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

Hydrogen sulfide (H 2 S) has been proposed to be a signaling agent. However, its mechanisms of action are usually unknown. H 2 S can modify cysteine residues to persulfides (RSSH), poorly characterized species proposed to mediate the signaling effects of H 2 S. The successful intracellular pathogen Mycobacterium tuberculosis (Mt), the causative agent of tuberculosis disease, is able to produce H 2 S and to import it from the host, although the effects of H 2 S 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 H 2 S 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 (10 3  M -1 s -1 at pH 7.4, 25oC) by two independent competition assays. In addition, MtAhpE catalyzed the reduction of H 2 O 2 by H 2 S indicating that H 2 S was able to complete the catalytic cycle of MtAhpE. However, H 2 S 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 (H 2 O 2 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.