Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 May 22;104(21):8743-8.
doi: 10.1073/pnas.0702081104. Epub 2007 May 14.

A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR

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

A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR

Jin-Won Lee et al. Proc Natl Acad Sci U S A. .

Abstract

Oxidation of protein thiolates is central to numerous redox-regulated processes. Bacillus subtilis OhrR is an organic peroxide sensor that represses expression of an inducible peroxiredoxin, OhrA. Here, we present evidence that oxidation of the sole cysteine residue in OhrR leads to a sulfenic acid-containing intermediate that retains DNA-binding activity: further reaction to generate either a mixed disulfide (S-thiolation) or a protein sulfenamide (sulfenyl-amide) derivative is essential for derepression. Protein S-thiolation protects OhrR from overoxidation and provides for a facile regeneration of active OhrR by thiol-disulfide exchange reactions. The sulfenamide can also be reduced by thiol-disulfide exchange reactions, although this process is much slower than for mixed disulfides. Recovery of oxidized OhrR from B. subtilis identifies three distinct S-thiolated species, including mixed disulfides with a novel 398-Da thiol, cysteine, and CoASH. Evidence for in vivo formation of the sulfenamide derivative is also presented.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Kinetic analysis of OhrR inactivation. (A) DNA-binding activity of purified OhrR (300 nM) was monitored by FA with labeled DNA (50 nM). Cysteine accelerates the CHP-dependent dissociation of OhrR from operator DNA. Samples were treated at the indicated time with a 10-fold molar excess (3 μM) of CHP in the presence of 0, 3 μM, 10 μM, 30 μM, 100 μM, or 1 mM Cys (in order of increasing dissociation rate). (B) OhrR was oxidized by CHP in either the presence or absence of the indicated LMW thiols. At the time indicated, 10 mM DTT was added to the thiol-containing reactions and the regain of DNA-binding activity was monitored. (C) In vitro oxidation products of OhrR were analyzed by ESI-MS. Purified OhrR (300 nM; trace 1) was treated with 3 μM CHP for 30 min in the presence of LMW thiols as indicated. Protein mass changes were monitored by ESI-MS.
Fig. 2.
Fig. 2.
Kinetics of OhrR oxidation and re-reduction in the presence of Cys. (A) OhrR (300 nM) and DNA were incubated in buffer with 1 mM Cys, and then 1 molar equivalent CHP was added at 5 min and 1 h. (arrows). (B) OhrR oxidation and re-reduction rates were measured as a function of Cys concentration. In order of increasing reactivation rate (and decreasing extent of net inactivation), the concentration of Cys was 0, 0.5, 1, 1.5, 2, 3, and 4 mM. (C) Pseudofirst-order rate constants (k′) for reactivation of OhrR were plotted against Cys concentration, where k′ = kreact[Cys]. (D) The AMS reactivity of Cys15 was monitored by SDS/PAGE in the absence or presence of 2 mM Cys. In the absence of Cys, OhrR is oxidized to an AMS-insensitive species, whereas in the presence of Cys this species appears transiently. See SI Fig. 9 for an image of the entire gel.
Fig. 3.
Fig. 3.
Kinetics of OhrR oxidation and re-reduction in the absence of Cys. (A–D) AMS reactivity of OhrR after CHP treatment was monitored as a function of time. (A) OhrR oxidized in the absence of Cys. The accumulation of the faster migrating band as a function of time indicates a loss of AMS reactivity. (B) Samples shown in A were treated with DTT for 60 min before AMS alkylation. Oxidized OhrR was largely restored to an AMS-reactive form, consistent with the OhrR sulfenamide as the major product. (C and D) Shown are the parallel reactions in the presence of 1 mM Cys. Only the region of the gel corresponding to the OhrR-AMS and unalkylated OhrR bands is shown (see SI Fig. 9 for an image of the entire gel). (E) The molecular mass of OhrR was monitored by ESI-MS before and 30 min after CHP (3 μM) treatment (see SI Fig. 10 for the time series data). (F) Comparison of OhrR inactivation (by CHP) and reactivation (by DTT) in reactions with or without Cys.
Fig. 4.
Fig. 4.
In vivo oxidation products of OhrR. OhrR-FLAG was recovered from B. subtilis and alkylated with IA, and the oxidation state of Cys15 was monitored by MALDI-TOF MS analysis of tryptic digests. (A) In untreated cells, the T3 tryptic peptide is fully alkylated by IA to generate the CysCAM derivative (T3+57 Da; white triangle). (B) In cells treated with 100 μM CHP for ≈2 min, oxidation leads to the appearance of an S-cysteinylated peak (T3+119 Da), an S-thiolated peak (T3+396 Da), and an alkylation-resistant peak (T3∗) with mass similar to fully reduced (CysSH) T3 (black triangle). (C) In cells treated with diamide for 1 min followed by 100 μM CHP, the yield of S-thiolated products is greatly decreased and the Cys15-SO2H derivative (T3+32) is now detected. Note that the intensities of other OhrR tryptic peptides (the peaks between 1,000 and 1,600 m/z) are not affected by oxidation.
Fig. 5.
Fig. 5.
Mechanism of sensing organic hydroperoxides by OhrR. OhrR is normally present in the cell in the fully reduced state and (①) reacts rapidly with organic hydroperoxides (R′-OOH) to generate a sulfenic acid intermediate. The OhrR sulfenate is still active for DNA-binding (see text), but can be rapidly S-thiolated (②), which inactivates OhrR and allows derepression of its target gene (encoding the OhrA peroxiredoxin). Re-reduction, by either spontaneous or enzyme-catalyzed thiol-disulfide exchange reactions regenerates the active repressor (③). The sulfenate can also spontaneously react to generate the sulfenamide (④), which accounts for the slow loss of DNA-binding activity observed in vitro in the absence of thiols. Sulfenamides can be reduced by thiol-disulfide exchange reactions (⑤) as described (23, 24). When intracellular thiols are depleted, overoxidation of Cys15 to the sulfinic and sulfonic acid derivatives can occur (⑥).
See this image and copyright information in PMC

References

    1. Imlay JA. Annu Rev Microbiol. 2003;57:395–418. - PubMed
    1. Fang FC. Nat Rev Microbiol. 2004;2:820–832. - PubMed
    1. Torres MA, Dangl JL. Curr Opin Plant Biol. 2005;8:397–403. - PubMed
    1. Mongkolsuk S, Helmann JD. Mol Microbiol. 2002;45:9–15. - PubMed
    1. Toledano MB, Delaunay A, Monceau L, Tacnet F. Trends Biochem Sci. 2004;29:351–357. - PubMed

Publication types

MeSH terms

LinkOut - more resources