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 journalArticlepeer-review

18 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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