OxyR2 Functions as a Three-state Redox Switch to Tightly Regulate Production of Prx2, a Peroxiredoxin of Vibrio vulnificus

Abstract

The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H2O2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H2O2 exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H2O2 with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO3H) by high H2O2 in vitro and in vivo, where the modification deterred the transcription of prx2 The DNA binding preferences of OxyR25CA-C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the -35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H2O2 sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism.

Keywords: OxyR; Vibrio vulnificus; antioxidant; gene regulation; hydrogen peroxide; overoxidation; peroxiredoxin; reactive oxygen species; sensing cysteine; three-state redox switch.

FIGURE 1.

MALDI-TOF mass spectra of OxyR2 and OxyR2-C206S oxidized by 500 μm H₂O₂in vitro. His₆-tagged OxyR2 (A) and OxyR2-C206S (B) proteins were reduced with 100 mm DTT for 1 h and desalted by gel filtration chromatography under anaerobic conditions. Then the OxyR2 proteins were reacted (bottom panels, +H₂O₂) or unreacted (top panels, −H₂O₂) with 500 μm H₂O₂ for 10 min. Each of the OxyR2 proteins was then incubated with iodoacetamide to alkylate reduced cysteine residues, digested with trypsin, and analyzed by MALDI-TOF MS in negative (left panels) and positive (right panels) ion reflector modes. A, mass spectra of peptides containing Cys²⁰⁶ of OxyR2 are indicated. ▾, a peptide with alkylated Cys²⁰⁶ and Cys²¹⁵; ▿, a peptide with S-sulfonated Cys²⁰⁶ and alkylated Cys²¹⁵. B, mass spectra of peptides containing Ser²⁰⁶ of OxyR2-C206S are indicated. *, a peptide with alkylated Cys²¹⁵. The form of each observed peptide is indicated in the box inside each panel. Theoretical (Th) and observed (Ob) monoisotopic masses ([M − H]⁻ for negative mode or [M + H]⁺ for positive mode) of the peptides are indicated in the bottom left corner of each panel. N.A., the corresponding peptide was not observed.

FIGURE 2.

Overoxidation of OxyR2 Cys²⁰⁶ in V. vulnificus cells exposed to various levels of H₂O₂. A, OH0703 (pDY1025) was grown anaerobically to an A₆₀₀ of 0.3 and exposed to various H₂O₂ concentrations for 3 min. Cellular proteins were precipitated with TCA and alkylated with fresh AMS buffer for 1 h at 37 °C. Proteins (3.5 μg for the top panel and 7 μg for the bottom panel) were resolved by non-reducing SDS-PAGE and immunoblotted using anti-OxyR2 antibody (top) or anti-OxyR2-Cys²⁰⁶-SO₃H antibody (bottom). The predicted numbers of AMS that alkylated each OxyR2 molecule and their redox states are indicated at the ends of the gel. Negative control (NC) was OH0703 (pJH0311), 6AMS control was OH0703 (pBANG1416), and 7AMS control was OH0703 (pDY1025). B, the specificity of anti-OxyR2-Cys²⁰⁶-SO₃H antibody to overoxidized and reduced OxyR2 was determined using ELISA. The microtiter 96-well plates were coated with 0.1 μg of synthetic peptides corresponding to either OxyR2 active site with overoxidized (S-sulfonated) Cys²⁰⁶ (EKEHC²⁰⁶(SO₃H)LTEHA) (○) or with reduced Cys²⁰⁶ (EKEHC²⁰⁶(SH)LTEHA) (●), and the peptides were reacted with various concentrations of the antibody as indicated. C, the S-sulfonated OxyR2 peptide (0.1 μg) was attached to the microtiter 96-well plates and then reacted with 4 μg of anti-OxyR2-Cys²⁰⁶-SO₃H antibody. As a binding competitor, either S-sulfonated or reduced OxyR2 peptides (12.5 ng) were added to the reaction with the antibody as indicated. Black bar, control where no competitor was added. Relative binding of the antibody to the specific peptides is presented as A₄₅₀. All data in B and C represent mean ± S.D. (error bars).

FIGURE 3.

Expression levels of prx2 in response to various levels of H₂O₂ and a working model for OxyR2 as a three-state redox switch. A, wild-type V. vulnificus was grown anaerobically to an A₆₀₀ of 0.3 and exposed to various concentrations of H₂O₂ as indicated for 3 min. Total RNAs were isolated, and the relative levels of prx2 (black bars) and oxyR2 (gray bars) transcripts were determined by quantitative real-time PCR analyses. The level of each transcript from wild type unexposed to H₂O₂ is presented as 1. *, statistically significant difference (p < 0.05) between groups. B, transcriptional activity of OxyR2-C206D. Cultures were grown aerobically to an A₆₀₀ of 0.5, total RNAs were isolated, and the relative levels of prx2 transcript were determined by quantitative real-time PCR analyses. The level of transcript from negative control (NC) is presented as 1. Negative control was OH0703 (pJH0311). OxyR2, OH0703 (pDY1025); OxyR2-C206D, OH0703 (pBANG1416). All data in A and B represent mean ± S.D. (error bars). C, OxyR2 shifts to three different redox states, revealing different transcriptional activities in response to various levels of H₂O₂ in cells (solid line) as indicated. Concurrently, the prx2 expression (dotted line) varies, depending on the redox states of OxyR2. C₂₀₆-SO_2/3H, overoxidized Cys²⁰⁶ with Cys²⁰⁶-SO₂H and/or Cys²⁰⁶-SO₃H.

FIGURE 4.

Binding sequences of OxyR2_5CA-C206D within the prx2 promoter region. A, a 260-bp DNA fragment of the upstream region of P_prx2 was radioactively labeled and then used as probe DNA. The radiolabeled probe DNA (25 nm) was incubated with increasing amounts of the purified OxyR2_5CA-C206D as indicated and then digested with DNase I. The regions protected by OxyR2_5CA-C206D are indicated by open boxes, whereas the nucleotides showing enhanced cleavage are indicated by a black box. Lanes C, T, A, and G, nucleotide sequencing ladder of P_prx2. Nucleotide numbers shown are relative to the transcription start site of P_prx2. B, the sequences for binding of OxyR2_5CA-C206D are indicated below the P_prx2 sequences as diagonal-filled boxes. The sequences for binding of oxidized OxyR2 (white box) and reduced OxyR2 (gray boxes) determined previously (11) are presented above the P_prx2 sequences. The nucleotides showing enhanced cleavage by binding of OxyR2_5CA-C206D and the reduced OxyR2 are indicated as black boxes. The transcription start site of P_prx2 identified previously (11) is indicated by a bent arrow, and the positions of the putative −10 and −35 regions are underlined. The ATG translation initiation codon and the putative ribosome-binding site (SD) are also indicated in boldface type.

FIGURE 5.

Expression levels of prx1 and katG in response to various levels of H₂O₂. Wild-type V. vulnificus was grown anaerobically to an A₆₀₀ of 0.3 and exposed to various concentrations of H₂O₂ as indicated for 3 min. Total RNAs were isolated, and the relative levels of prx1 (A) and katG (B) transcripts were determined by quantitative real-time PCR analyses. The level of each transcript from wild type unexposed to H₂O₂ is presented as 1. *, statistically significant difference (p < 0.05) between groups. All data represent mean ± S.D. (error bars).

FIGURE 6.

Proposed overoxidation mechanism of OxyR. A, modeled structures of OxyR, focusing on the sensing cysteine (C_S). S-Hydroxylated sensing cysteine (C_S-SOH) is oxidized by the bound H₂O₂ (left), resulting in S-sulfinated sensing cysteine (C_S-SO₂H; middle), which is in turn further oxidized to S-sulfonated sensing cysteine (C_S-SO₃H; right). The models were constructed based on the crystal structure of the P. aeruginosa OxyR-C199D variant complexed with a H₂O₂ molecule. The H₂O₂ molecule was bound by the polar interactions with the side chain and the backbone amide group of Thr¹³⁶, the backbone carbonyl group of Glu²⁰⁴, and the backbone amide group of Cys²⁰⁶, as indicated by the dotted lines. The lone pair of electrons are displayed as the sp³ orbital shapes, and the bound water molecules are shown as red spheres. The blue arrows indicate the nucleophilic attack by Sγ of sensing cysteine on an oxygen atom of H₂O₂. B, schematic reaction mechanism of oxidation of sensing cysteine and the concomitant three redox states of OxyR. Sensing cysteine of OxyR in the reduced state is converted to S-hydroxylated sensing cysteine by H₂O₂, as a reaction intermediate (black arrow). S-Hydroxylated sensing cysteine rapidly makes a disulfide with the other redox-sensitive cysteine (C_R), resulting in the disulfide state of OxyR (blue arrow). Alternatively, S-hydroxylated sensing cysteine is further oxidized consecutively by H₂O₂ to the final S-sulfonated sensing cysteine in the presence of excess H₂O₂, resulting in the overoxidized state of OxyR (red arrows).