We also report the rate constants for the i nactivation of E. coli dihydroxy-acid dehydratase, fumarase A, fumaras e B, and mammalian aconitase by O2 are close to 2 x 10(2) M-1 s-1, and the rate constants for the inactivation of E. coli dihydroxy-acid deh ydratase and mammalian aconitase by H2O2 are about 10(3) M-1 s-1. E. c oli dihydroxy-acid dehydratase has been reported previously to be inac tivated in vivo when cells are grown in hyperbaric O2, presumably due to the increased O2- generated under these conditions. We report here that E. coli fumarase A, fumarase B, and aconitase are also inactivate d in vivo by hyperbaric O2. Thermodynamic parameters for the oxidation of the cluster of aconitase by O2- and O2 are calculated.