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. 2008 Sep;190(17):5738-45.
doi: 10.1128/JB.00576-08. Epub 2008 Jun 27.

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide sensor

Sumarin Soonsanga  1 , Jin-Won Lee, John D Helmann
Affiliations

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide sensor

Sumarin Soonsanga et al. J Bacteriol. 2008 Sep.

Abstract

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.

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Figures

FIG. 1.
FIG. 1.
(A) Alignment of OhrR homologs. The sequences corresponding to tryptic peptides T3, T16, and T17 of B. subtilis OhrR are shown with Cys residues highlighted. The first two sequences shown are representative of the 1-Cys family of OhrR proteins, and the second pair are sequences that have a second Cys at the position corresponding to the redox active C127 of Xanthomonas campestris (last line). The next two have a more distal Cys residue corresponding to C131 in Xanthomonas. The last two sequences contain two Cys residues in this carboxyl-terminal region. Bacillus subtilis, CAA05594; Streptomyces coelicolor A3(2), CAB87337; Vibrio cholerae MZO-2, ZP_01977344; Pseudoalteromonas atlantica T6c, YP_659686; Agrobacterium tumefaciens strain C58, AAK86653; Bradyrhizobium sp. strain BTAi1, YP_001236314; Erwinia carotovora subsp. atroseptica SCRI1043, CAG76066; Xanthomonas campestris pv. Phaseoli, AAK62673. (B) B. subtilis OhrR (6) was modeled with the G120C and Q124C mutations. The predicted positions and distances between C15 and C120′ and C124′ are shown. Monomers are colored blue and green.
FIG. 2.
FIG. 2.
In vitro oxidation of mutant OhrR proteins is accompanied by disulfide bond formation. (A) FA assays monitoring inactivation of wild-type (WT) and G120C treated with 3 μM CHP in the presence or absence of 1 mM Cys. Oxidant was added at 5 min (arrow), and 10 mM DTT was added at 15 min. (B) The same analysis was performed using 3 μM LHP. Oxidant was added at 5 min, and 10 mM DTT was added at 30 min.
FIG. 3.
FIG. 3.
(A) Disulfide bond formation in OhrR as monitored by nonreducing SDS-PAGE. Samples of protein either were left untreated or were treated with 3 μM CHP for 10 min prior to analysis. (B) Reactions were performed as for panel A, except that the reaction mixtures contained 1 mM cysteine. Protein bands indicated by letters were selected for structural analysis using MALDI-TOF MS, as shown in Fig. 4 and 5. The first lane in each panel contains molecular mass standards corresponding to 15, 20, 25, 37, and 50 kDa (bottom to top).
FIG. 4.
FIG. 4.
Analysis of structural changes accompanying oxidation for wild-type and G120C OhrR proteins. Samples A through H (from Fig. 3) were analyzed by MALDI-TOF MS after tryptic digestion. The T3 peptide (Fig. 1A) with a reduced Cys residue was modified with IA and is indicated by a white triangle. S-cysteinylated T3 peptide is indicated with a gray triangle. Note that the mass of the T16 peptide is larger in the G120C mutant protein as predicted [indicated as T16(C)+IA].
FIG. 5.
FIG. 5.
Analysis of structural changes accompanying protein oxidation for OhrR Q124C and the G120C Q124C double mutant. Samples I through Q (from Fig. 3) were analyzed by MALDI-TOF MS after tryptic digestion. The T3 peptide (Fig. 1A) with a reduced Cys residue was modified with IA and is indicated by a white triangle. S-cysteinylated T3 peptide is indicated with a gray triangle. Note that the mass of the T16 peptide is larger in the G120C mutant protein as predicted [indicated as T16(C)+IA]. Note that the disulfide-linked peptides indicated as T16(C)+T17(C) are detected as two peaks of m/z values of 2,060 and 2,078 Da, as described in the text.
FIG. 6.
FIG. 6.
In vivo oxidation of the G120C and Q124C OhrR proteins. (A) β-Galactosidase activity assays of OhrR activity in strains expressing either the G120C or the Q124C variant. OhrR activity was monitored using a PohrA-cat-lacZ reporter fusion. Empty and filled bars represent the samples that were left untreated (-CHP) and treated with 100 μM CHP for 15 min (+CHP), respectively. (B) Immunoblot analysis of disulfide bond formation upon CHP oxidation. Cells were left untreated (−) or were treated with 100 μM CHP for 1 min (+) before immunoprecipitation in a buffer containing IA. Mon., monomeric.
FIG. 7.
FIG. 7.
MALDI-TOF analysis of oxidation products for OhrR G120C in vivo. The inset shows the Coomassie-stained gel of immunoprecipitated OhrR-FLAG samples used for analysis. Cells were left untreated (−) or were treated with 100 μM CHP for 1 min (+) before immunoprecipitation of OhrR-FLAG proteins in a buffer containing IA. Proteins were resolved by nonreducing 16% Tris-Tricine SDS-PAGE. The Coomassie-stained band of ∼24 Da corresponds to the light chain of the anti-FLAG antibody. The indicated bands (A to D) were used for MALDI-TOF MS analysis after digestion with trypsin. The resulting MALDI-TOF spectra are shown as spectra A through D, with peaks labeled as described for Fig. 4. Upon oxidation, wild-type OhrR forms mixed disulfides with a 398-Da thiol (shown as T3+396; red arrow) and with cysteine (T3+Cys; gray arrow). In addition, some of the protein is converted to an IA-resistant form (presumably the sulfenamide), as indicated by the blue arrow (T3*).
FIG. 8.
FIG. 8.
The G120C OhrR protein can form an intersubunit disulfide bond under conditions that irreversibly overoxidize the wild-type protein. Immunoblot analysis of G120C-FLAG treated with strong oxidants Cells (0.45 ml) either were left untreated (−) or were treated with 100 μM CHP, 1 mM CHP, or 5 μM LHP (as indicated) for 2 min before sonication in the presence of 10% trichloroacetic acid. The precipitated proteins were separated by nonreducing SDS-PAGE and analyzed using anti-FLAG antibody to visualize the monomeric (Mon.) and disulfide-cross-linked (dimer) forms of OhrR. Note that in contrast to that shown in Fig. 6, this immunoblot was done using crude-cell extracts rather than immunoprecipitated samples.
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