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Self-assembly of the oxy-tyrosinase core and the fundamental components of phenolic hydroxylation

Nature Chemistry volume 4, pages 317–322 (2012)Cite this article

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Abstract

The enzyme tyrosinase contains two CuI centres, trigonally coordinated by imidazole nitrogens of six conserved histidine residues. The enzyme activates O2 to form a µ-η2:η2-peroxo–dicopper(II) core, which hydroxylates tyrosine to a catechol in the first committed step of melanin biosynthesis. Here, we report a family of synthetic peroxo complexes, with spectroscopic and chemical features consistent with those of oxygenated tyrosinase, formed through the self-assembly of monodentate imidazole ligands, CuI and O2 at −125 °C. An extensively studied complex reproduces the enzymatic electrophilic oxidation of exogenous phenolic substrates to catechols in good stoichiometric yields. The self-assembly and subsequent reactivity support the intrinsic stability of the Cu2O2 core with imidazole ligation, in the absence of a polypeptide framework, and the innate capacity to effect hydroxylation of phenolic substrates. These observations suggest that a foundational role of the protein matrix is to facilitate expression of properties native to the core by bearing the entropic costs of assembly and precluding undesired oxidative degradation pathways.

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Figure 1: Oxygenated copper complexes.
Figure 2: Absorption spectra of Cu2O2 complexes.
Figure 3: Self-assembly of Cu2O2 complexes.
Figure 4: Kinetic analysis of substrate reactivity.
Figure 5: Peroxide core transfer.

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Acknowledgements

Financial support was provided by the National Institutes of Health (NIH; GM50730). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program. The authors thank E.I. Solomon for use of the EPR instrument, X. Ottenwaelder for the syntheses of the phenols, and a reviewer for the suggestion of the inclusion of the imidazole Cu–N–C multiple scattering vector at ∼4.2 Å for the EXAFS analyses.

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Authors and Affiliations

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

    Cooper Citek, Christopher T. Lyons & T. Daniel P. Stack

  2. Department of Chemistry and Biochemistry, California State University, Chico, 95929, California, USA

    Erik C. Wasinger

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  1. Cooper Citek
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Contributions

C.C. conducted all the described experiments involving self-assembly, quantification, stability, ligand variation and substrate reactivity. C.T.L. aided in the synthesis of imidazoles and in the XAS and EPR experiments. E.C.W. collected and analysed the XAS data. T.D.P.S. performed DFT and TD-DFT calculations, and C.T.C. and T.D.P.S. were responsible for the preparation of the manuscript.

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Correspondence to T. Daniel P. Stack.

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Citek, C., Lyons, C., Wasinger, E. et al. Self-assembly of the oxy-tyrosinase core and the fundamental components of phenolic hydroxylation. Nature Chem 4, 317–322 (2012). https://doi.org/10.1038/nchem.1284

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