Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine

Jason Crack, Melissa Stewart, Nicolas Le Brun

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The ability to specifically label the sulphide ions of protein-bound iron–sulphur (FeS) clusters with 34S isotope greatly facilitates structure–function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the O-acetyl-L-serine sulfhydrylase CysK to generate 34S-substituted L-cysteine and subsequently use it as a substrate for the L-cysteine desulfurase NifS to gradually supply 34S2− for in vitro FeS cluster assembly in an otherwise standard cluster reconstitution protocol.
Original languageEnglish
Article numberbpy015
JournalBiology Methods and Protocols
Issue number1
Publication statusPublished - 27 Jan 2019


  • cysteine
  • iron-sulphur
  • mass spectrometry
  • resonance Raman spectroscopy

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