Probing the mechanism of the dedicated NO sensor [4Fe-4S] NsrR: the effect of cluster ligand environment

Erin L. Dodd, Nick E. Le Brun

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NsrR from Streptomyces coelicolor is a bacterial nitric oxide (NO) sensor/nitrosative stress regulator as its primary function, and has been shown to have differential response at low, mid, and high levels of NO. These must correspond to discrete structural changes at the protein-bound [4Fe-4S] cluster in response to stepwise nitrosylation of the cluster. We have investigated the effect of the monohapto carboxylate ligand in the site differentiated [4Fe-4S] cluster cofactor of the protein NsrR on modulating its reactivity to NO with a focus on indentifying mechanistic intermediates. We have prepared a synthetic model [4Fe-4S] cluster complex with tripodal ligand and one single site differentiated site occupied by either thiolate or carboxylate ligand. We report here the mechanistic details of sequential steps of nitrosylation as observed by ESI MS and IR spectroscopy. Parallel non-denaturing mass spectrometry analyses were performed using site-differentiated variants of NsrR with the native aspartic acid, cysteine, or alanine in the position of the forth ligand to the cluster. A mono-nitrosylated synthetic [4Fe-4S] cluster was observed for the first time in a biologically-relevant thiolate-based coordination environment. Combined synthetic and protein data give unprecedented clarity in the modulation of nitrosylation of a [4Fe-4S] cluster.

Original languageEnglish
Article number112457
JournalJournal of Inorganic Biochemistry
Early online date21 Dec 2023
Publication statusPublished - Mar 2024


  • Biomimetic model complex
  • Mass spectrometry coordination chemistry
  • NO sensor protein
  • [4Fe-4S] cluster nitrosylation

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