*4.5. Membrane Preparation and Characterization*

Membrane preparations were isolated as previously described [22,24]. Briefly, cultures were harvested at 2772× *g* for 20 min at 4 ◦C. The pellet was resuspended in Tris-sucrose buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.8). Lysozyme was added to a final concentration of 0.5 mg/mL, EDTA (0.5 mM), phenylmethanesulfonyl fluoride (0.5 mM) and benzonase (12.5 U/mL). Cells were incubated on a roller bench for 30 min at 4 ◦C. Cell lyses was performed by freezing (−80 ◦C) and thawing (1 cycle) and by subsequent several short rounds (30 s) of low-intensity sonication, interspersed with 60 s of ice-bath submersion. The suspension was centrifuged at 2772× *g*, for 20 min at 4 ◦C to eliminate unbroken cells. Membranes were pelleted by ultracentrifugation of the supernatant at 100,000× g at 4 ◦C for 60 min. Membranes were resuspended in TGE buffer (75 mM Tris-HCl, 10% (*v*/*v*) glycerol, 25 mM EDTA, pH 7.5) using a Potter homogenizer and stored at −80 ◦C. Protein concentrations were determined using the method described by Bradford, following the manufacturer's protocol from Bio-Rad (San Francisco, CA, USA), using bovine serum albumin as the standard. CYP contents of membrane preparations were determined using CO-difference spectrophotometry. Membrane proteins were separated by SDS–PAGE gel electrophoresis (10% polyacrylamide gel) and either stained with Coomassie blue or electro-transferred to PVDF membranes and further processed. CPR content of membrane fractions was quantified by immunodetection against a standard curve of purified human, full-length WT CPR, using polyclonal rabbit anti-CPR primary antibody and biotin-goat anti-rabbit antibody in combination with the fluorescent streptavidin conjugate (WesternDot 625 Western Blot Kit; Invitrogen, Eugene, OR, USA) (see Figure S1). Densitometry of CPR signals was performed using LabWorks 4.6 software (UVP, Cambridge, UK).

#### *4.6. CYP-Enzyme Assays*

Using membrane preparations, CYP-activities were assessed through determination of product formation by EthR O-deethylation (EROD; CYP1A2) (excitation 530 nm; emission 580 nm), coumarin 7-hydroxylation (CYP2A6) (excitation 330 nm; emission 460 nm) or *O*-debenzylation of DBF (CYP3A4) (excitation 485 nm; emission 535 nm) [20,22,26]. Assays were performed in 96-well format with a multi-mode microtiter plate reader (SpectraMax®i3x, Molecular Devices, USA) using SoftMax Pro 2.0 Software. Experiments were conducted with 8 nM CYP1A2, 100 nM CYP2A6, and 25 nM CYP3A4 (final well concentrations). Reactions were performed in 100 mM potassium phosphate buffer (without NaCl) (pH 7.6) supplemented with 3 mM MgCl2 and an NADPH regenerating system (NADPH 200 μM, glucose 6-phosphate 500 <sup>μ</sup>M and glucose 6-phosphate dehydrogenase 40 U·L<sup>−</sup>1, final concentrations). Stock solutions of EthR were prepared in DMSO, while coumarin and DBF were prepared in acetonitrile (ACN). Final solvents concentrations were maintained constant throughout the experiment (0.2% (*v*/*v*) DMSO or 0.1% (*v*/*v*) ACN). Product formation was followed for 10 min at 37 ◦C and rates were calculated by using a standard curve of the products. Reactions were performed in triplicate with substrate concentrations ranging up to 5 μM EthR (CYP1A2), 20 μM coumarin (CYP2A6) or 10 μM DBF (CYP3A4). Velocity data were plotted according to the Michaelis–Menten equation with high confidence (*r*<sup>2</sup> > 0.95) using GraphPad Prism 5.01 Software (La Jolla, CA, USA) and kinetic parameters (*k*cat and *K*M) were derived [20,22,47]. Variance in data was analyzed using one-way ANOVA with Bonferroni's multiple comparison tests, with 95% confidence interval—GraphPad Prism 5.01 Software (La Jolla, CA, USA).

#### *4.7. Ionic Strength Effect*

Catalytic activity of CYP1A2, 2A6, and 3A4, sustained by WT CPR and CPR mutants, was assessed at various NaCl concentrations (0–1.25 M), using 5 μM EthR, 20 μM coumarin and 10 μM DBF, respectively, in 100 mM potassium phosphate buffer (pH 7.6), and NADPH regenerating system (NADPH 200 μM, glucose 6-phosphate 500 μM and glucose 6-phosphate dehydrogenase 40 U L−1, final concentrations). Velocities were measured in triplicate in 96-well format using multi-mode microtiter plate reader (SpectraMax®i3x, Molecular Devices, San José, CA, USA; SoftMax Pro 2.0). Initial rates (picomoles of fluorescent product formed per picomoles of CYP per minute) were derived from the linear part of the kinetic traces using a standard curve of the respective products. Control experiments were conducted to assess the effect of ionic strength on the pH of the reaction matrix and the fluorescence of products.
