Comparative electron paramagnetic resonance study of radical intermediates in turnip peroxidase isozymes.
The occurrence of isozymes in plant peroxidases is poorly understood. Turnip roots contain seven season-dependent isoperoxidases with distinct physicochemical properties. In the work presented here, multifrequency electron paramagnetic resonance spectroscopy has been used to characterize the Compound I intermediate obtained by the reaction of turnip isoperoxidases 1, 3, and 7 with hydrogen peroxide. The broad (2500 G) Compound I EPR spectrum of all three peroxidases was consistent with the formation of an exchange-coupled oxoferryl-porphyrinyl radical species. A dramatic pH dependence of the exchange interaction of the [Fe(IV)=O por(*+)] intermediate was observed for all three isoperoxidases and for a pH range of 4.5-7.7. This result provides substantial experimental evidence for previous proposals concerning the protein effect on the ferro- or antiferromagnetic character of the exchange coupling of Compound I based on model complexes. Turnip isoperoxidase 7 exhibited an unexpected pH effect related to the nature of the Compound I radical. At basic pH, a narrow radical species ( approximately 50 G) was formed together with the porphyrinyl radical. The g anisotropy of the narrow radical Delta(g) = 0.0046, obtained from the high-field (190 and 285 GHz) EPR spectrum, was that expected for tyrosyl radicals. The broad g(x) edge of the Tyr* spectrum centered at a low g(x) value (2.00660) strongly argues for a hydrogen-bonded tyrosyl radical in a heterogeneous microenvironment. The relationship between tyrosyl radical formation and the higher redox potential of turnip isozyme 7, as compared to that of isozyme 1, is discussed.
Ivancich
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Section de Bioénérgétique and Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, URA 2096 CNRS, CEA Saclay, 91191 Gif-sur-Yvette, France. ivancich@dsvidf.cea.fr
Mazza
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Desbois
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D016880:Anisotropy; D001937:Brassica / Q000201:enzymology*; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry*; D011166:Porphyrins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D005609:Free Radicals; D007527:Isoenzymes; D011166:Porphyrins; D014443:Tyrosine; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010544:Peroxidases