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Kinetic and product distribution analysis of NO· reductase activity in Nitrosomonas europaea hydroxylamine oxidoreductase

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Abstract

Hydroxylamine oxidoreductase (HAO) from the ammonia-oxidizing bacterium Nitrosomonas europaea normally catalyzes the four-electron oxidation of hydroxylamine to nitrite, which is the second step in ammonia-dependent respiration. Here we show that, in the presence of methyl viologen monocation radical (MVred), HAO can catalyze the reduction of nitric oxide to ammonia. The process is analogous to that catalyzed by cytochrome c nitrite reductase, an enzyme found in some bacteria that use nitrite as a terminal electron acceptor during anaerobic respiration. The availability of a reduction pathway to ammonia is an important factor to consider when designing in vitro studies of HAO, and may also have some physiological relevance. The reduction of nitric oxide to ammonia proceeds in two kinetically distinct steps: nitric oxide is first reduced to hydroxylamine, and then hydroxylamine is reduced to ammonia at a tenfold slower rate. The second step was investigated independently in solutions initially containing hydroxylamine, MVred, and HAO. Both steps show first-order dependence on nitric oxide and HAO concentrations, and zero-order dependence on MVred concentration. The rate constants governing each reduction step were found to have values of (4.7 ± 0.3) × 105 and (2.06 ± 0.04) × 104 M−1 s−1, respectively. A second reduction pathway, with second-order dependence on nitric oxide, may become available as the concentration of nitric oxide is increased. Such a pathway might lead to production of nitrous oxide. We estimate a maximum value of (1.5 ± 0.05) × 1010 M−2 s−1 for the rate constant of the alternative pathway, which is small and suggests that the pathway is not physiologically important.

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Abbreviations

AOB:

Ammonia-oxidizing bacteria

HAO:

Hydroxylamine oxidoreductase

Mb:

Ferromyoglobin

MbNO:

Nitrosomyoglobin

MVox :

Methyl viologen dication

MVred :

Methyl viologen monocation radical

SVD:

Singular value decomposition

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Acknowledgments

This publication was made possible by support from the National Institutes of Environmental Health Sciences (NIEHS, grant no. 1 R15 ES013955-01), and from the University of Wisconsin-Milwaukee’s Research Growth Initiative (101X076).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, 53211, USA

    Joshua Kostera, Matthew D. Youngblut, Jeffrey M. Slosarczyk & A. Andrew Pacheco

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  1. Joshua Kostera
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  2. Matthew D. Youngblut
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  4. A. Andrew Pacheco
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Correspondence to A. Andrew Pacheco.

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Kostera, J., Youngblut, M.D., Slosarczyk, J.M. et al. Kinetic and product distribution analysis of NO· reductase activity in Nitrosomonas europaea hydroxylamine oxidoreductase. J Biol Inorg Chem 13, 1073–1083 (2008). https://doi.org/10.1007/s00775-008-0393-4

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