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

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.