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

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Abstract

We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (KD = 0.48 μM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or α-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator.

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.).

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