The use of deuterated camphor as a substrate in 1H ENDOR studies of hydroxylation by cryoreduced oxy P450cam provides new evidence of the involvement of compound i

Roman Davydov, John H. Dawson, Roshan Perera*, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Electron paramagnetic resonance and 1H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H2-camphor) and (1R)-5,5-dideuterocamphor (D2-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O 2-substrate complex. Hydroxylation of H2-camphor produced a primary product state in which 5-exo-hydroxycamphor is coordinated with Fe(III). ENDOR spectra contained signals derived from two protons [Fe(III)-bound C5-OHexo and C5-Hendo] from camphor. When D 2-camphor was hydroxylated under the same condition in H2O or D2O buffer, both ENDOR Hexo and Hendo signals are absent. For D2-camphor in H2O buffer, H/D exchange causes the C5-OHexo signal to reappear during relaxation upon annealing to 230 K; for H2-camphor in D2O, the magnitude of the C5-OHexo signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.

Original languageEnglish (US)
Pages (from-to)667-671
Number of pages5
JournalBiochemistry
Volume52
Issue number4
DOIs
StatePublished - Jan 29 2013

ASJC Scopus subject areas

  • Biochemistry

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