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Reversible S-nitrosylation in an engineered azurin

Nature Chemistry volume 8, pages 670–677 (2016)Cite this article

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Abstract

S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalysed by haem proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Here, we report reversible insertion of NO into a copper–thiolate bond in an engineered copper centre in Pseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential by deleting a hydrogen bond in the secondary coordination sphere. The results not only provide the first direct evidence of S-nitrosylation of Cu(II)-bound cysteine in metalloproteins, but also shed light on the reaction mechanism and structural features responsible for stabilizing the elusive Cu(I)–S(Cys)NO species. The fast, efficient and reversible S-nitrosylation reaction is used to demonstrate its ability to prevent NO inhibition of cytochrome bo3 oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.

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Figure 1: Geometries of Cu(II) binding sites and Fourier transforms of EXAFS data with fitting.
Figure 2: UV–vis absorption, MS and EPR spectroscopic characterization of S-nitrosylation in engineered red copper protein.
Figure 3: Electrophilic and radical pathways for (Cys)S–NO bond formation and possible isomers of the Cu(I)/(Cys)S–NO product.
Figure 4: Effect of Cu(II)-M121H/H46E/F114PAz on NO inhibition of E. coli cytochrome bo3 oxidase.

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Acknowledgements

This material is based on work supported by the US National Science Foundation (award no. CHE 14-13328 to Y.L.) and the National Institutes of Health (NIH award no. DK31450 to E.I.S. and award no. GM054803 to N.J.B.). The authors thank H. Matsumura and P. Moënne-Loccoz for performing an initial investigation using resonance Raman spectroscopy, Z. Ding for providing E. coli cytochrome bo3 oxidase and K. Hwang for discussions and revisions of the manuscript. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-76SF00515). The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH and the National Institute of General Medical Sciences (NIGMS, including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, 61801, Illinois, USA

    Shiliang Tian, Jing Liu, Nicholas M. Marshall, Mark J. Nilges & Yi Lu

  2. Department of Chemistry, Stanford University, Stanford, 94305, California, USA

    Ryan E. Cowley & Edward I. Solomon

  3. Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, 61801, Illinois, USA

    Parisa Hosseinzadeh & Yi Lu

  4. Center of Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, 61801, Illinois, USA

    Yang Yu & Yi Lu

  5. Department of Biology, Brookhaven National Laboratory, Upton, 11973, New York, USA

    Howard Robinson

  6. Institute of Environmental Health, Oregon Health & Sciences University, Portland, 97239, Oregon, USA

    Ninian J. Blackburn

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Contributions

S.T., J.L., N.M.M. and Y.L. conceived and designed the experiments. S.T., J.L., P.H., Y.Y. and H.R. performed the experiments. S.T., R.E.C., P.H., M.J.N., N.J.B., E.I.S. and Y.L. analysed the data. S.T., J.L., R.E.C., E.I.S. and Y.L. co-wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Ninian J. Blackburn, Edward I. Solomon or Yi Lu.

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Tian, S., Liu, J., Cowley, R. et al. Reversible S-nitrosylation in an engineered azurin. Nature Chem 8, 670–677 (2016). https://doi.org/10.1038/nchem.2489

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