Structural and functional effects of cytochrome b5 interactions with human cytochrome P450 enzymes

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5. Mutations of key b5 residues suggest not only that different b5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

The small heme-containing protein cytochrome b 5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b 5 residues interacting with different human drugmetabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b 5 ␣4-5 (CYP2A6 and CYP2E1) or this region and ␣2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b 5 . Mutations of key b 5 residues suggest not only that different b 5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.
Cytochrome P450 (P450) 2 monooxygenases have vital roles both in the metabolism of xenobiotics, including drugs, and in the biosynthesis of endogenous compounds, such as steroids, vitamins, fatty acids, and eicosanoids. Although the ability of P450 enzymes to oxidize such substrates requires interaction with a redox partner protein, catalysis can additionally be modulated by interactions with the membrane-bound heme protein cytochrome b 5 (b 5 ). Cytochrome b 5 has a complex influence, such that P450 catalysis can be stimulated, not affected, or even inhibited, prompting numerous investigations into the mechanism(s) by which these varied responses are elicited (1,2). Proposals have largely focused on b 5 functioning 1) in a purely redox role of electron delivery or 2) as an allosteric modulator of P450 conformation. Metal-substituted, redox-silent forms of b 5 still stimulate certain P450 reactions (4,5), supporting a purely allosteric role for b 5 , at least in some cases. Allosteric modulation of P450 conformation could result in alterations in substrate binding, electron or proton delivery, or coupling of NADPH consumption to metabolite formation (1)(2)(3).
Regardless of the mechanism, the capability of b 5 to modulate P450 catalysis relies on direct binding between b 5 and a P450. Insights into individual P450/b 5 complexes gained from mutagenesis (3,4), chemical modification (5), and cross-linking studies (6,7) are consistent with the convex, negatively charged, heme-exposed face of b 5 (Fig. 1A) transiently interacting with the concave, largely positively charged, proximal face of P450 enzymes (Fig. 1B). This proximal P450 face is the same surface to which the required redox partners bind (NADPH-cytochrome P450 oxidoreductase (CPR) for microsomal P450 enzymes), underscoring the necessity of transient interactions between P450 and its protein partners (3,8).
Fundamental questions persist with regard to P450/b 5 interactions. How variable is the binding interface between b 5 and different P450 enzymes? Do the affinities vary? Could differences in the b 5 /P450 interaction underlie the different effects b 5 has on P450 catalysis? To complicate matters, the effects that b 5 has on catalysis can depend not only on the P450 enzyme, but also the P450 substrate being metabolized (9 -11) and experimental parameters, including the ratio of P450 to b 5 (10). Differences in b 5 effects with different substrates have fueled interest in whether conformational linking exists between the P450 active site cavity and the P450 proximal face interacting with b 5 and CPR (8,12,13).
Because there are no X-ray structures available of b 5 interacting with any P450, solution NMR is an attractive high-resolution approach to decoding these transient binding interactions. Previous studies of 15 N-labeled b 5 binding to increasing concentrations of unlabeled human steroidogenic CYP17A1 enzyme or rabbit CYP2B4 revealed only small chemical shift perturbations, but more pronounced decreases in the intensity of b 5 resonances (8,14). These line-broadening effects may result from a number of potential sources. Binding of the smaller ϳ16-kDa b 5 to a larger P450 enzyme (ϳ50 -55 kDa) would enhance the transverse relaxation rate, but could also result in paramagnetic effects from the P450 heme and/or alter the protein-protein interaction to fall within the intermediateexchange time scale (8,14). Thus, whereas progressive line broadening occurs upon formation of increasing amounts of the reversible P450:b 5 complex, there is not necessarily a 1:1 correspondence between signal loss and amount of complex formed, which prevents the determination of meaningful K d values. Regardless, the degree of broadening has varied between these two different P450 enzymes, the P450 ligands present, and experimental conditions, such as the presence of lipid mimic systems (8,14,15), providing various insights into these P450/b 5 interactions. Both CYP17A1 and CYP2B4 studies reported that certain b 5 resonances are broadened substantially more than the average. Because most of the b 5 amino acids have been assigned to specific resonances (16), such differential line broadening has been used to successfully map out specific b 5 residues that transiently interact with CYP17A1 and CYP2B4 (8,14).
The current study employed such solution NMR experiments to identify and compare b 5 residues interacting with the human xenobiotic cytochrome P450 enzymes 2A6, 2D6, 2E1, and 3A4. Results indicate that both shared and distinct interaction surfaces on b 5 are present in these respective complexes. Additionally, substantial differences between CYP2D6 and the other P450 enzymes examined are consistent with different relative affinities of the complex with b 5 . Key b 5 residues identified using this approach were subsequently validated by performing mutagenesis and evaluating both complex formation and effects on catalysis. This comparison of P450:b 5 complexes provides a physical basis to begin understanding the ability of b 5 to differentially modulate P450 catalytic activities.

Rationale for experimental scope
The specific human P450 enzymes included in this study were selected for their importance in clinical drug metabolism, their diversity across the xenobiotic P450 subfamily, and the range of b 5 effects on metabolite production as reported in the literature. Cytochrome b 5 reportedly stimulates CYP2E1-mediated oxidation of a number of different substrates, including acetaminophen, aniline, N-nitrosodimethylamine, and chlorzoxazone (17,18), but may play a redox role, as apo-b 5 does not increase product formation (19,20). The influence of b 5 on the major drug-metabolizing P450 enzyme CYP3A4 has been widely documented, with numerous CYP3A4-mediated reactions showing stimulation in the presence of both holo-and apo-b 5 (21). CYP2A6 coumarin 7-hydroxylation has also been reported to be stimulated 1.5-2.5-fold by b 5 (19,(22)(23)(24). CYP2D6 is a clinically significant P450 enzyme because of its ability to metabolize a wide array of pharmaceuticals and its high degree of polymorphism (25), but its metabolite production is reportedly less influenced by b 5 , at least with in vitro experiments (19,20,26,27). All of the human P450 enzymes in this study were the N-terminally truncated, C-terminally Histagged forms used to determine X-ray crystallographic structures. They are expressed recombinantly in the significant quantities required to make multiple NMR samples, are highly purified, and are functionally active without lipid addition. The latter aspect provides a common environment across NMR and catalytic assays and avoids variation due to variability in P450 incorporation into lipid.
Each P450 enzyme was saturated with a substrate to additionally stabilize the P450 proteins under NMR-compatible conditions. From significant previous experience with P450 enzymes produced for crystallographic studies, it is clear that high-affinity ligands often greatly improve the stability of these flexible, promiscuous enzymes. They also did so under condi-tions where the NMR experiments could be collected: 10 -100 M P450 in glycerol-free, low-ionic-strength buffer at 25°C for several hours. Additionally, some previous reports on P450/b 5 interactions have indicated a greater affinity of the complex between the two proteins when P450 substrate is present (5). In this study, classical substrates with a single or at least one major metabolite were preferred, as this might be expected to simplify interaction modes by promoting a more homogeneous P450 conformation as well as permitting quantification of a single product in catalytic assays. Thus, CYP2E1 was saturated with the muscle relaxant chlorzoxazone (denoted as CYP2E1 (CZN)), CYP3A4 with the hypotensive drug nifedipine (denoted as CYP3A4(NFP)), CYP2D6 with the cough suppressant dextromethorphan (denoted as CYP2D6(DXM)), and CYP2A6 with coumarin and p-nitrophenol (denoted as CYP2A6(pNP)). Of these substrates, the addition of coumarin, chlorzoxazone, and dextromethorphan to CYP2A6, CYP2E1, and CYP2D6, respectively, yielded shifts in the difference spectra indicating a transition from low spin to high spin, facilitating the ability to monitor protein saturation. The intrinsic absorbance of nifedipine and p-nitrophenol impaired such observations for CYP3A4 and CYP2A6, respectively. Under these conditions, after collection of NMR spectra none of the samples resulted in visible precipitation, and the reduced-carbon monoxide difference spectra were unchanged from the freshly purified P450 protein.
Whereas NMR-observed titrations necessarily involved various concentrations of P450, other experiments were designed to reflect ratios of P450:CPR:b 5 often used in P450 functional assays in the literature. This is important, as thoroughly demonstrated in studies on the rabbit CYP2B4 by Waskell and coworkers (10). Herein, all catalytic assays used 1:2:2 ratios of P450 to CPR to b 5 , rather than optimizing these ratios for maximal turnover with individual P450/substrate combinations as is frequently done. In NMR experiments, this same ratio of P450 to b 5 was used to evaluate the b 5 mutants (0.5:1 P450:b 5 , which is the same as 1:2 in the assays). To ensure that these ratios are accurate, special precautions were taken to avoid frequent problems with the quality and quantification of reductase and b 5 . Cytochrome b 5 was reconstituted with heme during purification to avoid large amounts of apo-b 5 that can otherwise occur when polypeptide production exceeds heme production and that could also possibly bind P450. Reductase was purified to remove as much as possible of the proteolyzed form that can reduce cytochrome c and might be able to bind P450 but is not able to reduce P450 and promote catalysis. Thus, reductase was quantitated by flavin content instead of cytochrome c reduction. Reductase was also quantitated by total protein from the bicinchoninic acid assay using bovine serum albumin as a standard and was no more than 30% higher than quantitation by flavin absorbance. Both b 5 and reductase were the human forms to match the human P450 enzymes, rather than the rat versions often employed. Finally, because electrostatics are probably involved in both steering and binding between b 5 and P450 enzymes, to provide additional consistency, the same buffer was used in NMR experiments and all catalytic assays, with the exception of the CYP3A4 nifedipine Cytochrome P450/b 5 interactions metabolism assay, which required slightly different conditions (see below).
Finally, although the effects of b 5 on P450 catalysis are often simply evaluated by monitoring differences in metabolite formation in the presence and absence of b 5 , b 5 may also have other effects not readily detected by this approach. It has been reported that b 5 can change the amount of NADPH consumed per amount of product formation (percent coupling) (1), which would not necessarily alter the amount of product formed. For this reason, NADPH consumption and its coupling to product formation were also evaluated in the absence and presence of wild-type and mutant b 5 proteins.

Interaction and catalytic effects of b 5 on CYP2A6
When the 2D HSQC spectrum of 15 N-labeled b 5 alone is collected (e.g. see Fig. 1C (black)), the intensity of each resonance is by definition 100%. As reported previously for CYP17A1 and CYP2B4, titrations of [ 15 N]b 5 with increasing concentrations of 2A6(coumarin) result in few chemical shifts, but line broadening occurs and the intensity of most resonances decreases (Fig. 1C), with more marked reductions for specific residues (Fig. 1D). Because the dominant features are changes in intensity and because such overlaid spectra are difficult to comprehend visually, each resonance in each series of spectra was analyzed, and the percentage of intensity remaining was plotted graphically (e.g. Fig. 2A).
For CYP2A6(coumarin), reductions in the average b 5 signal intensity occur even at very low P450:b 5 ratios. At a CYP2A6(coumarin):b 5 ratio of 0.1:1, an average of ϳ68% of the original signal remained. As expected with increasing formation of the complex, the average intensities continued to decrease as more and more CYP2A6(coumarin) was added. At 0.2:1, on average, ϳ41% of the original signal intensity remained ( Fig. 2A, black dashed line). At 0.3:1, 0.4:1, and 0.5:1, ϳ35%, 26%, and 22% of the original signal remained.
At titration points with ϳ40 -60% reduction in intensity, there is enough P450:b 5 complex formed to clearly identify specific b 5 amino acids whose resonances are differentially broadened compared with the average. For CYP2A6(coumarin), this corresponds to the 0.2:1 ratio, which reveals that a number of b 5 resonances were broadened more than one S.D. value ( Fig. 2A, red dashed line) from the average ( Fig. 2A, black dashed line), consistent with the involvement of these b 5 residues in binding CYP2A6(coumarin). These b 5 resonances correspond to residues Thr-60, Asp-65, His-68, Ser-69, Thr-70, and Arg-73, which cluster on b 5 helices 4 and 5 and the loop connecting them (Fig. 2B, red).
To further examine the role of specific b 5 residues interacting with CYP2A6(coumarin), a series of mutations were examined. Residues Asp-65 and Glu-49 were selected based on 1) their location on the b 5 surface, 2) previous evidence that electrostatic pairing is important in b 5 /P450 complex formation (4,8), 3) locations on distinct b 5 faces (Fig. 1A), and 4) their location in or near binding interfaces identified herein (see below). Asp-65 is located in b 5 ␣4, whereas Glu-49 is in b 5 ␣3. The effects of the charge-neutralizing E49Q and D65N mutations on P450 interactions with b 5 were evaluated at a P450:b 5 ratio of 0.5:1 for all P450 enzymes. As indicated above, under these conditions with wild-type b 5 , the intensity for the average b 5 resonance decreases to ϳ22% of the original signal (Fig. 2C, left). However, when the b 5 mutant D65N was mixed with CYP2A6(coumarin) at the same ratio, the overall line broadening was much less, to only 76% of the original (Fig. 2C, middle), consistent with decreased complex formation between b 5 D65N and CYP2A6 (coumarin) compared with wild-type b 5 . Conversely, mixing the b 5 mutant E49Q with CYP2A6(coumarin) yielded average line broadening (to ϳ25%; Fig. 2C, right) similar to wild-type b 5 (22%; Fig. 2C, left), suggesting that this mutation did not adversely affect formation of the b 5 /CYP2A6(courmarin) complex. These results suggest that whereas the anionic charge on Asp-65 in b 5 helix 4 has a significant role in b 5 binding to CYP2A6(coumarin), Glu-49 in b 5 helix 3 has little to no effect on complex formation.
To relate these structural observations to enzymatic function, coumarin metabolism assays were subsequently employed. Under our conditions, there was no significant effect of b 5 on CYP2A6 7-hydroxycoumarin formation. Neither k cat nor K m was substantially altered in the presence of b 5 (Fig. 2D). Thus, it was not surprising that neither the D65N nor E49Q b 5 mutants altered coumarin metabolism (Fig. 2D).
Although the presence of wild-type b 5 did not alter formation of the 7-hydroxycoumarin product, coupling studies revealed that NADPH consumption was reduced by ϳ60% (Fig. 2E, red). Thus, wild-type b 5 increased productive coupling of the reaction by ϳ1.9-fold (Fig. 2F, red). The b 5 D65N mutant, which appeared to decrease b 5 binding to CYP2A6(coumarin) in the NMR experiments, yielded a much smaller decrease in NADPH consumption (Fig. 2E, green) and had coupling similar to that when b 5 was not present (Fig. 2F, green versus black). The E49Q b 5 , which did not appear to alter b 5 binding to CYP2A6 (coumarin) in the NMR experiments, functioned more like wild-type b 5 , with a similar NADPH consumption (Fig. 2E, blue versus red) and productive coupling (Fig. 2F, blue versus red). Overall, wild-type and E49Q b 5 acted similarly, with 1.9-and 1.6-fold increases in coupling, respectively, whereas D65N has little effect on coupling (a 1.2-fold increase in coupling over reactions without b 5 ).
Whereas the effects of b 5 on NADPH consumption observed herein are consistent with the ϳ50% reduction previously reported for CYP2A6 coumarin 7-hydroxylation (22,24), there have also been reports that b 5 can increase product formation in the range of 1.5-2.5-fold (19,(22)(23)(24). However, no such increase in 7-hydroxycoumarin was observed herein. This apparent discrepancy could be due to other differences in experimental conditions. Of particular importance are the protein ratios used for P450:CPR:b 5 in catalytic assays. Experimental evidence to date strongly supports mutually exclusive, overlapping binding sites for CPR and b 5 on the proximal face of P450 enzymes. As a result, the ratio and relative affinities of reductase and b 5 for a particular P450 would dictate the observed effects (1). At high relative ratios and/or affinities, b 5 could inhibit required reduction of ferric P450 by reductase, whereas at lower relative ratios and/or affinities, b 5 may exert stimulatory effects on other steps of the P450 catalytic cycle to increase coupling (13). That b 5 significantly inhibits NADPH consumption without changes in product formation suggests

Cytochrome P450/b 5 interactions
that for this enzyme(substrate) combination, the 1:2:2 ratio may balance the inhibitory and stimulatory effects of b 5 . However, other significant differences, for example in the species and quantitation of b 5 and reductase, the presence of lipid, etc., may also contribute to different observations across the literature with respect to b 5 effects on product formation.
Regardless, NMR and functional assays herein consistently suggest that D65N in b 5 helix 4 is important in both binding to CYP2A6(coumarin) and in improving coupling of NADPH consumption to product formation. More broadly, the NMR data suggest that b 5 surface residues in both helix 4 and helix 5 are involved in binding to CYP2A6(coumarin). The heme is again shown as sticks. As a reference, the I helix is highlighted (orange) on the distal side of the heme where the active site is located. C, the 1 H-15 N HSQC NMR spectra of the soluble domain of human cytochrome b 5 titrated with increasing amounts of an unlabeled CYP2A6 saturated with coumarin are representative of the type of changes seen for all P450 enzymes examined herein. Progressive signal loss or line-broadening of most b 5 resonances occurs with increasing concentrations of P450, indicating progressive complex formation between the two proteins. D, a selected region of the NMR spectra (the region indicated as a dashed box in C) is shown with increasing P450 to demonstrate differential line broadening effects. In this example with CYP2A6, some resonances are broadened to a greater extent (Thr-70 and Arg-73) than others (Leu-14, Glu-48, and Glu-49).  (x axis) is plotted as a percentage of that resonance's intensity in the absence of P450 (y axis). Fine lines between circles indicate continuous assignments for sequential b 5 amino acids. Gaps in the line between circles indicate that one or more intervening b 5 amino acids have not been assigned to an individual resonance in the spectrum (also shown in white in B). The average () and average minus one S.D. (1) are indicated on the plot by black and red dashed lines, respectively, and constitute the selection criteria of b 5 residues involved in P450 binding (red circles). B, on the surface of the human soluble domain b 5 structure (PDB entry 2I96), residues displaying differential broadening effects are colored red, whereas residues that have assigned NMR resonances that are not differentially affected by P450 addition are colored gray, and unassigned residues are colored white. C, resonance intensity plots as in A, but comparing the effects of line broadening between WT b 5 and b 5 mutants D65N and E49Q at a fixed 0.

Cytochrome P450/b 5 interactions Interaction and catalytic effects of b 5 on CYP2E1
CYP2E1-mediated oxidation of many different substrates is reportedly stimulated by b 5 . These include chlorzoxazone, acetaminophen, aniline, and N-nitrosodimethylamine (17,18). Studies suggest that b 5 may play a redox role with CYP2E1, as apo-b 5 does not increase product formation (19,20).
NMR experiments performed in this study revealed that titration of b 5 with CYP2E1(chlorzoxazone), like CYP2A6 (coumarin), also caused fairly substantial overall broadening at moderate P450:b 5 ratios. At 0.25:1, for example, an average of ϳ55% of signal remained (Fig. 3A), and by 0.5:1, only ϳ28% of the original signal was present (Fig. 3C). The resonances for b 5 residues Val-34, His-68, Ser-69, Thr-70, and Ser-76 had signal losses more than one S.D. below this average (Fig. 3A). Each of these residues except for Val-34 reside on ␣5 or the preceding loop (Fig. 3B). This result is consistent with a monofacial interaction between b 5 and CYP2E1(CZN) involving ␣5 and nearby residues.
Consistent with the above idea, when evaluated at a consistent P450:b 5 ratio of 0.5:1, the b 5 D65N mutant had less loss of average signal intensity (to 66%; Fig. 3C, middle) than wild-type b 5 (Fig. 3C, left). This suggests that the D65N b 5 mutant reduces binding to CYP2E1(CZN). Notably, the E49Q mutant on the opposite b 5 surface had an intermediate effect on average signal intensity to ϳ41% of the original signal.
Comparisons of the mutational effects are clearer when evaluating chlorzoxaone 6-hydroxylation. Consistent with previous literature reports (28,29), the effect of wild-type b 5 on 2E1 chlorzoxazone 6-hydroxylation was pronounced, with both an increased k cat and decreased K m (Fig. 3D). Specifically, experiments herein revealed an ϳ1.7-fold increase in k cat and a 2-fold decrease in K m , yielding an ϳ3.5-fold increase in k cat /K m . The E49Q b 5 mutant performed very similarly to wild-type b 5 (Fig.  3D), suggesting that this residue is not critical. However, using the b 5 mutant D65N in these reactions yielded substrate metabolism and steady-state kinetic parameters more similar to when b 5 was not present at all (Fig. 3D). Thus, the surface near Asp-65 is important in both b 5 binding to CYP2E1(CZN) and stimulation of product formation.
Coupling between NADPH consumption and product formation is typically very poor for CYP2E1 in general. Under the current conditions in the absence of b 5 , CYP2E1 coupling was only ϳ3%. The addition of wild-type b 5 significantly decreased the NADPH consumption by 48% (Fig. 3E) while increasing product formation, resulting in increased coupling of the reaction by ϳ2.5-fold (Fig. 3F). The b 5 mutant E49Q behaved similarly to wild-type b 5 in that NADPH consumption was decreased to a similar extent (Fig. 3E), and combined with increased product formation, the coupling increased ϳ3.1-fold (Fig. 3F), slightly more than in the presence of wild-type b 5 . The b 5 mutant D65N also reduced NADPH consumption (ϳ38%; Fig. 3E) but had little effect on product formation, thus increasing coupling by ϳ1.7-fold when compared with reactions in the absence of b 5 (Fig. 3F).
Interactions between b 5 and CYP2E1 have previously been studied using cross-linking of the complex coupled with mass spectrometry. Those studies also identified residues on the same surface of b 5 : Asp-58 and Glu-61 in one study (6) and Asp-58 and Asp-65 in another study (30). In the latter study, these interactions were further tested for their functional role, and both residues appeared to be important in b 5 stimulation of the 6-hydroxylation of CZN (30). Unfortunately, the resonance for Asp-58 in b 5 has not been assigned, but it would be predicted to undergo differential line broadening. Overall, the current experiments and previous studies support the concept that the region surrounding Asp-65 is involved in both physical interaction between b 5 and CYP2E1 and increases observed in product formation.

Interaction and catalytic effects of b 5 on CYP3A4
Influence of b 5 on CYP3A4 has been reported broadly. A number of CYP3A4-mediated reactions are stimulated by both holo-and apo-b 5 (21), which may support a more allosteric role for b 5 . The current studies evaluated interactions and the effects of b 5 on the hypotensive drug NFP.
To evaluate the significance of these two surfaces, the b 5 mutants D65N and E49Q were used to further evaluate the physical interaction between b 5 and CYP3A4(NFP). As with the other enzymes, this was conducted at the P450:b 5 ratio of 0.5:1. Whereas CYP3A4(NFP) resulted in a signal reduction to an average of 29% for wild-type b 5 (Fig. 4C, left), this effect was dampened for both b 5 mutants. At this same ratio, ϳ45% of the average signal remained for D65N (Fig. 4C, middle) and ϳ60% for E49Q (Fig. 4C, right). Thus, both mutations appeared to reduce CYP3A4(NFP):b 5 complex formation. Notably, although the overall average intensities were higher for each b 5 mutant compared with wild type, distinctive differential broadening still occurred (Fig. 4C, middle and right), suggesting that specific interactions between b 5 and 3A4(nifedipine) were not completely disrupted by either single point mutation.
CYP3A4 converts nifedipine to dehydronifedipine. The addition of wild-type b 5 to such reactions resulted in inhibition of this reaction (Fig. 4D). The addition of b 5 both decreased the k cat and increased the K m such that the k cat /K m was almost half that when b 5 was not present. Literature has reported varied stimulation of nifedipine by b 5 , typically ϳ1.3-3-fold increased product formation (19,31). The differences in these observations may lie in the protein ratios used to perform the experi-   Fig. 2A. B, mapping of differentially affected residues (red) on human b 5 . The color code is as described in the legend to Fig. 2B. C, resonance intensity plots comparing line broadening for wild-type b 5

Cytochrome P450/b 5 interactions
icant differences between how the assays were accomplished in different reports, including the presence of lipid or the constructs or buffer used. CYP3A4-mediated metabolism seems to be more sensitive to these environmental parameters than many other P450 enzymes. Notably, the single b 5 mutants D65N and E49Q had effects on nifedipine oxidation most similar to wild-type b 5 .
Thus, the interactions between CYP3A4(NFP) and b 5 are not disrupted enough by either single-point mutation to ameliorate the effect of b 5 on product formation. Unfortunately, NADPH consumption and coupling of the 3A4(nifedipine) reaction were not successful due to low turnover rates of the reaction under the conditions employed for all of the assays herein.  5 ) ratio normalized to the free b 5 resonance intensity. The color code is as described in the legend to Fig. 2B. B, human soluble domain b 5 structure (PDB entry 2I96) with residues displaying differential broadening effects colored red, residues that are assigned in the NMR spectrum colored gray, and unassigned residues colored white. C, b 5 resonance intensity plots comparing the effects of line broadening between WT b 5 and b 5 mutants D65N and E49Q at a fixed 0.

Cytochrome P450/b 5 interactions
Overall, the differential line broadening of NMR signal suggests that CYP3A4(NFP) interacts with b 5 over the widest surface area for any of the P450/b 5 complexes in this study. Interactions between CYP3A4 and b 5 have been reported previously in cross-linking and mutational studies. Zhao et al. (7) used mass spectrometry to identify cross-links between the two proteins; residues on human b 5 identified were Glu-42, Glu-48, and Glu-61, which are located on ␣2, ␣3, and the start of ␣4. Another study reported similar residues, where mutations in human b 5 residues Glu-48, Glu-49, Asp-58, and Asp-65 had significant decreases in the ability of b 5 to enhance CYP3A4mediated testosterone and nifedipine oxidation (31). Moreover, the b 5 double mutant D58G/D65G significantly impaired the ability to form cross-links with CYP3A4, whereas the double mutant E48G/E49G had reduced stimulation but was highly dependent on the type of phospholipid in the assay (31). Overall, the current NMR data of the CYP3A4/b 5 complex are consistent with reported interactions in these previous studies but highlight additional residues.

Interaction and catalytic effects of b 5 on CYP2D6
CYP2D6 metabolizes numerous pharmaceuticals, including many antidepressants and antipsychotics. It exhibits substantial polymorphism in humans (25). To assess whether 2D6 forms a complex with b 5 , NMR-monitored titrations of [ 15 N]b 5 with CYP2D6 were carried out using a saturating amount of its common antitussive substrate DXM. The titrations resulted in line broadening, consistent with complex formation between the two proteins, but required much higher concentrations of CYP2D6 than any of the other drug-metabolizing enzymes in this study. For example, it takes a 1:1 CYP2D6(DXM):b 5 ratio before the average signal intensity drops to about half (48%; Fig. 5A). In comparison, CYP2A6(coumarin) had more signal reduction by a 0.2:1 ratio.
Within these data, the resonances for four residues are broadened more than one S.D. from the average. Gly-47, Glu-49, His-68, and Ser-69 are all differentially broadened (Fig. 5A). On the b 5 surface, these residues fall into two disconnected regions of b 5 : the start of ␣3 (Gly-47 and Glu-49) and the loop between ␣4 and ␣5 (His-68 and Ser-69) (Fig. 5B). These data suggest that when a complex is formed at higher concentrations of CYP2D6(DXM), both faces of b 5 are involved.
To probe the importance of these distinct faces, the E49Q and D65N b 5 mutants were also evaluated for their ability to bind CYP2D6(DXM). At the uniform ratio of 0.5:1, wild-type b 5 retained an average signal intensity of 69% (Fig. 5C, left), but both mutants retained even more signal intensity. E49Q and D65N retained ϳ80 and ϳ84% of the signal intensity, respectively (Fig. 5C, right and middle). This is consistent with both charges contributing to the CYP2D6(DXM):b5 complex when it does occur.
To link these structural observations with the effects of b 5 on CYP2D6 dextromethorphan metabolism, both wild-type and mutant b 5 proteins were employed in steady-state turnover assays. Similar to other in vitro reports on CYP2D6 reactions (19,32), the presence of wild-type b 5 had no significant effects on kinetics of metabolite formation from dextromethorphan ( Fig. 5D). Thus, not surprisingly, neither b 5 mutant altered product formation.
When NADPH consumption was measured, however, the presence of b 5 did result in a two-thirds reduction (Fig. 5E) and therefore an increase in coupling (from ϳ23 to ϳ72%; Fig. 5F). Each of the single E49Q and D65N b 5 mutants also reduced NADPH consumption (to ϳ59 and ϳ65%, respectively; Fig.  5E), but not as effectively as wild-type b 5 . Thus, the ϳ2.0-fold increases in coupling with E49Q and D65N b 5 were less than the ϳ3-fold increase in coupling observed for wild-type b 5 (Fig. 5F).
CYP2D6(DXM) interaction with b 5 appeared to be similar to CYP3A4(NFP) in that opposite surfaces of b 5 were implicated, but extensive broadening of b 5 resonances did not occur until an equal molar (1:1) ratio of 2D6(DXM):b 5 , a feature that would be consistent with a weaker interaction with CYP2D6(DXM) compared with the other P450:b 5 pairs studied. Most previous studies have reported no b 5 stimulation of CYP2D6-mediated in vitro bufuralol 1Ј-hydroxylation, tamoxifen 4-hydroxylation, or acetaminophen conversion to its toxic metabolite N-acetylp-benzoquinone imine (19,20,26,27). There is one conflicting report indicating that b 5 could modulate CYP2D6 metabolite formation in vivo, as mice humanized for CYP2D6 have decreased bufuralol and debrisoquine turnover upon hepatic deletion of the b 5 gene. This could be ameliorated by the addition of membranes containing b 5 (33).

Interaction and catalytic effects of b 5 on CYP2A6 saturated with p-nitrophenol
To begin to probe the effects that the identity of a given P450 substrate might have on interactions with b 5 , the interactions of CYP2A6 saturated with pNP were also investigated using NMR and functional assays and compared with earlier results for CYP2A6(coumarin).
The importance of Asp-65 in forming the complex between b 5 and CYP2A6(pNP) was similar to that observed with the substrate coumarin. At a uniform 0.5:1 P450:b 5 ratio, the b 5 D65N mutation retained more signal average intensity (ϳ86%; Fig. 6C, middle) than wild-type b 5 (49% signal remaining; Fig.  6C, left), consistent with reduced complex formation for the mutant. By comparison, under the same conditions, the b 5 E49Q mutant had an average intensity (54%; Fig. 6C, right) much more similar to that of wild-type b 5 , suggesting that this b 5 residue does not play a significant role in binding CYP2A6(pNP).
The effects of these b 5 mutants on formation of a CYP2A6 (pNP)/b 5 complex correlated with observations of the kinetics

Cytochrome P450/b 5 interactions
of CYP2A6 para-nitrophenol metabolism to 4-nitrocatechol. Whereas wild-type b 5 modestly stimulated the reaction with both an increase in k cat and decrease in K m , resulting in an overall ϳ1.5-fold increase in catalytic efficiency (Fig. 6D), the b 5 mutant E49Q performed very similarly (Fig. 6D), suggesting that this residue is not critical for b 5 simulation of CYP2A6-  Fig. 2B. B, human soluble domain b 5 structure (PDB entry 2I96) with residues displaying differential broadening effects colored red, residues that are assigned in the NMR spectrum colored gray, and unassigned residues colored white. C, b 5 resonance intensity plots comparing the effects of line broadening between WT b 5 and b 5 mutants D65N and E49Q at a fixed 0.

Cytochrome P450/b 5 interactions
mediated pNP metabolism. In contrast, reactions substituting the b 5 mutant D65N resulted in steady-state kinetic parameters more similar to reactions containing no b 5 at all (Fig. 6D), suggesting that Asp-65 is critical for the b 5 stimulation of CYP2A6-mediated pNP oxidation. It has previously been reported that CYP2A6-mediated pNP oxidation is not significantly altered by the presence of b 5 (34). However, these results were obtained at a single pNP concentration that was relatively low (5 M) and at  5 ) ratio normalized to the free b 5 resonance intensity. The color code is as described in the legend to Fig. 2B. B, human soluble domain b 5 structure (PDB entry 2I96) with residues displaying differential broadening effects colored red, residues that are assigned in the NMR spectrum colored gray, and unassigned residues colored white. C, b 5 resonance intensity plots comparing the effects of line broadening between WT b 5 and b 5 mutants D65N and E49Q at a fixed 0.

Cytochrome P450/b 5 interactions
a CYP2A6:CPR ratio of 1:5.8 (34). The CYP2A6 pNP assays performed in the current study used a range of pNP concentrations (25-1600 M) and a CYP2A6:CPR ratio of 1:2. Thus, differences in experimental parameters could contribute to observed differences in b 5 stimulation and again suggest that the ratio of b 5 to CPR is likely to determine the outcome because these two proteins both bind, and probably compete for, the P450 proximal surface.
Evaluation of NADPH coupling herein revealed that wildtype b 5 decreased NADPH consumption by ϳ30% (Fig. 6E) and increased coupling of NADPH consumption to formation of the 4-nitrocatechol product formation 1.8-fold (Fig. 6F). By comparison, neither the E49Q or D65N mutation significantly altered NADPH consumption (Fig. 6E). However, the increased product formation observed for the b 5 E49Q results in a ϳ1.4fold increase in coupling, whereas the absence of effect of D65N b 5 on product formation and NADPH consumption meant coupling was the same as in the absence of b 5 (Fig. 6F). Thus, these studies also support a significant role for Asp-65, but less so for Glu-49.
Overall, the NMR and mutation data herein suggest that the b 5 surface involved in binding to CYP2A6(pNP) (Fig. 6B) is very similar to the b 5 surface binding CYP2A6(coumarin) (Fig. 2B). Mutation studies confirmed that Asp-65 on one face of b 5 plays a significant role in both CYP2A6/b 5 interactions, whereas Glu-49 on the opposing b 5 face had relatively little contribution. The greater degree of line broadening occurring with the substrate coumarin compared with pNP is potentially consistent with a stronger b 5 interaction with CYP2A6(coumarin) compared with CYP2A6(pNP). However, we cannot exclude effects due to possible differences in CYP2A6 paramagnetism when bound to different ligands. CYP2A6(coumarin) results in almost complete conversion to high spin, whereas the CYP2A6 (pNP) spin state cannot be readily assessed because of significant pNP absorbance in the region of interest. Using immobilized CYP2A6 and biotinylated b 5 , Guengerich and co-workers (27) found no difference in binding for unliganded CYP2A6 versus CYP2A6 bound to coumarin. Like the structural data, the effects of b 5 mutants in turnover assays support an important role for D65N in coupling and metabolite formation. In contrast, E49Q performed more similarly to wild-type enzyme, suggesting that this residue is not critical for changes in CYP2A6-mediated pNP metabolism. In both CYP2A6-mediated coumarin and pNP metabolism, b 5 increased coupling ϳ2-fold. However, this mostly stemmed from a decrease in NADPH utilization for coumarin hydroxylation, whereas for pNP oxidation, both decreased NADPH consumption and increased metabolite formation contributed. Thus, although the b 5 residues binding CYP2A6 are conserved across these two substrates, the effects on CYP2A6 catalysis vary.

Comparison of P450/b 5 interactions across P450 enzymes and substrates
Of the four human P450 enzymes surveyed, all of them were found to interact with cytochrome b 5 and to do so predominately on at least one of the surfaces surrounding the hemeexposed face of b 5 . This is in general agreement with studies assessing b 5 interactions with CYP17A1 (4, 8), CYP2B4 (14), CYP3A4 (7,31), and CYP2E1 (6,30). However, the use of NMR allows one to simultaneously probe all possible b 5 residues involved in the interaction without modifying either interacting partner aside from isotopic labeling. As a result, this study identified two b 5 surfaces differentially interacting with different xenobiotic P450 enzymes. All four P450 enzymes in this study interacted with His-68 and Ser-69 on the loop between ␣4 and ␣5. Three of them (CYP2A6, CYP2E1, and CYP3A4) additionally interacted with adjacent residues in b 5 ␣5. In addition to this patch on one side of the heme, CYP2D6 and CYP3A4 can also bind to the opposite surface of b 5 , ␣3, with CYP3A4 having the broadest interaction surface for both regions. Notably, CYP2A6 saturated with either coumarin or pNP interacted with the same face of b 5 , but it remains to be seen whether this is true for other P450/substrate pairs. It is clear that across these P450/substrate pairs and across the different binding interfaces observed, b 5 consistently increases coupling but may or may not alter product formation, as was also seen for rabbit CYP2B4 (10).
Although the current NMR studies provide detailed structural information about the P450/b 5 interaction, they are not well suited to the determination of dissociation constants. However, it is notable that CYP2D6(DXM) was a very distinct outlier in terms of the amount of line broadening observed for b 5 resonances compared with CYP3A4(NFP), CYP2A6(coumarin or pNP), CYP2E1(CZN), and even CYP17A1 in a previous parallel study (8). Although other explanations are possible, the simplest explanation is that this observation is consistent with less complex formation. A study evaluating the physical interaction between P450 enzymes immobilized on a plastic plate and biotinylated b 5 ranked the affinities as unliganded CYP3A4 Ͼ CYP2A6 ϳ CYP2D6 Ͼ CYP2E1 but noted that much less CYP2D6/b 5 complex was formed in these experimental conditions as well (27).
In conclusion, it appears that cytochrome b 5 interacts with different drug-metabolizing P450 enzymes with both shared and distinct surfaces. Disruption of these surfaces correlates with functional effects on metabolite production and/or NADPH consumption. Thus, in the absence of X-ray structures, solution NMR is a high-resolution technique to examine these transient P450 interactions with other proteins. Further work remains to map the P450 residues involved in binding b 5 , to compare b 5 and reductase competition for binding different P450 enzymes, and to determine the mechanism(s) by which b 5 modulates P450 catalysis.

Generation of the soluble domain of human cytochrome b 5 with 15 N-labeling for NMR experiments
A synthetic, codon-optimized gene encoding the soluble domain (residues 1-108) of human microsomal cytochrome b 5 with a C-terminal His 6 tag (Genewiz) was cloned into the NcoI and BamHI restriction sites of pET15b and transformed into E. coli C41 (DE3) cells already containing the pGro7 plasmid (Takara Bio) for expression of GroEL/GroES chaperones. Transformed cells were selected by growing cells for ϳ18 h at 37°C on a non-inducing minimal medium plate (MDAG-11) Cytochrome P450/b 5 interactions (35) supplemented with carbenicillin (100 g/ml) and chloramphenicol (20 g/ml) to select for the b 5 and chaperone plasmids, respectively. All subsequent cultures contained these antibiotics. A single colony was picked and grown for ϳ16 h at 37°C with shaking (250 rpm) in a 50-ml liquid culture of noninducing minimal medium MDAG-135 (35). Expression cultures consisted of 1 liter of a defined minimal medium (48 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 9 mM NaCl, 19 mM 15 N NH 4 Cl, 53 mM glucose, 4 mM MgSO 4 , and trace metals) inoculated with 5 ml of the liquid starter culture. Cells were grown with shaking (250 rpm) at 37°C, to an optical density at 600 nm (A 600 ) of 0.3, at which point the heme precursor ␦-aminolevulinic acid (to 1 mM) and the chaperone inducer L-arabinose (to 13 mM) were added. After A 600 reached 0.7-0.8, b 5 expression was induced by adding 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside, and the temperature and shaking were reduced to 30°C and 225 rpm, respectively. Cultures were subsequently grown for 20 h before harvesting and freezing the cell pellet at Ϫ80°C. Purification was initiated by resuspending cells in lysis buffer (500 mM potassium phosphate, 100 mM NaCl, 15% glycerol, 1 mM EDTA, pH 7.4) with the addition of 1 mM PMSF. Resuspended cells were lysed by a French press, and heme reconstitution was performed as described (36). Lysed cells were clarified by ultracentrifugation at 140,000 ϫ g and loaded onto a pre-equilibrated 25-ml Ni-NTA column (Qiagen) with loading buffer (100 mM potassium phosphate, 20% glycerol, 200 mM NaCl, 20 mM imidazole, pH 7.4), washed with an additional 8 column volumes of loading buffer, and eluted using a 6 column volumes of elution buffer (loading buffer with 200 mM imidazole). Eluted fractions with A 413 /A 280 Ͼ 4.0 were pooled and concentrated before loading on a Superdex 200 Increase 10/300 GL column (GE Healthcare) and run with gel filtration buffer (50 mM potassium phosphate, 20% glycerol, 100 mM NaCl, pH 7.4). The protein sample was then exchanged into a storage buffer (50 mM potassium phosphate, 10% glycerol, pH 7.4) using a HiTrap desalting column (GE Healthcare). Final protein preparation was evaluated by SDS-PAGE and UV-visible spectroscopy (A 413 /A 280 Ͼ 6.0) and quantified using an extinction coefficient of 117 mM Ϫ1 cm Ϫ1 at 413 nm (37). Aliquots were stored at Ϫ80°C until use. Samples for NMR were created by exchanging labeled b 5 into NMR buffer (50 mM potassium phosphate, 50 mM NaCl, 10% D 2 O, pH 6.5) through dilution and centrifugation.

Generation of full-length human cytochrome b 5 for catalytic assays
A synthetic, codon-optimized DNA sequence encoding fulllength human cytochrome b 5 plus a C-terminal His 6 tag was generated (GenScript) and cloned into pET-15b vector using restriction enzymes NcoI and BamHI. Expression of full-length b 5 was similar to the soluble domain of b 5 , except 1) expression cultures were grown in Terrific broth, 2) induction was performed with 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside at an A 600 of 1.1, and 3) induced cultures were grown at 20°C with 250 rpm shaking for 44 h. Purification of full-length b 5 was initiated by resuspending cells in resuspension buffer (100 mM potassium phosphate, 20% glycerol, 1 mM EDTA, pH 7.4) supplemented with 1ϫ HALT protease inhibitor mixture (Thermo Fisher) and 1 mM PMSF. Cells were lysed and reconstituted with heme as described above for the soluble domain. Membrane fractions were isolated by ultracentrifugation at 140,000 ϫ g for 30 min, and b 5 was extracted using resuspension buffer with the addition of 0.2 M NaCl and 1% (w/v) CHAPS. Extracted membranes were pelleted by a second ultracentrifuge step at 140,000 ϫ g for 30 min, and the b 5 -containing supernatant was loaded onto a Ni-NTA column (Qiagen) and washed as described for the soluble domain of b 5 but with the addition of 0.5% (w/v) CHAPS. Eluted fractions with A 413 /A 280 Ͼ 2.0 were pooled, concentrated, and loaded on a Sephacryl S-200 HR column (GE Healthcare) equilibrated and run with gel filtration buffer (50 mM potassium phosphate, 20% glycerol, 100 mM NaCl, 0.05% (w/v) CHAPS, pH 7.4) and exchanged into storage buffer as described above. Protein purity was assessed on SDS-PAGE, and UV-visible spectroscopy was used to confirm degree of heme incorporation (A 413 /A 280 ϭ 3.0) and quantify dithionite-reduced b 5 (36).

Generation of full-length human NADPH-cytochrome P450 reductase for catalytic assays
A synthetic, codon-optimized gene for full-length human NADPH-cytochrome P450 reductase (Integrated DNA Technologies) preceded by the ompA signal peptide and followed by a His 6 tag was cloned into pET-29a(ϩ) using NdeI and HindIII. Expression of reductase was performed as described previously for a truncated CPR construct (38) with modifications. Modifications to the expression included 1) transformation and generation of the starter culture as described for soluble b 5 , 2) supplementation of expression cultures with 2 mg/liter riboflavin, and 3) shaking at 200 rpm after induction. Full-length CPR was also purified in a manner similar to a previously described method (38), with modifications: 1) membrane fractions after cell lysis were isolated by ultracentrifugation at 140,000 ϫ g for 25 min before detergent extraction, 2) omission of the ammonium sulfate precipitation step, and 3) the addition of a second nickel affinity column after elution from Octyl-Sepharose 4 Fast Flow (GE Healthcare) resin to deplete detergent. Final reductase samples were evaluated on SDS-PAGE and by UVvisible spectroscopy. CPR was quantitated by flavin absorbance of the fully oxidized protein at 454 nm with an extinction coefficient of 21.4 mM Ϫ1 cm Ϫ1 (39).

Generation of P450 enzymes for NMR and catalytic assays
All cytochrome P450 enzymes were the forms used previously to generate crystallographic structures and resulted in catalytically active protein omitting the N-terminal transmembrane helix and adding a C-terminal His tag. Constructs and expression and purification of human CYP2A6 (40) and CYP2E1 (41) were reported previously. Synthetic, codon-optimized genes encoding CYP2D6 and CYP3A4 were generated (GenScript) to match the reported constructs (42,43), cloned into the pCWori vector, and expressed and purified as described in these same publications with some modifications. Briefly, E. coli DH5␣ containing the pGro7 plasmid (Takara Bio) was used to express the P450 enzymes. Transformation and starter cultures were performed as described for soluble b 5 . Proteins were purified by isolating spheroplasts (44), lysis by

Cytochrome P450/b 5 interactions
French press, and detergent extraction using 4.8 mM CYMAL-5 (CYP2A6 and CYP2E1) or 14 mM CHAPS (CYP2D6 and CYP3A4). After extraction, lysate was clarified by ultracentrifugation at 104,000 ϫ g and purified through Ni-NTA, carboxymethyl cellulose ion-exchange, and size-exclusion chromatography. All proteins were depleted of detergent during ion-exchange chromatography, exchanged into storage buffer (50 mM potassium phosphate, 10% glycerol, pH 7.4), and frozen as aliquots at Ϫ80°C. P450 samples were evaluated by SDS-PAGE and UV-visible spectroscopy. The reduced-carbon monoxide difference spectra revealed, at most, only trace amounts of P420.

Mutagenesis
Single-amino acid mutants of cytochrome b 5 E49Q and D65N were produced by the QuikChange Lightning site-directed mutagenesis kit (Stratagene) for the full-length construct and commercially (GENEWIZ) for the soluble-domain construct. The complete genes were verified by sequencing, and protein was expressed and purified as described for the respective wild-type constructs.

NMR spectroscopy
NMR experiments were acquired at 298 K using a Bruker Avance III HD 800 MHz with a QCI cryogenic probe. Data were processed using NMRPipe (45) and visualized and analyzed using Sparky version 3 (T. D. Goddard and D. G. Kneller, University of California, San Francisco). Assignments for the backbone amide signals for human soluble domain of b 5 were transferred from the deposited chemical shifts in the Biological Magnetic Resonance Data Bank (accession number 6921) (16). All of the 2D 15 N HSQC experiments were obtained on 15 Nlabeled, soluble domain b 5 (0.1 mM) in NMR buffer (50 mM potassium phosphate, 50 mM NaCl, 10% D 2 O, pH 6.5) acquired with 64 scans and 128 increments, which took about 2.5 h. The signal for each individual resonance was measured as peak height, with the value at 0:1 P450:b 5 defined as 100% signal. For each titration point within a series, a new sample of labeled b 5 was prepared, and a defined molar amount of an unlabeled P450 enzyme was added to the sample. For samples including P450 enzyme, the peak height for each resonance was expressed as a percentage of the resonance peak height in the absence of the P450. Concentration of the P450 substrate was saturating and constant between samples (500 M coumarin for 2A6, 500 M CZN for 2E1, 2 mM DXM for 2D6, 5 mM pNP for 2A6, or 200 M NFP for 3A4). NMR experiments were performed with both b 5 and P450 in their oxidized states. At the conclusion of each NMR experiment, samples were collected from the NMR tube, centrifuged to determine whether any precipitation had occurred, and evaluated in terms of the reduced-carbon monoxide difference spectrum.

Cytochrome P450 catalytic assays
All catalytic assays were performed by incubating a P450 with full-length human P450 reductase and full-length human b 5 (when applicable) at a 1:2:0 ratio for minus b 5 reactions or a 1:2:2 ratio for b 5 containing reactions for 20 min at room temperature. The amounts of P450 enzyme were 10 pmol (couma-rin assay) or 50 pmol (pNP assay) of CYP2A6, 50 pmol of CYP2D6 (DXM assay), 50 pmol of CYP2E1 (CZN assay), and 50 pmol of CYP3A4 (NFP assay). This protein mixture was added to the same buffer used for NMR experiments, except for the nifedipine assay, which used an assay buffer consisting of 40 mM HEPES, 30 mM MgCl 2 , pH 7.4. The reactions also contained the respective P450 substrate (0 -128 M coumarin, 0 -600 M CZN, 0 -1600 M pNP, 0 -1000 M DXM, 0 -250 M nifedipine). For the nifedipine assay, all reactions were performed in amber vials. The reactions were preincubated at 37°C for 3 min and then initiated with 1 mM NADPH and allowed to proceed at 37°C for 10 min (coumarin, CZN, and pNP assays), 15 min (DXM assay), or 20 min (nifedipine assay). Reactions were terminated by the addition of diluted perchloric acid (acetonitrile for the nifedipine assay), placed on ice, and centrifuged at 5000 ϫ g for 5 min before injection onto a Luna C18 (5 m, 150 ϫ 4.60 mm; Phenomenex) column at a flow rate of 1 ml/min (0.75 ml/min for the nifedipine assay). Separation on HPLC was obtained using the following mobile phase solutions for each of the respective assays. For separation of coumarin and its 7-OH metabolite, the mobile phase consisted of 50%/ 50% 20 mM potassium phosphate, pH 2.8/methanol. For CZN and its 6-OH metabolite, the mobile phase consisted of 75%/ 25% 20 mM potassium phosphate, pH 2.8/acetonitrile to elute product and then a sharp gradient to 40%/60% to elute substrate, followed by reequilibration to 75%/25%. For pNP and its 4-nitrocatechol metabolite, the mobile phase consisted of 73%/ 27% 10 mM potassium phosphate, pH 3.5/acetonitrile. For DXM and its O-demethylated metabolite dextrorphan, the mobile phase consisted of 50%/50% 10 mM potassium phosphate, pH 3.5/50% acetonitrile and 100% methanol (250:200, v/v). For nifedipine and its metabolite dehydronifedipine, the mobile phase consisted of 45%/55% water/methanol. Metabolite detection occurred by fluorescence for coumarin (355-nm excitation, 460-nm emission) and DXM (280-nm excitation, 310-nm emission) assays and by UV absorbance for CZN (287 nm), pNP (345 nm), and NFP (270 nm) assays. The amounts of the different metabolites produced were calculated using authentic standards prepared by the same method as the samples. Each reaction at each substrate concentration was performed at least in duplicate. Data were fit to the Michaelis-Menten equation using GraphPad Prism.

Measurement of NADPH consumption
NADPH consumption during the various P450 reactions was measured by creating reaction samples similar to the catalytic assays except for the following: 1) 100 pmol P450 was used to increase signal/noise, 2) reactions were scaled up to 1 ml in the respective assay buffer containing the maximum substrate concentration used in turnover reactions, 3) NADPH concentration was reduced to 0.5 mM to reduce background, and 4) reactions were allowed to proceed for 10 min. CYP2A6(pNP) samples required the use of 2-mm path length cuvettes due to high substrate absorbance, so these reactions used 50 pmol of P450 and a final volume of 500 l. Rates of NADPH consumption were measured by monitoring linear decreases in absorbance at 340 nm. The amount of NADPH consumed was calculated using an extinction coefficient of 6.22 mM Ϫ1 cm Ϫ1 .