The Use of Deuterated Camphor as a Substrate in <sup>1</sup>H ENDOR Studies of Hydroxylation by Cryoreduced Oxy P450cam Provides New Evidence of the Involvement of Compound I

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