Davydov, Roman Dawson, John H. Perera, Roshan Hoffman, Brian M. 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. product state;D 2O;ENDOR Hexo;1 H electron;Hydroxylation;Hendo signals;Compound IElectron;1 H ENDOR Studies;Cryoreduced Oxy P 450cam Provides New Evidence;ternary ferrous cytochrome P 450cam;reactive species;resonance;Fe;230 K;D 2O buffer;H 2O;Deuterated Camphor;H 2O buffer;hydroxylation;annealing;camphor 2016-02-20
    https://acs.figshare.com/articles/journal_contribution/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/2449000
10.1021/bi301527c.s001