Direct Zinc Finger Protein Persulfidation by H2S Is Facilitated by Zn2

H2S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P‐SSH). One family of proteins modified by H2S are zinc finger (ZF) proteins, which contain multiple zinc‐coordinating cysteine residues. Herein, we report the reactivity of H2S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2S was observed by low‐temperature ESI‐MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2S to form superoxide, as detected by ESI‐MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA. ZINCing about it: Rapid persulfidation of the zinc finger protein tristetraprolin (TTP) was observed to lead to complete thiol oxidation and loss of activity (RNA binding; see scheme). Persulfidation was facilitated by O2 and zinc. When the same reaction was carried out with the zinc finger TTP bound to RNA, the zinc finger/RNA complex was found to be impervious to persulfidation, thus suggesting that the RNA shields the zinc–cysteine bonds.