Abstract
The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E. coli 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 cluster 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. coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine. The purification of the proteins responsible for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and beta-cystathionase. The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles 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 beta-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 these 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. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions.
Highlights
The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis
Apoprotein Formation—In our early experiments, the state of the apoprotein used as a substrate was of concern because we realized that if the apoprotein substrate could not be obtained in a state consistently competent for Fe-S cluster synthesis, reproducible results would be difficult to obtain
An example of the kind of variability that might affect Fe-S cluster synthesis is the different number of sulfur atoms in the S0 oxidation state that remain attached to sulfhydryl groups of the apoprotein of aconitase when it is prepared under various conditions (Kennedy and Beinert, 1988)
Summary
The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. In crude extract and partially purified preparations from E. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions. Since their discovery over 3 decades ago (Davenport et al., 1952; Arnon et al, 1957; San Pietro and Lang, 1958; Mortenson et al, 1962), proteins containing Fe-S cluster prosthetic groups have been found to play an expanding number of roles in biology. These roles include electron transport (Johnson, 1994; Lovenberg, 1973a, 1973b, 1977; Spiro, 1982), catalysis (Emptage, 1988; Emptage et al, 1983; Robbins and Stout, 1989; Flint and Emptage, 1988; Flint et al, 1993a), stabilization of protein structure (Grandoni et al, 1989; Kuo et al, 1992), regulation of metabolic pathways (Bernlohr and Switzer, 1981; Rouault et al, 1991), and a possible role in the formation of radicals (Frey and Reed, 1993; Reichard, 1993)
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