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Biochemistry. 2008 Jun 24;47(25):6637-49. doi: 10.1021/bi800305d.

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

Author information

1
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, 20000 Northwest Walker Road, Beaverton, Oregon 97006-8921, USA.

Abstract

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 ((2)H, (13)C, (17)O) into galactose oxidase using an engineered tyrosine auxotroph strain of Pichia pastoris . (33)S 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.

PMID:
18512952
PMCID:
PMC2844931
DOI:
10.1021/bi800305d
[Indexed for MEDLINE]
Free PMC Article

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