Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

We report a multifrequency (9.6-, 94-, 190-, and 285-GHz) EPR study of a freeze-quenched intermediate obtained from reaction of substrate-free cytochrome P450cam (CYP101) and its Y96F and Y96F/Y75F mutants with peroxy acids. It is generally assumed that in such a shunt reaction an intermediate [Fe(I...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-03, Vol.279 (12), p.10919-10930
Main Authors: Schünemann, Volker, Lendzian, Friedhelm, Jung, Christiane, Contzen, Jörg, Barra, Anne-Laure, Sligar, Stephen G., Trautwein, Alfred X.
Format: Article
Language:eng
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Summary:We report a multifrequency (9.6-, 94-, 190-, and 285-GHz) EPR study of a freeze-quenched intermediate obtained from reaction of substrate-free cytochrome P450cam (CYP101) and its Y96F and Y96F/Y75F mutants with peroxy acids. It is generally assumed that in such a shunt reaction an intermediate [Fe(IV)=O, porphyrin-π-cation radical] is formed, which should be identical to the species in the natural reaction cycle. However, for the wild type as well as for the mutant proteins, a porphyrin-π-cation radical is not detectable within 8 ms. Instead, EPR signals corresponding to tyrosine radicals are obtained for the wild type and the Y96F mutant. Replacement of both Tyr-96 and Tyr-75 by phenylalanine leads to the disappearance of the tyrosine EPR signals. EPR studies at 285 GHz on freeze-quenched wild type and Y96F samples reveal g tensor components for the radical (stretched gx values from 2.0078 to 2.0064, gy = 2.0043, and gz = 2.0022), which are fingerprints for tyrosine radicals in a heterogeneous polar environment. The measurements at 94 GHz using a fundamental mode microwave resonator setup confirm the 285-GHz study. From the simulation of the hyperfine structure in the 94-GHz EPR spectra the signals have been assigned to Tyr-96 in the wild type and to Tyr-75 in the Y96F mutant. We suggest that a transiently formed Fe(IV)=O porphyrin-π-cation radical intermediate in P450cam is reduced by intramolecular electron transfer from these tyrosines within 8 ms.
ISSN:0021-9258
1083-351X