Resonance Raman Characterization of the Peroxo and Hydroperoxo Intermediates in Cytochrome P450

Authors

Ilia G. Denisov, University of Illinois at Urbana-Champaign
Piotr J. Mak, Marquette UniversityFollow
Thomas M. Makris, University of Illinois at Urbana-Champaign
Stephen G. Sligar, University of Illinois at Urbana-Champaign
James R. Kincaid, Marquette UniversityFollow

Document Type

Article

Language

eng

Format of Original

8 p.

Publication Date

12-18-2008

Publisher

American Chemical Society

Source Publication

Journal of Physical Chemistry A

Source ISSN

1089-5639

Original Item ID

DOI: 10.1021/jp8017875

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 ∼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 ν(O−O) mode decreases from a superoxo-like frequency near ∼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 ν(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 ν(O−O) mode at 774 cm⁻¹, whereas the ν(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.

Comments

Accepted version. Journal of Physical Chemistry A, Vol. 112, No. 50 (December 18, 2008): 13172-13179. DOI. © 2008 American Chemical Society. Used with permission.

Recommended Citation

Denisov, Ilia G.; Mak, Piotr J.; Makris, Thomas M.; Sligar, Stephen G.; and Kincaid, James R., "Resonance Raman Characterization of the Peroxo and Hydroperoxo Intermediates in Cytochrome P450" (2008). Chemistry Faculty Research and Publications. 393.
https://epublications.marquette.edu/chem_fac/393