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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 P450⅐reductase 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.

To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO*-(2) anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.

RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (~1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.

The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of ϳ30 ms at 5°C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.Cytochrome P450 CYP3A4 is the most prevalent P450 monooxygenase in human liver and is responsible for the metabolism of almost half of xenobiotics encountered by man (1). This isozyme has a large and flexible active site and is able to bind and catalytically convert multiple substrates, often displaying homotropic and heterotropic cooperativity in substrate binding and product formation. As a central player in human drug metabolism, CYP3A4 is one of the most intensely studied P450s, either in isolated human liver microsomes (2-5), detergent-solubilized form (6), or purified soluble aggregates (7,8). Recently, two groups have reported the crystal structure of a truncated CYP3A4 (9, 10). Despite this structural information, precise chemical and biophysical characterization of the human P450s has lagged that of the monomeric, soluble P450 isozymes isolated from bacteria. In particular, the critical intermediates in the reaction cycle of human P450s have not been precisely documented, due in part to the inability to form a robust, monomeric, and soluble entity that is amenable to the rapid reaction and spectroscopic methodologies successfully applied to the bacterial P450s. This lacuna is significant, as CYP3A4 and other human P450s display complex aspects of substrate recognition and catalytic mechanism that are not present in the simpler P450s such as CYP101 from Pseudomonas.The current version of the catalytic reaction cycle of cytochrome P450 monooxygenases involves intermediate reduction states of heme iron and atmospheric dioxygen and represents the result of over 30 years of intensive research (11,12). Traditionally, the cyclic process begins with the ferric low spin species and a water molecule occupying the sixth coordination site of the heme. In many cases the complementary fit of the native substrate into the pocket displaces this water ligand allowing the system to assume a predominantly hig...

Allosteric mechanisms in human cytochrome P450 3A4 (CYP3A4) in oligomers in solution or monomeric enzyme incorporated into Nanodiscs (CYP3A4ND) were studied by high-pressure spectroscopy. The allosteric substrates 1-pyrenebutanol (1-PB) and testosterone were compared with bromocriptine (BCT), which shows no cooperativity. In both CYP3A4 in solution and CYP3A4ND we observed a complete pressure-induced high-to-low spin shift at pressures <3 kbar either in the substrate-free enzyme or in the presence of BCT. In addition, both substrate-free and BCT-bound enzyme revealed a pressure-dependent equilibrium between two states with different barotropic parameters designated "R" for relaxed and "P" for pressure-promoted conformations. This pressureinduced conformational transition was also observed in the studies with 1-PB and testosterone. In CYP3A4 oligomers the transition was accompanied by an important increase in homotropic cooperativity with both substrates. Surprisingly, at high concentrations of allosteric substrates the amplitude of the spin shift in both CYP3A4 in solution and in Nanodiscs was very low, demonstrating that hydrostatic pressure induces neither substrate dissociation nor an increase in heme pocket hydration in the complexes of the pressure-promoted conformation of CYP3A4 with 1-PB or testosterone. These findings suggest that the mechanisms of interactions of CYP3A4 with 1-PB and testosterone involve an effector-induced transition that displaces a system of conformational equilibria in the enzyme towards the state(s) with decreased solvent accessibility of the active site, so that water flux into the heme pocket is impeded, and the high-spin state of the heme iron is stabilized. † This research was supported by NIH grants GM54995 (JRH), GM33775 (SGS), GM31756 (SGS), Center grant ES06676 (JRH), and The effect of hydrostatic pressure on tryptophan fluorescence of CYP3A4-containing Nanodiscs, the pressure-induced changes in the fluorescence of DCVJ incorporated into the Nanodiscs, the effect of HPCD on the interactions of 1-PB and testosterone with CYP3A4 and the effect of hydrostatic pressure on the fluorescence of 1-PB complexes with HPCD. These materials are available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 30. Published in final edited form as:Biochemistry. Current advances in studies of substrate interactions of cytochromes P450 by biophysical techniques (1-3) and recently resolved X-ray structures of microsomal cytochromes P450 (4-6) have revealed an important conformational flexibility of cytochromes P450 3A4 (CYP3A4) and 2B4 (CYP2B4). The striking conformational rearrangements exhibited by these versatile drug-metabolizing enzymes are thought to play an important role in the adaptation of the geometry of the active site to a diverse set substrate structures. Further investigation of the molecular mechanisms and functional consequences of these conformational transitions are n...

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