S7-5 The mechanism(s) of protein persulfide formation: Detection of protein S-sulfhydration by a tag-switch assay

Abstract

Hydrogen sulfide (H 2 S) has been recently classified as a critical cell-signaling molecule. Literature published in the past few years increasingly suggests that H 2 S is a mediator of many physiological and/or pathological processes. Some of these effects are ascribed to the formation of protein persulfides, or protein S -sulfhydration (i.e. conversion of cysteine residues -SH to persulfides -S-SH). Two different approaches have been widely used to detect protein persulfides, but the selectivity of these methods have been challenged, as they do not stand on solid chemical ground. In addition, the underlying mechanisms of S -sulfhydration mediated by H 2 S are still unclear. In this study we report the first selective, “tag-switch” method, which can directly label protein S -sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H 2 S alone cannot lead to S -sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) or the involvement of metal centers, which would facilitate the oxidation of H 2 S to HS . Persulfides are predominantly located in endoplasmic reticulum and mitochondria (the organelles richest in PSOH and metal centers, respectively) and their formation appears to be highly dependant on MST enzymatic activity. While the clean Na 2 S solution has no effect on protein structure, polysulfides, commercially available NaHS or Na 2 S solutions supplemented with metal ions have dramatic effects on protein structure, leading to cleavage of disulfide bonds with concomitant persulfide formation. Physiological consequences of these reactions will be discussed.