Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy

Roman Davydov, Roshan Perera, Shengxi Jin, Tran Chin Yang, Thomas A. Bryson, Masanori Sono, John H. Dawson*, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

81 Scopus citations

Abstract

EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by γ-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; 14N, 1H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H2O2, as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.

Original languageEnglish (US)
Pages (from-to)1403-1413
Number of pages11
JournalJournal of the American Chemical Society
Volume127
Issue number5
DOIs
StatePublished - Feb 9 2005

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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