Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Abstract

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen's antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

“…In fact, the rate constant of peroxynitrite reduction by Ohr is comparable to the rate constants reported for Prxs and thiolor selenol-dependent Gpxs (10,12,13,(53)(54)(55). Therefore, it is feasible that Ohr plays a relevant role in vivo in the reduction of peroxynitrite.…”

“…A competition method was developed here, taking advantage of the fact that Ohr from X. fastidiosa and P. aeruginosa does not possess Trp residues, and consequently displays low intrinsic fluorescence. In contrast, MtAhpE has intrinsic fluorescence that decreases upon oxidation (8,55). As expected, XfOhr practically lacks intrinsic fluorescence when excited at 295 nm, in contrast to MtAhpE (Fig.…”

“…The t-BHP was obtained from Sigma-Aldrich Co. CHP and H 2 O 2 were obtained Merck KGaA. XfOhr and PaOhr, as well as MtAhpE, were obtained and, in some experiments, reduced as previously described (8,55). The 15(S)-HpETE and 5(S)-HpETE were purchased from Cayman Chemical.…”

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

“…The pK a of RSOH has been determined in only a few proteins (7,8). The experimentally determined pK a of some small-molecule sulfenic acids is 1-2 orders of magnitude lower than that of the corresponding thiols (9,10); however, it is not clear whether such compounds are appropriate models of cysteine sulfenic acid in proteins.…”

Characterization of the Vibrio vulnificus 1-Cys Peroxiredoxin Prx3 and Regulation of Its Expression by the Fe-S Cluster Regulator IscR in Response to Oxidative Stress and Iron Starvation

“…In this context, two 2-Cys Prxs, Prx1 and Prx2, in which the expression patterns and kinetic properties are differentially optimized to decompose distinct levels of H 2 O 2 were previously identified in Vibrio vulnificus, a facultative aerobic pathogen (16,17). However, studies on the bacterial 1-Cys Prxs are presently limited (13,18,19); therefore, the regulatory mechanisms used by bacteria to sense environmental signals and modulate their expression have not yet been addressed at the molecular levels.…”