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

Electron paramagnetic resonance and (1)H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H(2)-camphor) and (1R)-5,5-dideuterocamphor (D(2)-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex. Hydroxylation of H(2)-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 D(2)-camphor was hydroxylated under the same condition in H(2)O or D(2)O buffer, both ENDOR H(exo) and H(endo) signals are absent. For D(2)-camphor in H(2)O buffer, H/D exchange causes the C5-OH(exo) signal to reappear during relaxation upon annealing to 230 K; for H(2)-camphor in D(2)O, 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.