2.2.1. Whole-Cell Bioactivation Assays

A whole cell/bioactivation assay was used for the first evaluation of the effect of the three hinge mutations on the activity of the three CYPs. This approach made use of the applicability of the BTC-CYP bacteria in mutagenicity testing [26,28]. The levels in CYP-dependent bioactivation of different pre-carcinogens, namely 2AA (2-aminoanthracene), IQ (2-amino-3-methylimidazo(4,5-*f*)quinolone), NNdEA (*N*-nitrosodiethylamine), NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and AfB1 (aflatoxin B1)) were determined (Table 2; Figure 1). Interestingly, two of the three CPR hinge mutants lead to bioactivation capacities, which were either stimulated or equal, in comparison when CYPs were sustained by WT CPR, except for mutant I245P. This hinge mutant demonstrated a decrease for CYP1A2 and CYP3A4 mediated bioactivation of 2AA and AfB1, respectively. In contrast, CYP1A2 and CYP2A6 bioactivation capacities (for IQ and NNdEA, respectively) were increased when assayed with this mutant, with no significant differences in CYP2A6 bioactivation of NNK. Seemingly, the effect of CPR mutant I245P was CYP isoform and substrate dependent. The bioactivation capacity of all three CYPs was consistently augmented when assayed with CPR mutant R246A, i.e., all tested compounds demonstrated increased mutagenicity levels, in comparison with CYPs sustained by WT CPR. Mutant S243P demonstrated no significant differences in the bioactivation capacity of the three CYPs for the tested compounds, when compared with WT CPR.


**Table 2.** CYP-mediated bioactivation of pre-carcinogens.

Values are mean ± sd of three independent experiments, expressed as the number of revertant colonies per nmol <sup>1</sup> or per μmol <sup>2</sup> of pre-carcinogen.

**Figure 1.** Bioactivation of pre-carcinogens mediated by CYP 1A2, 2A6 or 3A4 when combined with the three CPR hinge mutants. Bioactivation capacities were normalized with the one observed when CYPs were combined with WT CPR. (2AA: 2-aminoanthracene; IQ: 2-amino-3-methylimidazo(4,5-*f*)quinoline; NNdEA: *N*-nitrosodiethylamine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; AfB1: aflatoxin B1).

#### 2.2.2. Membrane Preparations

#### CYP-Enzyme Kinetic Analysis

Enzyme activities of CYP1A2, 2A6 and 3A4 were measured using specific fluorogenic probe substrates ethoxyresorufin (EthR), coumarin and dibenzylfluorescein (DBF), respectively). Reaction velocities could be plotted according to the Michaelis-Menten equation and kinetic parameters (*k*cat and *K*M) could be derived (Table 3). In general, CYP activities promoted by CPR mutants showed lower turn-over rates (*k*cat) and affinities (*K*M) when compared with WT that were also CYP form dependent. However, differences were minor in both constants and *k*cat/*K*<sup>M</sup> values were not significantly different from WT values for all tested CYPs (Table 3).


**Table 3.** Michaelis-Menten kinetic parameters of CYP activities.

<sup>1</sup> *<sup>k</sup>*cat and *<sup>K</sup>*<sup>M</sup> values are expressed as mean values of three independent experiments ± sd, determined at 0 M NaCl.

#### Ionic Strength Effect on CYP:CPR Interaction

We previously demonstrated that mutations in the hinge region of human CPR strongly influences ionic strength profiles of ET to cytochrome *c* [24]. Ionic strength dependency of CPR's ET was thus analyzed via CYP activities measurement in presence of increasing NaCl concentrations (0–1.25 M) (Figures 2 and 3). Control experiments showed no effect of the salt concentration on the fluorescence of the formed products (Figure S2) or on the pH of the reaction mixture (data not shown). Interestingly, the maximum velocities of the three CYPs seem to occur at lower ionic strength conditions for all CPR (WT or variants) when compared to their maximum velocities in cytochrome *c* reduction reported in our previous study [24].

The salt dependence of CYP1A2 activity demonstrated a bell-shaped EthR O-deethylase activity curve with all CPR tested (Figure 2A), analogous to the ones described in our former study when measuring cytochrome *c* reduction [24]. The maximum activity (maximum *kobs*) was obtained, on average, at 100 mM NaCl. CYP1A2 activities dropped close to zero at the highest salt concentrations (1.25 M) for all CPR variants. CPR mutant S243P was more active than the WT, while other mutants showed lower activities when compared with WT.

For CYP2A6 (Figure 2B), the coumarin hydroxylase activity profiles showed less dependence on ionic strength as with CYP1A2 and showed no drop in activity at the highest salt concentrations. Maximum velocities were obtained approximately at 150 mM NaCl. All CPR mutants presented lower activities when compared with WT CPR.

CYP3A4 DBF O-debenzylase activity profiles (Figure 2C) were also bell-shaped, like CYP1A2, except for the CPR mutant R246A. The maximum activity was obtained, on average, at 100 mM NaCl. The activity dropped close to zero at the highest salt concentrations as was observed with CYP1A2. Interestingly, the CPR mutant R246A revealed a different salt profile with CYP3A4, leading to a continuous decrease of activity with increasing salt concentration, the maximal activity being even greater that the one obtained with WT CPR.

**Figure 2.** CYP reaction velocity (*k*obs) in function of the NaCl concentration, for the WT and mutant forms of CPR with CYP1A2 (**A**), CYP2A6 (**B**) or CYP3A4 (**C**).

**Figure 3.** Relative CYP-reaction velocities (*k*obs) of CYP1A2, 2A6 and 3A4 in function of the NaCl concentration, when combined with CPR WT (**A**) or mutants S234P (**B**), I245P (**C**) and R246A (**D**). Velocities were normalized with the one observed when combined with WT CPR at 0 M NaCl.

#### CYB5 Effect on Activity of CYP1A2, 2A6 and 3A4

CYB5 may act as an alternative donor of the second electron in the CYP catalytic cycle. We thus studied the effect of CPR hinge mutations when associated with CYB5, to determine if the presence of an alternative electron donor partner, capable of competing with CPR for CYPs is an important issue in the function of the hinge segment. The effects of CYB5 on CPR/CYP combinations were analyzed through enzyme activity assays in the presence of increasing concentrations of CYB5 and thus different CYB5:CPR ratios (Figure 4 and Table 4). Enzyme activities were normalized to the activity measured in the absence of CYB5. While the effect of CYB5 on CYP activities was not major, some interesting differences could be seen, notably in the CYB5 concentration giving the best stimulus. While for CYP1A2, the maximal effect was observed at 150 nM of CYB5, for CYP2A6 the concentration of CYB5 giving this maximal effect was dependent on the mutation, ranging from 50 nM with the WT CPR to 400 nM for the R246A mutant of CPR. For CYP3A4, the effect was relatively constant between all CPR mutants, but the concentration of CYB5 needed to obtain the maximal stimulation was much higher (400 nM). Overall, while CYP1A2 does not seem very sensitive to the presence of CYB5, a slight inhibition could be observed at high concentrations of CYB5. For the two other CYPs, the stimulation was quite pronounced, ranging from 2.6 to 3.4 or 1.8 to 2.6 for CYP2A6 and CYP3A4, respectively (Figure 4). No major differences were observed between CPR WT and mutants for each CPR/CYP combinations in term of the concentration of CYB5 to achieve the maximal effect, however, the intensity of the stimuli was different between CPR mutants.

**Figure 4.** Effect of CYB5 concentration on maximum reaction velocities of CYP1A2 (**A**), 2A6 (**B**) and 3A4 (**C**) when sustained by WT CPR and the three hinge mutants. Activities were normalized to the one without CYB5 for each CPR mutant in comparison to WT.


<sup>1</sup> Normalized maximum enzyme activity: (*k*obs)/(*k*obs at 0 nM CYB5). Values are expressed as the mean of three independent experiments ± sd. <sup>2</sup> Maximum CYB5 stimulus of enzyme activity when CYPs were combined with mutant CPRs, relative to the ones obtained with CPR WT. <sup>3</sup> The ratio at which maximum *k*obs was reached. Grey shaded values: Maximum fold stimulation of enzyme activity by CYB5 when CYPs were combined with WT CPR.

#### **3. Discussion**

Microsomal CYP-mediated metabolism is dependent on ET through protein:protein interaction with its primary redox partner CPR. Traditionally, membrane anchoring and a negatively-charged surface patch of CPR have been considered to be major determinants of the proper alignment in this interaction, in which hydrophobic interactions have also been implicated [32,33]. Still, the view on CPR:CYP interaction for ET has become increasingly more complex with the recognition of CPR protein dynamics in ET, presenting an equilibrium between closed/locked and open/unlocked conformers in its function, in which the flexible hinge region seems to have a determinant role [10,16–18]. In a recent report we have shown the importance of this hinge segment and the effect of ionic strength in ET to the non-physiological redox partner cytochrome *c*, using eight different CPR mutants targeting four critical residues [24]. Data from this report lead us to hypothesize a hydrogen H-bond network around R246, important for the function of the open/closed dynamics.

Although the use of the soluble surrogate redox partner cytochrome *c* has been informative, the open/closed dynamics and thus ET may occur differently with CPR's natural membrane-bound electron acceptors, such as CYPs. Three hinge mutants were selected from our former set, namely mutant R246A, for R246's role in the suggested H-bond network, and mutants S243P and I245P demonstrating augmented cytochrome *c* reduction capabilities when compared with WT CPR. The CPR variants were combined with three different CYPs, representatives of three major CYP-families involved in drug metabolism. CYP families 1, 2 and 3 are responsible for 75% of all phase I metabolism of clinically used drugs: CYP3A4 is the major enzyme, and together these three CYPs are involved in almost 50% of metabolism of drugs [34]. Different experimental approaches were used to probe the effect of these hinge mutations.

The first general observation is that none of the three mutations caused a complete inactivation of CYP activity (i.e., obliterated CPR electron transfer to CYP), consistent with the data obtained using cytochrome *c* as electron acceptor [24]. We thus confirm that the three targeted positions in the hinge region are certainly part of a set of residues capable of controlling the CPR function and thus the effects of the mutations on internal and external ET are partially compensated by a larger network involved in the structural transitions (opening/closing).

The second interesting feature that these mutants demonstrated was their relative selectivity in inducing CYP-isoform dependent effects. This was first observed with CYP mediated bioactivation of pre-carcinogens, which seemed CYP isoform and in some cases substrate dependent. Still, the measurement of mutagenicity might be a quite indirect measurement of CYP activity, in generating

DNA damaging metabolites. However, the specific features of CPR hinge mutants were more clearly demonstrated by the ionic strength dependency of CYP activities. Salt activity profiles were deviated differently by the three CPR hinge mutations, in a CYP-isoform dependent manner (Figures 2 and 3). We previously demonstrated that the salt concentration at which the overall CPR to acceptor ET occurs depends on the relative difference in the rate of reduction of CPR and the rate of reduction of the electron acceptor [17]. The fact that all hinge mutations affect the salt concentration at which this maximal activity occur indicate that these CPR mutations modify the conformational equilibria. However, as noted above, all mutations have distinct salt-dependent signatures (Figure 3). We can thus hypothesize that the induced differences in conformational equilibria affect differently the three tested CYPs. Interestingly, all *V*max and *K*<sup>M</sup> values measured for the three CYPs were nearly identical between CPR mutants. This reinforces our current hypotheses addressing the conformational equilibria: The single hinge CPR mutants modify the salt-dependent conformational equilibria and not the intrinsic CYP properties. As such, they uncover the dependence of CYPs toward these conformational equilibria and provide a potential explanation of the mechanism by which CPR could, in certain conditions, favor one CYP over another.

An additional interesting feature of these salt profiles concerns the optimal salt concentration for CYP activity. Previously, when membrane-bound, WT CPR and the three mutants demonstrated very different salt concentrations for optimal cytochrome *c* reduction (ranging from 220–550 mM NaCl [24]). However, in the current study when combined with CYP1A2, 2A6 or 3A4, optimal activities were found at 100–200 mM NaCl, which together with the K/P reaction buffer approaches the ionic strength of physiological serum (154 mM KCl). This current data set exemplifies the difference in ET between the soluble surrogate electron acceptor cytochrome *c* and CPR's natural membrane-bound redox partners. Maximum ET rates of soluble WT CPR in cytochrome *c* reduction was found to occur when CPR was equally distributed between open and closed conformers at approximately 375 mM NaCl [17]. With membrane bound WT CPR, maximum cytochrome *c* reduction rates were shifted above 500 mM NaCl, namely at 527 mM [24]. As such, data from our previous and current study suggests that optimal ET to various acceptors, either natural or artificial, occurs at very different ionic strengths. However, under physiological conditions, membrane bound CPR is present in an equilibrium, which is mostly in the locked state, however maintaining a very fast rate in alternating between open and closed conformations. This reinforce the idea that additional factors, such as the presence of membrane bound redox-partners, with stoichiometry's favoring the electron acceptors (as is the case for CYPs, outnumbering CPR by a factor of 5–10), modulate the open/closed dynamics, as we put forward in our previous study [24].

As mentioned above, our data showed differences in the salt effect of the three different CYPs when combined with WT CPR, corroborating the study by Yun et al. [35] that ascribed the ionic strength effect observed mostly to CPR:CYP "interaction" while having relative minor effects on CYP protein conformation [36]. Still, results of the study of Voznesensky and Schenkman [37] indicated that charge pairing between CPR and CYP may not be the major determinant of the salt effect. Our current data" as well as that of our previous study [24] confirms that the CPR:CYP interaction (i.e., electronic flow) dependency on ionic strength is mainly determined by its effect on the conformational equilibrium between locked and unlocked states of CPR and only in a minor manner by electrostatic interactions (affinity) between the FMN domain and the acceptor. Due to this major salt effect on CPR's open/closed dynamics, the identification of potential electrostatic interactions between CPR and CYP have therefore been obscured. In retrospect, the seminal studies of Voznesensky and Schenkman [37,38] and of others (reviewed in [32]) on the salt effect of CYP catalysis in the quest for electrostatic interactions between CPR and CYP have been hampered by the lack of knowledge, at that time, on the salt-dependent protein dynamics of CPR.

In vivo, under constant ionic strengths conditions, affinity parameters may become determining in the CPR:CYP interaction, modulating the open/closed dynamics as indicated above. CYB5, the optional electron donor, demonstrated also stimulation/inhibition profiles that were dependent on

CYPs, as well as on the various CPR used. This may also sign a relatively minor role for the affinity between CPR/CYP and CYB5/CYP interactions. Although CPR and CYB5 share similar, but not identical, binding-sites on the proximal side of CYP [39], the competition between the two electron donors does not seem to be a major factor in controlling CYP activities. Still, subtle differences in electrostatic interactions between the CPR/CYP and CYB5/CYP complexes have been shown [40,41], which seem even to be depending on substrate binding [42], indicating specific features in the sampling of the ensemble of open conformers of CPR.

Overall, our data point out that salt profiles are specific to CYP isoforms dependent interaction with CPR. The idea emerges for the existence of an ensemble of different unlocked CPR conformers that may be required for CYP-specific interactions. The highly flexible hinge region allows for a large ensemble of open conformations from which only a few or a subset may be required for ET to CYPs. CPR hinge mutants, by modifying the conformational equilibrium (as seen in salt profiles) may either promote or hinder specific conformations of the unlocked state, thus allowing or preventing interactions with (structurally) different redox partners. Such a selection of specific open conformations could explain the differential effect of the three hinge mutations on the activity of the different CYPs. Moreover, the soluble cytochrome *c* will sample these conformers quite differently when compared with the membrane-bound CYP redox partner, a plausible explanation for the difference in effects of the three hinge mutants when measuring ET to cytochrome *c* [24] or CYPs (this study).

From a structural perspective, it is clear that different CYP isoforms must share a common functional CPR binding surface. Although mitochondrial CYPs seem to have a signature of key basic amino acids on the proximal side for their interaction with the iron–sulfur protein adrenodoxin, such signature sequences do not exist for microsomal CYPs in their interaction with CPR [43]. This implies diversity in critical amino acids on their proximal side and suggests the possibility of affinity differences of microsomal CYPs for CPR, a plausible key element in the sampling of the open CPR conformers.

The conformational plasticity of CYPs could additionally play a role in this respect. Substrate binding has been shown to cause (subtle) conformational changes at the proximal site of CYPs, reviewed in Kandel and Lampe, 2014 [33], which may influence the affinity and thus sampling of open conformers of CPR for effective ET. In fact, this seems to be corroborated by our data of the hinge mutations, causing beside CYP-isoform dependent seemingly also substrate dependent effects, as described above.

It is tempting to speculate that CPR's protein dynamics, containing different ensembles of closed and open conformations, was Nature's way to enable CPR to be the "degenerated" electron supplier of so many (structural and functional) different redox-partners. In this respect it would be of interest to obtain insight on the evolutionary gain and thus the physiological relevance of such a universal electron donor for enzymes, involved in some many different and crucial metabolic pathways. In parallel to other central enzymes, the possibility may exist of a central hub- or central controller-function of CPR, for the fine tuning of multiple metabolic pathways and energy (NADPH) usage.

#### **4. Materials and Methods**

#### *4.1. Reagents*

L-Arginine, thiamine, chloramphenicol, ampicillin, kanamycin sulfate, isopropyl β-D-thiogalactoside (IPTG) (dioxane-free), δ-aminolevulinic acid, cytochrome *c* (horse heart), glucose 6-phosphate, glucose 6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH), 2-aminoanthracene (2AA), *N*-nitrosodiethylamine (NNdEA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), aflatoxin B1 (AfB1), resorufin, 7-hydroxy coumarine and fluorescein were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) was obtained from Toronto Research Chemicals (North York, ON, Canada). LB Broth (Formedium, Norfolk, UK), bacto tryptone and bacto peptone

were purchased from BD Biosciences (San Jose, CA, USA). Bacto yeast extract was obtained from Formedium (Norwich, England). EthR and coumarin were obtained from BD Biosciences (San Jose, CA, USA) and DBF from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A polyclonal antibody from rabbit serum raised against recombinant human CPR obtained from Genetex (GTX101099) (Irvine, CA, USA) was used for immunodetection of the membrane-bound CPR.
