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Tellurite-mediated disabling of [4Fe-4S] clusters of Escherichia coli dehydratases

Universidad de Santiago de Chile

ABSTRACT

The tellurium oxyanion tellurite is toxic for most organisms and it seems to alter a number of intracellular targets. In this work the toxic effects of tellurite upon Escherichia coli [4Fe-4S] cluster-containing dehydratases was studied. Reactive oxygen species (ROS)-sensitive fumarase A (FumA) and aconitase B (AcnB) as well as ROS-resistant fumarase C (FumC) and aconitase A (AcnA) were assayed in cell-free extracts from tellurite-exposed cells both in the presence and absence of oxygen. While over 90% of FumA and AcnB activities were lost in the presence of oxygen, no enzyme inactivation was observed in anaerobiosis. This result was not dependent upon protein biosynthesis, as determined using translation-arrested cells. Enzyme activity of purified FumA and AcnB was inhibited when exposed to an in vitro superoxide-generating, tellurite-reducing system (ITRS). No inhibitory effect was observed when tellurite was omitted from ITRS. In vivo and in vitro reconstitution experiments with tellurite-damaged FumA and AcnB suggest that tellurite effects involve [Fe-S] cluster disabling. In fact, after exposing FumA to ITRS, released ferrous ion from the enzyme was demonstrated by spectroscopic analysis using the specific Fe2+ chelators ferene and 2,2'-bipyridyl. Subsequent spectroscopic paramagnetic resonance analysis of FumA exposed to ITRS showed the characteristic signal of an oxidatively-inactivated [3Fe-4S]1+ cluster. These results suggest that tellurite inactivates this kind of enzymes through a superoxide-dependent disabling of their [4Fe-4S] catalytic clusters.

¹ E-mail: claudio.vasquez{at}usach.cl