Interactions of hydrogen sulfide with AhpE from Mycobacterium tuberculosis: formation and reactions of a model peroxiredoxin persulfide

Hydrogen sulfide (H2S) has been proposed to be a signaling agent. However, its mechanisms of action are usually unknown. H2S can modify cysteine residues to persulfides (RSSH), poorly characterized species proposed to mediate the signaling effects of H2S. The successful intracellular pathogen Mycobacterium tuberculosis (Mt), the causative agent of tuberculosis disease, is able to produce H2S and to import it from the host, although the effects of H2S on Mt physiology and virulence are still unknown. In this work, we utilized the one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mt (MtAhpE-S-) as a model to study the formation and reactions of protein persulfides. The peroxidatic cysteine in MtAhpE is oxidized by different peroxides to a stable sulfenic acid (MtAhpE-SOH), which reacts with H2S to form a persulfide derivative (MtAhpE-SS-). The formation of MtAhpE-SS- was evidenced by treatment with iodoacetamide and detection of the alkyl derivative (MtAhpE-SS-R) by mass spectrometry. The kinetics of persulfide formation reaction was determined (103 M-1s-1 at pH 7.4, 25oC) by two independent competition assays. In addition, MtAhpE catalyzed the reduction of H2O2 by H2S indicating that H2S was able to complete the catalytic cycle of MtAhpE. However, H2S is not expected to be the preferred reducing substrate in vivo, since it would not compete with mycothiol/mycoredoxin-1. Reactivity of MtAhpE-SS- was compared with that of MtAhpE-S-: MtAhpE-SS- reacted faster in non-specific nucleophilic reactions (such as with the disulfide 4,4’-dithiodipyridine) but lost the specific fast reactivity with hydroperoxides (H2O2 and peroxynitrite). Computational simulations indicated that persulfidation of peroxidatic cysteine leads to a disruption of the hydrogen bond network at the active site, which is required for fast hydroperoxide reduction. Overall, the data indicate that persulfuration is a posttranslational modification that can alter the reactivities of cysteine residues in different directions depending on their specific roles and protein microenvironments.