Dataset Information

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe-4S Cluster (†).

ABSTRACT: In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here, we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S](2+) cluster, which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O(2). These results, together with the fact that there are only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a noncysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and cross-linking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S](2+) holo form as well as in the apo form. These findings provide a first stepping stone to investigate the mechanism of NO sensing in NsrR.

SUBMITTER: Yukl ET

PROVIDER: S-EPMC2891187 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe-4S Cluster (†).

Yukl Erik T ET Elbaz Mohamed A MA Nakano Michiko M MM Moënne-Loccoz Pierre P

Biochemistry 20081201 49

In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here, we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S](2+) cluster, which reacts with NO to form dinitrosyl iron complexes. This me ...[more]

PMID: 19006327

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Project description:Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

Dataset Information

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe-4S Cluster (†).

Publications

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe-4S Cluster (†).

Similar Datasets

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