Peroxidase-oxidase Reaction Research Articles

Human myeloperoxidase catalyzes an oscillating peroxidase–oxidase reaction

We have studied the peroxidase–oxidase reaction catalyzed by human myeloperoxidase in an open system where both substrates—molecular oxygen and NADH—are supplied continuously to the reaction mixture. The reaction shows oscillatory kinetics at pH values around 5, provided that the reaction medium in addition to the enzyme and the substrates also contains an aromatic electron mediator such as melatonin or 4-hydroxybenzoic acid and chloride ions at concentrations >1 mM. The experimental findings can be simulated by a detailed model of the reaction. The results are important for our understanding of oxidant production in neutrophils.

Concerted Simulations Reveal How Peroxidase Compound III Formation Results in Cellular Oscillations

A major problem in mathematical modeling of the dynamics of complex biological systems is the frequent lack of knowledge of kinetic parameters. Here, we apply Brownian dynamics simulations, based on protein three-dimensional structures, to estimate a previously undetermined kinetic parameter, which is then used in biochemical network simulations. The peroxidase-oxidase reaction involves many elementary steps and displays oscillatory dynamics important for immune response. Brownian dynamics simulations were performed for three different peroxidases to estimate the rate constant for one of the elementary steps crucial for oscillations in the peroxidase-oxidase reaction, the association of superoxide with peroxidase. Computed second-order rate constants agree well with available experimental data and permit prediction of rate constants at physiological conditions. The simulations show that electrostatic interactions depress the rate of superoxide association with myeloperoxidase, bringing it into the range necessary for oscillatory behavior in activated neutrophils. Such negative electrostatic steering of enzyme-substrate association presents a novel control mechanism and lies in sharp contrast to the electrostatically-steered fast association of superoxide and Cu/Zn superoxide dismutase, which is also simulated here. The results demonstrate the potential of an integrated and concerted application of structure-based simulations and biochemical network simulations in cellular systems biology.

Response of the Peroxidase-Oxidase Oscillator to Light Is Controlled by MB+−NADH Photochemistry

Although the peroxidase-oxidase (PO) oscillator has been widely studied [Scheeline et al. Chem. Rev. 1997, 97, 739], 1 the continued lack of quantitative agreement between experiment and theory suggests that the molecularcomponents and reactions essential for oscillation dynamics are not fully understood. Particularly, the role of photochemical reactions has been largely overlooked even though the photosensitizer methylene blue (MB + ) has been routinely added to PO reaction mixes by investigators since the first demonstration of sustained oscillations [Nakamura et al. Nature 1969, 222, 794]. 2 We reasoned that the presence of MB + in the PO reaction should make oscillations sensitive to visible light exposure. We tested this possibility and observed that both the frequency and the amplitude of O 2 oscillations obtained with a free-running periodic PO oscillator were suppressed in a rapid and reversible manner when exposed to visible light. The effect occurred at illumination wavelengths between 600 and 700 nm, was greatest at 670 nm, and was consistent with the absorbance spectrum of MB + . Measurements of the rate of NADH oxidation by MB + during illumination with red light showed a dose dependence consistent with the response curve observed for periodic PO oscillations. We concluded that PO oscillations were influenced by light through a photochemical effect on MB + , with the photosensitive reaction being the oxidation of NADH by MB + . Given these results, photoinduced suppression effects may be a common experimental bias in PO experiments where broadband spectroscopic illumination has been used. Future laboratory work involving the PO oscillator will need to control for MB + -dependent photochemical effects and theoretical modeling efforts should account for photochemistry involving MB + and NADH.

Mechanism of protection of peroxidase activity by oscillatory dynamics.

The peroxidase-oxidase reaction is known to involve reactive oxygen species as intermediates. These intermediates inactivate many types of biomolecules, including peroxidase itself. Previously, we have shown that oscillatory dynamics in the peroxidase-oxidase reaction seem to protect the enzyme from inactivation. It was suggested that this is due to a lower average concentration of reactive oxygen species in the oscillatory state compared to the steady state. Here, we studied the peroxidase-oxidase reaction with either 4-hydroxybenzoic acid or melatonin as cofactors. We show that the protective effect of oscillatory dynamics is present in both cases. We also found that the enzyme degradation depends on the concentration of the cofactor and on the pH of the reaction mixture. We simulated the oscillatory behaviour, including the oscillation/steady state bistability observed experimentally, using a detailed reaction scheme. The computational results confirm the hypothesis that protection is due to lower average concentrations of superoxide radical during oscillations. They also show that the shape of the oscillations changes with increasing cofactor concentration resulting in a further decrease in the average concentration of radicals. We therefore hypothesize that the protective effect of oscillatory dynamics is a general effect in this system.

Implications of enzyme kinetics.

Of the many examples of oscillatory kinetic behaviour known, several are briefly reviewed, including those of glycolysis, the peroxidase-oxidase reaction and oscillations in cellular calcium concentration. It is shown that simple mathematical models employing allosteric rate laws are sufficient to explain the instability of the steady state and the appearance of sustained oscillations. The cAMP-signalling systems of cellular slime moulds and the dynamics of intracellular calcium oscillations illustrate the importance of such oscillophores to inter- and intra-cellular communication and differentiation.

CONTINUING EMERGENCE IN LIVING SYSTEMS

As discussed in other articles in this issue, chemical emergence may have led to the appearance of life on the pre-biotic earth, but it is even more obviously clear that emergence continues in living systems, producing complex phenomena such as ordering, biorhythms and even, possibly, consciousness. The role of continuing emergence in living systems is reviewed here with special attention to the Peroxidase–Oxidase reaction and neurochemical systems. For the latter, we review the role of subnetwork dynamics in epilepsy and an intriguing new possiblity that calcium waves in fields of astrocytes in the brain may be involved in the spread of epileptic seizures.

A new method for studying caffeine?s antioxygenic property: Peroxidase-Oxidase biochemical reaction

The effect of Caffeine on Peroxidase-Oxidase (PO) reaction was studied systematically in this paper. We proved that the valley of PO oscillation is the best phase angle which was used to research antioxygenic property by the Analyte Pulse Perturbation Technique (APP), based on investigating the mechanism. Area integral calculus was proposed to use in quantitative analysis for the first time. There is a good linear relationship (R = 0.9950) between the ratio of amplitude changes of PO oscillation and the concentration of caffeine in the range 4.61 × 10−7 mol/L–1.84 × 10−5 mol/L. A new method for analysis by PO oscillation was set up. We also investigated two-dimensional projections and Fourier spectrum of nonlinear complicate system—PO reaction which was perturbed by caffeine, in order to provide a theoretical basis for studying effects of kinds of antioxidants on life system.

A Model of the Oscillatory Metabolism of Activated Neutrophils

We present a two-compartment model to explain the oscillatory behavior observed experimentally in activated neutrophils. Our model is based mainly on the peroxidase-oxidase reaction catalyzed by myeloperoxidase with melatonin as a cofactor and NADPH oxidase, a major protein in the phagosome membrane of the leukocyte. The model predicts that after activation of a neutrophil, an increase in the activity of the hexose monophosphate shunt and the delivery of myeloperoxidase into the phagosome results in oscillations in oxygen and NAD(P)H concentration. The period of oscillation changes from >200 s to 10–30 s. The model is consistent with previously reported oscillations in cell metabolism and oxidant production. Key features and predictions of the model were confirmed experimentally. The requirement of the hexose monophosphate pathway for 10 s oscillations was verified using 6-aminonicotinamide and dexamethasone, which are inhibitors of glucose-6-phosphate dehydrogenase. The role of the NADPH oxidase in promoting oscillations was confirmed by dose-response studies of the effect of diphenylene iodonium, an inhibitor of the NADPH oxidase. Moreover, the model predicted an increase in the amplitude of NADPH oscillations in the presence of melatonin, which was confirmed experimentally. Successful computer modeling of complex chemical dynamics within cells and their chemical perturbation will enhance our ability to identify new antiinflammatory compounds.

Mechanism of melatonin-induced oscillations in the peroxidase–oxidase reaction

Melatonin induces oscillations in the peroxidase–oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.

Simulations of temperature sensitivity of the peroxidase–oxidase oscillator

The influence of temperature on the oscillatory kinetics of the peroxidase–oxidase reaction was studied theoretically. Assuming Q 10=2 for elementary reactions, the effect of multiplying the rate constants of the model by factors between 0.5 and 2 (corresponding to a 10 °C decrease and increase, respectively, of temperature) was investigated. First, the individual rate constants were successively multiplied by 0.5 or 2 while all other rate constants were kept unchanged. This resulted in either a longer or a shorter period, depending on the rate constant being changed. Multiplication by 0.5 or by 2 generally resulted in opposite effects on the period length. However, the absolute value of this deviation differed. Also, the dynamics changed when halving the dimerization rate of NAD as well as when doubling the rate constant for the reduction of ferric peroxidase by NAD . Next, simulations were performed multiplying all rate constants by one and the same factor, which increased progressively from 0.5 to 2. Intervals were found corresponding to temperature dependency, compensation, and over-compensation, respectively.

Secondary quasiperiodicity in the peroxidase–oxidase reaction

Secondary quasiperiodicity (period-doubled oscillations modulated by an incommensurate frequency), or “Q2”, is the temporal manifestation of quasiperiodic motion on period-doubled tori. The existence of this regime in a chemical reaction was first predicted (T. V. Bronnikova, W. M. Schaffer and L. F. Olsen, J. Chem. Phys., 1996, 105, 10 849) in the course of numerical explorations of a detailed model of the peroxidase–oxidase system. Subsequent analysis (T. V. Bronnikova, W. M. Schaffer, M. J. B. Hauser and L. F. Olsen, J. Phys. Chem. B, 1998, 102, 632) suggested the possibility of homoclinic transitions (“fat torus” bifurcation) to chaos involving Q2. In the present paper, we present the first experimental evidence for secondary quasiperiodicity and fat torus bifurcations in a chemical oscillator. We also identify a second (“thin torus”) route to chaos involving Q2. The relationship of these two bifurcation scenarios to each other and to the experimental findings is discussed.

Oscillatory dynamics protect enzymes and possibly cells against toxic substances.

We have used the oscillating peroxidase-oxidase (PO) reaction as a model system to study how oscillatory dynamics may affect the influence of toxic reaction intermediates on enzyme stability. In the peroxidase-oxidase reaction reactive intermediates, such as hydrogen peroxide, superoxide, and hydroxyl radical are formed. Such intermediates inactivate many cellular macromolecules such as proteins and nucleic acids. These reaction intermediates also react with peroxidase itself to form an inactive enzyme. The fact that the PO reaction shows bistability between an oscillatory and a steady state gives us a unique possibility to compare such inactivation when the system is in one of these two states. We show that inactivation of peroxidase is slower when the system is in an oscillatory state, and using numerical simulations we provide evidence that oscillatory dynamics lower the average concentration of the reactive intermediates.

Melatonin Activates the Peroxidase–Oxidase Reaction and Promotes Oscillations

We have studied the peroxidase–oxidase reaction with NADH and O2 as substrates and melatonin as a cofactor in a semibatch reactor. We show for the first time that melatonin is an activator of the reaction catalyzed by enzymes from both plant and animal sources. Furthermore, melatonin promotes oscillatory dynamics in the pH range from 5 to 6. The frequency of the oscillations depends on the pH such that an increase in pH was accompanied by a decrease in frequency. Conversely, an increase in the flow rate of NADH or an increase in the average concentration of NADH resulted in an increase in oscillation frequency. Complex dynamics were not observed with melatonin as a cofactor. These results are discussed in relation to observations of oscillatory dynamics and the function of melatonin and peroxidase in activated neutrophils.

Nonlinear Dynamics of the Peroxidase−Oxidase Reaction. II. Compatibility of an Extended Model with Previously Reported Model-Data Correspondences

In the course of formulating detailed models of complex chemical reactions, it is sometimes the case that modifications intended to account for one set of experimental observations wind up destroying a model's ability to account for other results. Here, we consider a recently proposed model of the peroxidase-oxidase reaction which derives from an earlier scheme via the addition of NADH oxidation by superoxide anion or its protonated form, hydroperoxyl radical. This modification was introduced to account for the observation of bistability and bursting at enzyme concentrations less than 0.5 μM. Left unanswered in our previous paper was the matter of whether the proposed “fix” invalidates previously published examples of model-data agreement at higher enzyme concentrations. In the present paper, we show that under these latter circumstances, the new mechanism is as good as, and in some instances superior to, its predecessor. More generally, we argue that the consequences of NADH oxidation by O2- or HO2• should be manifest principally at low enzyme concentrations, thereby offering a “global” explanation of our findings. Neither our original model, nor the derivative scheme treated here, provides for reactions involving NAD dimers, a species in which there has recently been renewed interest. Most importantly, it has been proposed to replace the reduction of coIII (an enzyme intermediate) by NAD radicals, a reaction for which there is no direct evidence, with the corresponding reaction involving NAD2. While a detailed assessment of the consequences of dimer chemistry to theoretical peroxidase-oxidase dynamics is beyond the scope of the present investigation, it is easily documented that this substitution, by itself, abolishes oscillatory behavior for all model parametrizations previously considered. Moreover, this result appears to obtain for arbitrarily small values of the associated rate constant. Whether or not the inclusion of additional dimer reactions can restore the model's ability to account for experimental observations of complex dynamics remains to be determined.

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.

Principal component analysis of dynamical features in the peroxidase-oxidase reaction

Inherent variance due to oscillations in the peroxidase-oxidase (PO) reaction was studied using principal component analysis (PCA). The substrates were oxygen and reduced nicotinamide adenine dinucleotide (NADH). Horseradish peroxidase (HRP) catalyzed the reaction. The concentration of a cofactor, methylene blue (MB), was varied, and 2,4-dichlorophenol was kept constant. Increase in the NADH influx was used to change the reaction dynamics from periodic to chaotic. The reaction space was abstracted to the most significant, mutually independent, pairs of absorption and kinetic basis vectors (principal components). Typically, two significant principal components were extracted from the periodic time series and three from the chaotic data. The PCA models accounted for 70-97% of experimental variance. The greatest fraction of the total variance was accounted for in experiments exhibiting periodic dynamics and less than 25 nM MB. More MB induced an increased contribution of NADH to the PO oscillator variance, as did increased NADH influx. A simulated absorption time series, computed from a mass-action model of the chemistry, was analyzed by PCA as well. The comparison of simulation with experiment indicates that the chemical model renders the time series for HRP oxidation forms with fidelity, but incompletely represents NADH chemistry and other salient processes underlying the observed dynamics.

Further studies of the effect of magnetic fields on the oscillating peroxidase–oxidase reaction

We have performed an experimental study of the effect of DC magnetic fields on the oscillating peroxidase–oxidase reaction to obtain additional information about the mechanism of the action of the magnetic field. The changes in oscillation amplitude and frequency were measured at low (5.1) and high (6.3) pH, respectively. Furthermore, the effect of a magnetic field with a field strength of 1000 G on the oscillation amplitude was measured at different concentrations of enzyme and the modifiers methylene blue and 4-hydroxybenzoic acid. Changes in pH and the concentration of methylene blue seemed to have little or no effect on the reaction's magnetosensitivity. However, increases in the enzyme concentration or the concentration of 4-hydroxybenzoic acid were accompanied by increases in magnetosensitivity. These results lend support to a previously proposed mechanism in which the magnetosensitive steps involve the transfer of electrons to two enzyme intermediates, compounds I and II.

On the role of methylene blue in the oscillating peroxidase–oxidase reaction

The role of methylene blue (MB) and its interaction with various enzymic and nonenzymic intermediates in the oscillating peroxidase–oxidase reaction were investigated. Time dependent spectral changes were deconvoluted into time series of the different intermediates. Ferric peroxidase, ferrous peroxidase, and peroxidase compound III account for most of the spectral changes, while the two remaining oxidation states of the enzyme (compound I and II) are only present in very low amounts during any phase of the oscillations. The presence of MB is required for the generation of sustained oscillations and complex dynamics. The amount of ferrous peroxidase formed during an oscillatory cycle decreases with increasing concentrations of MB. At ‘‘high ’’ concentrations of MB only damped oscillations are observed. These findings are interpreted in terms of either a competition between MB and other reactants for NAD• radicals, or of a reaction between MB and ferrous peroxidase. In the absence of MB only damped oscillations can be observed due to slow and irreversible degradation of the enzyme. This suggests that MB also protects the enzyme against degradation.

Perturbations of Simple Oscillations and Complex Dynamics in the Peroxidase−Oxidase Reaction Using Magnetic Fields

The effect of dc magnetic fields of the order of 0−4000 G on the oscillating peroxidase−oxidase (PO) reaction was studied. Specifically the effects of magnetic fields on the amplitude of simple periodic oscillations and on complex dynamic states were investigated. Magnetic fields induce a biphasic change in oscillation amplitude: At field strengths up to 1500 G, the amplitude decreases gradually by 11%. At higher field strengths the decrease in amplitude is less, and it vanishes at field strengths of 4000 G and higher. The effect of magnetic fields on complex dynamics is generally to shift the dynamics from one state to a neighboring state. Numerical simulations of a detailed model of the PO reaction support the hypothesis that the magnetosensitive step is the reduction of compound II to ferric peroxidase or the reduction of compound I to compound II or both.

Magnetic field perturbations as a tool for controlling enzyme-regulated and oscillatory biochemical reactions

The feasibility of magnetic field perturbations as a tool for controlling enzyme-regulated and oscillatory biochemical reactions is studied. Our approach is based on recent experimental results that revealed magnetic field effects on the in vitro activity of enzyme systems in accordance with the radical pair mechanism. A minimum model consisting of two coupled enzyme-regulated reactions is discussed that combines, in a self-consistent manner, magnetic field-sensitive enzyme kinetics with non-linear dynamical principles. Furthermore, a simple detector mechanism is described that is capable of responding to an oscillatory input. Results reveal that moderate-strength magnetic fields ( B=1–100 mT) may effectively alter the dynamics of the system. In particular, a response behavior is observed that depends on: (1) the combination of static and time-varying magnetic fields; (2) the field amplitude; and (3) the field frequency in a non-linear fashion. The specific response behavior is critically determined by the biochemical boundary conditions as defined by the kinetic properties of the system. We propose an experimental implementation of the results based on the oscillatory peroxidase–oxidase reaction controlled by the enzyme horseradish peroxidase.