Iron–sulfur proteins are the major source of protein-bound dinitrosyl iron complexes formed in Escherichia coli cells under nitric oxide stress

Abstract

Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not the proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not the proteins without iron-sulfur clusters, are modified forming protein-bound DINCs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of “chelatable iron pool” in the wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of “chelatable iron pool” in cells.

A Novel Protein Protects Bacterial Iron-Dependent Metabolism from Nitric Oxide

“…The dehydratase family of enzymes is a unique family in which the iron-sulfur cluster reacts directly with its substrate. Dehydratases are thus exquisitely sensitive to NO due to the solventexposed nature of their iron-sulfur clusters: NO binds and forms dinitrosyl iron complexes at these sites, inactivating the enzyme (8). On the other hand, substrates of nondehydratase enzymes, such as citrulline and 1,6-fructose bisphosphate, that accumulated in all three strains did not accumulate any further in the absence of nnrS (see Fig.…”

“…It is thought that the chelatable-iron pool is composed of both free and protein-bound iron (8,9). Other groups have shown that chelation of iron with 2,2=-dipyridyl prevents the formation of DNICs (8). Thus, we hypothesized that chelation of iron might complement the NO-dependent toxic effect of the deletion of nnrS by depleting the free iron available to react with NO.…”

“…The inhibition of dehydratases by DNIC formation occurs through the reaction of NO with the "chelatable-iron pool" (CIP), which is not a chemically defined mixture but is thought to be the cellular iron that is loosely coordinated and can thus be bound by chelators (9). It is thought that the chelatable-iron pool is composed of both free and protein-bound iron (8,9). Other groups have shown that chelation of iron with 2,2=-dipyridyl prevents the formation of DNICs (8).…”

“…The mechanisms whereby NO inhibits bacterial growth are diverse, but one of the most important properties of NO is its ability to bind iron and form dinitrosyl iron complexes (DNICs) bound to iron-sulfur cluster proteins, inhibiting their function (8,9). DNICs have also been shown to mediate the formation of nitrosothiols, another form of nitrosative stress that inhibits thiolcontaining proteins (10).…”

Recent advances in multinuclear metal nitrosyl complexes

“…The bactericidal effect on the food-spoilage bacterium Clostridium sporogenes is effective in the millimolar range [54]. Some water-soluble RREs act as much slower yet higher stoichiometric NO-release agents with low cytotoxicity towards immortalized vascular endothelial cells [55–56]. Because of many recent discoveries of the biological activities of RRS and RRE, there is a renewed interest in preparing new types of RRS and RRE and investigating their properties.…”