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

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–O₂–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-OH_exo and C5-H_endo] from camphor. When D₂-camphor was hydroxylated under the same condition in H₂O or D₂O buffer, both ENDOR H_exo and H_endo signals are absent. For D₂-camphor in H₂O buffer, H/D exchange causes the C5-OH_exo signal to reappear during relaxation upon annealing to 230 K; for H₂-camphor in D₂O, the magnitude of the C5-OH_exo signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.