Mechanistic studies on the binding of nitric oxide to a synthetic heme-thiolate complex relevant to cytochrome p450

The synthetic heme-thiolate complex (SR) in methanol binds nitric oxide (k(on) = (2.7 +/- 0.2) x10(6) M(-)(1) s(-)(1) at 25 degrees C) to form SR(NO). The binding of NO to the SR complex in a noncoordinating solvent, such as toluene, was found to be almost 3 orders of magnitude faster than that in methanol. The activation parameters DeltaH(), DeltaS(), and DeltaV() for the formation of SR(NO) in methanol are consistent with the operation of a limiting dissociative mechanism, dominated by dissociation of methanol in SR(MeOH). In the presence of an excess of NO, the formation of SR(NO) is followed by subsequent slower reactions. The substantially negative activation entropy and activation volume values found for the second observed reaction step support an associative mechanism which involves attack of a second NO molecule on the thiolate ligand in the initially formed SR(NO) complex. The following slower reactions are strongly accelerated by a large excess of NO or by the presence of NO(2)(-) in the SR/NO reaction mixture. They can be accounted for in terms of dynamic equilibria between higher nitrogen oxides (NO(x)()) and reactive SR species, which lead to the formation of a nitrosyl-nitrite complex of SR(Fe(II)) as the final product. This finding is clearly supported by laser flash photolysis studies on the SR/NO reaction mixture, which do not reveal simple NO photolabilization from SR(Fe(III))(NO), but rather involve the generation of at least three photoinduced intermediates decaying with different rate constants to the starting material. The species formed along the proposed reaction pathways were characterized by FTIR and EPR spectroscopy. The results are discussed in terms of their relevance for the biological function of cytochrome P450 enzymes and in context of results for the reaction of NO with imidazole- and thiolate-ligated iron(III) hemoproteins.     

Low-temperature rapid-scan detection of reactive intermediates in epoxidation reactions catalyzed by a new enzyme mimic of cytochrome p450

The use of synthetic iron(III) porphyrins as models for heme-type catalysts in biomimetic cytochrome P450 research has provided valuable information on the nature and reactivity of intermediates produced in the "peroxide shunt" pathway. This article reports spectroscopic detection of reactive intermediates formed in the epoxidation reaction of cis-stilbene with m-chloroperoxybenzoic acid catalyzed by a new mimic of cytochrome P450 with a substituted RSO3- group (1). The application of low-temperature rapid-scan stopped-flow techniques enabled the determination of equilibrium and rate constants for the formation and decay of all intermediates in the catalytic cycle of 1, including the rate constant for the formation (1*+)FeIV=O and for oxygen transfer to the substrate. Noteworthy, the reaction of (1*+)FeIV=O with cis-stilbene leads to an almost complete re-formation (95%) of the starting complex 1. The results show that complex 1 is a valuable catalyst with promising properties for further applications in a biomimetic approach toward mimicking oxygenation reactions of cytochrome P450.     

Nitric oxide binding to ferric cytochrome P450: a computational study

The interaction between nitric oxide (NO) and the active site of ferric cytochrome P450 was studied by means of density functional theory (DFT), at the generalized gradient approximation level, and of the SAM1 semiempirical method. The electrostatic effects of the protein environment were included in our DFT scheme by using a hybrid quantum classical approach. The active-site model consisted of an iron(III) porphyrin, the adjacent cysteine residue, and one coordinated water molecule. For this system, spin populations and relative energies for selected spin states were computed. Interestingly, the unpaired electron density, the HOMO, and the LUMO were found to be highly localized on the iron and in an appreciable extent on the sulfur coordinated to the metal. This provides central information about the reactivity of nitric oxide with the active site. Since the substitution of a molecule of H2O by NO has been proposed as being responsible for the inhibition of the cytochrome in the presence of nitric oxide, we have analyzed the thermodynamic feasibility of the ligand exchange process. The structure of the nitrosylated active site was partially optimized using SAM1. A low-spin ground state was obtained for the nitrosyl complex, with a linear Fe-N-O angle. The trends found in Fe-N-O angles and Fe-N lengths of the higher energy spin states provided a notable insight into the electronic configuration of the complex within the framework of the Enemark and Feltham formalism. In relation to the protein environment, it was assessed that the electrostatic field has significant effects on several computed properties. However, in both vacuum and protein environments, the ligand exchange reaction turned out to be exergonic and the relative orders of spin states of the relevant species were the same.     

Mechanistic studies on the reversible binding of nitric oxide to metmyoglobin.

The ferriheme protein metmyoglobin (metMb) in buffer solution at physiological pH 7.4 reversibly binds the biomessenger molecule nitric oxide to yield the nitrosyl adduct (metMb(NO)). The kinetics of the association and dissociation processes were investigated by both laser flash photolysis and stopped-flow kinetics techniques at ambient and high pressure, in three laboratories using several different sources of metMb. The activation parameters DeltaH, DeltaS, and DeltaV were calculated from the kinetic effects of varying temperature and hydrostatic pressure. For the "on" reaction of metMb plus NO, reasonable agreement was found between the various techniques with DeltaH(on), DeltaS(on), and DeltaV(on) determined to have the respective values approximately 65 kJ mol(-1), approximately 60 J mol(-1) K(-1), and approximately 20 cm(3) mol(-1). The large and positive DeltaS and DeltaV values are consistent with the operation of a limiting dissociative ligand substitution mechanism whereby dissociation of the H(2)O occupying the sixth distal coordination site of metMb must precede formation of the Fe-NO bond. While the activation enthalpies of the "off" reaction displayed reasonable agreement between the various techniques (ranging from 68 to 83 kJ mol(-1)), poorer agreement was found for the DeltaS(off) values. For this reason, the kinetics for the "off" reaction were determined more directly via NO trapping experiments, which gave the respective activation parameters DeltaH(off) = 76 kJ mol(-1), DeltaS(off) = approximately 41 J mol(-1) K(-1), and DeltaV(off) = 20 cm(3) mol(-1)), again consistent with a limiting dissociative mechanism. These results are discussed in reference to other investigations of the reactions of NO with both model systems and metalloproteins.     

Insights on cytochrome p450 enzymes and inhibitors obtained through QSAR studies

The cytochrome P450 (CYP) superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR), and three-dimensional quantitative structure activity relationships (3D-QSAR) represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions.     

Spectra and kinetic studies of the compound I derivative of cytochrome P450 119

The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which was first prepared by reaction of the resting enzyme with peroxynitrite. The UV-vis spectrum of the Compound I species contained an asymmetric Soret band that could be resolved into overlapping transitions centered at approximately 367 and approximately 416 nm and a Q band with lambda(max) approximately 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxidized (alkene oxidation) and hydroxylated (C-H oxidation) products, as demonstrated by product studies and oxygen-18 labeling studies. The kinetics of oxidations by CYP119 Compound I were measured directly; the reactions included hydroxylations of benzyl alcohol, ethylbenzene, Tris buffer, lauric acid, and methyl laurate and epoxidations of styrene and 10-undecenoic acid. Apparent second-order rate constants, equal to the product of the equilibrium binding constant (K(bind)) and the first-order oxidation rate constant (k(ox)), were obtained for all of the substrates. The oxidations of lauric acid and methyl laurate displayed saturation kinetic behavior, which permitted the determination of both K(bind) and k(ox) for these substrates. The unactivated C-H positions of lauric acid reacted with a rate constant of k(ox) = 0.8 s(-1) at room temperature. The CYP119 Compound I derivative is more reactive than model Compound I species [iron(IV)-oxo porphyrin radical cations] and similar in reactivity to the Compound I derivative of the heme-thiolate enzyme chloroperoxidase. Kinetic isotope effects (kH/kD) for oxidations of benzyl alcohol and ethylbenzene were small, reflecting the increased reactivity of the Compound I derivative in comparison to models. Nonetheless, CYP119 Compound I apparently is much less reactive than the oxidizing species formed in the P450 cam reaction cycle. Studies of competition kinetics employing CYP119 activated by hydrogen peroxide indicated that the same oxidizing transient is formed in the photochemical reaction and in the hydrogen peroxide shunt reaction.     

Resonance surface plasmon spectroscopy: low molecular weight substrate binding to cytochrome p450

A new detection mechanism has been developed for low molecular weight substrate binding to heme proteins based on resonance localized surface plasmon spectroscopy. Cytochrome P450 has strong electronic transitions in the visible wavelength region. Upon binding of a substrate molecule (e.g., camphor), the absorption band of cytochrome P450 shifts to shorter wavelength. The event of camphor binding to a nanoparticle surface modified with cytochrome P450 protein receptors is monitored using UV-vis spectroscopy. It is observed for the first time that the binding of the substrate molecules to the protein receptor induces a blue-shift in the localized surface plasmon resonance (LSPR) of the nanosensors. The coupling between the molecular resonance of the substrate-free and substrate-bound cytochrome P450 proteins and the nanoparticles' LSPR leads to a highly wavelength-dependent LSPR response. When the LSPR of the nanoparticles is located at a wavelength distant from the cytochrome P450 resonance, an average of approximately 19 nm red-shift is observed upon cytochrome P450 binding to the nanoparticles and a approximately 6 nm blue-shift is observed upon camphor binding However, this response is significantly amplified approximately 3 to 5 times when the LSPR of the nanoparticles is located at a slightly longer wavelength than the cytochrome P450 resonance, that is, a 66.2 nm red-shift upon cytochrome P450 binding and a 34.7 nm blue-shift upon camphor binding. This is the first example of the detection of small molecules binding to a protein modified nanoparticle surface on the basis of LSPR.     

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide prodrug

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide (NO) prodrug, in an overall yield of 26% in four steps from prolinol is reported; the previously published yield of this transformation was 1%. Using this revised strategy, the sarcosine analogue (14) of V-PROLI/NO was prepared. Finally, the methyl ester of V-PROLI/NO (15) was found to be an esterase-activated prodrug form of V-PROLI/NO.     

Time-resolved gas-phase kinetic and quantum chemical studies of the reaction of silylene with nitric oxide

Time-resolved kinetic studies of the reaction of silylene, SiH2, generated by laser flash photolysis of phenylsilane, have been carried out to obtain rate constants for its bimolecular reaction with NO. The reaction was studied in the gas phase over the pressure range 1-100 Torr in SF6 bath gas at five temperatures in the range 299-592 K. The second-order rate constants at 10 Torr fitted the Arrhenius equation log(k/cm3 molecule(-1) s(-1)) = (-11.66 +/- 0.01) + (6.20 +/- 0.10 kJ mol(-1))/RT ln 10 The rate constants showed a variation with pressure of a factor of ca. 2 over the available range, almost independent of temperature. The data could not be fitted by RRKM calculations to a simple third body assisted association reaction alone. However, a mechanistic model with an additional (pressure independent) side channel gave a reasonable fit to the data. Ab initio calculations at the G3 level supported a mechanism in which the initial adduct, bent H2SiNO, can ring close to form cyclo-H2SiNO, which is partially collisionally stabilized. In addition, bent H2SiNO can undergo a low barrier isomerization reaction leading, via a sequence of steps, ultimately to dissociation products of which the lowest energy pair are NH2 + SiO. The rate controlling barrier for this latter pathway is only 16 kJ mol(-1) below the energy of SiH2 + NO. This is consistent with the kinetic findings. A particular outcome of this work is that, despite the pressure dependence and the effects of the secondary barrier (in the side reaction), the initial encounter of SiH2 with NO occurs at the collision rate. Thus, silylene can be as reactive with odd electron molecules as with many even electron species. Some comparisons are drawn with the reactions of CH2 + NO and SiCl2 + NO.     

Thermodynamic and kinetic aspects of metal binding to the histidine-rich protein, Hpn

The histidine-rich protein, Hpn, binds to essential metals Ni2+, Cu2+, Zn2+ and a therapeutic metal Bi3+ with the in vitro affinities in the order of Cu2+ > Ni2+ > Bi3+ > Zn2+. In contrast, the in vivo (in E. coli) protection by the protein is in the order of Ni2+ > Bi3+ > Cu2+ approximately Zn2+. The release of Ni2+ from the protein follows a two-step process consisting of a rapidly established equilibrium and subsequently a rate-determining step (dissociation of Hpn-Ni...EDTA to Ni-EDTA). Our work suggests the nickel storage and homeostasis in H. pylori as the primary role of Hpn.     

Reorganization of immobilized horse and yeast cytochrome c induced by pH changes or nitric oxide binding

The redox properties of horse and yeast cytochrome c electrostatically immobilized on carboxylic acid-terminated self-assembled monolayers (SAMs) have been determined over a broad pH range (pH 3.5-8) in the absence and presence of nitric oxide. Below pH 6, both proteins exhibit comparable midpoint potentials, coverages, and electron-transfer rate constants, which suggests that they are adsorbed on the SAM in a similar fashion. Above pH 6, a sharp decrease in electron-transfer rate constants is observed for immobilized yeast cytochrome c, which is indicative of a change in the electron tunneling pathway between the heme and the electrode and hence suggests that the protein reorients on the surface. Such a decrease is not observed for horse cytochrome c and therefore must be related to the specific charge distribution on yeast cytochrome c. Apart from the charge distribution on the protein, the reorientation also seems to be related to the charge on the SAM surface. The presence of nitric oxide causes a decrease in electron-transfer rate constants of both yeast and horse cytochrome c at low pH. This is probably due to the fact that nitric oxide induces a conformational change of the protein and also changes the reorganization energy for electron transfer.     

Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian cytochrome P450

Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates: bromocriptine (BC), erythromycin (ERY), and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses as compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome.     

Formation of the aureothin tetrahydrofuran ring by a bifunctional cytochrome p450 monooxygenase

The polyketide antibiotic aureothin, produced by Streptomyces thioluteus, is equipped with a rare exomethylene-tetrahydrofuran moiety. Cloning, heterologous expression, and inactivation experiments reveal that AurH, a bifunctional cytochrome P450 monooxygenase, is required and sufficient for the stereoselective synthesis of the furan ring, involving the subsequent formation of two new C-O bonds.     

Fluorescence detection of ligand binding to labeled cytochrome P450 BM3

The cytochrome P450 superfamily of monoxygenases are highly relevant for pharmaceutical, environmental and biocatalytical applications. The binding of a substrate to their catalytic site is usually detectable by UV-vis spectroscopy as a low-to-high spin state transition of the heme iron. However, the discovery of potential new substrates is limited by the fact that some compounds do not cause the typical spin-shift even if they are oxidised by P450 enzymes. Here we report a fluorescence-based method able to detect the binding of such substrates to the heme domain of cytochrome P450 BM3 from Bacillus megaterium. The protein was labeled with the fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-Yl)-ethylenediamine (IANBD). Arachidonic and lauric acids are substrates of P450 BM3 and were used to validate the method, as their binding can be detected both by a spin-shift of the Soret peak from 419 to 397 nm and by the fluorescence change of the labelled protein. The fluorescence emission of the probe linked to the protein increased by a value corresponding to 121 ± 9% and 52 ± 5% with respect to the initial one, upon titration with arachidonic or lauric acids respectively. The dissociation constants were calculated by both UV-vis and fluorescence spectroscopy. Three drugs, propranolol, chlorzoxazone and nifedipine, known to be oxidized by P450 BM3 and that bind without causing spin-shift, were also tested and the fluorescence emission of IANBD was found to decrease by 29 ± 5%, 21 ± 2% and 23 ± 3%, respectively, allowing the measurement of their dissociation constants.     

Investigation on binding of nitric oxide to horseradish peroxidase by absorption spectrometry

Binding of nitric oxide to horseradish peroxidase (HRP) has been investigated by absorption spectrometry in 0.2 M anaerobic phosphate buffer solution (pH 7.4). Based on this binding equilibrium, a model equation for evaluating the binding constant of nitric oxide to HRP is developed and the binding constant is calculated to be (1.55 ± 0.06) × 10⁴ M^?1, indicating that HRP can form a stable complex with nitric oxide. The type of inhibition by nitric oxide is validated on the basis of studying initial reaction rates of HRP-catalyzed oxidation of guaiacol in the presence of hydrogen peroxide and nitric oxide. The inhibition mechanism is found to follow an apparent non-competitive inhibition by Lineweaver–Burk method. Based on this kinetic mechanism, the binding constant is also calculated to be (5.22 ± 0.06) × 10⁴ M^?1. The values of the binding constant determined by the two methods are almost identical. The non-competitive inhibition model is also applicable to studying the effect of nitric oxide on other metalloenzymes, which catalyze the two-substrate reaction with the “ping-pong” mechanism.     

Optical biosensing of nitric oxide using the metalloprotein cytochrome c'

The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO). The metalloprotein was encapsulated in a porous silicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements. Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre. Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period. The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO. However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%). An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate working range of 10-400 mumol l-1.     

单重态CCl~2与O~3反应的热力学及动力学性质研究

在量化计算的基础上运用统计热力学和Wigner校正的Eyring过渡态理论研究了不同温度下单重态CCl~2和臭氧O~3反应的热力学及动力学性质。计算结果表明该反应在低温下具有热力学优势,而在高温下具有动力学优势。     

Development of immobilized enzyme reactors based on human recombinant cytochrome P450 enzymes for phase I drug metabolism studies

Two cytochrome P450 (CYP)-based immobilized enzyme reactors (IMERs) were developed to perform automated on-line phase I drug metabolism studies. For this purpose, biotinylated recombinant CYP2D6 or CYP3A4 reconstituted systems were anchored to the surface of two monolithic mini-columns (2mmx6mm I.D.), which had been covalently grafted with NeutrAvidin. After optimization of immobilization conditions, the obtained IMERs were integrated on-line into a LC hyphenated to an electrospray ionization MS/MS system. Studies with probe substrates and a known competitive inhibitor were performed, showing the potential of CYP-based IMERs in drug metabolism. In the optimized conditions, ca. 15 experiments were carried out with each bioreactor.     

Dissecting the thermodynamics and cooperativity of ligand binding in cytochrome P450eryF

Conformational flexibility and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. In this study, we dissect the ligand binding stoichiometry and energetics of the soluble bacterial P450eryF by isothermal titration calorimetry (ITC) using three allosteric and two non-allosteric ligands of diverse chemistry. Complementary spectral binding studies and sequential, two-ligand docking simulations were performed to help assign the binding sites. Binding of 4-phenylpyridine (4-PP) or 4-(4-chlorophenyl)imidazole (4-CPI) showed 1:1 stoichiometry in ITC, consistent with the lack of cooperativity observed in spectral binding studies. The larger ligands 9-aminophenanthrene (9-AP), 1-pyrenebutanol (1-PB), and alpha-naphthoflavone (ANF) show cooperative spectral binding and yielded 2:1 stoichiometry. The associated thermodynamic parameters for the sites were calculated using a sequential binding mechanism. The binding constant (KD) for the first site was almost two times lower than that of the second site for all three compounds. Ligand binding at site 1 was entropically favored, whereas binding at site 2 was weak and entropically unfavorable. Simulations showed that two molecules of 9-AP, ANF or 1-PB can be adequately docked to two individual sub-sites within a large binding pocket. The absence of hydrophobic tethering and ligand stacking are consistent with the single low affinity binding site observed for 4-CPI and 4-PP. Competitive binding studies with P450eryF preloaded with either 1-PB or ANF showed a decrease in the affinities for 9-AP at both the sites, with large entropy-enthalpy compensation, indicating the ability of the binding pocket to accommodate two ligands of diverse chemistry and enable cooperativity.