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Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues
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    Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues
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    The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Osaka, Ibaraki 567-0047, Japan
    *E-mail: [email protected]. Telephone: +81-6-6879-8501.
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    Biochemistry

    Cite this: Biochemistry 2017, 56, 2, 403–410
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    https://doi.org/10.1021/acs.biochem.6b01096
    Published December 19, 2016
    Copyright © 2016 American Chemical Society

    Abstract

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    In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O2•–) has a direct role as a signal for E. coli SoxR and that the sensitivity of the E. coli SoxR response to O2•– is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O2•– activation. To investigate the determinants of SoxR’s sensitivity to O2•–, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O2•– using pulse radiolysis. The substitution of E. coli SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein’s reaction with O2•–. The second-order rate constant of the reaction was 3.3 × 107 M–1 s–1, which was 10 times smaller than that of wild-type SoxR. Conversely, the corresponding substitution of Ala90 with Lys in P. aeruginosa SoxR increased the rate approximately 10-fold. In contrast, introductions of the Arg127Ser128Asp129 → Leu127Gln128Ala129 substitution into E. coli SoxR, and the corresponding substitution (Leu125Gln126Ala127 → Arg125Ser126Asp127) in P. aeruginosa SoxR, did not affect the reaction rates. In addition, the Lys mutation in E. coli SoxR (K89AK92A) showed a defect in vivo transcriptional activity by measuring β-galactosidase expression in response to paraquat. Our findings clearly support the idea Lys is critical to the response to O2•– and further transcriptional activity of SoxR.

    Copyright © 2016 American Chemical Society

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b01096.

    • Comparison of absorption spectra of various variants of E. coli (A) and P. aeruginosa SoxR (B) (Figure S1), absorbance changes after pulse radiolysis of E. coli SoxR and its variants in the absence and presence of SOD [(A) K89RK92R, (B) K89EK92E, and (C) D129A (Figure S2)], and comparison of SOD effects on the oxidation of E. coli SoxR and its variants measured after pulse radiolysis [(A) effect of Lys89 and Lys92 mutations, (B) effect of a three-residue hydrophilic mutation, and (C) kinetic isotope effect (Figure S3)] (PDF)

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    Cited By

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    This article is cited by 9 publications.

    1. Kazuo Kobayashi. Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions. Chemical Reviews 2019, 119 (6) , 4413-4462. https://doi.org/10.1021/acs.chemrev.8b00405
    2. Kazuo Kobayashi . Sensing Mechanisms in the Redox-Regulated, [2Fe–2S] Cluster-Containing, Bacterial Transcriptional Factor SoxR. Accounts of Chemical Research 2017, 50 (7) , 1672-1678. https://doi.org/10.1021/acs.accounts.7b00137
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    Biochemistry

    Cite this: Biochemistry 2017, 56, 2, 403–410
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.biochem.6b01096
    Published December 19, 2016
    Copyright © 2016 American Chemical Society

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