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Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs

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Abstract

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with kon 2.82×107 M−1 s−1 and koff 4.49×103 s−1. The dissociation constant of the primary O2 compound was 160 μM (25°C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05  and 0.7 mM oxygen, suggesting a Km <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.

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Acknowledgements

This work was supported in RKP’s laboratory by BBSRC Research Grant P18939.

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Authors and Affiliations

  1. Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK

    Guanghui Wu, Hazel Corker & Robert K Poole

  2. Department of Public Health, Graduate School of Medicine, Kyoto University, Kyoto, 606, Japan

    Yutaka Orii

Authors
  1. Guanghui Wu
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  2. Hazel Corker
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  3. Yutaka Orii
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  4. Robert K Poole
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Correspondence to Robert K Poole.

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This paper is dedicated to Professor Dr Hans G. Schlegel, on the occasion of his 80th birthday.

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Wu, G., Corker, H., Orii, Y. et al. Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs. Arch Microbiol 182, 193–203 (2004). https://doi.org/10.1007/s00203-004-0699-8

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  • DOI: https://doi.org/10.1007/s00203-004-0699-8

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