Mass spectrometric detection of iron nitrosyls, sulfide oxidation and mycothiolation during nitrosylation of the NO sensor [4Fe-4S] NsrR.

Jason C Crack,Chris J Hamilton,Nick E Le Brun

doi:10.1039/c8cc01339j

Abstract

The bacterial nitric oxide (NO)-sensing transcriptional regulator NsrR binds a [4Fe-4S] cluster that enables DNA-binding and thus repression of the cell's NO stress response. Upon exposure to NO, the cluster undergoes a complex nitrosylation reaction resulting in a mixture of iron-nitrosyl species, which spectroscopic studies have indicated are similar to well characterized low molecular weight dinitrosyl iron complex (DNIC), Roussin's Red Ester (RRE) and Roussin's Black Salt (RBS). Here we report mass spectrometric studies that enable the unambiguous identification of NsrR-bound RRE-type species, including a persulfide bound form that results from the oxidation of cluster sulfide. In the presence of the low molecular weight thiols glutathione and mycothiol, glutathionylated and mycothiolated forms of NsrR were readily formed.

Highlights

The bacterial nitric oxide (NO)-sensing transcriptional regulator NsrR binds a [4Fe–4S] cluster that enables DNA-binding and repression of the cell’s NO stress response
Upon exposure to NO, the cluster undergoes a complex nitrosylation reaction resulting in a mixture of ironnitrosyl species, which spectroscopic studies have indicated are similar to well characterized low molecular weight dinitrosyl iron complex (DNIC), Roussin’s Red Ester (RRE) and Roussin’s Black Salt (RBS)
We report mass spectrometric studies that enable the unambiguous identification of NsrR-bound RRE-type species, including a persulfide bound form that results from the oxidation of cluster sulfide

Summary

Introduction

The bacterial nitric oxide (NO)-sensing transcriptional regulator NsrR binds a [4Fe–4S] cluster that enables DNA-binding and repression of the cell’s NO stress response. Exposure to an excess of NO (8 NO per [4Fe–4S] cluster) prior to analysis by LC-MS resulted in a 2 Da decrease in the mass of the apo-protein to 15 951 Da, suggesting that NO-mediated oxidation of apo-NsrR, resulting in a disulfide bond, occurred upon reaction with NO (Fig. S1, ESI†).

Results

Conclusion