Kinetics of electron transfer in the complex of cytochrome P450 3A4 with the flavin domain of cytochrome P450BM-3 as evidence of functional heterogeneity of the heme protein
Section snippets
Materials
Bromocriptine (BCT) mesylate, glucose oxidase, catalase, glucose-6-phosphate dehydrogenase, glucose-6-phosphate, NADPH, protocatechuic acid and protocatechuate-3,4-dioxygenase were from Sigma Chemicals (St. Louis, MO). 1-Pyrenebutanol was a product of Invitrogen (Carlsbad, CA). α-Naphthoflavone (ANF), testosterone, and glucose were obtained from Aldrich. All other chemicals used were of the highest grade available from commercial sources and were used without further purification.
Expression and purification of CYP3A4 and BMR
CYP3A4 was
Interaction of BMR with CYP3A4
Fig. 1a shows a series of absorbance spectra obtained in a counter-flow continuous variation experiment. As seen from the inset, the first principal spectrum obtained from the application of PCA to this dataset reveals a distinct low-to-high-spin shift of CYP3A4, which reflects the formation of the complexes of the heme protein with BMR, as observed earlier for the interactions of cytochromes P450 with CPR [72], [73]. The titration curve is represented by a one-half of a symmetric bell-shaped
Discussion
Our absorbance spectroscopy experiments with a counter flow Job’s titration setup confirmed that BMR forms a stoichiometric 1:1 complex with CYP3A4 with a KD of 0.48 μM, where the spin equilibrium of the heme protein is shifted towards the high-spin state, similar to observations with other microsomal cytochromes P450 and CPR [72], [73]. Consistent with this observation, BMR was able to reduce ferric CYP3A4 with the formation of the ferrous carbonyl complex of the enzyme. The lack of dependence
Acknowledgments
The authors thank Dr. T. M. Poulos at University of California-Irvine for the kind donation of plasmid DNA of BMR and Ms. N. Davydova for her assistance in the purification of CYP3A4. This research was supported by NIH Grant GM54995 (J.R.H.) and Center grant ES06676 (J.R.H.).
References (100)
- et al.
Biochem. Biophys. Res. Commun.
(2001) - et al.
Trends Pharm. Sci.
(2003) - et al.
Arch. Biochem. Biophys.
(2006) - et al.
Arch. Biochem. Biophys.
(2005) - et al.
J. Biol. Chem.
(2006) - et al.
J. Biol. Chem.
(2007) - et al.
Biophys. Chem.
(2006) - et al.
Arch. Biochem. Biophys.
(2004) - et al.
Arch. Biochem. Biophys.
(2003) - et al.
J. Biol. Chem.
(2007)
Arch. Biochem. Biophys.
Biochem. Pharm.
J. Biol. Chem.
Biochem. Biophys. Res. Commun.
J. Biol. Chem.
Arch. Biochem. Biophys.
FEBS Lett.
FEBS Lett.
J. Biol. Chem.
Biochim. Biophys. Acta
J. Biol. Chem.
Arch. Biochem. Biophys.
Biochem. Biophys. Res. Commun.
Arch. Biochem. Biophys.
Biochem. Biophys. Res. Commun.
Arch. Biochem. Biophys.
J. Biol. Chem.
Arch. Biochem. Biophys.
Arch. Biochem. Biophys.
Biochimie
Arch. Biochem. Biophys.
Biochem. Biophys. Res. Commun.
J. Biol. Chem.
J. Biol. Chem.
J. Inorg. Biochem.
J. Biol. Chem.
Biochimie
J. Biol. Chem.
J. Biol. Chem.
Arch. Biochem. Biophys.
J. Biol. Chem.
Biochem. Biophys. Res. Commun.
J. Biol. Chem.
J. Biol. Chem.
Pharm. Ther.
Biochem. Pharmacol.
Toxicol. Appl. Pharm.
Acta Biol. Med. Ger.
Biochemistry (Moscow)
Annu. Rev. Pharmacol. Toxicol.
Cited by (21)
Assembling the P450 puzzle: on the sources of nonadditivity in drug metabolism
2021, Trends in Pharmacological SciencesCitation Excerpt :The fact that this heterogeneity is eliminated by protein monomerization [24,27] suggests that it originates from some specific structural features of the P450 oligomers. Persistent heterogeneity also reveals itself in the kinetics of dithionite- and NADPH-dependent reduction of microsomal P450s [28–30]. Both processes followed multiexponential kinetics in P450 oligomers, either in solution or in the membrane.
Spectroscopic studies of the cytochrome P450 reaction mechanisms
2018, Biochimica et Biophysica Acta - Proteins and ProteomicsInteractions among cytochromes P450 in microsomal membranes: Oligomerization of cytochromes P4503A4,3A5, and 2E1 and its functional consequences
2015, Journal of Biological ChemistryCitation Excerpt :Opposite to the case of dithionite, the fast phase of the NADPH-dependent reduction is preferential for the high-spin heme protein. Selective reduction of the high-spin P450 in the fast phase of the NADPH-dependent process has been demonstrated with CYP2C11 (68), CYP2B4 (69), and most recently with CYP3A4 (22, 40). The contrast in the kinetics of reduction between the high- and the low-spin states seemingly contradicts the very high rate of P450 spin transitions (62, 70–72), which implies that the position of equilibrium between the two states must remain unaffected throughout the reduction.
Cytochrome b<inf>5</inf> and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions
2014, ToxicologyCitation Excerpt :One of the most prominent consequences of oligomerization is given by “persistent heterogeneity” or apparent divergence of a pool of a single CYP species into non-interconverting and functionally separate fractions (Davydov, 2011). This suggestion was supported by several investigations for substrate oxidation by various CYP enzymes in the reconstituted systems (Kaminsky and Guengerich, 1985; Backes and Eyer, 1989; Iwase et al., 1991; Fernando et al., 2007, 2008; Davydov, 2011). The reasons for the results found in the present work seem to be even more complex, as BaP oxidation by the CYP1A1 reconstituted system was substantially stimulated by cytochrome b5.
Interactions of cytochrome P450s with their ligands
2011, Archives of Biochemistry and BiophysicsElectron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium
2010, Biochimica et Biophysica Acta - BioenergeticsCitation Excerpt :The latter were refined by studying the pressure-induced P450-to-P420 conversion of the CYP3A4 ferrous carbonyl complex, similarly to the technique used for CYP2B4 and CYP1A2 [45,46]. The complete set of the standards used in this study are available on-line as Supporting Information to our recent publication [29]. These kinetic curves were fitted to a multiexponential equation using a combination of Marquardt and Nelder-Mead algorithms.