Abstract

Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m-1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.

Highlights

  • Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals

  • We focused on the kinetic characterization of the reaction between MtAhpE– SOH and H2S to form a persulfide

  • To compare the reactivity of the persulfide in the peroxidatic cysteine to that of the thiol, we evaluated the kinetics with specific substrates and unspecific reactants of Prxs

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Summary

The abbreviations used are

Peroxiredoxin; MtAhpE, alkyl hydroperoxide reductase E of M. tuberculosis; DTT, 1,4-dithiothreitol; TNB, 5-thio-2nitrobenzoic acid; DTDPy, 4,4Ј-dithiodipyridine; MD, molecular dynamics; DTNB, 5,5Ј-dithiobis-(2-nitrobenzoic acid); DTPA, diethylenetriaminepentaacetic acid; PDB, Protein Data Bank. The oxidizing substrate (H2O2, organic hydroperoxide or peroxynitrite) reacts with the thiolate at the peroxidatic cysteine in the reduced enzyme to form a sulfenic acid [44]. The reactivities of the thiolates in peroxidatic cysteines of Prx with hydroperoxides are several orders of magnitude faster than those of typical low- or high-molecular-weight thiols This can be explained by the decrease in the energy of activation of the reaction by an exquisite network of electrostatic and hydrogen-bonding interactions involving the functional groups of an arginine and a threonine among others [45,46,47]. H2S is another possible reducing substrate for MtAhpE– SOH; it is not clear how effective its contribution could be Both the ability of the resulting persulfide (MtAhpE–SSH) to react with typical Prx substrates or, alternatively, the capacity to be transferred to acceptor thiols remain unexplored. The possibility of Prx assistance in persulfidation reactions (transpersulfidation) was explored

Results
Discussion
Experimental procedures
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