Cytochrome P450 119 Compounds I Formed by Chemical Oxidation and Photooxidation Are the Same Species

Compound I from cytochrome P450 119 prepared by the photooxidation method involving peroxynitrite oxidation of the resting enzyme to Compound II followed by photooxidation to Compound I was compared to Compound I generated by m-chloroperoxybenzoic acid (MCPBA) oxidation of the resting enzyme. The two methods gave the same UV/Visible spectra, the same products from oxidations of lauric acid and palmitic acid and their (ω-2,ω-2,ω-3,ω-3)-tetradeuterated analogues, and the same kinetics for oxidations of lauric acid and caprylic acid. The experimental identities between the transients produced by the two methods leave no doubt that the same Compound I species is formed by the two methods.     

Cytochrome p450 compound I

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate. The Compound II derivative in turn was produced by oxidation of the P450 with peroxynitrite, which effected a net one-electron, oxo-transfer reaction to the iron(III) atom of the resting enzyme. For the P450 studied in this work, CYP119 from the thermophile Sulfolobus solfactaricus, the P450 Compound II derivative was stable for seconds at ambient temperature, and the Compound I transient decayed with a lifetime of ca. 200 ms.     

Caldariomyces fumago chloroperoxidase catalyzes the oxidative dehalogenation of chlorophenols by a mechanism involving two one-electron steps

We have employed rapid scan stopped-flow spectroscopy to examine whether the mechanism of oxidative dehalogenation catalyzed by C. fumago chloroperoxidase (CCPO) involves two consecutive one-electron steps or a single two-electron oxidation. First, we optimized the formation of CCPO compound I (CCPO-I) [Fe(IV)=O/porphyrin radical] and CCPO compound II (CCPO-II) [Fe(IV)=O] for use in double mixing rapid scan stopped-flow experiments. Reaction of CCPO-I with 2,4,6-trichlorophenol (TCP) quickly yielded CCPO-II. Reaction of CCPO-II, a one-electron oxidant, with TCP rapidly regenerated the ferric resting state of the enzyme. The rates of the reaction of both CCPO-I and -II with TCP are first-order with respect to [TCP]. In the absence of organic substrate, CCPO-I is slowly reduced to CCPO-II and then the ferric state. The ability of both CCPO-I and -II to carry out the oxidative dehalogenation reaction is consistent with a mechanism involving two consecutive one-electron oxidations. In contrast, reaction of CCPO-I with thioanisole generated the ferric enzyme with no evidence of CCPO-II, consistent with a single two-electron oxidation by insertion of an oxygen atom. The relative stability of CCPO-I and -II has allowed us to differentiate between one- and two-electron substrate oxidations using rapid scan stopped-flow techniques.     

Luminol Oxidation by Hydrogen Peroxide Catalyzed by Tobacco Anionic Peroxidase: Steady-State Luminescent and Transient Kinetic Studies

The properties of a newly isolated anionic tobacco peroxidase from transgenic tobacco plants overexpressing the enzyme have been studied with respect to the chemiluminescent reaction of luminol oxidation. These were compared to the properties of horseradish peroxidase in the cooxidation of luminol and p -iodophenol, the enhanced chemiluminescence reaction. The pH, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the tobacco enzyme. The detection limit for the latter under the optimal conditions (2.5 m M luminol, 2 m M hydrogen peroxide, 100 m M Naborate buffer, pH 9.3) was about 0.1 p M , which is at least five times lower than that for horseradish peroxidase in enhanced chemiluminescence with p -iodophenol. The rate constants for the elementary steps of the enzyme-catalyzed reaction have been determined: k ₁= 4.9 × 10⁶ M ⁻¹ s¹, k ₂= 7.3 × 10⁶ M ⁻¹ s⁻¹, k ³= 2.1 × 10⁶ M ⁻¹ s⁻¹ (pH 9.3). The similarity of these rate constants is unusual for plant peroxidases. The high catalytic activity of tobacco peroxidase in the luminescent reaction is explained by the high reactivity of its Compound II toward luminol and the high stability of the holoenzyme with respect to heme dissociation. This seems to be a unique property of this particular enzyme among other plant peroxidases.     

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.     

Reaction of Aplysia limacina metmyoglobin with hydrogen peroxide

Myoglobin (Mb) from gastropod mollusc Aplysia limacina shows only 20% sequence homology to the 'prototype' sperm whale Mb but exhibits a typical Mb fold and can reversibly bind oxygen. An intriguing feature of aplysia Mb is that it lacks the distal histidine and displays a ligand stabilisation based on an arginine. Here we report the reaction of aplysia metMb with hydrogen peroxide studied by optical and electron paramagnetic resonance (EPR) spectroscopies. Two electron oxidation of the protein by H2O2 results in formation of two intermediates typical for this class of reactions, the oxoferryl haem state and a globin-bound free radical. An unusual characteristic of the aplysia Mb reaction is formation, prior to haem oxidation, of an optically distinct compound with an EPR spectrum typical of the low spin Fe3+ haem state. This compound is interpreted as the complex between H2O2 and the ferric haem state (Compound), formed prior to cleavage of the dioxygen bond. We conclude that H2O2 is singly deprotonated in Compound which can thus be notated as [Fe3+--OOH]. A new low spin ferric haem state has been observed over the period of Compound decay, and hypotheses have been formulated as to its identity and role. The location of the protein bound radical observed in aplysia Mb is discussed in light of the fact that the protein does not have any tyrosine residues, the most common site of free radical formation in the haem protein/peroxide systems. All intermediates of the reaction are kinetically characterised.     

An efficient proton-coupled electron-transfer process during oxidation of ferulic acid by horseradish peroxidase: coming full cycle

Quantum mechanics/molecular mechanics calculations were utilized to study the process of oxidation of a native substrate (ferulic acid) by the active species of horseradish peroxidase (Dunford, H. B. Heme Peroxidases; Wiley-VCH: New York, 1999), Compound I and Compound II, and the manner by which the enzyme returns to its resting state. The results match experimental findings and reveal additional novel features. The calculations demonstrate that both oxidation processes are initiated by a proton-coupled electron-transfer (PCET) step, in which the active species of the enzyme participate only as electron-transfer partners, while the entire proton-transfer event is being relayed from the substrate to and from the His42 residue by a water molecule (W402). The reason for the observed (Henriksen, A; Smith, A. T.; Gajhede, M. J. Biol. Chem. 1999, 274, 35005-35011) similar reactivities of Compound I and Compound II toward ferulic acid is that the reactive isomer of Compound II is the, hitherto unobserved, Por(*)(+)Fe(III)OH isomer that resembles Compound I. The PCET mechanism reveals that His42 and W402 are crucial moieties and they determine the function of the HRP enzyme and account for its ability to perform substrate oxidation (Poulos, T. L. Peroxidases and Cytochrome P450. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: New York, 2000; Vol. 4, pp 189). In view of the results, the possibility of manipulating substrate oxidation by magnetic fields is an intriguing possibility.     

Participation of the photosensitizer alpha-terthienyl in the peroxidase-catalyzed oxidation of indole-3-acetic acid

The plant photosensitizer alpha-terthienyl (alpha T) is toxic toward a variety of organisms, and normally requires exposure to ultraviolet-A radiation for activation and singlet molecular oxygen formation. However, some toxicity has also been reported to occur in the dark. One hypothesis that has been proposed to account for this light-independent toxicity is that the sensitizer becomes activated by energy transfer from the excited-state products of enzymatic reactions. We have investigated this hypothesis using the horseradish peroxidase (HRP)-catalyzed oxidation of indole-3-acetic acid (IAA), which generates indole-3-aldehyde in an excited triplet state. Light is emitted during the IAA/HRP reaction at acidic pH, is increased by inclusion of alpha T and is not observed with heat-denatured HRP. The rates of both the oxidation of IAA and the subsidence of light emission are more rapid in the IAA/alpha T/HRP system than with IAA and HRP alone, indicating that the presence of alpha T accelerates the reaction. Bleaching occurs at the wavelength of maximal alpha T absorbance and is promoted by the inclusion of IAA. Readdition of both IAA and alpha T to a spent reaction mixture is required to restore light emission after it has subsided, further suggesting that both are consumed in the reaction. We were unable to detect measurable quantities of singlet molecular oxygen formation in this system. These results do not support the energy transfer hypothesis, but instead are more compatible with a model proposed by Krylov and Chebotareva [Krylov, S. N. and A. B. Chebotareva (1993) FEBS Lett. 324, 6-8] for the co-oxidation of IAA and xanthene dyes.     

金属卟啉存在下芳醛氧化反应的研究

本文研究了在金属四苯基卟啉[Co(II)TPP,Fe(III)TPPCl,Mn(III)TPPCl,Zn(II)TPP,Cu(II)TP.TPP=四苯基卟啉]存在下,用氧气氧化芳醛的过程.测定了反应体系的吸氧动力学曲线;观察了氧化过程中金属卟啉的可见光谱的变化;研究了底物,金属卟啉在反应体系中的浓度以及溶剂等因素对反应的影响.结果发现,除能可逆键合分子氧的Co(II)TPP外,不具此种功能的Fe(III)TPPCl和Mn(III)TPPCl也能加速芳醛的氧化反应.然而,它们的催化作用是在金属四苯基卟啉与反应过程中积累起来的过酸作用,卟啉环遭到破坏后观察到的,此时可能形成了某种新的催化活性中心.金属卟啉本身对反应起抑制作用,它只是表观上的催化剂,其催化作用看来不应归结为对分子氧的活化.     

Catalytic Oxidation of Iron(II) Aqua Complex by Oxygen in the Presence of Palladium(II) Tetraaqua Complex

The catalytic oxidation of iron(II) with oxygen occurs along with an autocatalytic reaction between palladium(II) tetraaqua complex and iron(II) aqua complex in an oxygen atmosphere. The reaction is catalyzed by a compound of palladium in an intermediate oxidation state, presumably by a small palladium cluster formed in the course of the reduction of palladium(II) tetraaqua complex with iron(II) aqua complex.     

Ferric superoxide and ferric hydroxide are used in the catalytic mechanism of hydroxyethylphosphonate dioxygenase: a density functional theory investigation

Hydroxyethylphosphonate dioxygenase (HEPD) is a mononuclear nonheme iron enzyme that utilizes an O(2) molecule to cleave a C-C bond in 2-hydroxyethylphosphonate and produce hydroxymethylphosphonate (HMP) and formic acid. Density functional theory calculations were performed on an enzyme active-site model of HEPD to understand its catalytic mechanism. The reaction starts with H-abstraction from the C2 position of 2-HEP by a ferric superoxide-type (Fe(III)-OO(?-)) intermediate, in a similar manner to the H-abstraction in the reaction of the dinuclear iron enzyme myo-inositol oxygenase. The resultant Fe(II)-OOH intermediate may follow either a hydroperoxylation or hydroxylation pathway, the former process being energetically more favorable. In the hydroperoxylation pathway, a ferrous-alkylhydroperoxo intermediate is formed, and then its O-O bond is homolytically cleaved to yield a complex of ferric hydroxide with a gem-diol radical. Subsequent C-C bond cleavage within the gem-diol leads to formation of an R-CH(2)(?) species and one of the two products (i.e., formic acid). The R-CH(2)(?) then intramolecularly forms a C-O bond with the ferric hydroxide to provide the other product, HMP. The overall reaction pathway does not require the use of a high-valent ferryl intermediate but does require ferric superoxide and ferric hydroxide intermediates.     

C-H bond activation by a ferric methoxide complex: modeling the rate-determining step in the mechanism of lipoxygenase.

Lipoxygenases are mononuclear non-heme iron enzymes that regio- and stereospecifcally convert 1,4-pentadiene subunit-containing fatty acids into alkyl peroxides. The rate-determining step is generally accepted to be hydrogen atom abstraction from the pentadiene subunit of the substrate by an active ferric hydroxide species to give a ferrous water species and an organic radical. Reported here are the synthesis and characterization of a ferric model complex, [Fe(III)(PY5)(OMe)](OTf)(2), that reacts with organic substrates in a manner similar to the proposed enzymatic mechanism. The ligand PY5 (2,6-bis(bis(2-pyridyl)methoxymethane)pyridine) was developed to simulate the histidine-dominated coordination sphere of mammalian lipoxygenases. The overall monoanionic coordination provided by the endogenous ligands of lipoxygenase confers a strong Lewis acidic character to the active ferric site with an accordingly positive reduction potential. Incorporation of ferrous iron into PY5 and subsequent oxidation yields a stable ferric methoxide species that structurally and chemically resembles the proposed enzymatic ferric hydroxide species. Reactivity with a number of hydrocarbons possessing weak C-H bonds, including a derivative of the enzymatic substrate linoleic acid, scales best with the substrates' bond dissociation energies, rather than pK(a)'s, suggesting a hydrogen atom abstraction mechanism. Thermodynamic analysis of [Fe(III)(PY5)(OMe)](OTf)(2) and the ferrous end-product [Fe(II)(PY5)(MeOH)](OTf)(2) estimates the strength of the O-H bond in the metal bound methanol in the latter to be 83.5 +/- 2.0 kcal mol(-1). The attenuation of this bond relative to free methanol is largely due to the high reduction potential of the ferric site, suggesting that the analogously high reduction potential of the ferric site in LO is what allows the enzyme to perform its unique oxidation chemistry. Comparison of [Fe(III)(PY5)(OMe)](OTf)(2) to other coordination complexes capable of hydrogen atom abstraction shows that, although a strong correlation exists between the thermodynamic driving force of reaction and the rate of reaction, other factors appear to further modulate the reactivity.     

2-[4-Bis(2-chloroethyl)aminophenyl]indane-1,3-dione and its dehydrodimer

The reaction of the phthalic anhydrides with phenylacetic acids in acetic anhydride and triethylamine was used for the synthesis of 2-[4-bis(2-chloroethyl)aminophenyl]indane-1,3-dione (II). The oxidation of (II) by ferric chloride or DDQ gave the corresponding dehydrodimer (IV).Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 4, pp. 943–944, April, 1990.     

多铜氧化酶/中介体催化氧还原反应动力学分析

采用分光光度法、Clark型氧电极以及循环伏安法结合旋转圆盘电极技术,分别测定了游离多铜氧化酶在扩散型电子中介体存在时,催化氧还原循环中每一个组成步骤的速率并进行了比较,试图确定这个催化反应的决速步骤。 实验结果表明,漆酶分子内部的电子迁移速率(103/s)最高,酶催化氧气化学还原的速率次之(91/s),酶催化中介体氧化产物在电极上电化学还原的速率再次之(0.19/s或7.8×10-3 cm/s),底物O2气以及氧化态/还原态电子中介体2,2′-连氮-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)的传质系数分别为1.7×10-3、4.4×10-4 和6.3×10-4 cm/s,相应地酶催化中介体氧化的化学反应速率为0.047/s,酶催化中介体氧化的化学反应步骤以及中介体的传质步骤是影响催化反应速率的关键。 在此基础上,通过系统改变体系中酶的种类、活力以及浓度、中介体种类及浓度、溶液温度及pH值等参数,研究了酶电催化氧还原活力与这些参数之间的依赖关系,进一步确证了前述的结论。     

血红蛋白过氧化物模拟酶胶束催化显色体系

在胶束Tween80介质中，研究了以血红蛋白（hemoglobin,Hb)作为过氧化物模拟酶、隐性亮绿（recessive brilliant green,RGB)为氢供体底物、溶解氧为受氢体的酶催化反应特性。在pH5.64(NH4)2HPO4-KH2PO4缓冲溶液中，利用模拟酶对溶解氧作为受氢体催化RBG氧化生成亮绿（brilliant green,BG)而拟定了测定Hb含量的新方法。讨论了胶束介质对酶体系催化反应的影响及酶催化反应的可能机理。     

DFT Calculations Suggest a New Type of Self‐Protection and Self‐Inhibition Mechanism in the Mammalian Heme Enzyme Myeloperoxidase: Nucleophilic Addition of a Functional Water rather than One‐Electron Reduction

The mammalian heme enzyme myeloperoxidase (MPO) catalyzes the reaction of Cl^? to the antimicrobial‐effective molecule HOCl. During the catalytic cycle, a reactive intermediate “Compound I” (Cpd I) is generated. Cpd I has the ability to destroy the enzyme. Indeed, in the absence of any substrate, Cpd I decays with a half‐life of 100 ms to an intermediate called Compound II (Cpd II), which is typically the one‐electron reduced Cpd I. However, the nature of Cpd II, its spectroscopic properties, and the source of the additional electron are only poorly understood. On the basis of DFT and time‐dependent (TD)‐DFT quantum chemical calculations at the PBE0/6‐31G* level, we propose an extended mechanism involving a new intermediate, which allows MPO to protect itself from self‐oxidation or self‐destruction during the catalytic cycle. Because of its similarity in electronic structure to Cpd II, we named this intermediate Cpd II′. However, the suggested mechanism and our proposed functional structure of Cpd II′ are based on the hypothesis that the heme is reduced by charge separation caused by reaction with a water molecule, and not, as is normally assumed, by the transfer of an electron. In the course of this investigation, we found a second intermediate, the reduced enzyme, towards which the new mechanism is equally transferable. In analogy to Cpd II′, we named it Fe^II′. The proposed new intermediates Cpd II′ and Fe^II′ allow the experimental findings, which have been well documented in the literature for decades but not so far understood, to be explained for the first time. These encompass a) the spontaneous decay of Cpd I, b) the unusual (chlorin‐like) UV/Vis, circular dichroism (CD), and resonance Raman spectra, c) the inability of reduced MPO to bind CO, d) the fact that MPO‐Cpd II reduces SCN^? but not Cl^?, and e) the experimentally observed auto‐oxidation/auto‐reduction features of the enzyme. Our new mechanism is also transferable to cytochromes, and could well be viable for heme enzymes in general.     

Highly selective ferric ion sorption and exchange by crystalline metal phosphonates constructed from tetraphosphonic acids

With the motivation of searching for highly selective ferric ion sorbents, two open-framework and microporous materials, {[Pb7(HEDTP)2(H2O)] x 7H2O}n (1) and {[Zn2(H4EDTP)] x 2H2O}n (2) [H8EDTP = N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)], have been synthesized and structurally characterized. The structure of compound 1 results from the seven crystallographically different lead atoms that are bridged by two HEDTP(7-) ligands to yield a three-dimensional microporous framework with tunnels along the a and b axes. Compound 2 features a layer architecture built of square waves along the a axis. The layers are connected by hydrogen bonds between uncoordinated phosphonate oxygen atoms to form a three-dimensional supramolecular network, with one-dimensional tunnels along the a axis. Both compounds 1 and 2 exhibited high ion sorption and exchange capacities for millimolar concentrations of Fe(III). Specifically, when 0.01 g of 1 (or 2) was added to 5 mL of a 1 mM metallic chloride aqueous solution and the mixture was allowed to stand for 2 days at room temperature, compound 1 adsorbed nearly 100% of Fe(III) and compound 2 adsorbed 96.8% of Fe(III). They were also found to adsorb ferric ions selectively over other metal ions, such as Ca(II), Cr(II), Mn(II), Cu(II), Zn(II), Cd(II), etc. Their special ferric ion uptake capacities may be attributed to the cation exchange, coordination bonding, and electrostatic attraction between ferric ions and metal phosphonates.     

The possible production of natural flavours by amino acid degradation

Abstract  

This work describes the degradation of phenylalanine and tryptophane catalysed by their complexes with Fe(II), Co(II), and Cu(II). The influences of the central atom and of the reaction conditions on the degradation of the amino acids were observed. The necessary condition of the degradation is the possibility of a redox reaction on the central atom between M(II) and M(III). Moreover, the coordination sphere of the central cation of the transition metal must not be sterically shielded. The necessary conditions are fulfilled only in the Fe(II) complexes. The degradation is strictly anaerobic because due to the influence of oxygen, an irreversible oxidation of Fe(II) to Fe(III) proceeds. This reaction results in 5-hydroxy-1H-indol instead of the mixture of the degradation products, such as benzaldehyde, phenylacetaldehyde, and phenylacetic acid. The influence of the temperature on the catabolism is very important because the reaction accelerates with temperature increase. The phenylalanine anion acts as a reducing agent, and Fe(II) is spontaneously reduced to Fe(0).     

Probing a complex of cytochrome c and cardiolipin by magnetic circular dichroism spectroscopy: implications for the initial events in apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH(-)-ligated. The ferrous state is predominantly high-spin and, most likely, His/-. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form.