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


ABSTRACT: 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.

SUBMITTER: Crack JC 

PROVIDER: S-EPMC7200944 | BioStudies | 2019-01-01

REPOSITORIES: biostudies

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