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N2O binding at a [4Cu:2S] copper–sulphur cluster in nitrous oxide reductase

Nature volume 477, pages 234–237 (2011)Cite this article

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Abstract

Nitrous oxide (N2O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO2 300-fold1,2. In bacterial denitrification, N2O is reduced to N2 by the copper-dependent nitrous oxide reductase (N2OR)3. This enzyme carries the mixed-valent CuA centre and the unique, tetranuclear CuZ site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its CuZ site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N2OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N2O-pressurized crystals. The active CuZ cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N2O bound side-on at CuZ, in close proximity to CuA. With the substrate located between the two clusters, electrons are transferred directly from CuA to N2O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N2OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N2O bound to its reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase4.

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Figure 1: N2OR from Pseudomonas stutzeri.
Figure 2: The copper sites of purple P. stutzeri N 2 OR.
Figure 3: Substrate binding in N 2 OR.
Figure 4: Changes in the electron excitation spectrum of N 2 OR form I upon binding of the substrate N 2 O.

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Protein Data Bank

Data deposits

Atomic coordinates for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 3SBP (native1), 3SBQ (native2) and 3SBR (N2O adduct).

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (IRTG 1422) and the Center for Biological Signalling Studies (bioss, EXC 294). We thank the beam line staff at the Swiss Light Source for their assistance with data collection, T. Spatzal, K. Dörner and T. Friedrich for recording EPR spectra and F. Neese for discussions.

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Authors and Affiliations

  1. Lehrstuhl für Biochemie, Institut für organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104 Freiburg, Germany

    Anja Pomowski & Oliver Einsle

  2. Abteilung Molekulare Mikrobiologie, Institut für Angewandte Biowissenschaften, Karlsruher Institut für Technologie, Hertzstr. 16, 76187 Karlsruhe, Germany

    Walter G. Zumft

  3. Fachbereich Biologie, Universität Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany

    Peter M. H. Kroneck

  4. Centre for Biological Signalling Studies (bioss), Albert-Ludwigs-Universität Freiburg, Hebelstr. 25, 79104 Freiburg, Germany

    Oliver Einsle

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Contributions

A.P., W.G.Z. and O.E. performed the experiments, A.P. and O.E. built and refined the structural model, P.M.H.K. and O.E. designed the experiments, O.E., P.K. and W.G.Z. wrote the manuscript.

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Correspondence to Oliver Einsle.

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The authors declare no competing financial interests.

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The file contains Supplementary Figures 1-7 with legends, Supplementary Methods, Supplementary Table 1, a Supplementary Discussion and additional references. (PDF 2844 kb)

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Pomowski, A., Zumft, W., Kroneck, P. et al. N2O binding at a [4Cu:2S] copper–sulphur cluster in nitrous oxide reductase. Nature 477, 234–237 (2011). https://doi.org/10.1038/nature10332

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