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Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path

Nature Structural & Molecular Biology volume 11, pages 1179–1185 (2004)Cite this article

Abstract

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s−1. The disulfide bond–mediated conformation switch results in a metastable form that is locally strained by ∼3 kcal mol−1. On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

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Figure 1: MALDI-TOF spectra for the tryptic digests of reduced (top) and oxidized (bottom) wild-type OxyR.
Figure 2: AMS analysis of OxyR cysteines in cells treated with H2O2.
Figure 3: Characterization of OxyR oxidation by chemical modification.
Figure 4: Steady-state fluorescence spectra of OxyR.
Figure 5: Stopped-flow measurement of conformational switch of OxyR during oxidation.
Figure 6: Intermediates in the structural transitions of OxyR.
Figure 7: Urea-induced equilibrium unfolding transition of OxyR.

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Acknowledgements

We thank A. Matouschek for helpful suggestions on the equilibrium unfolding study, and W. Outten for comments on the manuscript. This research was supported by the National Creative Research Initiative Program (MOST, Korea) and the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program.

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Authors and Affiliations

  1. Center for Cellular Switch Protein Structure, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yuseong-gu, Daejeon, 305-806, Korea

    Cheolju Lee, Soon Mi Lee, Seung Jun Kim & Seong Eon Ryu

  2. Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yuseong-gu, Daejeon, 305-806, Korea

    Seung Jun Kim, Sang Chul Lee & Seong Eon Ryu

  3. Life Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, 130-650, Korea

    Cheolju Lee & Woo-Sung Ahn

  4. Functional Proteomics Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, 130-650, Korea

    Myeong-Hee Yu

  5. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Partha Mukhopadhyay & Gisela Storz

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  1. Cheolju Lee
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Supplementary information

Supplementary Table 1

Sensitivities of OxyR C199S and C208S single mutant strains and C199S C208S double mutant strain to H2O2. (PDF 51 kb)

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Lee, C., Lee, S., Mukhopadhyay, P. et al. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat Struct Mol Biol 11, 1179–1185 (2004). https://doi.org/10.1038/nsmb856

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