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. 2020 Mar 13;295(11):3664-3677.
doi: 10.1074/jbc.RA119.012438. Epub 2020 Jan 28.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Maria-Armineh Tossounian  1 , Anh-Co Khanh Truong  1 , Lieven Buts  2 , Khadija Wahni  1 , Álvaro Mourenza  3 , Martine Leermakers  4 , Didier Vertommen  5 , Luis Mariano Mateos  3 , Alexander N Volkov  2 , Joris Messens  6
Affiliations

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Maria-Armineh Tossounian et al. J Biol Chem. .

Abstract

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Keywords: biochemistry; enzyme mechanism; enzyme structure; hydrogen peroxide; kinetics; methionine sulfoxide; nuclear magnetic resonance (NMR); redox regulation.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
NMR solution structure of reduced Cd-MsrB and its active site. A, an overlay of the 20 lowest-energy structures of reduced Cd-MsrB. The secondary structural elements (yellow for β-sheets and red for α-helices) and the zinc (gray) are labeled (PDB code 6TR8). B, Cd-MsrB monomeric structure in a semi-transparent surface is presented in green cartoon. The location of the conserved Cys-66 and Cys-122 (thiols are surface exposed) and the nonconserved Cys-127 (buried thiol) are shown in red, and the cysteines involved in Zn2+ coordination are colored yellow. The distance between Cys-122 and Cys-66, and Cys-122 and Cys-127 are 8 and 15 Å, respectively. The N and C termini are indicated. C, the active site of Cd-MsrB has the hydrophobic pocket (Trp-68), and the H-bond donors (conserved His-103, His-106, and Asn-124). The amino acids are indicated in a green stick representation. D, the active site of C. diphtheriae MsrA (PDB 4D7L) is shown in wheat color. It has a mirror-like image of the active site of Cd-MsrB, with a hydrophobic pocket (Tyr-53 and Trp-54) and conserved H-bond donors (Tyr-83, Glu-95, and Tyr-135, and Asp-130 via a water molecule (W, black)). The cacodylate molecule mimicking the interactions a substrate would make with the active site residues is colored purple and labeled CAC.
Figure 2.
Figure 2.
Cys-122 is required for substrate reduction. A, RPC-HPLC chromatograms of Cd-MsrB WT, C66S, C122S, and C127S mutants are shown, where Met peak formation is observed at a retention time 7.45 min. In the presence of MetSO and DTT, as a reducing agent, the C122S mutant does not convert MetSO to Met, whereas the WT and the other mutants (C66S and C127S) show Met production. B, the concentration of Met produced by the WT and Cys mutants are shown in a dot plot. WT and the C127S mutant show similar Met production, whereas the C66S mutant shows 10-fold excess Met production. The data are presented as a mean ± S.E. of at least two independent technical repeats and the graphs were generated using Prism8.
Figure 3.
Figure 3.
MsrB oxidation with MetSO leads to pronounced changes in the NMR spectrum. The [1H,15N]-HSQC spectra of (A) reduced MsrB, (B) freshly prepared MetSO-oxidized MsrB, and (C) MetSO-oxidized MsrB incubated at 25 °C for 19 h are shown. The spectra can be attributed to distinct redox species with different Cys-66, Cys-122, and Cys-127 oxidation status (see text), schematically shown above the plots.
Figure 4.
Figure 4.
Two disulfide forms of the oxidized MsrB can be discerned in the NMR spectra. The semi-transparent MsrB molecular surface shows solvent-exposed Cys-66 (cyan) and Cys-122 (orange), and buried Cys-127 (green sticks). The panels contoured in orange and green show HSQC spectral regions that report on the local chemical environment of Cys-122 and Cys-127, respectively. The spectra of reduced MsrB (black), freshly prepared MetSO-oxidized MsrB (red), and MetSO-oxidized MsrB incubated at 25 °C for 19 h (blue), correspond to those shown in Fig. 3. Note the absence of the red resonance for Ile-123 (orange panel), indicating a large shift to a new position lying outside of the plotted spectral region. Two possible disulfide forms are schematically shown above the plots, and the cysteine residue reported upon in a given panel is colored.
Figure 5.
Figure 5.
Thioredoxin only reduces the Cys-66–Cys-122 disulfide. A, progress curves of WT Cd-MsrB and the cysteine mutants (C66S, C122S, and C127S) coupled to the Trx/TrxR pathway, following MetSO reduction, are shown. The C66S and C122S mutants do not consume NADPH, whereas mutating Cys-127 has almost no effect on the reaction rate. B, the reduction of Cys-66–Cys-122 disulfide bond by Trx follows the Michaelis-Menten steady-state kinetics. Increasing concentrations of Met-SO–oxidized Cd-MsrBS-S were used as substrate for thioredoxin. From the Michaelis-Menten curve, the rate of disulfide bond reduction was determined to be 1.7 × 104 m−1 s−1. The data are presented as a mean ± S.D. of three independent technical repeats and the graphs were generated using Prism8.
Figure 6.
Figure 6.
Catalytic mechanism of Cd-MsrB coupled to the Trx/TrxR pathway. (I) Cd-MsrB Cys-122 performs a nucleophilic attack on the sulfoxide of MetSO, which results in the formation of a sulfenic acid on Cys-122 and the release of Met. Then, the catalytic cysteines, Cys-66 or Cys-127, attack the sulfur of the sulfenic acid, which results in the formation of the Cys-122–Cys-127 or Cys-122–Cys-66 disulfides (II or III). For Cys-122–Cys-127, a disulfide exchange occurs and Cys-66–Cys-122 is formed (IV), which is recognized by the Trx and reduced (V).
Figure 7.
Figure 7.
Mycothiol protects Cys-127 in the presence of hydrogen peroxide. A, Cd-MsrB is not recycled via the mycothiol reduction pathway. Progress curves show no consumption of NADPH when Cd-MsrB is coupled to the MSH/Mrx1/Mtr pathway after adding MetSO as a substrate. B, in the presence of mycothiol and H2O2, Cd-MsrB gets S-mycothiolated and becomes substrate of the MSH/Mrx1/Mtr reduction pathway. The rate of demycothiolation is 17.0 ± 0.4 milli-absorbance units/min. Data are presented as a mean ± S.D. of three independent technical repeats and the graphs were generated using Prism8.
Figure 8.
Figure 8.
Cys-66 and Cys-127 are involved in Cd-MsrB dimerization in the presence of H2O2. Size-exclusion chromatograms of Cd-MsrB (A) WT, and its Cys mutants, (B) C66S, (C) C127S, and (D) C66S/C127S), in the presence and absence of H2O2 are shown. Elution peaks 1 and 2 correspond to the elution position of the monomer (17.4 kDa) and the dimer (34.8 kDa), respectively. These results indicate that Cys-66 and Cys-127 are involved in the dimerization of Cd-MsrB. The data presents the union of two independent technical repeats.
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