SummaryThe suf and isc operons of Escherichia coli have been implicated in Fe-S cluster assembly. However, it has been unclear why E. coli has two systems for Fe-S cluster biosynthesis. We have examined the regulatory characteristics and mutant phenotypes of both operons to discern if the two operons have redundant functions or if their cellular roles are divergent. Both operons are similarly induced by hydrogen peroxide and the iron chelator 2,2 ¢ ¢ ¢ ¢ -dipyridyl, although by different mechanisms. Regulation of the isc operon is mediated by IscR, whereas the suf operon requires OxyR and IHF for the response to oxidative stress and Fur for induction by iron starvation. Simultaneous deletion of iscS and most suf genes is synthetically lethal. However, although the suf and isc operons have overlapping functions, they act as distinct complexes because the SufS desulphurase alone cannot substitute for the IscS enzyme. In addition, suf deletion mutants are more sensitive to iron starvation than isc mutants, and the activity of the Fe-S enzyme gluconate dehydratase is diminished in the suf mutant during iron starvation. These findings are consistent with the model that the isc operon encodes the housekeeping Fe-S cluster assembly system in E. coli , whereas the suf operon is specifically adapted to synthesize Fe-S clusters when iron or sulphur metabolism is disrupted by iron starvation or oxidative stress.
The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established. To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized. Using this iscS deletion strain, we have observed decreased specific activities for proteins containing [4Fe-4S] clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase). A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS. These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis. We also have characterized a growth phenotype associated with the loss of IscS. Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required. The lack of IscS also had a general effect on the growth of E. coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied.NifS ͉ FNR ͉ oxidative stress repair ͉ sulfur metabolism
The [4Fe-4S]2؉ clusters of dehydratases are rapidly damaged by univalent oxidants, including hydrogen peroxide, superoxide, and peroxynitrite. The loss of an electron destabilizes the cluster, causing it to release its catalytic iron atom and converting the cluster initially to an inactive [3Fe-4S] 1؉ form. Continued exposure to oxidants in vitro leads to further iron release. Experiments have shown that these clusters are repaired in vivo. We sought to determine whether repair is mediated by either the Isc or Suf cluster-assembly systems that have been identified in Escherichia coli. We found that all the proteins encoded by the isc operon were critical for de novo assembly, but most of these were unnecessary for cluster repair. IscS, a cysteine desulfurase, appeared to be an exception: although iscS mutants repaired damaged clusters, they did so substantially more slowly than did wild-type cells. state in vivo when stress is prolonged. Under the conditions of our experiments, mutants that lacked other repair candidates-Suf proteins, glutathione, and NADPH: ferredoxin reductase-all repaired clusters at normal rates. We conclude that the mechanism of cluster repair is distinct from that of de novo assembly and that this is true because mild oxidative stress does not degrade clusters in vivo to the point of presenting an apoenzyme to the de novo cluster-assembly systems.Proteins employ iron-sulfur clusters for a variety of purposes. Although clusters were first recognized for their participation in electron-transfer reactions, they also serve as catalytic Lewis acids in a family of dehydratases (1-3). In enzymes such as serine dehydratase, fumarase, and aconitase, the [4Fe-4S] clusters ligand the leaving hydroxyl group of the dehydration substrate.To bind substrate, these clusters must be situated in the protein so that they are exposed to solvent. A consequence is that the clusters are vulnerable to oxidation by any small univalent oxidants that can penetrate into the active site (4). Oxidation converts the cluster to a metastable form that rapidly degrades as in Reactions 1 and 2,The iron atom that is lost is essential for catalysis, and so the oxidized enzymes are inactive. Indeed, mutants of Escherichia coli and Saccharomyces cerevisiae that lack superoxide dismutases struggle to grow in aerobic media (5-7), because superoxide rapidly oxidizes these dehydratases, thereby disabling the biosynthetic and catabolic pathways to which they belong (8 -11). Hydrogen peroxide and peroxynitrite are also physiological oxidants that inactivate these enzymes (4, 12, 13). The rate constant for inactivation by superoxide is so high (ϳ10 6 -10 7 M Ϫ1 s Ϫ1 ) that it is likely that even wild-type cells contain enough superoxide to continually damage these enzymes. Their half-life has been estimated to be 40 min in aerobic E. coli (14). In response, bacterial cells have acquired the ability to repair the damaged clusters. This repair capacity becomes evident when superoxide-stressed cells are restored to an anaerobic environmen...
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