Direct electrochemistry of immobilized human cytochrome P450 2E1

This communication reports the first electrochemical study of the human P450 2E1 either absorbed or covalently linked to different electrode surfaces. Glassy-carbon and gold electrodes gave reversible electrochemical signals of an active P450 2E1. Molecular modeling of the enzyme helped to rationalize the results. A monolayer coverage was obtained on gold modified with cystamine/maleimide that covalently linked surface accessible cysteines of P450 2E1. The midpoint potential measured for the oriented P450 2E1 was -177 +/- 5 mV comparable to that of the FeIII/FeII of other P450 enzymes. The observed electron-transfer rate for this electrode was 10 s-1. The turnover of the active enzyme was measured with the P450 2E1 specific substrate p-nitrophenol, resulting in a KM of 130 +/- 3 muM and the formation of 2.2 muM of the p-nitrocatechol product upon application of a -300 mV bias.     

Electrochemistry of Cytochrome P450 2B6 on Electrodes Modified with Zirconium Dioxide Nanoparticles and Platin Components

The direct electrochemical and electrocatalytic behavior of the immobilized cytochrome P450 2B6 (CYP2B6) on zirconium dioxide nanoparticles (ZrO₂) was investigated. The film of nano‐structured ZrO₂ that incorporated cytochrome P450 2B6 (CYP2B6) with colloidal paltin, which was stabilized by poly‐lysine (Pt‐PLL), was prepared on glassy carbon electrodes. In anaerobic solutions, the immobilized CYP2B6 exhibited a reversible electron transfer between the heme electroactive center of CYP2B6 and electrodes with a formal potential of ?(0.449±0.004) V at pH 7.4. In air‐saturated solutions, an increased bioelectrocatalytic reduction current could be obtained with the CYP2B6‐modified electrode with the addition of anticancer drugs, such as lidocaine. This leads to the construction of disposable biosensors for drugs by utilizing the electrochemical activity and catalytic reactions of the immobilized CYP2B6.     

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid-induced Unfolding

Transitions among various heme coordination/spin states, heme environments and protein conformations of human cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it's kind. Our results indicated that the thermal and acid‐induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe‐S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X‐ray structure of P450 2C8 suggested that the specific structure of the β‐bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid‐induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.     

Modulating the coupling efficiency of human cytochrome P450 CYP3A4 at electrode surfaces through protein engineering

This study shows that regulating the electron flow to the heme of human cytochrome P450 CYP3A4, using artificial redox chains, can significantly enhance its coupling efficiency and catalytic activity at electrode surfaces. The human CYP3A4 was fused at the genetic level either to the reductase domain of CYP102A1 (BMR) to create the CYP3A4/BMR or to Desulfovibrio vulgaris flavodoxin (FLD) to create the CYP3A4/FLD. Direct electrochemistry of the CYP3A4, CYP3A4/BMR and CYP3A4/FLD on glassy carbon and gold electrodes showed that the BMR and FLD flavo-proteins reduced the electron transfer rate to the CYP3A4 heme. Electrocatalysis resulted in appreciably higher product formation with the immobilized CYP3A4/BMR and CYP3A4/FLD on both surfaces due to an increased coupling efficiency. Rotating disk electrode studies and quantification of hydrogen peroxide were consistent with the proposed mechanism of a longer lived iron-peroxy species in the immobilized CYP3A4/BMR and CYP3A4/FLD. The approaches in this study provide a better understanding of cytochrome P450 uncoupling at electrode surfaces and aids in the construction of improved cytochrome P450 biosensors and bioelectrocatalysts.     

Gold sputtered electrode surfaces enhance direct electron transfer reactions of human cytochrome P450s

We immobilized human cytochrome P450 (CYP), a membrane-bound enzyme, onto both smooth and nanostructured surfaces of gold electrodes via a naphthalene thiolate monolayer film. Rapid electron transfer of CYP with an electrode as a redox partner took place when the enzyme was immobilized onto an electrode surface with nanostructures. This structure was easily prepared by conventional sputtering techniques. A well-defined pair of peaks was observed at ? 0.175 V (vs. SHE) with the largest heterogeneous electron transfer rate constant of 340 s^? 1 for human CYP. The positive redox potential shift of 45 mV upon drug (testosterone) binding was clearly detected, which corresponded to a change in the spin states of heme iron in CYP. The present study showed that gold sputtered surfaces are very useful for direct electron transfer reactions of human CYP isoforms.     

Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3

Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min(-1)) and support thousands of product turnovers. The regio- and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.     

Electrochemical study of the interaction between cytochrome P450sccK201E and cholesterol

Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme–substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection.

The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.     

Oxygen activation by cytochrome p450: a thermodynamic analysis

Electrode potentials for every intermediate in the cytochrome P450 cycle were estimated and evaluated by means of an oxidation state diagram. By this approach, and within the uncertainties of the approximations, the superoxide complex of cytochrome P450 at pH 7 is oxidizing: E degrees ' (P450FeO(2)2+, H+/P450FeOOH2+) = +0.93 V, and the Gibbs energy for the reaction of the hydroperoxo complex of cytochrome P450 to form compound I and water, P450FeOOH2+ + H+ = P450FeO2+ por(*+) + H2O, is 0 kJ/mol. Although cytochrome P450FeOOH2+ and cytochrome P450FeO2+ por(*+) are approximately isoenergetic, they are likely to react at different rates with substrates and may yield different products. Homolysis of the hydroperoxo complex of cytochrome P450 to compound II and the hydroxyl radical, P450FeOOH2+ = P450FeO2+ + HO(*), is unfavorable (DeltaG degrees ' = +92 kJ/mol), as is the dissociation into HOO- and cytochrome P450Fe3+ (+73 kJ/mol). It is shown that the sum of the Gibbs energy of association for cytochrome P450Fe3+ with the hydroperoxo anion and the Gibbs energy for the one-electron reduction of cytochrome P450FeOOH2+, relative to NHE, is constant (-203 kJ/mol). While the estimated E degrees ' (P450FeO(2)2+, H+/P450FeOOH2+) = +0.93 V at pH 7 is larger than necessary to effect reduction of cytochrome P450FeO(2)2+, the magnitude of this electrode potential implies that the binding constant for cytochrome P450Fe3+ with hydrogen peroxide is ca. 3 x 106 M(-1) at pH 7. An association constant of this magnitude ensures that a fraction of cytochrome P450FeOOH2+ is available to form compound I or to react with substrates directly, while a larger one would imply that compound I is too weak an oxidant. In general, the energetics of the reduction of dioxygen to water determines the energetics of catalysis of hydroxylations by cytochrome P450. These results enable calibration of energy levels obtained for intermediates in the cytochrome P450 reaction cycle obtained by ab initio calculations and provide insights into the catalytic efficiency of cytochrome P450 and guidelines for the development of competent hydroxylation catalysts.     

Spectroscopy and electrochemistry of cytochrome P450 BM3-surfactant film assemblies

We report analyses of electrochemical and spectroscopic measurements on cytochrome P450 BM3 (BM3) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of BM3-DDAB films on silica slides reveal the characteristic low-spin FeIII heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 FeII-CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at approximately 420 nm is present. Infrared absorption spectra of the BM3 FeII-CO complex in DDAB dispersions feature a time-dependent shift of the carbonyl stretching frequency from 1950 to 2080 cm(-1). Voltammetry of BM3-DDAB films on graphite electrodes gave the following results: FeIII/II E(1/2) at -260 mV (vs SCE), approximately 300 mV positive of the value measured in solution; DeltaS degrees (rc), DeltaS degrees , and DeltaH degrees values for water-ligated BM3 in DDAB are -98 J mol(-1) K(-1), -163 J mol(-1) K(-1), and -47 kJ mol(-1), respectively; values for the imidazole-ligated enzyme are -8 J mol(-1) K(-1), -73 J mol(-1) K(-1), and -21 kJ mol(-1). Taken together, the data suggest that BM3 adopts a compact conformation within DDAB that in turn strengthens hydrogen bonding interactions with the heme axial cysteine, producing a P420-like species with decreased electron density around the metal center.     

Immobilization of cytochrome P-450 and electrochemical control of its activity

P-450_cam (camphor-induced cytochrome P-450) was immobilized on an indium tin oxide (ITO) electrode by polypyrrole and its activity was controlled electrochemically. The results showed that P-450_cam was immobilized on the ITO electrode without denaturing and the amount of P-450_cam could be easily controlled. When, the electric potential was swept repeatedly between 0.4 and 0 V, the remarkable decrease of oxygen in the reaction mixture solution was observed only in the presence of camphor. In addition, hydroxycamphor was detected only in the same system by means of gas chromatography/mass spectroscopy. These results suggested that immobilized P-450_cam catalyzed the hydroxylation of camphor by the supply of electron from the electrode. The effect of pH and ionic strength on the activity was examined, and it was found that the high activity expressed at the pH of 6.0 – 7.0 and KCl concentration of 0.1 – 0.2 M . The paper is the first report that P-450 enzyme activity could be controlled artificially. © 1998 John Wiley & Sons, Ltd.     

Electrochemistry of cytochrome P450 BM3 in sodium dodecyl sulfate films

Direct electrochemistry of the cytochrome P450 BM3 heme domain (BM3) was achieved by confining the protein within sodium dodecyl sulfate (SDS) films on the surface of basal-plane graphite (BPG) electrodes. Cyclic voltammetry revealed the heme FeIII/II redox couple at -330 mV (vs Ag/AgCl, pH 7.4). Up to 10 V/s, the peak current was linear with the scan rate, allowing us to treat the system as surface-confined within this regime. The standard heterogeneous rate constant determined at 10 V/s was estimated to be 10 s-1. Voltammograms obtained for the BM3-SDS-BPG system in the presence of dioxygen exhibited catalytic waves at the onset of FeIII reduction. The altered heme reduction potential of the BM3-SDS-graphite system indicates that SDS is likely bound in the enzyme active-site region. Compared to other P450-surfactant systems, we find redox potentials and electron-transfer rates that differ by approximately 100 mV and >10-fold, respectively, indicating that the nature of the surfactant environment has a significant effect on the observed heme redox properties.     

Screening of a minimal enriched P450 BM3 mutant library for hydroxylation of cyclic and acyclic alkanes

A minimal enriched P450 BM3 library was screened for the ability to oxidize inert cyclic and acyclic alkanes. The F87A/A328V mutant was found to effectively hydroxylate cyclooctane, cyclodecane and cyclododecane. F87V/A328F with high activity towards cyclooctane hydroxylated acyclic n-octane to 2-(R)-octanol (46% ee) with high regioselectivity (92%).     

Direct electrochemistry of cytochrome P450 in a biocompatible film composed of an epoxy polymer and acetylene black

We report on a composite matrix composed of epoxy copolymers P (GMA-co-MPC) and acetylene black that can be used to entrap cytochrome P450. The composite provides a biocompatible microenviroment and can substantially accelerate the electron transfer between the cytochrome P450 and the electrode. The electrochemical response is characterized by a pair of well-defined redox peaks for the heme Fe(II/III) redox couples were observed at ?483?mV (vs. SCE). The immobilized cytochrome P450 exhibits excellent electrocatalytical activity to diethylstilbestrol (DES). The amperometric response varied linearly with the concentration of DES in the 0.2 to 2.8?μM concentration range. The biosensor displays a detection limit 5.9?×?10^-8?mol?L^-1 and thus represents a promising candidate for studying the electrochemistry of cytochrome P450s and its sensing applications.

Spectroscopic and Electrochemical Characterization of Cytochrome P450st‐DDAB Films on a Plastic‐Formed Carbon Electrode

We have studied the characterization of thermophilic cytochrome P450 (P450st)‐didodecyldimethylammonium bromide (DDAB) films by using UV‐vis absorption, resonance Raman spectroscopy, and electrochemical methods. The observed Raman spectrum indicated near‐native conformation of the heme iron in DDAB film on the surface of a glass slide, while on the surface of a plastic‐formed carbon (PFC) electrode, the conformation of P450st‐DDAB was very similar to that of heme‐DDAB film, suggesting the release of heme from P450st in DDAB films on PFC electrodes. When NaBr was added as salt to the casting solution, the result of Raman spectrum indicated near‐native conformation of P450st in DDAB film even on the PFC electrode, but no redox potential of P450st which has near native structure was observed. This study suggests the essential experimental conditions when working with heme protein‐DDAB films as, in some cases, heme iron from proteins is released on the surface of the electrode.     

Electrochemical decomposition of layer-by-layer thin films composed of TEMPO-modified poly(acrylic acid) and poly(ethyleneimine)

The decomposition of layer-by-layer (LbL) thin films composed of 2,2,6,6-tetramethylpiperidine-1-oxyl free radical-appended poly(acrylic acid) (TEMPO-PAA) and poly(ethylenimine) (PEI) was studied by using a quartz crystal microbalance (QCM) and cyclic voltammetry. The electrode potential of the (PEI/TEMPO-PAA)₄/PEI film-coated Au resonator was scanned from +0.2 to +0.8 V vs Ag/AgCl. The CV showed that the oxidation peak current decreased as the number of scans increased. The change in the resonance frequency of the QCM increased after electrolysis, indicating that the film was decomposed by electrolysis. The positive charges originating from the oxoammonium ions probably destabilized the (PEI/TEMPO-PAA)₄/PEI film. Furthermore, the release of 5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonic acid (TPPS) from TPPS-loaded (PEI/TEMPO-PAA)₄/PEI-coated ITO electrodes was investigated. TPPS was released at electrode potentials greater than +0.6 V by the decomposition of the film. The results suggest that TEMPO-PAA/PEI LbL films are suitable for electrochemically controlled drug delivery systems.     

Efficient bioelectronic actuation of the natural catalytic pathway of human metabolic cytochrome P450s

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein 30 nm nanometer-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations supports a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.     

Evidence for basic ferryls in cytochromes P450

Using a combination of M?ssbauer spectroscopy and density functional calculations, we have determined that the ferryl forms of P450(BM3) and P450cam are protonated at physiological pH. Density functional calculations were performed on large active-site models of these enzymes to determine the theoretical M?ssbauer parameters for the ferryl and protonated ferryl (Fe(IV)OH) species. These calculations revealed a significant enlargement of the quadrupole splitting parameter upon protonation of the ferryl unit. The calculated quadrupole splittings for the protonated and unprotonated ferryl forms of P450(BM3) are DeltaE(Q) = 2.17 mm/s and DeltaE(Q) = 1.05 mm/s, respectively. For P450cam, they are DeltaE(Q) = 1.84 mm/s and DeltaE(Q) = 0.66 mm/s, respectively. The experimentally determined quadrupole splittings (P450(BM3), DeltaE(Q) = 2.16 mm/s; P450cam, DeltaE(Q) = 2.06 mm/s) are in good agreement with the values calculated for the protonated forms of the enzymes. Our results suggest that basic ferryls are a natural consequence of thiolate-ligated hemes.     

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam

Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam). Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis. (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.     

Immobilization of Cytochrome P-450 and its Application in Ehzikb Electrodes

Abstract

For application in enzyme electrodes liver microsomal cytochrome P-450 was immobilized in a membraneous form. The immobilization yielded 60% of activity and did not impair the functional stability of the enzyme. By coimmobilization of glucose oxidase with P-450 the cofactor NADPH could be replaced by H₂O₂ formed from the enzymatic glucose oxidation. Fixed to a graphite electrode the obtained preparations were employed for quantitative substrate analysis. The P-450 substrate aniline was measured by anodic oxidation of its hydroqlation product at +250mV. A linear dependence of: the current on aniline concentration up to 0.5mM was obtained.     

Interfacing cytochrome c to electrodes with a DNA – carbon nanotube composite film

Multi-walled carbon nanotube (MWCNT) is successfully immobilized on the surface of platinum electrode by mixing with DNA. The DNA/MWCNT modified electrodes are characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Further research indicates that cytochrome c can strongly adsorbed on the surface of the modified electrode, and forms an approximate monolayer. The immobilized MWCNT can promote the redox of horse heart cytochrome c which gives reversible redox peaks with a formal potential of 81 mV vs SCE.