STUDIES ON THE SYNTHESIS OF THE FE-S CLUSTER OF DIHYDROXY-ACID DEHYDRATASE IN ESCHERICHIA-COLI CRUDE EXTRACT - ISOLATION OF O-ACETYLSERINE SULFHYDRYLASE-A AND SULFHYDRYLASE-B AND BETA-CYSTATHIONASE BASED ON THEIR ABILITY TO MOBILIZE SULFUR FROM CYSTEINE AND TO PARTICIPATE IN FE-S CLUSTER SYNTHESIS

The apoprotein of Escherichia coil dihydroxy-acid dehydratase, which c ontains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. T he inactive apoprotein could be reactivated in vitro by factors presen t in the crude extract of E. coil and to a much smaller extent in the presence of Fe3+, S2-, and dithiothreitol. This reactivation occurs as a result of Fe-S cluster synthesis. It is anticipated that the Fe-S c luster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo. The origin of the sulfur used to form Fe-S clusters was investigated. Four enzymatic activities in the crude extract of E. coil were found t hat can provide sulfur for Fe-S cluster synthesis in vitro by mobilizi ng the sulfur from cysteine, The purification of the proteins responsi ble for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-ac etylserine sulfhydrylase B, and beta-cystathionase. The rate and exten t of sulfide mobilization from cysteine in the reaction catalyzed by O -acetylserine sulfhydrylases A and B depend on the presence of nucleop hiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine. A new amino acid is formed when the nucleophiles add to the aminoacrylate. Sulfur mobilization by bet a-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of beta-cystathionine, with pyruvate, ammonia, and sulfide being the products. Once sulfur is mobilized by t hese enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract. In crude extract and partially purified preparations from E. coil where these factors are present, the rapidity with which Fe-S clu sters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typif ied by enzymatic reactions.