High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical

Gary J. Gerfen, Brendan F. Bellew, Robert G. Griffin, David J. Singel, Christopher A. Ekberg, James W. Whittaker

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

The activated form of galactose oxidase from the fungus Dactylium dendroides contains a single divalent copper ion which is antiferromagnetically coupled to a protein-based free radical. Chemical oxidation of the apoenzyme generates the free radical which is localized on a covalently cross-linked tyrosine - cysteine residue. This species, together with model radicals generated by UV irradiation of protonated and selectively deuterated o-(methylthio)cresol (MTC), has been studied by high-frequency EPR spectroscopy (139.5 GHz/5 T) in conjunction with molecular orbital calculations employing self-consistent local density functional (LDF) methods. The Zeeman interactions (g values) determined from the high-frequency spectra of the apogalactose oxidase and the MTC model radicals are remarkably similar and support the assignment of the protein radical to a sulfur-substituted tyrosyl moiety. Molecular orbital calculations accurately reflect the experimental data, including an increase in the axial symmetry of the Zeeman interaction for the MTC radical compared with the unsubstituted tyrosyl radical species. An explanation of this effect based on an analysis of individual atomic contributions to the molecular g values is presented. High-frequency echo-detected EPR spectroscopy of the apogalactose oxidase radical resolves hyperfine splittings. Based on the molecular orbital calculations and the EPR spectroscopic results presented here, the hyperfine splittings are assigned to two methylene protons - one derived from tyrosine and one from cysteine. These findings are consistent with the radical spin density being localized on the tyrosine - cysteine moiety, rather than delocalized throughout an extended π-network involving a nearby tryptophan as had been previously suggested as a possible explanation of the stability of the radical species.

Original languageEnglish (US)
Pages (from-to)16739-16748
Number of pages10
JournalJournal of Physical Chemistry
Volume100
Issue number41
StatePublished - Oct 10 1996
Externally publishedYes

Fingerprint

cresol
Orbital calculations
oxidase
Molecular orbitals
Cysteine
Paramagnetic resonance
Tyrosine
electron paramagnetic resonance
Oxidoreductases
Spectroscopy
Free radicals
Free Radicals
Galactose Oxidase
cresols
spectroscopy
Apoenzymes
Proteins
tyrosine
cysteine
Fungi

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Gerfen, G. J., Bellew, B. F., Griffin, R. G., Singel, D. J., Ekberg, C. A., & Whittaker, J. W. (1996). High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical. Journal of Physical Chemistry, 100(41), 16739-16748.

High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical. / Gerfen, Gary J.; Bellew, Brendan F.; Griffin, Robert G.; Singel, David J.; Ekberg, Christopher A.; Whittaker, James W.

In: Journal of Physical Chemistry, Vol. 100, No. 41, 10.10.1996, p. 16739-16748.

Research output: Contribution to journalArticle

Gerfen, GJ, Bellew, BF, Griffin, RG, Singel, DJ, Ekberg, CA & Whittaker, JW 1996, 'High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical', Journal of Physical Chemistry, vol. 100, no. 41, pp. 16739-16748.
Gerfen GJ, Bellew BF, Griffin RG, Singel DJ, Ekberg CA, Whittaker JW. High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical. Journal of Physical Chemistry. 1996 Oct 10;100(41):16739-16748.
Gerfen, Gary J. ; Bellew, Brendan F. ; Griffin, Robert G. ; Singel, David J. ; Ekberg, Christopher A. ; Whittaker, James W. / High-frequency electron paramagnetic resonance spectroscopy of the apogalactose oxidase radical. In: Journal of Physical Chemistry. 1996 ; Vol. 100, No. 41. pp. 16739-16748.
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