The cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide. Steady state kinetic mechanism

Initial velocities for the cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide have been measured as functions of both the ferrocytochrome c (0.27-104 microM) and hydrogen peroxide (0.25-200 microM) concentrations at 25 degrees C, 0.01 M ionic strength, and pH 7 in a cacodylate/KNO3 buffer system Eadie-Hofstee plots of the initial velocity as a function of ferrocytochrome c concentration at constant hydrogen peroxide are nonlinear. A mechanism is proposed which includes random addition of the two substrates to the enzyme and a single catalytically active cytochrome c binding site. The mechanism is consistent with prior studies on cytochrome c peroxidase and fits the steady state kinetic data well.     

Parametric modeling of reaction time experiment data

A simple parametric model is proposed for data from a point-process version of a reaction time experiment. It is used to statistically check for the presence and nature of nonlinear inhibition in the eye-brain-hand system, as well as to study the nature of the reaction time delay distribution. The model tells us that, in principle, the second-order intensity estimate can be used to determine whether the experimental subject is systematically observing the first or the second of two flashes transmitted in short succession. Nonparametric estimates of second-order intensity functions are used in conjunction with this model. In particular, the model allows for the computation of good bandwidths for intensity curve estimation. A parametric bootstrap can also be implemented. Our methods are illustrated with 12 runs of data from a real reaction time experiment. It is found that nonlinear inhibition is present in the eye-brain-hand system. However, there are insufficient data to distinguish between log-normality and normality in the reaction time distribution, due partly to confounding with the particular kind of nonlinear inhibition present in the system.     

Deactivation of immobilized beef liver catalase by hydrogen peroxide

Immobilized beef liver catalase has been used in a flow reactor to decompose hydrogen peroxide; at the same time the catalase is inactivated by its substrate. A model has been developed which predicts this rate of decomposition of peroxide and inactivation of catalase. First order dependence on peroxide concentration is assumed. The model was verified by experiment for a range of operating conditions and then used to predict the effects of a change in operating variables.     

THE KINETICS OF THE DECOMPOSITION OF PEROXIDE BY CATALASE

It has been shown that the experimental results obtained by Morgulis in a study of the decomposition of hydrogen peroxide by liver catalase at 20°C. and in the presence of an excess of a relatively high concentration of peroxide are quantitatively accounted for by the following mechanisms. 1. The rate of formation of oxygen is independent of the peroxide concentration provided this is greater than about 0.10 M. 2. The rate of decomposition of the peroxide is proportional at any time to the concentration of catalase present. 3. The catalase undergoes spontaneous monomolecular decomposition during the reaction. This inactivation is independent of the concentration of catalase and inversely proportional to the original concentration of peroxide up to 0.4 M. In very high concentrations of peroxide the inactivation rate increases. 4. The following equation can be derived from the above assumptions and has been found to fit the experiments accurately. See PDF for Equation in which x is the amount of oxygen liberated at the time t, A is the total amount of oxygen liberated (not the total amount available), and K is the inactivation constant of the enzyme.     

Steady-state kinetics of thyroid peroxidase. Evidence for a high degree rate equation using the F statistic

The initial-rate kinetics of bovine thyroid peroxidase are reported using 325 sets of concentrations of hydrogen peroxide and guaiacol. Extended ranges of concentrations are used and the v(S) profiles are fitted by rational functions of degree 2:2, 3:3 and +:4 by interactive non-linear regression analysis. Estimates of initial slopes in v(S) plots obtained by this regression are then replotted against the fixed substrate concentration and this confirms the need for a high-degree rate equation. Values of the F statistic indicate that the rate equation is 3:3 in guaiacol and 4:4 in hydrogen peroxide. It is concluded that the kinetics of peroxidase from bovine thyroid, like horse radish and human cervical mucus peroxidase and lactoperoxidase can be accommodated by the greater cyclic mechanism and that this is the minimal kinetic scheme for peroxidase In general.     

Amperometric biosensor for the detection of hydrogen peroxide using catalase modified electrodes in polyacrylamide

A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation

The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.     

Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases.

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2.4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A compound is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4 and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arhenius plots the activation energy for the reaction was calculated to be 2.9 +/- 0.1 kcal/mol for P1 and 5.4 +/- 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol-water mixtures up to 30% glycerol.     

Cytochrome c peroxidase catalyzed oxidation of ferrocytochrome c by hydrogen peroxide: ionic strength dependence of the steady-state rate parameters

The steady-state kinetics of the cytochrome c peroxidase catalyzed oxidation of horse heart ferrocytochrome c by hydrogen peroxide have been studied at both pH 7.0 and pH 7.5 as a function of ionic strength. Plots of the initial velocity versus hydrogen peroxide concentration at fixed cytochrome c are hyperbolic. The limiting slope at low hydrogen peroxide give apparent bimolecular rate constants for the cytochrome c peroxidase-hydrogen peroxide reaction identical with those determined directly by stopped-flow techniques. Plots of the initial velocity versus cytochrome c concentration at saturating hydrogen peroxide (200 microM) are nonhyperbolic. The rate expression requires squared terms in cytochrome c concentration. The maximum turnover rate of the enzyme is independent of ionic strength, with values of 470 +/- 50 s-1 and 290 +/- 30 s-1 at pH 7.0 and 7.5, respectively. The limiting slope of velocity versus cytochrome c concentration plots provides a lower limit for the association rate constant between cytochrome c and the oxidized intermediates of cytochrome c peroxidase. The limiting slope varies from 10(6) M-1 s-1 at 300 mM ionic strength to 10(8) M-1 s-1 at 20 mM ionic strength and extrapolates to 5 x 10(8) M-1 s-1 at zero ionic strength. The data are discussed in terms of both a two-binding-site mechanism and a single-binding-site, multiple-pathway mechanism.     

A possible origin of chemiluminescence in phagocytosing neutrophils. Reaction between chloramines and H2O2

A mixture of chloramines and hydrogen peroxide emits light. It was found that the reaction between taurine monochloramine and hydrogen peroxide is very slow. The stoichiometry of the reaction is 1:1 and taurine is detected as one of the products. The chlorinated proteins and bacteria, containing N-Cl groups, when reacting with hydrogen peroxide, are more effective in emitting light than low-molecular chloramines. Luminol enhances considerably light yield of the chloramine-hydrogen peroxide reaction. The chloramine-H2O2 reaction may account for light emitted by neutrophils during phagocytosis.     

THE PRODUCTION OF HYDROGEN PEROXIDE BY BLUE-GREEN ALGAE: A SURVEY1

A survey of 38 axenic isolates of blue-green algae indicated that over half the isolates produced hydrogen peroxide under defined growth conditions. Three kinetic profiles for the formation of hydrogen peroxide, were observed; these are described. The possible site or sites of hydrogen peroxide formation remain unknown.     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Utilization of peroxide and its relevance in oxygen insertion reactions catalyzed by chloroperoxidase.

Chloroperoxidase (CPO) catalyzed oxygen insertions are highly enantioselective and hence of immense biotechnological potential. A peroxide activation step is required to give rise to the compound I species that catalyzes this chiral reaction. A side reaction, a catalase type peroxide dismutation, is another feature of CPO's versatility. This work systematically investigates the utilization of different peroxides for the two reactions, i.e. the catalase type reaction and the oxygen insertion reaction. For the oxygen insertion reaction, indene and phenylethyl sulfide were chosen as substrate models for epoxidation and sulfoxidation respectively. The results clearly show that CPO is stable towards hydrogen peroxide and has a total number of turnovers near one million prior to deactivation. The epoxidation reactions terminate before completion because the enzyme functioning in its catalatic mode quickly removes all of the hydrogen peroxide from the reaction mixture. Sulfoxidation reactions are much faster than epoxidation reactions and thus are better able to compete with the catalase reaction for hydrogen peroxide utilization. A preliminary study towards optimizing the reaction system components for a laboratory scale synthetic epoxidation is reported.     

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

Oxidative destruction of estradiol after treatment with hydrogen peroxide catalyzed by horseradish peroxidase and methemoglobin]

It is shown that estradiol in the presence of horse radish peroxidase interacts with hydrogen peroxide, which is evidenced by an increase in its optical density at 280 nm. The photometering of samples containing estradiol and horse radish peroxidase upon their titration with hydrogen peroxide indicated that the increase in optical density stops after introducing hydrogen peroxide equimolar in concentration to estradiol. The stoichiometric ratio of estradiol consumed during oxidative destruction to hydrogen peroxide was 1:1. In the presence of ascorbate, the oxidative destruction of estradiol by the action of hydrogen peroxide, catalyzed by horse radish peroxidase, was observed only after a latent period and showed the same regularities as in the absence of ascorbate. It was found by calorimetry that, during the latent period, estradiol catalyzes the degradation of hydrogen peroxide and ascorbate without undergoing oxidative destruction. The substrates of the peroxidase reaction benzidine, 1-naphthol, and phenol interact with hydrogen peroxide in the presence of ascorbate and horse radish peroxidase in a similar way. Presumably, upon interaction with hydrogen peroxide in the presence of horse radish peroxidase, estradiol, like other substrates of this reaction, undergoes oxidative destruction by the mechanism of peroxidase reaction. It is shown that oxidative destruction of estradiol by the action of hydrogen peroxide can also be catalyzed by methemoglobin by the same mechanism. These data are important for understanding the role of estradiol in the organism and the pathways of its metabolic conversions.     

N-dealkylation of aminopyrine catalyzed by soybean lipoxygenase in the presence of hydrogen peroxide

A hypothesis that lipoxygenase may mediate N-dealkylation of xenobiotics was investigated using the prototype drug aminopyrine and soybean lipoxygenase as a model enzyme in the presence of hydrogen peroxide. Formaldehyde production as a result of N-demethylation of aminopyrine exhibited pH optimum of 6.5. The reaction was dependent on the incubation time, amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. Under the experimental conditions employed, the specific activity for N-demethylation of aminopyrine was found to be 823 ± 93 nmoles per min/mg protein or 89 ± 10 nmoles per min/nmole of enzyme. The reaction was significantly inhibited by nordihydroguaiaretic acid and gossypol, the classical inhibitors of lipoxygenase. Spectrophotometric analyses indicated the generation of a nitrogen-centered free-radical cation as the initial oxidation product of aminopyrine. The rate of accumulation of this radical species was also dependent on pH, the amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. The radical production was markedly suppressed by ascorbate, glutathione, and dithiothreitol in a concentration-dependent manner. Preliminary data gathered for the oxidation of other chemicals indicated that the lipoxygenase exhibits a unique substrate specificity. Collectively, the evidence presented suggests for the first time that lipoxygenase pathway may be involved in N-demethylation of aminopyrine and other chemicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 175–183, 1998     

Evaluation of relative contributions of two enzymes supposed to metabolise hydrogen peroxide in Paracoccus denitrificans

A biosensor exploiting an electrochemically mediated enzyme-catalysed reaction was used to quantify relative contributions of cytoplasmic catalase and periplasmic cytochrome c peroxidase to the overall rate of hydrogen peroxide breakdown in cells of Paracoccus denitrificans. The effects of antimycin (an inhibitor of electron flow to cytochrome c peroxidase), the reaction rate versus substrate concentration profiles for the whole cells and subcellular fractions, and the time courses of oxygen concentration demonstrated a profound decrease in the capacity of cytochrome c peroxidase to reduce H2O2 under in vivo conditions. The reason is suggested to be a competition for available electrons between the enzyme and terminal oxidases metabolising oxygen produced by catalase.     

Muscle function in animal movement: passive mechanical properties of leech muscle

We investigated passive properties of leech body wall as part of a larger project to understand better mechanisms that control locomotion and to establish mathematical models that predict such dynamical behavior. In tests of length-tension relationships in 2-segment-long preparations of body wall through step-stretch manipulations (step size = 1 mm), we discovered that these relationships are nonlinear, with significant hysteresis, even for the relatively small changes in length that occur during swimming. We developed a mathematical model comprising three nonlinear springs, two in series with nonlinear dashpots that describe well the tension statics and dynamics for step-stretch experiments. This model suggested that body wall dynamics are slow enough to be neglected when predicting the tension generated by imposed sinusoidal length changes (about ±10% of nominal) at 1–3 Hz, mimicking swimming. We derived a static model, comprising one nonlinear spring, which predicts sinusoidal data accurately, even when preparations were exposed to serotonin (0.1–10 μM). Preparations bathed in saline-serotonin had significantly reduced steady-state and peak tensions, without alterations in tension dynamics. Anesthetizing preparations (8% ethanol) reduced body wall tension by 77%, indicating that passive tension in the obliquely striated longitudinal muscles of leeches results primarily from a resting tonus.     

Killing of bacillus spores by aqueous dissolved oxygen,ascorbic acid,and copper ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.