The electronic structure of the Cys-Tyr free radical in galactose oxidase determined by EPR spectroscopy

Yuk Ki Lee, Mei M. Whittaker, James W. Whittaker

    Research output: Contribution to journalArticlepeer-review

    39 Scopus citations


    Galactose oxidase is a metalloenzyme containing a novel metalloradical complex in its active site, comprised of a mononuclear copper ion associated with a protein free radical. The free radical has been shown to be localized on an intrinsic redox cofactor, 3′-(S-cysteinyl)tyrosine (Cys-Tyr), formed by a posttranslational covalent coupling of tyrosine and cysteine side chains in a self-processing reaction. The role of the thioether linkage in the function of the Cys-Tyr cofactor is unresolved, and some computational studies have suggested that the thioether substituent has a negligible effect on the properties of the tyrosyl free radical. In order to address this question experimentally, we have incorporated site-selectively labeled tyrosine ( 2H, 13C, 17O) into galactose oxidase using an engineered tyrosine auxotroph strain of Pichia pastoris. 33S was also incorporated into the protein. EPR spectra for the Cys-Tyr free radical in each of these isotopic variants were analyzed to extract nuclear hyperfine parameters for comparison with theoretical predictions, and the unpaired spin distribution in the free radical was reconstructed from the hyperfine data. These labeling studies allow the first comprehensive experimental evaluation of the effect of the thioether linkage on the properties of Cys-Tyr and indicate that previous calculations significantly underestimated the contribution of this feature to the electronic ground state of the free radical.

    Original languageEnglish (US)
    Pages (from-to)6637-6649
    Number of pages13
    Issue number25
    StatePublished - Jun 24 2008

    ASJC Scopus subject areas

    • Biochemistry


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