Polysulfides Link H2S to Protein Thiol Oxidation

Introduction

For 300 years, hydrogen sulfide (H2S) gas has been known for both its malodorous smell and toxicity, the latter being primarily related to its potent ability to inhibit cytochrome c oxidase (36, 44). Interestingly, recent research has revealed H2S to act as a signaling molecule involved in various physiological processes, including inflammation, apoptosis, vasorelaxation, and neuromodulation (44). While more functions of H2S are being uncovered, its molecular mechanism of action remains unclear.

H2S has a pKa1 of 6.77 at 37°C, which is why it exists as both H2S (∼20%) and HS− (∼80%) at pH 7.4 (32). Due to its second pKa2 of>12, the concentration of the completely deprotonated S2− is extremely low at physiological pH (32). For simplicity, H2S will be used henceforth to refer to the total sulfide pool (H2S+HS−+S2−). While some of the observed (patho-) physiological effects induced by H2S have been attributed to its antioxidative capacity or to the inhibition of cytochrome c oxidase, H2S appears to predominantly act via S-sulfhydration of target proteins (34). This concept describes the addition of H2S-derived sulfur to a cysteinyl thiolate (Cys-S−) to yield a cysteinyl persulfide (Cys-S-S−), which then leads to either activation or inhibition of protein activity (20, 28). Numerous proteins have been reported to be S-sulfhydrated by H2S, both in vitro and/or in intact cells, including actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nuclear factor κB (NF-κB), ATP-sensitive potassium channel (KATP), protein tyrosine phosphatase 1B (PTP1B), and Kelch-like ECH-associated protein-1 (Keap1) (11, 20, 28, 29, 38, 47). Surprisingly, none of these original publications have experimentally addressed the paradox that H2S, with its sulfur in the lowest possible oxidation state (−2), causes oxidation of thiols to persulfides, although by itself it should only be able to act as a reductant. The aim of this study was thus to elucidate the mechanism of H2S-induced oxidation of protein thiols.

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The lipid phosphatase and tensin homolog (PTEN) was chosen as a model protein to conduct these investigations. As the main antagonist of the phosphoinositide 3-kinase (PI3K)-Akt pathway, PTEN acts as a tumor suppressor, and is well established to be redox regulated in the course of growth factor signaling (21, 23). Specifically, upon oxidative challenge, PTEN was shown to form a disulfide bond between the active site cysteine Cys-124 and Cys-71, resulting in its immediate and reversible inhibition (22).

By monitoring the activity of PTEN in a newly developed real-time assay, we were able to continuously assess its redox state in response to H2S solutions. Our data demonstrate that dissolved sodium hydrosulfide (NaHS) leads to very rapid oxidation of the PTEN active site cysteine in vitro, surpassing even hydrogen peroxide (H2O2) in its oxidative efficiency. We identify polysulfides formed in neutral solutions of NaHS as the oxidizing species. Notably, we find evidence for the formation of polysulfides in solutions of all “H2S donors” tested, including sodium sulfide (Na2S), gaseous H2S, as well as morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Moreover, we show that polysulfides in the culture medium lead to oxidative PTEN modification in intact cells. Our study highlights the phenomenon of polysulfide formation in neutral and weakly alkaline H2S solutions and suggests that at least some of the previously reported findings in H2S research may have been mediated by polysulfides.

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