Catalytic Cycle Of Cytochrome P450 Research Articles

Alternative Electron Sources for Cytochrome P450s Catalytic Cycle: Biosensing and Biosynthetic Application

The functional significance of cytochrome P450s (CYP) enzymes is their ability to catalyze the biotransformation of xenobiotics and endogenous compounds. P450 enzymes catalyze regio- and stereoselective oxidations of C-C and C-H bonds in the presence of oxygen as a cosubstrate. Initiation of cytochrome P450 catalytic cycle needs an electron donor (NADPH, NADH cofactor) in nature or alternative artificial electron donors such as electrodes, peroxides, photo reduction, and construction of enzymatic “galvanic couple”. In our review paper, we described alternative “handmade” electron sources to support cytochrome P450 catalysis. Physical-chemical methods in relation to biomolecules are possible to convert from laboratory to industry and construct P450-bioreactors for practical application. We analyzed electrochemical reactions using modified electrodes as electron donors. Electrode/P450 systems are the most analyzed in terms of the mechanisms underlying P450-catalyzed reactions. Comparative analysis of flat 2D and nanopore 3D electrode modifiers is discussed. Solar-powered photobiocatalysis for CYP systems with photocurrents providing electrons to heme iron of CYP and photoelectrochemical biosensors are also promising alternative light-driven systems. Several examples of artificial “galvanic element” construction using Zn as an electron source for the reduction of Fe3+ ion of heme demonstrated potential application. The characteristics, performance, and potential applications of P450 electrochemical systems are also discussed.

Electrochemical generation of high-valent oxo-manganese complexes featuring an anionic N5 ligand and their role in O-O bond formation.

Generation of high-valent oxomanganese complexes through controlled removal of protons and electrons from low-valent congeners is a crucial step toward the synthesis of functional analogues of the native oxygen evolving complex (OEC). In-depth studies of the water oxidation activity of such biomimetic compounds help in understanding the mechanism of O-O bond formation presumably occurring in the last step of the photosynthetic cycle. Scarce reports of reactive high-valent oxomanganese complexes underscore the impetus for the present work, wherein we report the electrochemical generation of the non-heme oxomanganese(IV) species [(dpaq)MnIV(O)]+ (2) through a proton-coupled electron transfer (PCET) process from the hydroxomanganese complex [(dpaq)MnIII(OH)]ClO4 (1). Controlled potential spectroelectrochemical studies of 1 in wet acetonitrile at 1.45 V vs. NHE revealed quantitative formation of 2 within 10 min. The high-valent oxomanganese(IV) transient exhibited remarkable stability and could be reverted to the starting complex (1) by switching the potential to 0.25 V vs. NHE. The formation of 2via PCET oxidation of 1 demonstrates an alternate pathway for the generation of the oxomanganese(IV) transient (2) without the requirement of redox-inactive metal ions or acid additives as proposed earlier. Theoretical studies predict that one-electron oxidation of [(dpaq)MnIV(O)]+ (2) forms a manganese(V)-oxo (3) species, which can be oxidized further by one electron to a formal manganese(VI)-oxo transient (4). Theoretical analyses suggest that the first oxidation event (2 to 3) takes place at the metal-based d-orbital, whereas, in the second oxidation process (3 to 4), the electron eliminates from an orbital composed of equitable contribution from the metal and the ligand, leaving a single electron in the quinoline-dominant orbital in the doublet ground spin state of the manganese(VI)-oxo species (4). This mixed metal-ligand (quinoline)-based oxidation is proposed to generate a formal Mn(VI) species (4), a non-heme analogue of the species 'compound I', formed in the catalytic cycle of cytochrome P-450. We propose that the highly electrophilic species 4 catches water during cyclic voltammetry experiments and results in O-O bond formation leading to electrocatalytic oxidation of water to hydrogen peroxide.

Regression Modeling for the Prediction of Hydrogen Atom Transfer Barriers in Cytochrome P450 from Semi‐empirically Derived Descriptors

AbstractThe calculation of hydrogen atom transfer (HAT) barriers within the cytochrome P450 catalytic cycle is of central importance for the prediction of metabolites formed from medicinally relevant compounds. We report the accurate estimation of hydrogen atom transfer barriers using inexpensive descriptors computed for a panel of 21 compounds. By a simple univariate linear regression between barriers previously computed using density functional theory (DFT) and newly computed “frozen radical” bond dissociation energies using the GFN2‐xTB method, a mean absolute error of 1 kcal mol−1 is achieved. Other affordable levels of theory are studied to assess differences in performance and computational cost. Multiple linear regression incorporating additional descriptors using GFN2‐xTB is shown to predict HAT barriers with mean absolute errors of 0.82 kcal mol−1. With computing times in milliseconds on modest computing hardware, this systematic approach is accessible and extensible to large scale screening workflows.

Biomimetic Metabolism of Kaurenoic Acid Validated by Microsomal Reactions

The cytochrome P450 is a remarkable family of enzymes with widespread occurrence in animals and plants. In humans, they catalyze phase I metabolism reactions, such as oxidations and hydroxylations, playing a significant role in xenobiotics detoxification. Although mechanistic details of the catalytic cycle of cytochrome P450 are known, and human isoforms have their crystal structure solved, mimicking cytochrome P450 reactions using in vitro systems is still challenging. For this reason, biological assays using isolated enzymes or microsomal fractions are typically required to study the oxidative metabolism of new drugs before approval by regulatory agencies. Here we demonstrate that a series of metalloporphyrin catalysts generate the same oxidative product in vitro as a liver microsomal assay. The bioactive diterpene kaurenoic acid was chosen as a substrate model because it is a component of widely used phytomedicines in Brazil, such as guaco syrups and copaiba waxes. We evaluated the influence of reaction parameters such as catalyst type, oxygen donor type, solvent, and catalyst concentration on substrate consumption. Three products had their structures proposed by mass spectrometry, and among them, a dihydroxylated oxidation product was detected as a major metabolite after liver microsomal biotransformation and using porphyrin catalysts. We envision that the use of metalloporphyrins could be a useful strategy for biomimetic oxidation of kaurenic terpenes.

One small step for cytochrome P450 in its catalytic cycle, one giant leap for enzymology

The intermediates operating in the cytochrome P450 catalytic cycle have been investigated for more than half a century, fascinating many enzymologists. Each intermediate has its unique role to carry out diverse oxidations. Natural time course of the catalytic cycle is quite fast, hence, not all of the reactive intermediates could be isolated during physiological catalysis. Different high-valent iron intermediates have been proposed as primary oxidants: the candidates are compound 0 (Cpd 0, [FeOOH][Formula: see text]P450) and compound I (Cpd I, Fe(IV)[Formula: see text]O por[Formula: see text]P450). Among them, the role of Cpd I in hydroxylation is fairly well understood due the discovery of the peroxide shunt. This review endeavors to put the outstanding research efforts conducted to isolate and characterize the intermediates together. In addition to spectral features of each intermediate in the catalytic cycle, the oxidizing powers of Cpd 0 and Cpd I will be discussed along with most recent scientific findings.

Light-controlled switching of the spin state of iron(III)

Controlled switching of the spin state of transition metal ions, particularly of FeII and FeIII, is a prerequisite to achieve selectivity, efficiency, and catalysis in a number of metalloenzymes. Here we report on an iron(III) porphyrin with a photochromic axial ligand which, upon irradiation with two different wavelengths reversibly switches its spin state between low-spin (S = 1/2) and high-spin (S = 5/2) in solution (DMSO-acetone, 2:598). The switching efficiency is 76% at room temperature. The system is neither oxygen nor water sensitive, and no fatigue was observed after more than 1000 switching cycles. Concomitant with the spin-flip is a change in redox potential by ~60 mV. Besides serving as a simple model for the first step of the cytochrome P450 catalytic cycle, the spin switch can be used to switch the spin-lattice relaxation time T1 of the water protons by a factor of 15.

Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron

Cytochrome P450cam (CYP101Fe3+) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe3+ by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO2]2+ Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH]2+ (compound 0) accumulates within 5 μs and decays rapidly to CYP101Fe3+, with a k440 nm of 9.6 × 104 s-1 All processes are complete within 40 μs at 4 °C. Importantly, no transient absorbance bands could be assigned to CYP101[FeO2+por•+] (compound 1) or CYP101[FeO2+] (compound 2). However, indirect evidence for the involvement of compound 1 was obtained from the kinetics of formation and decay of a tyrosyl radical. 5-Hydroxycamphor was formed quantitatively, and the catalytic activity of the enzyme was not impaired by exposure to radiation during the pulse radiolysis experiment. The rapid decay of compound 0 enabled calculation of the limits for the Gibbs activation energies for the conversions of compound 0 → compound 1 → compound 2 → CYP101Fe3+, yielding a ΔG‡ of 45, 39, and 39 kJ/mol, respectively. At 37 °C, the steps from compound 0 to the iron(III) state would take only 4 μs. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time.

Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle.

The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, six-coordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme-thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, six-coordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80-90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.

ChemInform Abstract: Redox Cycling in the Activation of Peroxides by Iron Porphyrin and Manganese Complexes. ′Catching′ Catalytic Active Intermediates

AbstractReview: 261 refs.

Characterizing the Intermediates Compound I and II in the Cytochrome P450 Catalytic Cycle with Nonlinear X-ray Spectroscopy: A Simulation Study.

Cytochrome P450 enzymes are an important family of biocatalysts that oxidize chemically inert CH bonds. There are many unresolved questions regarding the catalytic reaction intermediates, in particular P450 Compound I (Cpd-I) and II (Cpd-II). By using simple molecular models, we simulate various X-ray spectroscopy signals, including X-ray absorption near-edge structure (XANES), resonant inelastic X-ray scattering (RIXS), and stimulated X-ray Raman spectroscopy (SXRS) of the low- and high-spin states of Cpd-I and II. Characteristic peak patterns are presented and connected to the corresponding electronic structures. These X-ray spectroscopy techniques are complementary to more conventional infrared and optical spectroscopy and they help to elucidate the evolving electronic structures of transient species along the reaction path.

Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation.

Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [10(3) M(-1) s(-1)], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.

Disruption of biomolecule function by nanoparticles: How do gold nanoparticles affect Phase I biotransformation of persistent organic pollutants?

The potential influence of nanoparticles on cytochrome P-450 (CYP) isozyme mediated Phase I biotransformation of persistent organic pollutants (POPs) in vitro was investigated using citrate-capped gold nanoparticles (AuNPs) and 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95) as the probe nanoparticle and compound, respectively. AuNPs affected the biotransformation activity of rat CYP2B1 and changed the atropisomeric composition of PCB 95, depending on the incubation time and the AuNP concentration. Electrostatic repulsion between citrate-coated AuNPs and rat CYP2B1 may influence the active conformation of the isozyme and consequently affect its activity and stereoselectivity. In addition, the effects of AuNPs on rat CYP2B1 activity also appeared to be through interference with the CYP catalytic cycle’s electron transfer chain. Incubations with AuNPs had a decline in buffer conductance and an absorbance band red shift of AuNPs, from electrostatic interactions of K+ with negatively-charged AuNP aggregates. These ionic strength changes affected the formation rate of nicotinamide adenine dinucleotide phosphate, which provides electrons for the oxidative reaction cycle, and the biotransformation activity and stereoselectivity of CYP. This study suggests that charged nanoparticles may be able to alter the functions of biomolecules directly, by electrostatic interaction, or indirectly, by changes to the surrounding ionic strength. These factors should be taken into account for further understanding and prediction of the environmental behavior and fate of POPs and nanoparticles.

Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4 reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase.

Peroxidase-like activity of uncoupled cytochrome P450: Studies with bilirubin and toxicological implications of uncoupling

The NADPH-dependent consumption of O2 by cytochrome P450 BM3 was stimulated by either laurate or perfluorolaurate, but the NADPH/O2 molar consumption ratios were approximately 1 and 2, respectively, indicating that perfluorolaurate does not become oxygenated by BM3 and oxygen undergoes full reduction to water. The nature of this catalytic cycle uncoupled to hydroxylation was explored using bilirubin as a molecular probe. During uncoupling with perfluorolaurate bilirubin was degraded and stimulated O2 uptake by an approximately equimolar amount. No stimulation of oxygen uptake was caused by bilirubin in presence of NADPH alone or in presence of laurate together with NADPH; under these conditions little degradation of bilirubin was observed. Mesobilirubin was also degraded during uncoupling with perfluorolaurate, whereas biliverdin (which lacks the central methene bridge present in rubins) was unaffected. It is suggested that the CYP ferryl oxygen species abstracts a hydrogen atom from the central methene bridge of bilirubin to generate a radical, which is further dehydrogenated to biliverdin or else binds O2 and undergoes fragmentation. We conclude that the uncoupled catalytic cycle of cytochrome P450 has properties resembling those of a peroxidase and that bilirubin is rapidly oxidized as a peroxidase substrate. The potential toxicological significance of cytochrome P450 uncoupling is considered.

Quantum mechanically derived AMBER‐compatible heme parameters for various states of the cytochrome P450 catalytic cycle

Molecular mechanics (MM) methods are computationally affordable tools for screening chemical libraries of novel compounds for sites of P450 metabolism. One challenge for MM methods has been the absence of a consistent and transferable set of parameters for the heme within the P450 active site. Experimental data indicate that mammalian P450 enzymes vary greatly in the size, architecture, and plasticity of their active sites. Thus, obtaining X-ray-based geometries for the development of accurate MM parameters for the major classes of hepatic P450 remains a daunting task. Our previous work with preliminary gas-phase quantum mechanics (QM)-derived atomic partial charges greatly improved the accuracy of docking studies of raloxifene to CYP3A4. We have therefore developed and tested a consistent set of transferable MM parameters based on gas-phase QM calculations of two model systems of the heme-a truncated (T-HM) and a full (F-HM) for four states of the P450 catalytic cycle. Our results indicate that the use of the atomic partial charges from the F-HM further improves the accuracy of docked predictions for raloxifene to CYP3A4. Different patterns for substrate docking are also observed depending on the choice of heme model and state. Newly parameterized heme models are tested in implicit and explicitly solvated MD simulations in the absence and presence of enzyme structures, for CYP3A4, and appear to be stable on the nanosecond simulation timescale. The new force field for the various heme states may aid the community for simulations of P450 enzymes and other heme-containing enzymes.

Propiconazole increases reactive oxygen species levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450 enzyme mediated process

Propiconazole induces hepatocellular carcinomas and hepatocellular adenomas in mice and promotes liver tumors in rats. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicated that propiconazole induced oxidative stress. Here we sought to identify the source of the reactive oxygen species (ROS) induced by propiconazole using both AML12 immortalized mouse hepatocytes in culture and liver tissues from mice. We also sought to further characterize the nature and effects of ROS formation induced by propiconazole treatment in mouse liver. ROS was induced in AML12 cells by propiconazole as measured by fluorescence detection and its formation was ameliorated by N-acetylcysteine. Propiconazole induced glutathione- S-transferase (GSTα) protein levels and increased the levels of thiobarbituric acid reactive substances (TBARS) in AML12 cells. The TBARS levels were decreased by diphenylene iodonium chloride (DPIC), a cytochrome P450 (CYP) reductase inhibitor revealing the role of CYPs in ROS generation. It has been previously reported that Cyp2b and Cyp3a proteins were induced in mouse liver by propiconazole and that Cyp2b and Cyp3a proteins undergo uncoupling of their CYP catalytic cycle releasing ROS. Therefore, salicylic acid hydroxylation was used as probe for ROS formation using microsomes from mice treated with propiconazole. These studies showed that levels of 2,3-dihydroxybenzoic acid (an ROS derived metabolite) were decreased by ketoconazole, melatonin and DPIC. In vivo, propiconazole increased hepatic malondialdehyde levels and GSTα protein levels and had no effect on hepatic catalase or superoxide dismutase activities. Based on these observations we conclude that propiconazole induces ROS in mouse liver by increasing CYP protein levels leading to increased ROS levels. Our data also suggest that propiconazole induces the hydroxyl radical as a major ROS form.

Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis

Cytochrome P450(BSβ), a H(2)O(2)-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid-base residue around the heme, which is indispensable for the efficient generation of the active species using H(2)O(2). On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450(BSβ), it was suggested that the role of the general acid-base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450(BSβ) can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a "decoy molecule". As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid-base catalyst. This result further confirms that the role of the acid-base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450(BSβ).

Common system setup for the entire catalytic cycle of cytochrome P450cam in quantum mechanical/molecular mechanical studies

We describe a system setup that is applicable to all species in the catalytic cycle of cytochrome P450(cam). The chosen procedure starts from the X-ray coordinates of the ferrous dioxygen complex and follows a protocol that includes the careful assignment of protonation states, comparison between different conceivable hydration schemes, and system preparation through a series of classical minimizations and molecular dynamics (MD) simulations. The resulting setup was validated by quantum mechanical/molecular mechanical (QM/MM) calculations on the resting state, the pentacoordinated ferric and ferrous complexes, Compound I, the transition state and hydroxo intermediate of the C--H hydroxylation reaction, and the product complex. The present QM/MM results are generally consistent with those obtained previously with individual setups. Concerning hydration, we find that saturating the protein interior with water is detrimental and leads to higher structural flexibility and catalytically inefficient active-site geometries. The MD simulations favor a low water density around Asp251 that facilitates side chain rotation of protonated Asp251 during the conversion of Compound 0 to Compound I. The QM/MM results for the two preferred hydration schemes (labeled SE-1 and SE-4) are similar, indicating that slight differences in the solvation close to the active site are not critical as long as camphor and the crystallographic water molecules preserve their positions in the experimental X-ray structures.

Stochastic Simulations of the Cytochrome P450 Catalytic Cycle

Cytochrome P450 enzymes involve a complex reaction cycle which has been described, for the first time, by a stochastic simulation of the system in the present work. A series of models are developed for a basic catalytic cycle, employing a set of microscopic rate constants for the oxidation of p-alkoxyacylanilides catalyzed by the cytochrome P450 1A2. By analyzing the effects of low concentrations of enzyme and substrate on the system, and the dependence of the system on several rate constants, it is discovered that the system evolves along relatively stable patterns from its initial state, as indicated from different runs of simulations. Strong fluctuations appear at the entrance and exit of the pathway, with very weak fluctuations in the middle sections of the cycle. Although noises are apparent when the reactant populations are very low, basically, the fundamental feature of the P450 cycle based on a microscopic view is that it is deterministic in nature. Meanwhile, the mathematical models we developed are qualitatively validated by a comparison with those experimental results of the P450 cycle. The findings of this work will be helpful for a further deeper understanding of the catalytic mechanism of cytochrome P450 enzymes.

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4: SUBSTRATE DEPENDENCE OF FORMATION AND DECAY KINETICS

The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of approximately 30 ms at 5 degrees C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.