Resonance Raman characterization of the peroxo and hydroperoxo intermediates in cytochrome P450

Abstract

Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450(cam) (CYP101) are reported. Owing to the fact that proton delivery to the active site is hindered in this mutant, the unprotonated peroxo-ferric intermediate is observed as the primary species after radiolytic reduction of the oxy-complex in frozen solutions at 77 K. In as much as previous EPR and ENDOR studies have shown that annealing of this species to approximately 180 K results in protonation of the distal oxygen atom to form the hydroperoxo intermediate, this system has been exploited to permit direct RR interrogation of the changes in the Fe-O and O-O bonds caused by the reduction and subsequent protonation. Our results show that the nu(O-O) mode decreases from a superoxo-like frequency near approximately 1130 cm(-1) to 792 cm(-1) upon reduction. The latter frequency, as well as its lack of sensitivity to H/D exchange, is consistent with heme-bound peroxide formulation. This species also exhibits a nu(Fe-O) mode, the 553 cm(-1) frequency of which is higher than that observed for the nonreduced oxy P450 precursor (537 cm(-1)), implying a strengthened Fe-O linkage upon reduction. Upon subsequent protonation, the resulting Fe-O-OH fragment exhibits a lowered nu(O-O) mode at 774 cm(-1), whereas the nu(Fe-O) increases to 564 cm(-1). Both modes exhibit a downshift upon H/D exchange, as expected for a hydroperoxo-ferric formulation. These experimental RR data are compared with those previously acquired for the wild-type protein, and the shifts observed upon reduction and subsequent protonation are discussed with reference to theoretical predictions.

Figure 1

(A) EPR spectra of the reduced oxy-ferrous complex of D251N CYP101. Data measured after irradiation at 77 K, and after all resonance Raman experiments and annealing at 185 K. The hydrogen atom doublet is marked by the star sign. Vertical bars indicate the features of peroxo-ferric (upper line) and hydroperoxo-ferric (bottom line) complexes with the corresponding g-values. Frozen samples are in 100 mM phosphate (H₂O or D₂O, as indicated), 100 mM KCl, 30% glycerol v/v, T = 15 K. Other details of EPR spectroscopy are described in Materials and Methods. (B) Absorption spectra of D251N CYP101: (1) Oxy-ferrous complex, T=85 K, before irradiation; (2) peroxo-ferric complex, 90 K, after irradiation at 77 K; (3) hydroperoxo-ferric complex, 180 K, after irradiation at 77 K and annealing at 185 K. Frozen samples are in 100 mM phosphate (H₂O), 100 mM KCl, pH 7.4, 65% glycerol.

Figure 1

(A) EPR spectra of the reduced oxy-ferrous complex of D251N CYP101. Data measured after irradiation at 77 K, and after all resonance Raman experiments and annealing at 185 K. The hydrogen atom doublet is marked by the star sign. Vertical bars indicate the features of peroxo-ferric (upper line) and hydroperoxo-ferric (bottom line) complexes with the corresponding g-values. Frozen samples are in 100 mM phosphate (H₂O or D₂O, as indicated), 100 mM KCl, 30% glycerol v/v, T = 15 K. Other details of EPR spectroscopy are described in Materials and Methods. (B) Absorption spectra of D251N CYP101: (1) Oxy-ferrous complex, T=85 K, before irradiation; (2) peroxo-ferric complex, 90 K, after irradiation at 77 K; (3) hydroperoxo-ferric complex, 180 K, after irradiation at 77 K and annealing at 185 K. Frozen samples are in 100 mM phosphate (H₂O), 100 mM KCl, pH 7.4, 65% glycerol.

Figure 2

The high frequency resonance Raman spectra of oxy D251N CYP101 measured at 77 K, and the difference spectra before irradiation (excitation at 413 nm). Inset shows low frequency difference spectra of ¹⁶O₂-¹⁸O₂in H ₂O (A) and in D ₂O (B) buffer.

Figure 3

Theresonance Raman spectra of irradiated P450 D251N samples in H ₂O buffer: spectra A (¹⁶O₂) and B (¹⁸O₂) and in the D₂O buffer: spectra C (¹⁶O₂) and D (¹⁸O₂). The two bottom traces shows the difference spectra of ¹⁶O₂-¹⁸O₂ in H₂O and ¹⁶O₂-¹⁸O₂ in D₂O buffer (excitation line 442 nm, temperature 77 K).

Figure 4

The resonance Raman spectra of irradiated and annealed at 185 K samples of P450 D251N in H₂O buffer: spectra A (¹⁶O₂) and B (¹⁸O₂) and in the D₂O buffer: spectra C (¹⁶O₂) and D (¹⁸O₂). The two bottom traces shows the difference spectra of ¹⁶O₂-¹⁸O₂ in H₂O and ¹⁶O₂-¹⁸O₂in D ₂O buffer (excitation at 442 nm, temperature 77 K).

Scheme 1