Peroxidase Compound Research Articles

A new concept for production and scaling up of bioactive compounds from Egyptian date palm (Zaghlool) cultivar using bioreactor

A promising and successful protocol for enhancement and production of total phenolic and peroxides compounds from Egyptian date palm cultivar Zaghlool cells in stirred tank reactor was established. The influence of cell culture cultivation in combination with Aspergillus niger extract, and methyl-jasmonate elicitors incorporation in the feeding medium on cell growth patterns and production of active compounds was investigated. The maximum value of cell growth parameters and the highest content of bioactive compounds were obtained from elicitation of modified MS-medium with Aspergillus niger extract at 0.1% in combination with methyl-jasmonate (100 µM), as compared with other concentrations after 10 days of cultivation. The chemical analyses of phenolic and peroxidase compounds were spectrophotometrically performed.

Reactivity of nonsteroidal anti-inflammatory drugs with peroxidase: a classification of nonsteroidal anti-inflammatory drugs

To improve understanding of the essential effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostaglandin H synthase (PGHS), the reactivity of NSAIDs with peroxidases and the tyrosyl radical derived from myoglobin was examined. Horseradish peroxidase and myoglobin were used as models of peroxidase and cyclooxygenase of PGHS, respectively. From the results, a new classification of NSAIDs has been proposed. Class 1 includes the majority of NSAIDs, which reacted with horseradish peroxidase compound I, thus causing a spectral change by PGHS peroxidase and also including diminished electron spin resonance signals of the tyrosyl radical of myoglobin. They reduced compound I of horseradish peroxidase and scavenged the tyrosyl radical. The branched-chain mechanism by which the porphyrin radical is transferred to the tyrosine residue of the protein might be blocked by these NSAIDs. Class 2 includes salicylic acid derivatives that reacted only with the porphyrin radical and not with horseradish peroxidase compound II (oxoferryl species). Class 3 includes aspirin, nimesulide, tolmetin, and arylpropionic acid derivatives, including ibuprofen and the coxibs of celecoxib and rofecoxib, which are not substrates for horseradish peroxidase or PGHS peroxidase. Understanding the essential mode of action of NSAIDs is particularly important for designing an effective therapeutic strategy against inflammatory diseases.

On the Reactivity of Mononuclear Iron(V)oxo Complexes

Ferric tetraamido macrocyclic ligand (TAML)-based catalysts [Fe{C(6)H(4)-1,2-(NCOCMe(2)NCO)(2)CR(2)}(OH(2))]PPh(4) [1; R = Me (a), Et (b)] are oxidized by m-chloroperoxybenzoic acid at -40 °C in acetonitrile containing trace water in two steps to form Fe(V)oxo complexes (2a,b). These uniquely authenticated Fe(V)(O) species comproportionate with the Fe(III) starting materials 1a,b to give μ-oxo-(Fe(IV))(2) dimers. The comproportionation of 1a-2a is faster and that of 1b-2b is slower than the oxidation by 2a,b of sulfides (p-XC(6)H(4)SMe) to sulfoxides, highlighting a remarkable steric control of the dynamics. Sulfide oxidation follows saturation kinetics in [p-XC(6)H(4)SMe] with electron-rich substrates (X = Me, H), but changes to linear kinetics with electron-poor substrates (X = Cl, CN) as the sulfide affinity for iron decreases. As the sulfide becomes less basic, the Fe(IV)/Fe(III) ratio at the end of reaction for 2b suggests a decreasing contribution of concerted oxygen-atom transfer (Fe(V) → Fe(III)) concomitant with increasing electron transfer oxidation (Fe(V) → Fe(IV)). Fe(V) is more reactive toward PhSMe than Fe(IV) by 4 orders of magnitude, a gap even larger than that known for peroxidase Compounds I and II. The findings reinforce prior work typecasting TAML activators as faithful peroxidase mimics.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol–disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV–Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pKa of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4±0.2)×107 M−1 s−1) and peroxynitrite ((3.7±0.4)×105 M−1 s−1) at pH 7.4 and 25°C.

ChemInform Abstract: Oxidation of Substituted Anilines by Horseradish Peroxidase Compound II

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Effect of tramadol on expression of 5-HT1A receptor in distal cerebrospinal fluid contacting neurons in mid-brain in a rat model of neuropatlic pain

Objective To investigate the effect of tramadol on the expression of 5-HT1A receptor in the distal'cerebrospinal fluid contacting neurons (CSF-CNs) in mid-brain in a rat model of neuropathic pain. Methods Forty male SPF SD rats weighing 220-280 g were randomly divided into 5 groups (n = 8 each): group Ⅰ normal control (group C); group Ⅱ normal saline (group NS); group Ⅲ tramodol (group T); group Ⅳ neuropathic pain + normal saline (group NP+ NS) and group Ⅴ neuropathic pain + tramadol (group NP + T). Neuropathic pain was induced by chronic constrictive injury (CCI) in group Ⅳ and Ⅴ . Four silk ligatures were placed on the sciatic nerve at 1 mm intervals. In group Ⅱ (NS) and group Ⅲ (T) the sciatic nerve was exposed but not ligated and NS 2 ml/kg and tramadol 10 mg/kg were injected IP respectively, while in group Ⅳ and Ⅴ NS 2 ml/kg and tramadol 10 mg/kg were injected IP respectively on the 7th day after CCI. Paw withdrawal threshold (PWT) to von Frey filament stimulation and paw withdrawal latency (PWL) to noxious thermal stimuli were measured before (T1) and after IP NS or tramadol injection (T2) in group Ⅱ-Ⅴ. The distal CSF-CNs in the mid-brain was labelled with 30% cholera toxin subunit B and horseradish peroxidase compound (CB-HRP) 3 μl injected in left lateral cerebral ventricle. The expression of 5-HT1A receptors was measured by immuno-histochemistry. Results PWT and PWL were significantly decreased after CCI in group Ⅳ (NP + NS) and tramadol significantly inhibited the mechanical and thermal hyperalgesia in group Ⅴ (NP + T). There was no significant difference in the number of distal CSF-CNs among the 5 groups. CCI significantly down-regulated the expression of 5-HT1A in distal CSF-CNs in group Ⅳ(NP+ NS) as compared with group Ⅰ , Ⅱ and Ⅲ and tramadol significantly inhibited the CCI-induced downregulation of 5-HT1A receptor expression. Conclusion Tramadol can ease neuropathic pain by down-regulating the expression of 5-HT1A receptor in distal CSF-CNs in mid-brain. Key words: Tramadol; Neurons; Receptor, serotonin, 5-HT1A; Neuralgia

Crystallographic and Single-Crystal Spectral Analysis of the Peroxidase Ferryl Intermediate

The ferryl [Fe(IV)O] intermediate is important in many heme enzymes, and thus, the precise nature of the Fe(IV)-O bond is critical in understanding enzymatic mechanisms. The 1.40 A crystal structure of cytochrome c peroxidase Compound I has been determined as a function of X-ray dose while the visible spectrum was being monitored. The Fe-O bond increases in length from 1.73 A in the low-X-ray dose structure to 1.90 A in the high-dose structure. The low-dose structure correlates well with an Fe(IV) horizontal lineO bond, while we postulate that the high-dose structure is the cryo-trapped Fe(III)-OH species previously thought to be an Fe(IV)-OH species.

Resonance Raman Study of a High-valent Fe=O Porphyrin Complex as a Model for Peroxidase Compound II

Abstract Resonance Raman spectroscopy is applied to a FeIV oxo porphyrin with imidazolate as the axial ligand. The νFe=O mode is observed at 792 cm−1, which is 23 cm−1 lower than that of the analogous 1–methylimidazole complex and similar to that of horseradish peroxidase compound II (787 cm−1) at alkaline pH, for which presence of an anionic histidine was previously postulated. This study thus provides a useful model compound of horseradish peroxidase compound II.

Analysis of oxidation of p-cresol catalyzed by horseradish peroxidase compound II.

Analysis of oxidation of p-cresol catalyzed by horseradish peroxidase compound II.

Evidence for Heme Oxygenase Activity in a Heme Peroxidase

The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H(2)O(2)-dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O(2)-dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase. Hence, we describe kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide and does not form the usual peroxidase Compound I intermediate; instead, structural data show that a product is formed in which the heme has been cleaved at the alpha-meso position, analogous to the heme oxygenase mechanism. We interpret this to mean that the Compound I (peroxidase) pathway is shut down, so that instead the reaction intermediate diverts through the alternative (heme oxygenase) route. A mechanism for formation of the product is proposed and discussed in the light of what is known about the heme oxygenase reaction mechanism.

Mechanism of and exquisite selectivity for O–O bond formation by the heme-dependent chlorite dismutase

Chlorite dismutase (Cld) is a heme b-dependent, O-O bond forming enzyme that transforms toxic chlorite (ClO(2)(-)) into innocuous chloride and molecular oxygen. The mechanism and specificity of the reaction with chlorite and alternate oxidants were investigated. Chlorite is the sole source of dioxygen as determined by oxygen-18 labeling studies. Based on ion chromatography and mass spectrometry results, Cld is highly specific for the dismutation of chlorite to chloride and dioxygen with no other side products. Cld does not use chlorite as an oxidant for oxygen atom transfer and halogenation reactions (using cosubstrates guaiacol, thioanisole, and monochlorodimedone, respectively). When peracetic acid or H(2)O(2) was used as an alternative oxidant, oxidation and oxygen atom transfer but not halogenation reactions occurred. Monitoring the reaction of Cld with peracetic acid by rapid-mixing UV-visible spectroscopy, the formation of the high valent compound I intermediate, [(Por(*+))Fe(IV) = O], was observed [k(1) = (1.28 +/- 0.04) x 10(6) M(-1) s(-1)]. Compound I readily decayed to form compound II in a manner that is independent of peracetic acid concentration (k(2) = 170 +/- 20 s(-1)). Both compound I and a compound II-associated tryptophanyl radical that resembles cytochrome c peroxidase (Ccp) compound I were observed by EPR under freeze-quench conditions. The data collectively suggest an O-O bond-forming mechanism involving generation of a compound I intermediate via oxygen atom transfer from chlorite, and subsequent recombination of the resulting hypochlorite and compound I.

Red-Excitation Resonance Raman Analysis of the νFe═O Mode of Ferryl-Oxo Hemoproteins

The Raman excitation profile of the nuFe O mode of horseradish peroxidase compound II exhibits a maximum at 580 nm. This maximum is located within an absorption band with a shoulder assignable to an oxygen-to-iron charge transfer band on the longer wavelength side of the alpha-band. Resonance Raman bands of the nuFe O mode of various ferryl-oxo type hemoproteins measured at 590 nm excitation indicate that many hemoproteins in the ferryl-oxo state have an oxygen-to-iron charge transfer band in the visible region. Since this red-excited resonance Raman technique causes much less photochemical damage in the proteins relative to blue-excited resonance Raman spectroscopy, it produces a higher signal-to-noise ratio and thus represents a powerful tool for investigations of ferryl-oxo intermediates of hemoproteins.

A comparison of catalytic site intermediates of cytochrome c oxidase and peroxidases

Compounds I and II of peroxidases such as horseradish peroxidase and cytochrome c peroxidase are relatively well understood catalytic intermediates in terms of their structures and redox states of iron, heme, and associated radical species. The intermediates involved in the oxygen reduction chemistry of the cytochrome c oxidase superfamily are more complicated because of the need for four reducing equivalents and because of the linkage of the oxygen chemistry with vectorial proton translocations. Nevertheless, two of these intermediates, the peroxy and ferryl forms, have characteristics that can in many ways be considered to be counterparts of peroxidase compounds I and II. We explore the primary factors that minimize the generation of unwanted reactive oxygen species products and ensure that the principal enzymological function becomes either that of a peroxidase or an oxidase. These comparisons can provide insights into the nature of biological oxygen reduction chemistry and guidance for the engineering of biomimetic synthetic materials.

How does the push/pull effect of the axial ligand influence the catalytic properties of Compound I of catalase and cytochrome P450?

Density functional theory (DFT) calculations on the chemoselective epoxidation versus hydroxylation reactions of propene by oxoiron porphyrin models mimicking the active sites of catalase, cytochrome P450 (P450) and horseradish peroxidase Compound I (CpdI) are presented. The catalase reactions are concerted and proceed via two-state reactivity patterns on competing doublet and quartet spin state surfaces, but the lowest barrier is the one leading to epoxide products on the doublet spin surface. The results are compared with earlier DFT studies of models of cytochrome P450, horseradish peroxide (HRP), taurine/α-ketoglutarate dioxygenase and some synthetic oxoiron catalysts. The catalase barriers are midway in between those obtained for HRP and P450 models, so that tyrosinate ligated heme systems should be able to catalyze C–H hydroxylation and C C epoxidation reactions. We show that for heme systems the barrier height of epoxidation linearly correlates with the electron affinity of Compound I as expected from the electron transfer mechanism of the rate determining step. Our studies show that the axial ligand does not influence the chemoselectivity of a reaction but that it does regulate the barrier heights and rate constants. Finally, we estimated the effect of the axial ligand on the oxoiron group and derived that it contributes from a field effect due to the charge of the ligand and a quantum mechanical effect as a result of orbital mixing. In catalase, the major component is the field effect, while the quantum mechanical effect is negligible. This is in contrast to P450 CpdI, where both effects are of similar order of magnitude.

Reaction of Mycobacterium tuberculosis Truncated Hemoglobin O with Hydrogen Peroxide: EVIDENCE FOR PEROXIDATIC ACTIVITY AND FORMATION OF PROTEIN-BASED RADICALS

In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H(2)O(2) to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme pi-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr(55) and Tyr(115), which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H(2)O(2) and the one-electron donor 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs.

Direct Electron Transfer Kinetics in Horseradish Peroxidase Electrocatalysis

The study of direct electron transfer between enzymes and electrodes is frequently hampered by the small fraction of adsorbed proteins that remains electrochemically active. Here, we outline a strategy to overcome this limitation, which is based on a hierarchical analysis of steady-state electrocatalytic currents and the adoption of the "binary activity" hypothesis. The procedure is illustrated by studying the electrocatalytic response of horseradish peroxidase (HRP) adsorbed on graphite electrodes as a function of substrate (hydrogen peroxide) concentration, electrode potential, and solution pH. Individual contributions of the rates of substrate/enzyme reaction and of the electrode/enzyme electron exchange to the observed catalytic currents were disentangled by taking advantage of their distinct dependence on substrate concentration and electrode potential. In the absence of nonturnover currents, adoption of the "binary activity" hypothesis provided values of the standard electron-transfer rate constant for reduction of HRP Compound II that are similar to those reported previously for reduction of cytochrome c peroxidase Compound II. The variation of the catalytic currents with applied potential was analyzed in terms of the non-adiabatic Marcus-DOS electron transfer theory. The availability of a broad potential window, where catalytic currents could be recorded, facilitates an accurate determination of both the reorganization energy and the maximum electron-transfer rate for HRP Compound II reduction. The variation of these two kinetic parameters with solution pH provides some indication of the nature and location of the acid/base groups that control the electronic exchange between enzyme and electrode.

Spectroscopic investigations of intermediates in the reaction of cytochrome P450 BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450 BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450 CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450 CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.

On the formation of Horseradish Peroxidase Compound I at high pH: New insights from ab initio molecular dynamics

Compound I formation at neutral or basic pH, starting from ferric peroxidase–peroxide complex has been described, for the first time, by ab initio molecular dynamics on doublet surface. Proton transfer involving distal residues occurs efficiently in a stepwise mechanism in which the O–O bond is broken. An energy barrier of 4.8 kcal/mol is found to obtain the final products when the Fe III[O–OH] (−) complex is formed on the doublet surface. In the final part of the reaction, just before Compound I formation, a spin crossing between doublet and quartet magnetic surfaces is observed in our model system.

Theoretical Evidence Favoring True Iron(V)-Oxo Corrole and Corrolazine Intermediates

Although many formally FeV intermediates are known in the form of peroxidase compound I intermediates and their synthetic models, "true" d3 FeVO intermediates have remained elusive and hence a Holy Grail of sorts for many bioinorganic chemists. Very recently, Newcomb and co-workers provided transient absorption spectroscopic evidence suggestive of FeVO corrole intermediates. Here, we report DFT calculations predicting nearly isoenergetic FeVO and FeIVO corrolato2-* states for Fe(corrolato)(O) intermediates. In the course of a theoretical search for systems in which a true FeVO state might be favored by a clear and substantial margin of energy, we have identified corrolazine as a promising supporting ligand; thus, we find that with corrolazine, the FeVO states are favored by at least 0.5 eV over FeIVO corrolazinato2-* states.

Interpretation of the reactivity of peroxidase compound II with phenols and anilines using the Marcus equation

The catalytic cycle of heme peroxidases involves three processes: the formation of compound I, its conversion to compound II and regeneration of the native enzyme. Each of the processes consists of a reversible binding stage followed by an irreversible transformation stage. Our group has proposed a continuous, sensitive and reliable chronometric method for measuring the steady-state rate of peroxidase activity. Furthermore, we have derived an analytical expression for the steady-state rate and simplified it, taking into consideration the experimental values of the rate constants of some stages previously determined by other authors in stopped-flow assays. We determined the value of the constant for the transformation of a series of phenols and anilines by compound II, and found that it involves a deprotonation step and an electron transfer step. Study of the solvent deuterium isotope effect on the oxidation of phenol revealed the non-rate-limiting character of the deprotonation step in a proton inventory study. Usage of the Marcus equation showed that the electronic transfer step is rate-limiting in both cases, while phenols and anilines were oxidised at different rates for the same potentials. This can be attributed to the shorter electron-tunnelling distance for electron transfer to the iron ion in the phenols than in the anilines.