Cooperativity in Cytochrome P450 3A4: LINKAGES IN SUBSTRATE BINDING, SPIN STATE, UNCOUPLING, AND PRODUCT FORMATION

Ilia G Denisov,Bradley J Baas,Yelena V Grinkova,Stephen G Sligar

doi:10.1074/jbc.m609589200

Ilia G Denisov, Bradley J Baas + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m609589200

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Abstract

Understanding the detailed metabolic mechanisms of membrane-associated cytochromes P450 is often hampered by heterogeneity, ill-defined oligomeric state of the enzyme, and variation in the stoichiometry of the functional P450.reductase complexes in various reconstituted systems. Here, we describe the detailed characterization of a functionally homogeneous 1:1 complex of cytochrome P450 3A4 (CYP3A4) and cytochrome P450 reductase solubilized via self-assembly in a nanoscale phospholipid bilayer. CYP3A4 in this complex showed a nearly complete conversion from the low- to high-spin state when saturated with testosterone (TS) and no noticeable modulation due to the presence of cytochrome P450 reductase. Global analysis of equilibrium substrate binding and steady-state NADPH consumption kinetics provided precise resolution of the fractional contributions to turnover of CYP3A4 intermediates with one, two, or three TS molecules bound. The first binding event accelerates NADPH consumption but does not result in significant product formation due to essentially complete uncoupling. Binding of the second substrate molecule is critically important for catalysis, as the product formation rate reaches a maximum value with two TS molecules bound, whereas the third binding event significantly improves the coupling efficiency of redox equivalent usage with no further increase in product formation rate. The resolution of the fractional contributions of binding intermediates of CYP3A4 into experimentally observed overall spin shift and the rates of steady-state NADPH oxidation and product formation provide new detailed insight into the mechanisms of cooperativity and allosteric regulation in this human cytochrome P450.

Highlights

Cytochrome P450 3A4 (CYP3A4)3 is the most abundant P450 in the human liver [1]
The fully purified CYP3A41⁄7CPR Nanodiscs were re-injected onto a size exclusion column, which showed a single homogeneous peak with a retention time corresponding to a Stokes diameter of 14.0 Ϯ 0.2 nm (Fig. 3)
One factor complicating the understanding of atypical kinetics observed with CYP3A4 is due to the population heterogeneity of CYP3A4 oligomers [18, 43, 50]

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Imidazole, sodium cholate, NADPH, testosterone (TS), and androstenedione were purchased from Sigma. 6␤-, 2␤-, and 15␤-OH-TS were from Steraloids, Inc. (Newport, RI). Three enzyme1⁄7substrate complexes must be used in this global analysis because it was impossible to adequately describe all available data with only one or two TS molecules bound to CYP3A4 Each of these binding intermediates is characterized by a specific fraction of high-spin state, the steady-state rate of NADPH oxidation, and the rate of product formation. The total turnover rate (k) was expressed as the linear combination of the fractional rates of substrate hydroxylation (ki, where i is the number of TS molecules bound to one molecule of CYP3A4 (i ϭ 1, 2, 3)), and the fractions of binding intermediates were calculated using dissociation constants (Ki) derived from the global fit as described above (Equation 4), FIGURE 1. The reconstitution proceeds from detergent-solubilized CYP3A4, CPR, phospholipid, and MSP as described under “Experimental Procedures.”

RESULTS

Statea Spin shift NADPH rate Turnover rate Couplingb

DISCUSSION