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

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2012 Dec 11

PMID 23215047

Citations 10

Authors

Roman Davydov

John H Dawson

Roshan Perera

Brian M Hoffman

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study.

Famulari A, Correddu D, Di Nardo G, Gilardi G, Mitrikas G, Chiesa M Molecules. 2024; 29(2).

PMID: 38276601 PMC: 10819608. DOI: 10.3390/molecules29020518.

An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network.

Petrik I, Davydov R, Kahle M, Sandoval B, Dwaraknath S, Adelroth P Biochemistry. 2021; 60(4):346-355.

PMID: 33464878 PMC: 7888536. DOI: 10.1021/acs.biochem.0c00852.

Linkage between Proximal and Distal Movements of P450cam Induced by Putidaredoxin.

Liou S, Chuo S, Qiu Y, Wang L, Goodin D Biochemistry. 2020; 59(21):2012-2021.

PMID: 32369344 PMC: 9749489. DOI: 10.1021/acs.biochem.0c00294.

Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron.

Erdogan H, Vandemeulebroucke A, Nauser T, Bounds P, Koppenol W J Biol Chem. 2017; 292(52):21481-21489.

PMID: 29109145 PMC: 5766938. DOI: 10.1074/jbc.M117.813683.

Spectroscopic studies of the cytochrome P450 reaction mechanisms.

Mak P, Denisov I Biochim Biophys Acta Proteins Proteom. 2017; 1866(1):178-204.

PMID: 28668640 PMC: 5709052. DOI: 10.1016/j.bbapap.2017.06.021.

References

Davydov R, Kappl R, Huttermann J, Peterson J . EPR-spectroscopy of reduced oxyferrous-P450cam. FEBS Lett. 1991; 295(1-3):113-5. DOI: 10.1016/0014-5793(91)81398-r. View

Sono M, Roach M, Coulter E, Dawson J . Heme-Containing Oxygenases. Chem Rev. 1996; 96(7):2841-2888. DOI: 10.1021/cr9500500. View

Spolitak T, Funhoff E, Ballou D . Spectroscopic studies of the oxidation of ferric CYP153A6 by peracids: Insights into P450 higher oxidation states. Arch Biochem Biophys. 2009; 493(2):184-91. PMC: 2812633. DOI: 10.1016/j.abb.2009.10.014. View

Denisov I, Makris T, Sligar S, Schlichting I . Structure and chemistry of cytochrome P450. Chem Rev. 2005; 105(6):2253-77. DOI: 10.1021/cr0307143. View

Davydov R, Kofman V, Fujii H, Yoshida T, Ikeda-Saito M, Hoffman B . Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants. J Am Chem Soc. 2002; 124(8):1798-808. DOI: 10.1021/ja0122391. View

Davydov R, Makris T, Kofman V, Werst D, Sligar S, Hoffman B . Hydroxylation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes. J Am Chem Soc. 2001; 123(7):1403-15. DOI: 10.1021/ja003583l. View

Davydov R, Ledbetter-Rogers A, Martasek P, Larukhin M, Sono M, Dawson J . EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine. Biochemistry. 2002; 41(33):10375-81. DOI: 10.1021/bi0260637. View

Rittle J, Green M . Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science. 2010; 330(6006):933-7. DOI: 10.1126/science.1193478. View

Davydov R, Perera R, Jin S, Yang T, Bryson T, Sono M . Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy. J Am Chem Soc. 2005; 127(5):1403-13. DOI: 10.1021/ja045351i. View

10.

ROBERTS E, Vaz A, Coon M . Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation. Proc Natl Acad Sci U S A. 1991; 88(20):8963-6. PMC: 52631. DOI: 10.1073/pnas.88.20.8963. View

11.

de Montellano P, De Voss J . Oxidizing species in the mechanism of cytochrome P450. Nat Prod Rep. 2002; 19(4):477-93. DOI: 10.1039/b101297p. View

12.

Bach R . The rate-limiting step in P450 hydroxylation of hydrocarbons a direct comparison of the "somersault" versus the "consensus" mechanism involving compound I. J Phys Chem A. 2010; 114(34):9319-32. DOI: 10.1021/jp1045518. View

13.

Hlavica P . Models and mechanisms of O-O bond activation by cytochrome P450. A critical assessment of the potential role of multiple active intermediates in oxidative catalysis. Eur J Biochem. 2004; 271(22):4335-60. DOI: 10.1111/j.1432-1033.2004.04380.x. View

14.

Davydov R, Hoffman B . Active intermediates in heme monooxygenase reactions as revealed by cryoreduction/annealing, EPR/ENDOR studies. Arch Biochem Biophys. 2010; 507(1):36-43. PMC: 3021649. DOI: 10.1016/j.abb.2010.09.013. View

15.

Jin S, Makris T, Bryson T, Sligar S, Dawson J . Epoxidation of olefins by hydroperoxo-ferric cytochrome P450. J Am Chem Soc. 2003; 125(12):3406-7. DOI: 10.1021/ja029272n. View

16.

Davydov R, Sudhamsu J, Lees N, Crane B, Hoffman B . EPR and ENDOR characterization of the reactive intermediates in the generation of NO by cryoreduced oxy-nitric oxide synthase from Geobacillus stearothermophilus. J Am Chem Soc. 2009; 131(40):14493-507. DOI: 10.1021/ja906133h. View

17.

Davydov R, Osborne R, Shanmugam M, Du J, Dawson J, Hoffman B . Probing the oxyferrous and catalytically active ferryl states of Amphitrite ornata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I. J Am Chem Soc. 2010; 132(42):14995-5004. PMC: 2978736. DOI: 10.1021/ja1059747. View

18.

Coon M, Vaz A, McGinnity D, Peng H . Multiple activated oxygen species in P450 catalysis: contributions To specificity in drug metabolism. Drug Metab Dispos. 1998; 26(12):1190-3. View

19.

Davydov R, Razeghifard R, Im S, Waskell L, Hoffman B . Characterization of the microsomal cytochrome P450 2B4 O2 activation intermediates by cryoreduction and electron paramagnetic resonance. Biochemistry. 2008; 47(36):9661-6. PMC: 2709102. DOI: 10.1021/bi800926x. View

20.

Kim S, Yang T, Perera R, Jin S, Bryson T, Sono M . Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant. Dalton Trans. 2005; (21):3464-9. DOI: 10.1039/b506764m. View