Tyrosine radical formation in the reaction of wild type and mutant cytochrome 
P450cam with peroxy acids - A multifrequency EPR study of intermediates on the 
millisecond time scale

Schünemann, V.; Lendzian, F.; Jung, C.; Contzen, J.; Barra, A. L.; Sligar, S. G.; Trautwein, A. X.

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Tyrosine radical formation in the reaction of wild type and mutant cytochrome P450cam with peroxy acids - A multifrequency EPR study of intermediates on the millisecond time scale

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Jung, C.
Department Solid State Quantum Electronics (Jochen Mannhart), Max Planck Institute for Solid State Research, Max Planck Society;

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Barra, A. L.
High Magnetic Field Laboratory, Former Departments, Max Planck Institute for Solid State Research, Max Planck Society;

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Citation

Schünemann, V., Lendzian, F., Jung, C., Contzen, J., Barra, A. L., Sligar, S. G., et al. (2004). Tyrosine radical formation in the reaction of wild type and mutant cytochrome P450cam with peroxy acids - A multifrequency EPR study of intermediates on the millisecond time scale. Journal of Biological Chemistry, 279(12), 10919-10930.

Cite as: https://hdl.handle.net/21.11116/0000-000E-F771-C

Abstract

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-pi-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-pi-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 g(x) values from
2.0078 to 2.0064, g(y) = 2.0043, and g(z) = 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-pi-cation radical intermediate in P450cam is reduced by
intramolecular electron transfer from these tyrosines within 8 ms.