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The di-flavoenzyme reductase directly activates oxygen for the metabolism of diverse drug molecules by liver microsomal Cytochrome P450s

Kelath Murali Manoj*

October19, 2006. Department of Chemistry, Washington State University, Pullman, WA-99163, USA.

& Department of Biochemistry & Molecular Biology, University of Arkansas for Medical

Sciences, Little Rock, AR-72205, USA.

Running Title: Reductase directly activates oxygen for drug metabolism by P450s

Address correspondence to- Kelath Murali Manoj, Department of Biochemistry & Molecular Biology, Biomedical Research Building I, 4301 W. Markham, Slot 516, Little Rock, Arkansas-72205, USA. Phone: +501-588-4458, Fax: +501-686-8169, Email: mkmanoj@uams.edu

Human liver microsomal cytochrome P450s (hlmCYPs) metabolize a wide variety of endogenous and xenobiotic molecules. It is argued herein that the currently accepted “heme activates oxygen” (HAO) hypothesis explaining the overall catalytic phenomenon of these versatile enzymes poses severe inconsistencies. Experimental evidence is provided here that the diflavo-enzyme cytochrome P450 reductase (CPR) catalyzes the activation of molecular oxygen to generate diffusible reduced oxygen species (DROS). It is also shown that the dynamics of DROS generation in the hlmCYP + CPR system is not linked obligatorily to the presence of substrate. These observations support the existence of a facile pathway in which the DROS generated by CPR reacts with hlmCYP to form the catalytic intermediate(s), which in turn transfers one oxygen atom to suitable substrates. This hypothesis is further supported by the following arguments- (a) catalytic amounts of DROS utilizing proteins critically affect the reaction chemistry where as moderate excesses of several redox cyclers do not, (b) the hydroxylation reaction is observed even in the absence of hlmCYP+CPR complexation and (c) the hydroxylation reaction can be efficiently simulated by hlmCYPs in conjunction with DROS alone. The loss of redox equivalents from the reduced nicotinamide nucleotides is accounted for by

the newly discovered regulatory role of CPR. The new “flavin activates oxygen” (FAO) pathway thus addresses several controversies and explains the evolutionary significance of CPR’s features, distribution of relevant proteins in situ, non-specificity of reductions by CPR, the variations of DROS in the reaction mixture, the role of cytochrome b5 and the substrate diversities of many hlmCYPs. Most importantly, this study reveals that DROS, hitherto considered an undesired manifestation of pathogenic states, is in fact an essential aspect of routine cellular oxidative machinery.

Cytochrome P450s (CYPs) are a family of ubiquitous heme-thiolate proteins exhibiting a characteristic 450 nm spectral signature, a feature which affords their traditional name and routine identification protocol1-3. When present along with cytochrome P450 reductase (CPR) and reduced nicotinamide adenine dinucleotides (HNADP & HNAD, standing for the 2’ phosphorylated and hydroxylated forms respectively), CYPs catalyze the oxidations of a wide variety of substrates4-5. Besides mediating the conversions of endogenous and xenobiotic molecules in all life forms, CYPs also metabolize the majority of drugs consumed by humans6-7.

The currently accepted “heme activates oxygen” (HAO) hypothesis explaining the activity of human liver microsomal (hlm) CYPs involves eight sequential steps, of which seven are

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chronologically ordered8-13 (Scheme A, supplementary material). The first step of HAO hypothesis seeks that hlmCYP binds to its substrate at a locus distinct from the heme center. This binary complex formation is necessary to afford a spin change of the heme iron, concomitant with an increase in redox potential of heme center by >100 mV. Such an enzyme-substrate binary complex further binds to molecular oxygen at the heme center of the CYP, giving a trimolecular complex. Subsequently, this trimolecular complex binds to reduced CPR and the latter transfers electrons to the heme center via a protein-protein interaction within the tetramolecular complex. In this HAO pathway, the generation of diffusible reduced oxygen species (DROS) in the reaction system is attributed to the “uncoupling” of the reduction process mediated at the heme center. The loss of redox equivalents from reduced nicotinamide adenine dinucleotide is hypothesized to be due to the “un-provable” production of water at the heme center.

Though an overwhelming volume of literature advocates the HAO hypothesis, I argue that the mechanistic scheme proposed therein seeks improbable requisites and presents fundamental paradoxes. Here, the dynamic production of DROS by CPR is proposed to be a more facile pathway to generate the catalytic intermediate of hlmCYPs, which finally catalyzes the typical hydroxylation reactions of these versatile oxidative enzymes. The new pathway is unordered and all steps are bimolecular. Therefore, it is more appealing to molecular dynamics and probability considerations. The existence of subtle single-electron redox equilibrium is revealed in this reaction system. It is also brought to the fore that some components of the new pathway were actually investigated in the early 1970s and subsequently sidelined without due considerations in the three decades of research that followed. Overall, a holistic attempt has been made to understand the complex chemistry involved.

Experimental Procedures

Materials: Pure human CYP2C9 and CPR were prepared using the cDNA available in Dr. Jeff Jones’ lab14. The SDS-PAGE of lab-made CPR preparations used in this study is shown in Figure A, supplementary material. CYP3A4 (P2377),

CYP2D6 (P2283) and CYP2C9 (P2378) baculosomes were purchased from Invitrogen (PanVera) and a CYP2C9 preparation was gifted from Merck (#011902). Other proteins and chemicals were from Sigma and Lancaster companies. Incubations: To minimize the consumption of HNADP in routine experimentations, stocks prepared on one day were used after a day or two. Therefore, the reactions may have up to 20% excess NADP+, in conjunction with the actual initial value of HNADP quoted. All incubations were done with aeration in open vials at 37±1 °C in 100mM phosphate buffer, pH 7.4. Unless otherwise mentioned, reconstituted systems had 10 µg/ml of 0.2 µm vesicles of dilauryl phosphatidylcholine (DLPC, Avanti Lipids). Monitoring reaction progress: Depletion of HNADP was traced by monitoring absorbance at 340 nm, using an extinction coefficient of 6220 M-

1cm-1. Absolute concentration of a commercial peroxide stock was determined by titanium complex formation15, which is more accurate than the UV spectrophotometric estimation procedure at ~250nm. The standardized peroxide stock was then used for preparing the standard plot of micromolar level peroxide and for estimation of unknown concentrations by the Peroxoquant method of Pierce chemicals. In CYP2C9 reactions with diclofenac as the substrate, termination of reaction for product quantification was achieved by adding 200 µl of a cold solvent mixture (94% acetonitrile + 6% acetic acid, containing tert-butyl phenol as the internal standard) to 500 µl of sample. Conversion of substrate (diclofenac) was determined using Alltima C18 5u HPLC column (150 mm x 3.2 mm) from Alltech. The solvent system of 70% (of 30% acetonitrile in water with 1 mM perchloric acid) and 30% methanol was pumped at 0.8 ml/min. Chromatography was monitored with a diode array detector at 267 and 275 nm. Elution times and areas of standard samples of diclofenac and its’ 3’, 4’ & 5’ hydroxylated derivatives were used for detecting and quantifying the products. Under the conditions employed, 4’hydroxydiclofenac eluted and diclofenac eluted at ~6 and 7.5 minutes respectively. Another HPLC method (optimized by Dr. Grover P. Miller at UAMS) using a

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Symmetry C18 3.5µm column from Waters was also employed to estimate p-nitrocatechol (pNC), the hydroxylated product of p-nitrophenol (pNP) oxidation by CYP2E. A mixture of 75% of 0.1% trifluoroacetic acid in water and 25% acetonitrile was pumped at 1.5ml/min. pNC, 2-nitro resorcinol (internal standard taken at 20 µM) and pNP eluted at 2.4, 3.3 and 4.4 minutes respectively, as detected by UV absorbance detector at 320 nm. For the quantification of 7-hydroxy trifluoromethylcoumarin (7-HFC), the de-ethylated product of CYP1A2 oxidation of 7-ethoxy trifluoromethylcoumarin (7-EFC), the same Symmetry column was employed with a binary solvent system- solvent A = 75% of 0.1% trifluoroacetic acid in water & solvent B = acetonitrile, using a gradient method. The details are- 0 min = 1 ml/min, 75%A+25%B; 4 min = 1.5 ml/min, 75%A+25%B; 9 min = 1.5 ml/min, 0%A+100%B; 11 min = 1.5 ml/min, 75%A+25%B &12 min = 1ml/min, 75%A+25%B. A fluorescence detector with excitation and emission wavelengths set at 385 nm and 500 nm respectively was used to quantify the reactants and products. The analytes of 7-hydroxycoumarin (internal standard at 20 µM), 7-HFC and 7-EFC eluted at 2.6, 8.2 and 9.6 minutes respectively Specific details:

Tracing peroxide in milieu for different CYP baculosome preparations: Initial concentrations of reactants were- [HNADP] = 2 mM & [CYPs] = 20 nM. Substrates, when present, were at 200 µM. The Panvera CYP2C9 reaction with diclofenac afforded a conversion of ~20/min, which was in agreement with Invitrogen’s certificate of analysis value of ~23/min for this preparation. The Merck CYP2C9 preparation gave a diclofenac hydroxylation rate of ≥40/min. Invitrogen guarantees a conversion rate of 226/min for CYP3A4 under identical reaction conditions for testosterone.

Tracing HNADP depletion and peroxide production with different substrates in a CYP2C9 reconstituted system: CYP2C9 and CPR were at 140 nM each, the final concentration of flurbiprofen and diclofenac were at 160 µM and pentafluorobenzoic acid was at ~1.6 mM, at ≤1% CH3CN. The reaction was initiated with ~180 µM HNADP.

Activation of oxygen by CPR in reconstituted systems: Initial concentration of components were- [CYP2C9] = 100 nM, [CPR] = 25 or 100 or 400 nM, [diclofenac] = 200 µM, [HNADP] = 210 µM. A control of 100 nM CYP with 210 µM HNADP gave no observable peroxide at 15min and depleted HNADP at the rate of ~0.2 µM/min, which is only slightly higher than the auto-degradation rate of HNADP under these conditions. Depletion rate of HNADP was determined from the slope of linear fit of 340nm OD at 16, 26 and 36 minutes. For the HNAD reaction, the initial concentrations were- [HNAD] = 135 µM. [CPR] = 25 or 100 or 400 nM. [CYP2C9] = 100 nM, [diclofenac] = 100 µM. The Merck baculosome reaction had CYP2C9 at 10 nM.

Probing the effect of redox sensitive small molecules on the CYP+CPR chemistry: Initial concentrations: 200 µM diclofenac, 1 mM HNADP and redox sensitive molecules were at 2 µM. Reconstituted system and Merck baculosome preparation reactions were carried out at 200 nM (CPR was ~400 nM) and 10 nM respectively for CYP2C9.

Probing the effect of DROS utilizing proteins on the CYP+CPR chemistry: Initial concentrations were- [HNADP] = 160 µM, [CYP2C9] ~50 nM, [CPR] ~150 nM, [diclofenac] = 200 µM. The extraneous proteins (from Sigma) were added at 0.1-0.5 mg/ml of reaction. HNADP consumption was determined from the slope of linear fit of 340nm OD at 0.25, 15, 30, 45 & 60 minutes (except for the HRP inclusive reaction, for which only the first three points were taken because all of the HNADP was practically depleted within thirty minutes). Hydroxydiclofenac production was determined at 15 and 30 minutes by termination of a suitable aliquot dispensed from the reaction mixtures.

Effect of chloroperoxidase on hydroxylation of diclofenac by CYP2C9 at different pH. Initial concentrations were- [HNADP] = 125 µM, [diclofenac] = 100 µM, Invitrogen CYP2C9 baculosomes at 5 nM & CPO was at ~ 30 nM.

Demonstration of diclofenac hydroxylation in the absence of CYP-CPR complexation: Stock solutions of 8 µM pure CYP2C9, 3 µM pure CPR, 200 mM diclofenac sodium, 100 mM HNADP & 20% v/v glycerol

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were made in 100 mM potassium phosphate buffer, pH 7.4. The test reaction was conducted in a 2+4 ml mixture. The 2ml taken in the dialysis tubing (Spectra/Por, 12-14 KD cutoff) had 150 µl of pure CYP2C9, 200 µl of diclofenac, 40 µl of HNADP, 300 µl of glycerol-buffer and 1310 µl of buffer. The 4 ml taken in free solution had 800 µl of pure CPR, 600 µl of diclofenac, 120 µl of HNADP, 900 µl of glycerol buffer and 3580 µl of buffer. The positive control (1ml) reaction had 18.8 µl of CYP2C9, 100 µl of CPR, 20 µl of HNADP, 150 µl of glycerol buffer and 611.3 µl of potassium phosphate buffer. The reactions were gently stirred with a magnetic paddle and incubations were done for 45 minutes at 37°C.

CYP2C9 mediated oxidation of the specific 4’hydroxylated product of the original diclofenac substrate: From a reaction mixture having an initial concentration of 150 nM CYP2C9, 300 nM CPR, 2 mM HNADP & 200 µM diclofenac, samples were drawn at 45 minutes and 4 hours of incubation at 37°C. Apparently, the 4’hydroxylated product was converted to more polar products (perhaps di- or poly- hydroxylated) as a result of which peaks were observed at earlier elution times (5.2 and 5.8 minutes). Another set of reactions were done to confirm that the more polar product eluting before the 4’hydroxylated product was indeed owing to the 4’hydroxylated product’s reaction with CYP2C9. The reaction incubations for 30 minutes at 37°C had 6.8 nM Invitrogen CYP2C9 and 1.25 mM HNADP.

Simulation of CYP-CPR activity with CYP-DROS for hlmCYPs: Initial concentrations of the reaction components were- [diclofenac or pNP] = 25µM, [DLPC] = 20 µM, [CYP2C9 or CYP2E1] = 100 nM. When present, the following components were initially at - [HNADP] = 200 µM, [CPR] = 200nM, [Cytb5] = 200nM, [H2O2] = 32 µM, [KO2] ≤16 µM. Superoxide stock for this reaction was prepared as follows- ~5mg of KO2 was weighed out and dissolved in 700 µl of 50-50 dimethyl sulfoxide (DMSO) and 18-crown ether to give an approximately 100 mM solution of superoxide. 2 µl of this was added to 300 µl of 50-50 DMSO and 15-crown ether to give a ~667 µM solution of superoxide. 10 µl of this superoxide stock was added to 400 µl of the reaction. Therefore, the reaction also had ~ 1% DMSO and 15-crown ether. The actual initial concentration of superoxide would be much lower than 16 µM

because superoxide readily absorbs moisture (while weighing out and from DMSO). The water molecules provide sufficient protons for the formation and stabilization of peroxide from superoxide16.

Probing the effect of N-terminus on the hydroxylation efficiency of CYP2C9 and CYP2E1: Three CPR preparations of rabbit reductase, with a K56Q mutation (to minimize proteolysis, a proposal of Dr. Grover Miller at UAMS) expressed in two different cell lines were compared to the CPR prepared from human cDNA. The concentration of CPR was accurately determined with a solution of 10 µM potassium ferricyanide, using an A455-A550 extinction coefficient of 21,200 for the reductase. The value thus found correlated with protein estimations and stained gels. The CYP2C9 reactions had- [CPR] = 400 nM, [CYP2C9] = 100 nM, [diclofenac] = 25 µM & when present [Cytb5] = 200nM. The CYP2E1 reactions had- [CPR] = 100 nM, [CYP2E1] = 25 nM, [pNP] = 25 µM & when present [Cytb5] = 50nM. DLPC was at 20 µM and HNADP was at 500 µM. Reactions were terminated after 8 and 16 minutes of incubation with 3:1 ratio of sample vs. quencher (acetonitrile).

Comparing the effect of Cytb5 on CYP2E1-pNP steady state reaction in two systems: The reactions contained 20 µM DLPC, 15 nM CYP2E1, 60 nM CPR, 0/50/500 nM Cytb5 and the appropriate concentrations of pNP. 200 µl of the regeneration system reaction mixture had- 0.3 µl of 2M MgCl2, 2.4 µl of 5 µg/µl of superoxide dismutase, 0.6 µl of 1000 Units/µl catalase, 0.6 µl of 1 Unit/µl glucose-6-phosphate dehydrogenase & 60 µl of glucose-6-phosphate. The reactions were initiated with the addition of 500 µM NADP+ in the regeneration system reaction and 500 µM HNADP in another set. Aliquots were taken at 6, 12 and 18 minutes and the rate of production of pNC was calculated by the slope of linear plot obtained.

Demonstration of competitive reactions in the hlmCYP system using the CYP1A2 mediated de-ethylation of 7-EFC: Initial concentrations- [CPR] = 200 nM, [CYP1A2] = 100 nM, [DLPC] = 20 µM, [HNADP] = 500 µM & varying [7EFC] stocks in DMSO added at a final concentration of 1% of the cosolvent. At designated incubation intervals (5, 10 &15

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minutes), 75 µl of the reaction sample was taken and quenched with 25 µl of acetonitrile containing the internal standard and centrifuged. An appropriate amount (usually 40 µl of the supernatant) was injected for HPLC. The rate was calculated from the slope of line formed by fitting a linear plot with the three time points.

Other specifics are given in the appropriate figure legends and pertinent portions of the text. If standard errors of measurement are not provided along with the data, the maximum value of ≤15-20% is implied. I have used DROS to collectively represent the species of superoxide and peroxide or any other reduced/protonated oxygen species that could exist in dynamic equilibrium with molecular oxygen16-17. Since the reaction pH and aqueous reaction environment favors the formation of peroxide from superoxide16, the concentration of peroxide in milieu was checked as an indicator for DROS production and “index of uncoupling”. Also, HNADP (and not NADPH) appeals to me as a more suitable name for the reduced form of nicotinamide adenine dinucleotide phosphate (NADP).

Results & Discussion

Several CYP+CPR reaction mixtures were investigated for substrate depletion and product formation, with appropriate controls. The basic goal was to understand the overall phenomenology with a minimum reductionist approach. The following salient observations and inferences were noted- 1. Peroxide formation is disconnected from substrate activation of CYP in the major hlmCYPs: Table 1 shows the peroxide profile in reaction milieu of some commercially available baculosome preparations of hlmCYPs. Baculosome preparations are supposed to mimic the more complex and natural conditions in situ, showing several folds higher hydroxylation activity than the reconstituted systems for a given amount of CYP. The baculosome preparations also give much higher efficiency for utilization of the redox equivalents from HNADP. When substrates were presented to the CYP+CPR reaction mixture and the milieu monitored over time, similar peroxide concentration profiles were obtained as

the controls lacking the substrate (Table 1). In fact, controls lacking the substrate showed higher peroxide than the substrate inclusive test reaction at almost all time frames in the three major CYP baculosomes’ reactions. Table 1 also shows that the peroxide in milieu was variable upon reaction composition and time frames. The HAO hypothesis states that peroxide is formed in the CYP+CPR reaction owing to a “decoupling shunt” in the presence of a relatively inefficient substrate, towards the last phase of the cycle4-5, 8-13. The observations found above are inconsistent with the foundation of HAO hypothesis which requires that- (1) peroxide is produced in the presence of substrate; (2) peroxide concentration builds up in time.

Table 2 presents the effect of some substrates on HNADP consumption and peroxide formation. It was seen that for CYP2C9, the substrates employed did not produce dramatic shifts in the HNADP consumption or peroxide generation profiles within this reconstituted system. High concentrations of a relatively non-oxidizable substrate like pentafluorobenzoic acid did not affect the reaction system markedly. Therefore, the data suggest that there is an alternate mode of oxygen activation involved. 2. CPR activates molecular oxygen to give diffusible reduced oxygen species (DROS): To account for the observations in Tables 1 & 2, a reconstituted system of pure components was investigated in detail with the phosphorylated and dephosphorylated forms of electron donors of reduced nicotinamide adenine dinucleotide (HNADP & HNAD). Figures 1 & 2 depict the rate of oxidation of the reduced nicotinamide adenine dinucleotides and production of peroxide in milieu. Both systems show a significant amount of reductant consumption and peroxide production with CPR alone. Also, the overall magnitude of peroxide production or the rate of reductant depletion positively correlated to the concentration of CPR alone. At initial times, peroxide production and HNAD(P) consumption were increased to various extents by the presence of CYP2C9. The presence of substrate did not bring about substantial increase to the amount of peroxide in milieu in both the reductant systems.

These observations for CPR indicate that it is an efficient reducer/activator of molecular

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oxygen. It is known that flavins could activate molecular oxygen by two ways in aqueous systems18- (Scheme B, supplementary material)- (1) Slow reaction with triplet oxygen (Eo′ ~ -160 mV), giving superoxide (2) Fast reaction with singlet oxygen (first electron transfer Eo′ ~ +640 mV), giving peroxide. In the early 1970s, Aust’s group had shown that cytochrome c reductase gave diffusible active oxygen species in the reaction milieu19-20. Cytochrome c reductase was the erstwhile name of CPR. Only, the isolation procedure involved a tryptic cleavage of its N-terminus to give a soluble protein. The significance of this aspect of CPR catalysis was sidetracked by researchers while considering the mechanism and stoichiometry of CYP+CPR reactions in the years that followed13. We can now explain the peroxide formation by CYP+CPR mixture in the presence/absence of substrate with the consideration that CPR is the primary reducer of molecular oxygen. Now, the observations from Tables 1 & 2 could also be interpreted to indicate that the DROS generated by CPR is used by hlmCYPs to hydroxylate the final substrates.

The auto equilibration/dismutation of superoxide, originally produced by CPR, is a probable route for the formation of peroxide in milieu. The frequently noted higher peroxides in the presence of CYPs could be due to the ability of the metal atom to facilitate the peroxide generation by multiple routes. The reducible metal center of hlmCYPs could also function as a superoxide dismutase in the presence of a reductant like HNADP, thereby yielding higher peroxide in milieu. Another lesser probable route could perhaps be- the partially filled d-orbitals could catalyze the endothermic process of singlet oxygen generation, which could react faster with CPR giving peroxide. The presence of singlet oxygen in such reaction mixtures have been documented earlier by Aust et al21-22, King et al23 and stressed on recently by Hayashi et al24. Both these processes of peroxide generation could perhaps be more efficient if the iron is in high spin state, like many native CYP2E1. 3. Inclusion of molecules of varying redox potentials do not show significant impact on the outcome of the CYP+CPR reaction: The next step was to probe if the reduction of molecular oxygen

by CPR is a more viable process than CYP-bound oxygen getting the electrons by a protein-protein interaction. Excess of small molecules of varying single and double electron redox potentials like- methyl viologen (Eo′~ -446 mV), phenosafranin (Eo′~ -273 mV), anthraquinone disulfonate (Eo′~ -184mV) and indigocarmine (Eo′~ -125 mV) were incorporated into baculosome and reconstituted systems exhibiting comparable hydroxylation efficiencies. The test samples incorporating the redox sensitive small molecules showed only ~10% variations from the control for the hydroxylation of diclofenac by CYP2C9 in both the reaction setups (Figure B, Supplementary material). Such an eventuality (that is- non-perturbation of the overall hydroxylation efficiency) is seen when these redox cycling molecules could also serve as efficient competitive substrates/inhibitors for CYP. If the HAO hypothesis were operative, we would expect- (a) these molecules to effectively compete with the CYP and shunt away the electrons from reduced CPR into futile cycles or (b) give enhanced hydroxylation efficiencies due to overall enhanced electron transfers. Both these aspects were not observed. This indicated that an electron transport mechanism was operative which was not perturbed by the introduction of such redox cyclers.

There is no evidence to support the argument that CPR is CYP-specific in its action because CPR is known to reduce a wide variety of dyes and cytochrome proteins like Cytc (Eo′ ~ +230mV) and Cytb5 (Eo′ ~ +20mV)25-27. Also, the CYP+CPR reaction functions effectively in spite of the addition of copious amounts of non-redox sensitive proteins like bovine serum albumin and chick albumin. If protein-protein electron transfer mechanisms were involved, one would expect some inhibition owing to non-specific binding of extraneous proteins on to the reactive proteins’ surfaces.

The prioritized considerations for an electrochemical process are- (1) redox potential, (2) mobility and (3) concentration. The concentration of oxygen (usually above 20µM) exceeds that of superoxide by many orders of magnitude in most “healthy” cellular environments. Therefore, Nernst equation would afford a much higher redox potential in situ for triplet oxygen above +200 mV28, as opposed to the normal equilibrium value of -150 mV. This would

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enhance the drive for direct electron transfer from CPR by many folds. The relevant HFMN-H2FMN and FAD-HFAD redox couples of CPR have mid-point potentials of -270mV and -290mV respectively29-30. Redox potentials of CYPs are in the range of ~-300 mV or even lower12, 27, 31. This aspect of hlmCYPs poses a thermodynamic “obstacle” with respect to the redox couples of CPR. Therefore, kinetically significant electron transfers between CPR and CYP would be significant only if the concentration of CPR exceeds the concentration of CYPs by many orders of magnitude. This is a seemingly impossible requirement because in the liver cells, the distribution of relevant proteins is ~100 CPR to 101-102 CYPs32. The small molecule of oxygen has a far more favorable potential, is more mobile and is available at several folds higher concentrations than the relatively localized and bulky CYPs with unfavorable redox potentials. Therefore, it is only forthright to hypothesize that oxygen gets reduced directly by CPR. 4. Incorporation of DROS-utilizing proteins establish DROS’ pivotal role in the dynamics of hlmCYP mediated reactions: The effect of inclusion of DROS utilizing proteins on CYP2C9 mediated reaction with diclofenac is presented in Figure 3. Incorporation of horseradish peroxidase (HRP) in the CYP+CPR reaction increased HNADP consumption by many folds and hydroxylation of diclofenac was completely inhibited. Inset of Figure 3 shows an investigation into the intriguing effect of HRP in presence of diclofenac. CPR gave high peroxide with low HNADP consumption but inclusion of HRP took away the peroxide, consuming excess HNADP. This shows that the production of DROS from HNADP by CPR is on a thermodynamically regulated demand-supply basis. Direct electron transfer from CPR to HRP would be minimal because- (a) HRP’s ferric center is already in a high spin state with an Eo′ ~ -275mV (but this feature would enable it to serve as an efficient DROS processor), (b) the active site offers little scope for binding of substrates like diclofenac and (c) the concentration ranges of the proteins in milieu are ~10-7 M. These considerations indicate that HRP’s utilization of DROS, not specific protein-protein interaction, is the critical factor that inhibits the CYP reaction. This conclusion is

overwhelmingly supported by another work on HRP from Adak et al33, which clearly establishes the essential roles of DROS (both superoxide and peroxide) in HRP catalyzed oxidative processes.

The deduction that DROS was crucial to hlmCYPs’ activity was further supported by observations from another experiment with chloroperoxidase (CPO, as shown in Figure C, supplementary material). At pH 7.4, CPO did not show any deleterious effect on hydroxylation by CYP2C9. At this pH, CPO is catalytically inactive, though having an intact hemoprotein structure34. At a pH of 6.2, the hydroxylation efficiencies of test and control reactions were markedly lower in comparison to the reaction at pH 7.4, though HNADP consumption was increased. CPO (Eo′ ~ -150mV) also showed an influence along the lines of HRP at the acidic pH where it is active, although the effect was only marginal in comparison to HRP.

In the CYP+CPR reaction, incorporation of small amounts of superoxide dismutase (SOD) afforded a marginal increase in hydroxylation efficiency at relatively lower consumption of HNADP (please refer Figure 3 and Figure D, supplementary material). The SOD containing sample also removed non-specific products when compared to the control reaction (Figure D, supplementary material). The above mentioned observations were also valid for catalase included reactions, although to a much lesser degree. The consumption of higher amounts of HNADP in the absence of DROS utilizing enzymes like catalase and SOD indicates that DROS not only generates the catalytic intermediate(s) of/from CYP, it could also serve as a competitive substrate for it. Such logic has already been proven for oxygen insertion reactions of a similar enzyme- chloroperoxidase (CPO)35. Higher amounts of SOD were deleterious to the hydroxylation process (result not shown) and this is an observation supported by the earlier works of Strobel and Coon36. Both HRP and CPO are highly polar proteins (and CPO is highly glycosylated and acidic), with very little sequence homology to hlmCYPs, thereby belittling the argument for a protein-protein interactive mechanism. It is known that HRP, CPO and catalase have low millimolar, high millimolar and above molar KM respectively for the DROS species of peroxide37, 35, 38. Catalase has also been proven to behave as an oxidase recently39. The

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ability of catalase to afford high efficiency of HNADP utilization is perhaps owing to its low redox potential of ~ -500 mV. This would make it non-reducible by HNADP, an aspect which could be deemed essential for superoxide utilization. CPO’s deep-seated heme has a distal glutamate 183 residue hovering just above the iron center, which would not enable it to efficiently utilize the negatively charged superoxide species. These arguments explain the phenomena observed by the incorporation of DROS utilizing proteins and corroborate the essential role of DROS in the hlmCYP mediated hydroxylations. 5. Demonstration of hydroxylation without CYP-CPR complexations and simulation of activity of hlmCYPs with DROS: When CYP2C9 was taken in dialysis tubing and CPR was presented in free solution, any direct complexation was prevented by the dialysis membrane. The relevant chromatograms are shown in Figure E, Supplementary material. In the test sample, the specific 4’hydroxylated product was noted within the tubing. The concentration of hydroxydiclofenac in the tubing was relatively higher than the products in free solution, indicating that equilibration had not occurred across the dialysis membrane. Therefore, diffusion and availability of DROS generated by CPR can be considered a limiting factor in this “segregated” system. The positive control in which the components were freely mixed showed ~102 higher hydroxylation in the same time frame (Figure E, supplementary material). The HAO hypothesis, which seeks an obligatory binding of CYP to CPR at two steps, is inconsistent with these observations. This experiment could be effectively taken as a limiting model for in situ conditions.

Also, analysis of product profiles within a few seconds of reaction initiation by substrate or HNADP addition gave identical conversion and product profiles in a reconstituted system (results not shown, profiles were similar to chromatogram B in the left panel of Figure E). The product profile distribution showed non-specific reactions, quite like the reactions mediated by peroxide and superoxide chemical controls. This is explained by the consideration that in the initial time frames, the surge in DROS production gives non-specific reactions, which is subsequently shadowed by

sustained usage of the DROS by CYPs to give the specific hydroxylated product.

Table 3 shows the simulation of hydroxylation activities of two hlmCYPs with two DROS species and a comparison with the natural CPR+HNADP system. The chemical control reactions (lacking the enzymes) of diclofenac and pNP with DROS were non specific, giving much lower products. In the enzyme catalyzed (and chemical control reactions also), peroxide was relatively inefficient in comparison to superoxide. Superoxide could efficiently simulate the enzyme process for both hlmCYPs. Controls with superoxide gave significant non-specific background reactions on its own merit (the extent of which was lowered by HNADP, results not shown) which can be explained on the basis of Fenton’s and Haber’s chemistry. In the pNP reaction, it was seen that incorporation of Cytb5 enhanced the enzyme mediated hydroxylations in the HNADP+CPR, peroxide or superoxide reaction setups.

These findings, together with the observations noted by the incorporation of various redox sensitive molecules, indicate that CPR primarily catalyzes the electron transfer from HNADP to molecular oxygen. This would be a more facile process than the inefficient long-range intermolecular electron transfer. The transport of electrons across twenty bonds requires millisecond time scales. Also, the process is not highly spontaneous with distances over 10-15 Angstroms, even within intramolecular systems. The non-specific nature of redox chemistry mediated by CPR is thus effectively explained by the intermediary role of redox relaying DROS molecules. 6. CPR possesses DROS regulating abilities: Figure 4 shows the novel ability of CPR to deplete peroxide. This is the first report of such an activity for any flavoenzyme. The observation indicates that CPR could also function as an effective peroxide scavenger. Peroxide concentration in the reaction system would be sensitive to CPR because H2O2 O2 + 2H+ + 2e- ; Eo′ ~ -280 mV, which falls in the redox range of the two flavins of CPR. So, this observation also accounts for the loss of HNADP redox equivalents in CYP+CPR reactions. Also, the fluctuations of peroxide concentration in the CYP+CPR reaction milieu

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can now be explained as a result of the DROS processing dynamics of CYP and CPR. From preliminary studies, it was noted that this peroxide depleting activity of CPR increased with the amount of initial peroxide in the milieu (results not shown). This is in agreement with the decrease in rate of peroxide depletion with increasing time in Figure 4. A comparison in Figure 5 shows that CPR is more efficient than HRP for depleting peroxide in the milieu. It is forthright to extrapolate that higher concentrations of superoxide produced by CPR generate higher concentrations of peroxide by equilibration. This in turn, is done away by the CPR itself. This inference is further corroborated from Figures 1 & 2 where the peroxide formation rate falls in the milieu after initial incubation times (without a concomitant drop in HNADP or HNAD consumption). This is because at higher peroxide concentrations, equilibrium considerations dictate that CPR dons its peroxide depleting role.

Thus, the regulation of oxidative metabolism of drugs by hlmCYPs is a rather extravagant process, in terms of cellular energetics. It is only natural for it to be so because an overall non-specific mechanism cannot be envisaged to evolve cost-effectively and remain functionally viable at the same time. It is now opportune to hypothesize a new “flavin activates oxygen” pathway (or FAO pathway), as shown in Scheme 1, in which the DROS, most probably superoxide, dynamically generated by CPR is used by CYP to hydroxylate the substrate. This pathway would agree well with all the observations hitherto presented. It is a well-known fact that DROS shunts, specifically using peroxide, can also support CYP mediated reactions40. It is established with this study that superoxide mediated reaction is even more efficient. 7. Evidence and arguments further supporting the FAO pathway and discrediting the HAO pathway:

(1) Analogy from CPO: The observations and deductions reported here indicate that the reaction mixture practically self-regulates in the absence of a DROS utilizing CYP in vicinity, quite like CPO mediated chlorinations41. There, in the absence of a suitable substrate (analogous to CYP in this case), components of the reaction mixture (peroxide, which activates the enzyme to

generate the diffusible reactive intermediate) consumed the diffusible chlorinating intermediate (analogous to DROS in this case) released from the active site of CPO (analogous to CPR). But if a suitable substrate is present in the milieu, it can efficiently out-compete the peroxide for this reaction. Similarly, proximity of hlmCYPs to CPR is required for it to compete efficiently for the favored DROS of superoxide produced by CPR. Otherwise, the superoxide produced by CPR quickly dismutates into peroxide, which is a relatively poor species for activation of hlmCYPs. Also, peroxide is further done away by CPR itself. This consideration is yet another reason explaining the lower hydroxylation efficiency obtained in the experiment where the CYP2C9 and CPR were separated by dialysis membrane. Also, the enhanced consumption of HNADP at pH 6.2 without a concomitant increase in hydroxylation efficiency (Figure C, supplementary material) can be explained by the consideration that lower pH shifts the equilibrium between superoxide and peroxide to the right.

Another aspect gained from my CPO chlorination study and very relevant in the analysis of this work is that at low nano-micromolar concentrations of a diffusible reactive species, the ability of any molecule to compete with other molecules and interact with the reactive species could be dramatically different from a scenario when the concentrations are into higher ranges41. In the CPO chlorination chemistry, the fact that reaction rates afforded by an intermediate like HClO was lower (in comparison to the higher rate afforded by the enzymatic reaction) was taken as an evidence that the diffusible species mediated catalysis was not operative. This turned out to be a fallacy from several perspectives. Further, detailed investigations had shown that the reaction components exerted multiple effects in the overall chemistry and chemical controls could approach the enzymatic reaction’s features. Similarly for hlmCYPs, (a) the rates seen in the reaction cannot be explained by protein-protein mediated electron transfers & (b) rates of CYPs with superoxide could efficiently approach rates afforded by the original CYP+CPR+HNADP system.

(2) Further oxidation of the hydroxylated product: As per the HAO scheme, upon the hydroxylation of a hydrophobic substrate, the molecule supposedly loses its “tight association”

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with CYP and diffuses out. It is well known that molecules of various dimensions and moieties, possessing hydroxylated structures, are efficiently utilized by hlmCYPs. (A random sampling of some of the substrates/inhibitors for CYP2C9 is shown in Scheme C, supplementary material.) In this study, the hydroxylated product of the original substrate, 4’hydroxydiclofenac, could be further oxidized by the same hlmCYP (center panel of Figure E, Supplementary material). The original substrate of diclofenac is not an efficient inhibitor of the second oxidation step (right panel of Figure E, Supplementary material), quite contrary to what we would expect if the logic of HAO hypothesis were operative.

(3) Addressing earlier literature which advocates the CYP-CPR reduction/complexation chemistry- To date, it is noteworthy that there exists no direct experimental evidence or justification regarding the necessity of CYP-CPR complexations for the overall catalysis in a dynamic state. Job titration data starts off with an assumption that there is an association of CYP & CPR and therefore, it cannot be used to establish a 1:1 complexation stoichiometry of CYP & CPR42-

43. Besides, the reaction system would have several aggregated (homo and hetero associations of CYP and CPR) and complexed (interaction with oxygen, substrate and reductant) states of the two enzymes. Also, I have observed that the law of mass action is not abided by the CYP+CPR systems in several concentration ranges for CYP2C9, CYP2E1 and CYP1A2. A typical titration for CYP1A2 mediated oxidation of 7-EFC is shown in Figure 6. These aspects of the reaction system flout the fundamental premise of conducting and interpreting the Job titrations on the basis of HAO hypothesis.

The deleterious effect of higher ionic strengths or mutations of key amino acids on the overall reaction cannot be interpreted to be owing to a disruption of electrostatic interactions between CYP and CPR alone44-45. Ionic strength could impact a wide variety of phenomena in an electron transfer process. In the work by Shen et al.45, the electron transfer rates increased sharply when ionic strength is increased at lower concentration ranges. I have confirmed this with experiments on Cytc reduction by intact CPR. Since pH determines the charged states of the amino acids touted to play roles in CYP-CPR interactions, this

enhancement of activity at lower ionic strengths does not go well with a protein-protein complexation process. I have noted (with Raman spectroscopy on met-myoglobin and CYP101) that increasing ionic strength is cumulatively detrimental to the structural integrity of hemoproteins above a few hundred millimolar concentrations. This could explain the relative lowering of electron transfer rates at higher ionic strengths that they observed. Next, mutations in residues located quite distant from the active site significantly affected the catalytic rates in a heme-thiolate protein like chloroperoxidase46. So, the effects attributed to a disruption of putative protein-protein interaction by mutation studies can be explained by other considerations too. It is energetically favorable and quite naturally expected that any mixture of hydrophobic proteins show complexations in aqueous systems. It would also be expected that the complexations would vary depending upon the environment specifics. The existence of various homo- or hetero- oligomers in CYP-CPR mixtures is not questioned, but the obligatory relevance of the same in the overall catalysis appears to be doubtful.

(4) The N-terminus of CPR- In the late 1970s, Coon’s group had observed that the hydroxylation activity of CYPs was inhibited by the N-terminus hydrophobic segment of CPR47-49. They also observed that Cytc could be efficiently reduced by this truncated CPR. This led the researchers to infer that the N-terminus played a critical role in CYP-CPR complexations, which in turn were inferred to be crucial for the hydroxylation process. Subsequently, Strobel’s group had found no such effects50, contradicting the observations and interpretations of Coon et al. To address this controversy, four preparations of mammalian CPR were compared in this study. As seen from Table 4, the N-terminus lacking CPR was quite capable of carrying out the hydroxylation reactions for both CYP2C9 and CYP2E1. In many cases (in particular, the reactions with CYP2E1), the inhibitory effect or lowered reaction rates could be alleviated or altered significantly by adding Cytb5 in the mixture (Table 4).

(5) The role of Cytb5- CYP2E1 reaction mixtures are well known for showing higher concentrations of DROS. The hitherto held belief

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was that Cytb5 serves as an electron donor to the CYPs in certain cases and in some others, it serves as a conformational modulator of CYPs25-27, 51. Here, I interpret the overall process by the following consideration- At certain regimes, Cytb5 helps in establishing a dynamic equilibrium condition that favors the hydroxylation reaction by- (a) prolonging the life of superoxide by affording a dynamic equilibrium between reduced Cytb5 and molecular oxygen & (b) minimizing the consumption of DROS by the activated CYP and regulatory role of CPR. In such regimes, the reaction of the substrate with activated CYP intermediate is favored. This consideration is held valid because in both CYP2C9 and CYP2E1, increasing the concentration of Cytb5 by some folds resulted in a relative lowering of the hydroxylation efficiency. Figure 7 shows the result for CYP2E1. This is supported by the observation that in the reaction mixtures with CYP2C9 where Cytb5 afforded an enhancement in hydroxylation efficiencies, the peroxide concentrations were markedly lower in milieu and HNADP consumption was relatively reduced (in comparison to a system lacking Cytb5, results not shown).

I have conducted several experiments and observed that Cytb5 either improves or lowers or minimally perturbs the hydroxylation efficiency in the CYP2C9 and 2E1 systems, depending upon slight variations in the overall reaction components’ composition. This is backed by literature51. Changing the reductant from regenerating HNADP system to providing an initial high concentration of HNADP also gives different effects. This is depicted in Figure 7, a steady state assay of CYP2E1 with pNP. Each reaction composition has an optimized range of Cytb5 below and above which the hydroxylation reaction is not optimal. At relatively higher concentrations of Cytb5, it can be seen that the regenerating system (which minimizes DROS species because it has catalase and SOD within)’s hydroxylation profile becomes comparable to the HNADP system. This is because under such circumstances, the competition for superoxide (by SOD and Cytb5) would become a limiting factor. The HAO hypothesis cannot account for the lowering of hydroxylation efficiency with increasing Cytb5 because if the latter served as a protein-protein electron transfer shuttler (which is

a rate-limiting step in the HAO cycle), the hydroxylation efficiency should only be increased by increasing Cytb5. As per the new hypothesis, at higher concentrations of Cytb5, the competition of Cytb5 for superoxide could be envisaged to become a limiting factor. The subtlety of this redox equilibrium could explain the conflicting reports of the effect of Cytb5 in different hlmCYP reactions.

These interpretations are once again supported by the report of competitive reactions in the CPO catalyzed chlorinations41 (a single enzyme reaction system which does not have protein-protein interactions) where the modulation of reaction components altered the competitive ability of various substrates. In that system, increasing peroxide or chloride concentration enhanced chlorination rates to a certain extent. Further increase diverted the enzyme to other competing reaction cycles. The data presented in Table 3 (which shows enhancement in the reactions of the enzyme with DROS species by the inclusion of Cytb5) can also be explained on the same basis. Ockham’s razor does not favor the interpretation that Cytb5 brings about subtle changes in the conformations of CYPs. Therefore, one could gather that the inhibition of hydroxylation by the hydrophobic peptide (or the relatively lower efficiency of the N-term lacking CPR for the hydroxylation reaction) has to be explained by an alternate mechanism. Only then can we reason out the diversity of results obtained by Coon’s group, Strobel’s group and this study. Perhaps, the hydrophobic N-terminus serves to repel the negatively charged superoxide away from any immediate or subsequent interactions with CPR, thereby greatly enhancing the probability of a nearby hlmCYP to react with the superoxide. The lethality of reductase lacking the N-terminus52 could more probably be because such a CPR would be soluble and delocalized without the signal peptide, as a result of which it could avail oxygen more readily, generating high amounts of DROS at unwarranted locations.

(6) Interfacial reaction chemistry- Lipids incorporated into the reaction medium help to localize the CYPs and CPR in proximity and perhaps afford an environment suitable for the pertinent chemistry involving DROS species. From initial studies, I have noted that a variety of surface active molecules could inhibit the

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hlmCYP+CPR reaction. For example- L-ascorbate (sodium salt, at 10 µM), a known superoxide scavenger, showed a marginal enhancement of CYP2C9 (5 nM of Invitrogen baculosomes) mediated hydroxylation (of diclofenac taken at 25 µM, at 1mM HNADP). Incorporation of 10 µM concentration of a derivative of the same molecule with a 6-palmitate substitution almost completely inhibited (>99%) the hydroxylation reaction. This is yet another conclusive evidence for- (a) the critical role of superoxide, (b) the existence of subtle equilibriums in the reaction system and (c) a hitherto improperly understood interfacial chemistry in the hlmCYP process.

(7) Historically, the elaborate scheme of HAO hypothesis was modeled along the observations from prokaryotic bacterial enzymes CYP10130 and CYP10253. Now, I shall critically analyze the overall phenomenology of the hypothesis with respect to the drug metabolizing hlmCYPs-

(A) Let us suppose that two structurally diverse substrates A and B get converted at rates of 1 molecule per second and 1 molecule per minute respectively by a given molecule of hlmCYP. The HAO hypothesis implies that the molecule B should be a very tight binder, showing a Type I interaction (which does not occur at the heme center). For the same, the substrate molecule should have a residence time of ~ 1 minute (the turnover time) within the active site of the given hlmCYP. This is not an appealing concept with respect to molecular interactions. Also, binding of even a strong ligand like carbon monoxide to reduced heme species (Type II interaction which occurs at the heme center) is subject to spontaneous dissociation processes, which are kinetically viable even at -100K. The HAO hypothesis stringently requires the sequential and deterministic formation of several tri- and tetra- molecular complexes of such Type I and Type II small molecule bindings with CYP, along with a protein-protein complexation, in a repetitive dynamic process. In any repetitive sequential reaction, the ping-pong scheme can be viable but not a concerted and deterministic course of aggregation. Therefore, the HAO hypothesis falls short on basic scales of probability. Nevertheless, let us analyze the different bindings involved.

The Type I interaction of CYP and its substrate comes first. A survey of substrates and

inhibitors7, 54 for the major hlmCYPs do not exhibit structural commonalities among the drug molecules. For example- Ibuprofen, Warfarin, Irbesartan, Sildenafil and Trabectedin are some drugs of diverse topologies and functionalities metabolized by CYP2C9 (Scheme C, supplementary material). The HAO hypothesis obligatorily requires that such diverse drug molecules remain bound for time periods exceeding a second (!) in the active site of hlmCYPs, until they are hydroxylated. (If the substrate is not bound, the heme-center redox potential does not increase and hence, the thermodynamic requisite for reduction of oxygen at the metal center is not met with.) Such a process would require a very tight binding between the enzyme and substrate, like that between avidin-biotin. With respect to the diverse structures of the substrates, this requisite could be brought about in hlmCYPS by two ways only- (1) the active site alters its topology dynamically for each one of its substrates or (2) there are specific binding sites within the active site for each of these diverse drug molecules. Both these options appear to be far-fetched because the published crystal structure of CYP2C9 shows a large active site of 470 Å3, devoid of any special features55-56. Unlike the hypsochromic Soret shift characteristic of the Type I binding of camphor with CYP10130, I could not note any visible electronic spectral changes for ~50-100 nM CYP2C9 with 103 excess of some of its well-known substrates like diclofenac, flurbiprofen and warfarin. Though presenting some drug molecules do lead to induction of the corresponding CYP genes in vivo, inductions of several hlmCYP genes are not well-connected with majority of their substrates7. This connotes to an absence of a highly tuned genetic regulation of drug metabolism mediated by specific substrate receptors. This makes perfect sense with an evolutionary perspective because a non-specific system geared to deal with xenobiotics need not be regulated via receptors. (The simple question- how many receptors can there be?) Therefore, one could entertain the idea that lowering of redox potential upon substrate addition could be a coincidental feature of only certain evolutionarily tailored CYPs with specific substrates and constrained active sites. Recently, experimental and theoretical evidences have been obtained to support this argument57-58. Quite contrarily, the

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evolutionary dictate of the hlmCYPs may not necessitate such active-site constraints. Therefore, the HAO hypothesis cannot explain the substrate diversity of hlmCYPs. By contrast, the FAO hypothesis, which requires the substrate only at a single step in a bimolecular reaction, can account for the substrate diversity of hlmCYPS.

Now, let us address the Type II binding at the heme center. It is known that Fe(III) has a low affinity for oxygen in comparison to Fe(II). The physiological conditions dictate an overwhelming tilt to the left in the equilibrium between ferric and ferrous species for thiolate ligated heme systems. Therefore, any ferrous species would be very short lived. The binding of molecular oxygen to Fe(III) or Fe(II) would therefore be a relatively inefficient probabilistic route. On the other hand, a relatively stable DROS species like superoxide would have better affinities for Fe(III). This is yet another aspect that favors the FAO hypothesis.

Next, the binding of CYP and CPR is to be addressed. Given the low density distributions of CPR (of ~100 CPR to 101-102 CYPs) and the constraints imposed in the membranous matrix in situ, it is difficult to visualize kinetically viable collisions with hlmCYP26, 32, 59. The fruitful cycle of HAO hypothesis seeks two such collisions/complexations per cycle, which also call for- (a) super-conserved mechanism of proton and electron shuttle/transfer machinery within all the diverse CYPs8 and (b) specific modes of interaction of all these CYPs with the CPR44-45. For the unaccountable/unverifiable production of water at the metal center (an explanation afforded by HAO hypothesis), it would take even more of such binding or collisions (Scheme A, supplementary material). The necessity of proton relaying machineries (protons are at ~40 nM in physiological milieu!) in the hydrophobic core of the hlmCYP active site is yet another seemingly impossible requisite for the HAO hypothesis. The newly presented FAO hypothesis can explain the oxidation of various drug molecules a minimal equation, without invoking protons in its catalytic cycle- RH + HNAD(P) + O2 ROH + NAD(P)+ + OH-. The fact that the single mammalian reductase can support the catalysis by a bevy of various CYPs is another testament to the inference that specific protein-protein interactions may not be involved. On the other hand, the ability of proteins like HRP and interfacial superoxide

scavengers (like ascorbic acid 6-palmitate) to critically affect the reaction chemistry clearly supports the intermediary role of DROS in the CYP+CPR chemistry.

(B) Regardless of the above considerations, even if we accept the HAO hypothesis and the events detailed in them to be relevant, then- four steps are purported to have second order rate constants of ~106 M-1s-1 and three have first order rate constants ~101 s-1[11]. I have noted that baculosome preparations of human CYP2C9 gave optimized conversions of ~100 s-1 with a catalytic efficiency of ~106 M-1s-1. It is unlikely that the seven slow chronologically ordered reactions could ever give such a hyper-concerted performance. When we see that some of the backward reactions are more favorable than the forward ones10 and note that there are also three “decoupling shunts” pilfering the forward flow12, the HAO hypothesis looks an improbable course of event. The HAO hypothesis, with rate limiting steps for CYP-CPR electron transfer, cannot explain the intermolecular and intramolecular kinetic isotope effects (KIEs) observed for a wide variety of substrates and reactions60-63. The KIE values usually are above 10 for the hydroxylations of non-activated carbons. To account for this, it was proposed that the substrates could virtually be visualized to tumble freely within the CYP’s distal pocket664-65. This could explain the intramolecular KIEs, but it would definitely put the HAO hypothesis into discredit. For, given such an “unbound state of binding”, hlmCYPs must possess a “supramolecular sense and will” to increase its redox potential on its own accord and the substrate molecule must possess a “supramolecular code” to remain “committed to catalysis”66. The newly proposed FAO hypothesis does not endorse such paradoxes. It is a relatively unordered ping-pong scheme that allows sufficient spatio-temporal and probabilistic scope for explanation of all hlmCYP reactions reported to date.

(C) The unaccountable loss of redox equivalents in CYP+CPR mediated hydroxylation was proposed to be due to the formation of water by Compound I of CYP, a hypothetical catalytic intermediate of CYP12. As per the HAO scheme, the highly electrophilic Compound I radical opts for the relatively slower two-electron and two-proton transfers to form water, consuming an

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excess molecule of HNADP (This is besides the one used to generate the Compound I. So, the water production at heme center actually does not explain redox equivalents loss at all!). This is when hydroxyl ion (which is available at six times the concentration of protons at physiological pH) could easily serve as a suitable nucleophile. Its’ oxidation to molecular oxygen requires a potential of ~ +820 mV only. If the HAO hypothesis was operative, then CYPs’ Compound I should be easily isolated. But in spite of several decades of efforts, Compound I from CYPs is yet to be isolated. Also, if CYP’s hydroxylating intermediate is Compound I, then it would most probably not have the potential to hydroxylate lesser reactive carbon atoms which require much higher potentials. CYPs are known to hydroxylate some of the most non-reactive carbon atoms. Therefore, the “un-provable” production of water by CYP appears to be inconsistent with the HAO hypothesis itself. The newly proposed FAO scheme obviates such situations. I have checked out that a few hundred nanomolar concentrations of CYP2C9 could not mediate any significant conversion of redox equivalents from peroxide to water within an hour of incubation. The lowering of peroxide in the milieu (and the loss of redox equivalents in the overall stoichiometry) is therefore explained by the regulatory role of CPR.

(8) Substrate inhibitions- Another interesting phenomenon observed in the reaction system is the inhibition of CYP’s activity by higher concentrations of substrate. Currently, this phenomenon is interpreted by a bi- or multi- substrate binding model67. Though it is quite probable that the large active hlmCYPs could accommodate two or more molecules of the substrate, it is highly unlikely that so many different hlmCYPs could have multiple substrate binding sites for a diverse array of molecules. I have preliminary results to indicate that certain substrates might affect the DROS equilibriums involved on their own merit. Figure 8 shows the 1A2 mediated steady state assay of an easily oxidizable substrate, 7-EFC. As seen, the 7’ hydroxylated product forms only a fraction of the substrate converted and the substrate is converted by alternate routes too. This could explain the “non-trendy” profiles in CYP2C9 and CYP2E1 steady state profiles with HNADP reaction system (Figure 7 shows an instance for CYP2E1, results

for CYP2C9 is not shown), which has been sidelined as experimental errors by many researchers. Such substrate-superoxide (or DROS) interactions could also explain the coincidental observation of increase in peroxide in milieu with some substrates at high concentrations. The lower KM obtained with reactions incorporating Cytb5 (for example- Figure 7) is owing to a lowering of competing side reactions. This inference is corroborated by the observations reported in Table 3, which shows significant side reactions for the superoxide containing controls and enzyme reactions.

Therefore, the erstwhile HAO hypothesis surely looks an improbable and inconsistent explanation to account for the CYP+CPR reaction chemistry. The presented observations, arguments and Ockham’s razor favor the FAO hypothesis (Scheme 1), which could be taken as the operative mechanism in situ. In the microscopic realm, the small molecules’ distribution may not be homogeneous in a dynamic state. Also, the competitive or preferential utilization of DROS molecules by hlmCYPs and the interfacial aspects of the overall phenomena are to be studied in detail, especially in the presence of an excess amount of a reductant like HNADP. So, the work reported here seeks a review of the CYP reaction chemistry and the competitive reactions involved therein with a fresh perspective. In particular, molecular probes to differentiate the dynamic nano-micro molar pools of superoxide, peroxide and singlet oxygen would prove crucial towards revealing more information about the intricate equilibriums involved in the reaction mixture.

At this juncture, an “esthetics” argument

could be posed that an enzyme generating reactive diffusible intermediates would be deleterious to cellular machinery. The relevant argument is that it is the dynamic concentration and localization of the DROS which matters, not its mere presence. Earlier, esthetic considerations, backed up by misinterpreted observations, had led to the sidetracking of the more probable explanation for chloroperoxidase catalyzed chlorinations originally proposed by some researchers41. Recently, it was established that selective and controlled biochemical processes could also be afforded by mechanistic routes involving reactive diffusible species41. Similarly, it is evident here

15

that P450 research took a wrong turn when esthetic considerations, the effect of CPR’s N-terminus, the coincidental observations of spin shifts and DROS production in some systems were taken to be the yardsticks for interpretation of the holistic phenomena involved. These factors paved way for the highly improbable HAO hypothesis.

Conclusion

The redox process of drug metabolism in the liver is mediated primarily by two proteins- CPR first and then CYP. The hydroxylation of substrates provides an overwhelmingly unidirectional electron sink, thereby shifting the oxygen-superoxide equilibrium to the right. This generates a “thermodynamic impulse or pull” for CPR to produce more superoxide from HNAD(P). The overall reaction is efficient at the membrane interface, where the two hydrophobic proteins are co-localized. In the absence of CYP & substrate in vicinity, CPR mediates the loss of redox equivalents from HNAD(P) to oxygen, subsequently yielding water. Thus, the di-flavoenzyme CPR also functions as a finely tuned “redox sensor and regulator” and the substrate serves as the potential generator. Overall, the CYP+CPR system functions as a pseudo-electrochemical cell. In this process, the native metal center of CYP does not play a major role in the initial oxygen reduction step but could hasten the process of converting superoxide to peroxide. Therefore, the HNAD(P) redox equivalents consumed in the reaction is accounted by substrate converted by hlmCYP and DROS produced/converted by CPR. Cytb5 plays a role by affecting the oxygen-superoxide equilibrium. The efficient production of the active hydroxylating species of hlmCYPs is most probably from

superoxide. Effective substrates get hydroxylated by CYPs using superoxide, thereby keeping peroxide concentrations low in the milieu. Some substrates (or certain concentration ranges of these substrates or certain reaction setups) show higher peroxide in milieu only because they do not affect the non-productive superoxide to peroxide conversions or alter the superoxide to peroxide equilibrium on their own merit.

The low density distributions of CPR in vivo is now accounted because- (1) the reaction cycle does not seek for them to bind or repeatedly collide with hlmCYPs & (2) higher CPR concentrations would generate greater amount of DROS, which is deleterious to the cell machinery. Thus, the FAO hypothesis addresses the hitherto problematic aspects of HAO hypothesis and explains the broad substrate specificities of mammalian hlmCYPs and the independence of induction of several specific human CYP genes by most of their substrates. The atypical kinetics observed at times in CYP reactions68 could also be explained owing to the dynamics of DROS. Preliminary stoichiometry data of CYP2C9 reactions did not show clear-cut correlation of the substrate’s topography to the peroxide in milieu because the same substrate gave varying peroxide to product ratios under different reaction conditions (Tables 1 & 2). It is therefore opportune to warn that endeavors to optimize drugs (through in vitro studies) affording “better redox coupling”69 could end up with inappropriate conclusions. Most importantly, this study- (a) reveals that sustained low concentrations of DROS is crucial to cellular oxidative metabolism of drugs involving hlmCYPs and (b) stresses on the relevance of self-regulating non-specific redox reactions in physiological milieu.

Footnotes

Acknowledgements: This paper is dedicated to Dr. Lowell P. Hager (UIUC), my postdoctoral mentor in the years 1999-2000. Dr. Jeffrey P. Jones (WSU) and the Department of Chemistry (WSU) extended the research facilities employed in this study. A small portion of this study was also conducted at the Department of Biochemistry and Molecular Biology (UAMS). I thank Michelle Hebner (WSU) and Arvind Jamakhandi (UAMS) for sharing their CPR preparations and Dr. Victor Samokyszyn (UAMS) for discussions. Dr. Manish Bharadwaj (Wake Forest University, North Carolina) analyzed the SDS-PAGE of CPR preps employed in the study. Abbreviations: CYP- cytochrome P450; hlm- human liver microsomal, CPR- cytochrome P450 reductase, HRP- horseradish peroxidase, CPO- chloroperoxidase, SOD- superoxide dismutase, Cytc- cytochrome C,

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cytochrome b5- Cytb5, DROS- diffusible reactive oxygen species, NAD(P)- nicotinamide adenine dinucleotide (phosphate), HNAD(P)- reduced nicotinamide adenine dinucleotide (phosphate), HAO- heme activates oxygen, FAO- flavin activates oxygen, Dic or Diclof- diclofenac, pNP- para-nitrophenol, pNC- para-nitrophenol, 7-EFC or 7EFC- 7-ethoxyfluoromethyl coumarin, 7-HFC or 7HFC- 7-hydroxycoumarin, DLPC- dilauryl phosphatidylcholine, DMSO- dimethylsulfoxide, Flu- flurbiprofen, Pfb- pentafluorobenzoic acid, regen- regenerating system, KIE- kinetic isotope effect & nd- not determined.

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21. Bus, J. S., Aust, S. D. & Gibson, J. E. (1974) Superoxide- and singlet oxygen-catalyzed lipid peroxidation as a possible mechanism for paraquat(methyl viologen) toxicity. Biochem. Biophys. Res. Comm. 58(3), 749-755. 22. Pederson, T. C. & Aust, S. D. (1975) Mechanism of liver microsomal lipid peroxidation. Biochim. Biophys. Acta: General Subjects 385(2), 232-241. 23. M. M. King, E. K. Lai & P. B. McCay. (1975) Singlet oxygen production associated with enzyme-catalyzed peroxidation in liver microsomes. J. Biol. Chem. 250, 6496-6502. 24. Hayashi, S. Yasui, H. & Sakurai, H. (2005) Essential role of singlet oxygen species in Cytochrome P450-dependent substrate oxygenation by rat liver microsomes. Drug Metab. Pharmacokinet. 20, 14-23. 25. Schenkman, J. B. & Jansson, I. The many roles of cytochrome b5. Pharamacol. & Therap. 97, 139-152. 26. Guengerich, F. P. (2005) Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase. Arch. Biochem. Biophys. 440, 204-211. 27. Porter, T. D. (2002) The roles of Cytochrome b5 in Cytochrome P450 reactions. J. Biochem. Molec. Toxicol. 16, 311-316. 28. Pierre, J. L., Fontecave, M. & Crichton, R. R. (2002) Chemistry for an essential biological process: the reduction of ferric iron. Biometals 15, 341-346. 29. Munro, A. M., Noble, M. A., Robledo, L., Daff, S. N. & Chapman, S. K. (2001) Determination of the redox properties of human NADPH-Cytochrome P450 reductase. Biochemistry 40, 1956-1963. 30. Porter, T. D. (2001) Enzymology of Cytochrome P450 reductase. http://www.uky.edu/Pharmacy/ps/porter/CPR_enzymology.htm. 31. Sligar, S. G. & Gunsalus, I. C. (1976) A thermodynamic model of regulation: modulation of redox equilibria in camphor monooxygenase. Proc. Natl. Acad. Sci. USA 73, 1078-1082. 32. Guengerich, F. P. Cytochrome P450: What have we learned and what are the issues? Drug Metab. Rev. 36, 159-197 (2004). 33. Adak, S., Bandyopadhyay, U., Bandyopadhyay, D. & Banerjee, R. K. (1998) Mechanism of Horseradish Peroxidase Catalyzed Epinephrine Oxidation: Obligatory Role of Endogenous O2- and H2O2. Biochemistry 37, 16922-16933. 34. Blanke, S. R., Martinis, S. A., Sligar, S. G., Hager, L. P., Rux, J. J. & Dawson, J. H. (1996) Probing the heme coordination structure of alkaline chloroperoxidase, Biochemistry 35, 14537–14543. 35. Kelath Murali Manoj & Hager, L. P. (2001) Utilization of peroxide and its relevance in oxygen insertion reactions catalyzed by chloroperoxidase, Biochim. Biophys. Acta. 1547, 408-417. 36. Strobel, H. W. & Coon, M. J. (1971) Effect of superoxide generation and dismutation on hydroxylation reactions catalyzed by liver microsomal cytochrome P450. J. Biol. Chem. 246, 7826-7829. 37. Baker, C. J., Deahl, K., Domek, J. & Orlandi, E. W. (2000) Scavenging of H2O2 and production of oxygen by horseradish peroxidase. Arch. Biochem. Biophys., 382, 232-237. 38. Ogura, Y. (1955) Catalase activity at high concentration of hydrogen peroxide. Arch. Biochem. Biophys. 57, 288-300. 39. Vetrano, A. M. Heck, D. E., Mariano, T. M., Mishin, V., Laskin, D. L. & Laskin, J. D. (2005) Characterization of the oxidase activity in mammalian catalase. J. Biol. Chem., 280, 35372-35381. 40. Joo, H., Lin, Z. & Arnold, F., H. (1999) Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399, 670-673. 41. Kelath Murali Manoj. (2006) Chlorinations catalyzed by chloroperoxidase occur via a diffusible intermediate and the reaction components play multiple roles in the overall process. Biochim. Biophys. Acta- Proteins & Proteomics 1764, 1325-1339. 42. Miwa, G. T., West, S. B., Huang, M-T. & Lu, A. Y. H. (1979) Studies on the association of cytochrome P-450 and NADPH-cytochrome c reductase during catalysis in a reconstituted hydroxylating system. J. Biol. Chem. 254, 5695-5700. 43. Fernando, H., Halpert, J. R. & Davydov, D. R. (2006) Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4: The Stoichiometry of the Enzyme-Substrate Complexes Probed by FRET and Job's Titration. Biochemistry 45, 4199 – 4209.

18

44. Voznesensky, A. I. & Schenkman, J. B. (1994) Quantitative analyses of electrostatic interactions between NADPH- cytochrome P450 reductase and cytochrome P450 enzymes. J. Biol. Chem. 269, 15724-15731. 45. Shen, A. L. & Kasper, C. B. (1995) Role of Acidic Residues in the Interaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome P450 and Cytochrome c J. Biol. Chem. 270, 27475-27480. 46. Rai, G. P., Zong, Q. & Hager, L. P. (2000) Isolation of directed evolution mutants resistant to suicide inactivation by primary olefins. Isr. J. Chem. 40, 63-70. 47. Black, S. D., French, J. S., Williams, C. H. & Coon, M. J. (1979) Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450LM. Biochem. Biophys. Res. Commun. 91, 1528-1535. 48. French, J. S., Black, S. D., Williams, C. H. & Coon, M. J. (1980) Studies on the association of P-450LM2 with NADPH-cytochrome P-450 reductase and with tryptic peptides derived from the reductase. In Microsomes, Drug oxidations and Chemical carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. W. Gelboin, H. V., Gillette, J. R. & O’Brien, P. J. Eds.) pp. 387-390. Academic Press, New York. 49. Black, S. D. & Coon, M. J. (1982) Structural features of liver microsomal NADPH-cytochrome P-450 reducase: Hydrophobic domain, hydrophilic domain and connecting region. J. Biol. Chem. 257, 5929-5938. 50. Gum, J. R. & Strobel, H. W. (1981) Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase: Charecterization of the peptide and its role in the interaction of reductase with cytochrome P450. J. Biol. Chem. 256, 7478-7486. 51. Kumar, S., Davydov, D. R. and Halpert, J. R. (2005) Role of cytochrome b5 in modulating peroxide-supported CYP3A4 activity: Evidence for a conformational transition and cytochrome P450 heterogeneity. Drug Metab. Dispos. 33, 1131-1136. 52. Shen, A. L., O’Leary, K. A. & Kasper, C. A. (2002) Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-Cytochrome P450 oxidoreductase. J. Biol. Chem. 277, 6536-6541. 53. Graham-Lorence, S. E. & Peterson, J. A. (1996) P450s: Structural similarities and functional differences. FASEB J. 10, 206-214. 54. Fontana, E., Dansette, P. M. & Poli, S. M. (2005) Cytochrome P450 enzymes mechanism based inhibitors: Common substructures and reactivity. Curr. Drug Metab. 6, 413-454. 55. Williams, P. A., Cosme, J., Ward, A., Angove, H. C., Vinkovic, D. M. & Jhoti, H. (2003) Crystal structure of human cytochrome P450 2C9 with bound warfarin. Nature 424, 464-468. 56. Wester, R. M., Yano, J. K., Schoch, G. A., Yang, C., Griffin, K. J., Stout, D. C. & Johnson, E. F. (2004) The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2 Å resolution. J. Biol. Chem. 279, 35630-35637. 57. Segall, M. (1997) An ab initio study of biological systems. Doctoral Thesis submitted at the University of Cambridge. The pertinent link is titled “Understanding cytochromeP450-ligand interactions”. http://www.tcm.phy.cam.ac.uk/~mds21/thesis/thesis.html. 58. Shukla, A., Gillam, E. M. Mitchell, D. J. & Bernhardt, P. V. (2005) Direct electrochemistry of enzymes from the cytochrome P450 2C family. Electrochem. Commun. 7, 437-442. 59. Ingelman-Sundberg, M. (1986) Cytochrome P-450 organization and membrane interactions. In: Ortiz de Montellano, P. R., ed. Cytochrome P-450 structure, mechanism and biochemistry. Plenum Press, New York. pp119-160. 60. Karki, S. B., Dinnocenzo, J. P., Jones, J. P. & Korzekwa, K. R. (1995) Mechanism of oxidative amine dealkylation of substituted N,N-Dimethylanilines by cytochrome P-450: Application of isotope effect profiles. J. Am. Chem. Soc. 117(13), 3657-64. 61. Higgins L., Bennett G. A., Shimoji, M. & Jones, J. P. (1998) Evaluation of cytochrome P450 mechanism and kinetics using kinetic deuterium isotope effects. Biochemistry 37(19), 7039-46.

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62. Jones, J.P., Rettie, A.E. & Trager, W.F. (1990) Intrinsic isotope effects suggest that the reaction coordinate symmetry for the cytochrome P-450 catalyzed hydroxylation of octane is isozyme independent. J. Med. Chem. 33, 1242-1246. 63. Jones, J. P., Korzekwa, K. R., Rettie, A. E. & Trager, W. F. (1986) Isotopically sensitive branching and its effect on the observed intramolecular isotope effects in cytochrome P-450 catalyzed reactions: A new method for the estimation of intrinsic isotope effects. J. Am. Chem. Soc. 108(22), 7074-7078. 64. Lee, H., Ortiz de Montellano, P. R. & McDermott, A. E. (1999) Deuterium magic angle spinning studies of substrates bound to cytochrome P450. Biochemistry 38, 10808-10813. 65. Audergon, C. Iyer, K. R., Jones, J. P., Darbyshire, J. F. & Trager, W. F. (1999) Experimental and theoretical study of the effect of active-site constrained substrate motion on the magnitude of observed intramolecular isotope effect for the P450 101 catalyzed benzylic hydroxylation of isomeric xylenes and 4,4’-dimethylbiphenyl. J. Am. Chem. Soc. 121, 41-47. 66. Miwa, G. T., Zweig, J. S., Walsh, J. S. & Lu, A. Y. H. (1980) Kinetic isotope effects in cytochrome P-450 catalyzed oxidation reactions. I. Substrate-cytochrome P-450 interactions. In Microsomes, Drug oxidations and Chemical Carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. J. Eds.) pp. 363-363. Academic Press, New York. 67. Shou, M. et al. (2000) Enzyme kinetics of cytochrome P-450 mediated reactions. Curr. Drug. Metab. 2, 17-36. 68. Atkins, W. M. (2005) The non-Michaelis-Menten kinetics observed in CYP catalyzed reactions. Ann. Rev. Pharmacol. Toxicol., 45, 291-310. 69. Narasimhulu, S. (2003) Vascular Surgery Research (at University of Pennsylvania Health System Research Program). http://www.uphs.upenn.edu/surgery/res/lab-shak.html.

Legends to Tables, Figures & Scheme

Table 1: Peroxide profile in milieu with different hlmCYPs. The upper values in each row are the peroxide in milieu (in µM) with the substrate where as the lower emboldened values are in the absence of substrates. Table 2: HNADP consumption and peroxide profiles with different substrates for CYP2C9. The values given are concentrations, in µM. Acronyms are: Flu- flurbiprofen, Dic- diclofenac & Pfb- pentafluorobenzoic acid. Table 3: Simulation of hydroxylating activities of hlmCYPs with DROS. Concentrations of hydroxylated products, formed after 10 minutes are given in nM. The asterisk indicates that these reactions also showed non-specific hydroxylations or side reactions. Table 4: Comparison of the hydroxylating activities of four different CPR preps. Values given are micromolar hydroxylated product formed in the milieu. Figure 1: Generation of peroxide by CPR from HNADP and its correlation to depletion of HNADP in a pure reconstituted system is presented. Inset shows the standard plot for H2O2 using the Peroxoquant method. Figure 2: Generation of peroxide by CPR from HNAD and its correlation to depletion of HNADP in a pure reconstituted system is presented. Figure 3: Effect of DROS utilizing proteins on hydroxylation of diclofenac and consumption of HNADP in the CYP+CPR reaction is shown. HRP shows dramatic influence by completely inhibiting

20

hydroxylations by CYP2C9. Inset probes the effect of HRP on HNADP consumption and peroxide in milieu, with controls. For the inset, the initial concentrations were [CPR] = 180 nM, [HRP] = 157 nM, [diclofenac] = 200 µM & [HNADP] = 215 µM. Figure 4: The DROS regulating nature of CPR is depicted. Initial rate of HNADP depletion in the CPR + HNADP was ~0.8 µM/min with or without H2O2, which was ~5 times the value in chemical controls. As the third trace in the figure shows, CPR does not “use” HNADP for depleting peroxide. The initial concentration of components were- [CPR] = 230 nM, [HNADP] = 164 µM & [H2O2] = 40 µM. Figure 5: Comparison of CPR’s peroxide depleting function with that of an equal concentration of HRP. CPR is seen to be a more effective peroxide lowering enzyme than HRP. Figure 6: The effect of variation of CYP1A2 Vs. CPR on the outcome of 7-EFC de-ethylation: The reaction was carried out at 100 mM potassium phosphate buffer, 20 µM DLPC, 50 µM 7-EFC & 500 µM HNADP. 50 nM CYP1A2 or CPR was taken and 0-500 nM of CPR or CYP1A2 were respectively varied. Sample for HPLC analysis was drawn at 10 minutes of reaction time. Nanomolar concentrations of 7-HFC formed in the milieu is plotted. In these conditions, the highest concentrations of titrants showed >20% of substrate conversion (as compared to the 0 nM titrant concentration control), which did not correspond to the <0.1% of 7-HFC formed. Figure 7: A comparison of HNADP vs. regenerating system is shown for the hydroxylation of pNP by CYP2E1. In the HNADP system, the presence of two regions of optimal activity can be seen and the presence of Cytb5 shows a quicker approach to the maximal rates. Figure 8: CYP1A2 mediated de-ethylation of 7-EFC: Data on the initial substrate concentrations, final substrate in milieu and hydroxylated product are shown. Since only a few tens of nanomolar concentrations of 7-EFC was converted in all reaction setups, practically, there should be very little difference in the initial amounts of 7-EFC taken. But the actual 7-EFC concentrations were much lower from the minimal expected values, which was in agreement with the observations in the experiment for Figure 6. Scheme 1: The newly proposed FAO hypothesis is shown. The mechanism has four minimal bimolecular reactions and the substrate is required only in the last step. From the active CYP (designated with an asterisk), the upward arrow is the non-productive process and the downward arrow is the productive step. The dotted lines designate probable schemes of interaction involving the various components.

21

Tables

Table 1, KMM: CYP ± substrate 0.3 min 10 min 20 min 30 min 75 min

Invitrogen 3A4 + or -Testosterone

1.5 ± 0.3 2.2 ± 0.2

5.9 ± 0.3 8.9 ± 0.1

3.5 ± 0.2 5.9 ± 0.2

2.4 ± 0.1 4.3 ± 0.4

0.4 ± 0.3 1.3 ± 0.3

Invitrogen 2D6 + or -Dextromethorphan

0.6 ± 0.4 0.7 ± 0.5

0.6 ± 0.4 1.0 ± 0.4

- 0.2 ± 0.1

- 0.3 ± 0.2

- -

Invitrogen 2C9 + or -Diclofenac

1.4 ± 0.3 1.6 ± 0.4

- 0.4 ± 0.2

- 1.0 ± 0.1

- 1.4 ± 0.2

- 0.2 ± 0.1

Merck 2C9 + or -Diclofenac

0.4 ± 0.2 1.7 ± 0.4

10.9 ± 0.9 13.8 ± 0.2

18.0 ± 0.2 25.2 ± 0.1

23.6 ± 0.2 34.3 ± 0.1

48.3 ± 0.6 61.5 ± 0.5

Table 2, KMM:

7.5 min 15 min 30 min Reaction Mixture HNADP H2O2 HNADP H2O2 HNADP H2O2

CPR only nd 5.4 12.9 10.6 27.2 11.1 CPR + CYP2C9 nd 7.5 17.2 10.7 34.7 12.8

CPR + CYP2C9 + Flu nd 5.3 17.9 10.1 36.2 12.4 CPR + CYP2C9 + Dic nd 6.4 17.8 11.3 39.4 14.6 CPR + CYP2C9 + Pfb nd 7.9 17.9 12.3 37.0 14.7 Table 3, KMM:

Diclofenac pNP Cytb5+pNP Reaction CYP2C9 Control CYP2E1 Control CYP2E1 Control

CPR+HNADP 911 ± 45 trace 450 ± 51 trace 786 ± 36 trace H2O2 ~41 <10 <10 nil ~74 nil O2

- ~995* ~112* ~104** ~88** ~499** ~70** Table 4, KMM:

CYP2C9 CYP2C9+Cytb5 CYP2E1 CYP2E1+Cytb5CPR source (Intact protein%) 8min 16min 8min 16min 8min 16min 8min 16min Top-3 (99%) 1.24 2.43 2.39 4.72 0.38 0.50 0.44 0.68

Top-3 (~68%) 0.45 0.91 0.62 1.3 0.16 0.26 0.33 0.61 C-41 (<32%) 0.17 0.33 0.85 1.75 0.11 0.15 0.26 0.49 C-1A (<28%) 0.59 0.92 0.45 0.76 0.09 0.15 0.41 0.59

22

Figure 1

0 10 20 30

1/4X CPR

CPR

CPR + CYP

CPR + Diclof

CPR + CYP +Diclof

4X CPR

Rates or concentrations

µM HNADP per 10 minµM peroxide at 30 minµM peroxide at 20 minµM peroxide at 10 min

Standard Plot

y = 0.2652xR 2 = 0.9993

0

0.2

0.4

0.6

0.8

0 1 2 3Peroxide (nmoles / ml)

OD

at 5

60nm

..

23

Figure 2

0 1 2 3 4

1/4X CPR

CPR

4X CPR

CPR + Diclof

CYP + CPR

1CYP+1CPR+diclof

Rates or concentrations

µM peroxide at 32minµM peroxide at 16minµM HNAD/min

24

Figure 3

0 1 2 3 4 5 6

2C9 only

2C9 + HRP

2C9 + CPO

2C9 + Catalase

2C9 + SOD

Rates or Concentrations

µM 4'hydroxydiclofenac at 15min

µM HNADP per min

010203040

HNADPonly

HNADP +CPR

HNADP +CPR +HRP R

ate

or C

once

ntra

tions

..

µM HNADP / 10min

µM peroxide, 15min

25

Figure 4

0

10

20

30

40

0 20 40 60 80

Time (min)

Pero

xide

(nm

oles

/ m

l)

HNADP + Peroxide (control)

HNADP + Peroxide + CPR

Peroxide + CPR

HNADP + CPR

26

Figure 5

0

20

40

60

80

100

0 15 30 45 60 75 90

Time (min)

Pero

xide

(nm

oles

/ml)

100 µM peroxide only

160 nM CPR + 100 µM peroxide

160 nM HRP + 100 µM peroixde

27

Figure 6

0

10

20

30

40

50

0 100 200 300 400 500

Titrant (nM CPR or CYP1A2)

7HFC

(nM

) CPRCYP1A2

28

Figure 7

0

0.1

0.2

0.3

0.4

0 200 400 600

pNP (nmoles/ml)

pNC

(nm

oles

/ml/m

in.)

Regen: No Cytb5

Regen: 50nM Cytb5

Regen: 500nM Cytb5

HNADP: No Cytb5

HNADP: 50nM Cytb5

HNADP: 500nM Cytb5

29

Figure 8

0

10

20

30

40

50

0 20 40 60 80 100

Initial 7-EFC (nmoles/ml)

7-H

FC (p

mol

es/m

l)

0

20

40

60

80

100

Rem

aini

ng 7

-EFC

(nm

oles

/ml)

nM 7HFC at 5minnM 7HFC at 10minnM 7HFC at 15minµM 7EFC at 5minµM 7EFC at 10minµM 7EFC at 15minMinimal 7EFC expected

30

Scheme 1

HCPR

ROH

O2

RH

HNADP

CYP*

CYP3

4

Cytb5

O2.-= H2O2 (DROS)

H2O

CPR

NADP+

12

31

Supplementary material for KMM-CYP Scheme A: Scheme 1: The existent HRO hypothesis is elaborate, sequential, has substrate obligatorily bound to the CYP throughout the cycle and requires ternary molecular reactions.

RH-CYP-Fe(III)

RH

CPR

NADPHCPR-H:

NADP+

RH-CYP-Fe(II)CPR-H.

1

2

3

RH-CYP-Fe(II)-O-O-

4

H2O

RH-CYP-Fe(II)-O-O

5

RH-CYP-Fe(III)-O-O2-

RH-CYP-Fe(III)-O-O-H

H+

RH-CYP-Fe(III)-O-O.

6

O=O.

H+

H2O2

H+ H2O7

(a)

(b)

R.-CYP-Fe(IV)-OH

R-OH

8

NADPH

NADP+

CPR cycle?

H+

(c)

H2O

CPR cycle O=O

Stoichiometry

RH + NADPH + O2 + H+ = ROH + NADP+ + H2O ........ (productive cycle)2O2 + NADPH = 2O2

.- + NADP+ + H+........ (a = superoxide shunt)O2 + NADPH + H+ = H2O2 + NADP+ ....... (b = peroxide shunt)O2+ 2NADPH + 2H+ =2H2O+2NADP+ ....... (c =water shunt)

amphiphilic

highly electrophilic

H2O

H2O

H2O

nucleophilic

highly nucleophilic

CYP cycle

CYP-Fe(III)-OH2

RH-CYP.Fe(IV)=O

32

Scheme B: Activation of oxygen by CPR to generate DROS (adapted from Silverman18).

NH

NH

O

NR

N

O

singlet oxygen

triplet oxygen

N

NH

O

NR

N

O

N

NH

O

NR

N

O

NH

NH

O

NR

N

O

O OH

. O=O.-

Reduced CPR Oxidized CPR

H

H2O2

Radicalrecombination

e- transfer, H

Externalbase stabilizn

Cagedradicals

33

Scheme C: Some of the substrates and non-mechanism based inhibitors of varying dimensions and topologies reported for CYP2C9 (References 70-82)

NO

NH2

S OH

Zileuton, C11H12N2O2S

O

OH

Ibuprofen, C13H18O2

O

OHNH

Cl

Cl

Diclofenac, C14H11Cl2NO2O

O

OH

OWarfarin, C19H16O4

OS

NN

FFF

H2N

O

Celecoxib, C17H14F3N3O2S

N

Amitriptyline, C20H23N

OHO OH

OH

N

F

Fluvastatin, C24H26FNO4

NO

Tamoxifen, C26H29NO

ON

NN

NHN

N

Irbesartan, C25H28N6O

N

NN

N

OOO

OH

S

O

ONH2

Bosentan, C27H31N5O6S

O I

I

O

N

O

Amiodarone, C25H29I2NO3

Trabectedin, C39H43N3O11S

NNH

NH

NH

O

O

OO

SN

HO

SN

Ritonavir, C37H48N6O5S2

O

NH

SO

O

O

N

HN

O

O

Zafirlukast, C31H33N3O6SN

N SO O

N

NN

NH

O

O

Sildenafil, C22H30N6O4S

34

References for Scheme C 70. de Groot, M. J., Alexander, A. A. & Jones, B. C. Development of a combined protein and pharmacophore model for Cytochrome P450 2C9. J. Med. Chem. 45, 1983-1993 (2002). 71. Crewe, H. K., Ellis S. W., Lennard, M. S. & Tucker, G. T. Variable contribution of cytochromes P450 2D6, 2C9 and 3A4 to the 4-hydroxylation of tamoxifen by human liver microsomes. Biochem. Pharmacol. 53, 171–8 (1997). 72. Jones, B. C., Hawksworth, G., Horne, V. A., Newlands, A., Morsman, J., Tute, M. S. & Smith, D. A. Putative active site template model for cytochrome P4502C9 (tolbutamide hydroxylase). Drug Metab. Dispos. 24, 260 (1996). 73. Stempak, D., Bukaveckas, B. L., Linder, M., Koren, G. & Burachel, S. CYP2C9 genotype: impact on celecoxib safety and pharmacokinetics in a pediatric patient. Clin. Pharmacol. Ther. 78, 309–310 (2005). 74. von Moltke, L. L., Greenblatt, D. J., Grassi, J. M., Granda, B. W., Duan, S. X., Fogelman, S. M., Daily, J. P., Harmatz, J. S. & Shader, R. I. Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with Ritonavir. J. Clin. Pharmacol. 38, 106-111 (1998). 75. Gorge, G., Fluchter, S., Kirstein, M. & Kunz, T. Sex, erectile dysfunction, and the heart: a growing problem. Herz 28, 284-90 (2003). 76. Cheng, J. W. M. Bosentan. Heart Disease 5, 161-169 (2003). 77. Brandon, E. F. A., Meijerman, I., Klijn, J. S., den Arend, D., Sparidans, R. F. W., Lopez, L. L., Beijnen, J. H. & Schellens, J. H. M. In-vitro cytotoxicity of ET-743 ( Trabectedin , Yondelis), a marine anti-cancer drug, in the Hep G2 cell line: influence of cytochrome P450 and phase II inhibition, and cytochrome P450 induction. Anti-Cancer Drugs 16, 935-943 (2005). 78. Olesen, O. V. & Linnet, K. Metabolism of the tricyclic antidepressant amitriptyline by cDNA-expressed human cytochrome P450 enzymes. Pharmacology 55, 235-243 (1997). 79. Scripture, C. D. & Pieper, J. A. Clinical pharmacokinetics of fluvastatin. Clinical Pharmacokinetics 40, 263-281 (2001). 80. Shader, R. I., Granda, B. W., Von Moltke, L. L., Giancarlo, G. M. & Greenblatt, D. J. Inhibition of human cytochrome P450 isoforms in vitro by zafirlukast. Biopharm. & Drug Dispos. 20, 385-388 (1999). 81. Bourrie, M., Meunier, V., Berger, Y. & Fabre, G. Role of cytochrome P-4502C9 in irbesartan oxidation by human liver microsomes. Drug Metab. Dispos. 27, 288-296 (1999). 82. Naganuma, M., Shiga, T., Nishikata, K., Tsuchiya, T., Kasanuki, H. & Fujii, E. Role of desethylamiodarone in the anticoagulant effect of concurrent amiodarone and warfarin therapy. J. Cardiovas. Pharmacol. Therap. 6, 363-367 (2001).

35

Figure A: SDS-PAGE of CPR purified in the lab, which were employed in this study. The two flanking lanes are Biorad high range molecular weight markers of 200, 116, 97, 66 and 45 KD respectively. Key to the four CPR lanes from right to left are- lane 1 = C41 prep (<32% intact), lane 2 = Top3 prepI (~68% intact), lane 3 = C1-A prep (<28% intact) & lane 4 = Top3 prepII (>99% intact).

36

Figure B: The effect of various redox sensitive molecules on CYP2C9 catalyzed hydroxylation of diclofenac is shown.

Structures of redox sensitive molecules employed in the study

N+N

NH2

NH2

PhenosafraninHN

NHS

O

O

-O

SO

O

O-O

OIndigo carmine

O

O

SO

OO-

SO

O-O

Anthraquinone disulfonate

N+ N+

Methyl viologen

37

Figure C: The comparison of reaction mixtures incorporating CPO at different pH.

38

Figure D: Standard HPLC chromatogram of diclofenac and its hydroxylation products is shown. Inset shows the product profiles of reaction incorporated with DROS utilizing proteins. A- CPO (the same as control reaction), B- catalase inclusive reaction and C- SOD inclusive reaction. The chromatogram of reaction mixture with HRP did not give any measurable peaks within 6 to 6.5 minutes of elution time. The peak(s) eluting hydroxydiclofenac in the reaction could also be the benzoquinone imine intermediate. (Ref: 82. Poon, G. K., Chen, Q., Teffera, Y., Ngui, J. S., Griffin, P. R., Braun, M. P., Doss, G. A., Freeden, C., Stearns, R. A., Evans, D. C., Baillie, T. A. & Tang, W. Bioactivation of diclofenac via benzoquinone imine intermediates- identification of urinary mercapturic acid derivatives in rats and humans. Drug Metab. Dispos. 29, 1608-1613 (2001).)

A

B

C

3’, 4’ & 5’ hydroxylated diclofenac

Side products

39

Figure E: HPLC chromatograms of some CYP2C9 reactions. (The 4’hydroxylated diclofenac elutes at 6.36 minutes in these set of chromatograms, owing to a minor lowering of the organic phase component of the elution solvent mixture.) Left panel: Physical separation of CYP2C9 and CPR gave specific hydroxylation of diclofenac. Profiles A & B shows the chromatograms of sample drawn at 45 minutes of reaction time from within the dialysis tubing and from the free solution in the test reaction respectively, in the test reaction in which CYP and CPR were separated. Clearly, A shows the specific product formation. Profile C is the sample drawn from the positive control reaction in which CYP and CPR were mixed. Center panel: CYP catalyzed hydroxylation of hydroxydiclofenac. Profiles M & N : M is the sample drawn at 45 minutes of incubation and N is a sample drawn after 4 hours of reaction. The lowering of hydroxylated diclofenac peak and the appearance of side products (as a result of further oxidation of diclofenac) is seen at later time frame. Right panel: CYP catalyzed hydroxylation of hydroxydiclofenac. Profiles X, Y & Z : X is the control reaction which had 80 µM diclofenac only and it does not show any significant formation of the polar product. Y is the test reaction with only 40 µM of 4’hydroxydiclofenac and it shows the increased production of the more polar product. Z had both 40 µM 4’hydroxydiclofenac and 80 µM diclofenac added initially in the reaction mixture. As seen, the hydroxydiclofenac is formed to the same extent, irrespective of the presence of excess diclofenac. It shows that the latter is not an effective inhibitor in CYP2C9’s oxidation of 4’hydroxydiclofenac.

M

N

X

Y

Z

A

B

C