Mass spectrometric identification of [4Fe-4S](NO)x intermediates of nitric oxide sensing by regulatory iron-sulfur cluster proteins

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Abstract

Nitric oxide (NO) can function as both a cytotoxin and a signalling molecule. In both cases, reaction with iron–sulfur (Fe–S) cluster proteins plays an important role because Fe–S clusters are reactive towards NO and so are a primary site of general NO-induced damage (toxicity). This sensitivity to nitrosylation is harnessed in the growing group of regulatory proteins that function in sensing of NO via an Fe–S cluster. Although information about the products of cluster nitrosylation is now emerging, detection and identification of intermediates remains a major challenge, due to their transient nature and the difficulty in distinguishing spectroscopically similar iron-NO species. Here we report studies of the NO-sensing Fe–S cluster regulators NsrR and WhiD using non-denaturing mass spectrometry, in which non-covalent interactions between the protein and Fe/S/NO species are preserved. The data provide remarkable insight into the nitrosylation reactions, permitting identification, for the first time, of protein-bound mono-, di- and tetranitrosyl [4Fe–4S] cluster complexes ([4Fe–4S](NO), [4Fe–4S])(NO) 2 and [4Fe–4S](NO) 4 ) as intermediates along pathways to formation of product Roussin's red ester (RRE) and Roussin's black salt (RBS)-like species. The data allow the nitrosylation mechanisms of NsrR and WhiD to be elucidated and clearly distinguished.

Original languageEnglish
Pages (from-to)3675-3684
Number of pages10
JournalChemistry - A European Journal
Volume25
Issue number14
Early online date2 Jan 2019
DOIs
Publication statusPublished - 7 Mar 2019

Keywords

  • gene regulation
  • mass spectrometry
  • nitric oxide
  • sulfur

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