The binding of cyanide to ferroperoxidase

1. The equilibrium and kinetics of cyanide binding to ferroperoxidase were investigated. At pH9.1 the equilibrium and kinetic measurements agree closely and disclose a single process with an affinity constant of 1.1x10(3)m(-1) and combination and dissociation velocity constants of 29m(-1).s(-1) and 2.5x10(-2)s(-1) respectively. 2. At pH values below 8 the affinity constant falls until at pH6.0 the ferroperoxidase.cyanide complex is no longer formed. This is shown to be associated with the formation of ferriperoxidase.cyanide complex in the mixture even in the presence of excess of sodium dithionite. 3. Rapid-pH-jump experiments show a fast pseudo-first-order interconversion between ferroperoxidase.cyanide complex at pH9.1 and ferriperoxidase.cyanide complex at pH6.0. 4. The kinetics of binding of cyanide to dithionite-reduced peroxidase at pH6.0 are complicated and radically different from those observed at pH9.1. 5. Above pH8 the change of affinity constant with pH is consistent with the undissociated species, HCN, being bound by the ferroperoxidase. The enthalpy for this process measured both by equilibrium and kinetic methods is about -8kcal/mol. 6. The binding of cyanide to reconstituted peroxidases, proto, meso and deutero, was investigated. 7. The results are discussed in relation to known data on cyanide binding to other haemoproteins.     

Tomato peroxidase: purification, characterization, and catalytic properties

A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.     

Ligational effects on reduction of myoglobin and horseradish peroxidase by inorganic reagents

Previous studies of the reduction of metmyoglobin and adducts by dithionite have been extended to horseradish peroxidase and its complexes. In addition, the reduction of metmyoglobin, horseradish peroxidase and adducts by a much bulkier reactant, cobalt(II) sepulchrate has been studied. Similar patterns of kinetic behavior were observed, namely, direct reduction of cyanide and imidazole adducts of the iron(III) proteins and indirect (via dissociation) reduction of the fluoride adduct. In the reduction of horseradish ferriperoxidase by cobalt(II) sepulchrate, three steps are observed and the spectral properties of the intermediate(s) and their kinetic behavior delineated. The final product is ferroperoxidase confirmed by spectral properties and its behavior on oxygenation. Reduction of cytochrome c(III) and Hipip by cobalt(II) sepulchrate appears to be a uniphasic reaction and second-order rate constants have been determined.     

Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

The binding of ethyl isocyanide to ferroperoxidase

The equilibrium and kinetics of ethyl isocyanide binding to ferroperoxidase were studied. At pH9.1 the results of both studies are consistent with a single-process model with an affinity constant of 95m(-1) and combination and dissociation constants of 2.2x10(3)m(-1).s(-1) and 23s(-1) respectively. Ethyl isocyanide is not bound significantly at pH values lower than 6.0, and in this behaviour and the pH-dependence of the affinity constant, similarities exist between isocyanide and cyanide binding. The enthalpy of the process measured by equilibrium methods is -59kJ/mol (-14kcal/mol). At pH values below 9, the ethyl isocyanide adduct changes in a slow time-dependent manner, giving rise to a new species. These changes are reversible on increasing the pH. The results are discussed in relation to other known information about ligand binding to ferroperoxidase and to myoglobin.     

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Does diphenylene iodonium chloride have any effect on the O2- -generating step of plant peroxidases?

The O2*- -generating step of plant peroxidases during their catalytic cycle is represented by the decay of compound III (CoIII) into ferriperoxidase, which most likely involves the dissociation of a ferric-O2*- complex to yield the ferric form of the enzyme and O2*-. Diphenylene iodonium chloride (DPI), at 50-100 microM, does not significantly enhance the stability of CoIII of peroxidase, as judged by the values of k(decay), and therefore, DPI appears to have no effect on the O2*- -generating step of plant peroxidases. From these results, it is concluded that caution should be exercised when considering peroxidase as a possible enzyme target of O2*- -mediated plant physiological processes sensitive to DPI inhibition.     

Titration study of guaiacol oxidation by horseradish peroxidase.

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.     

The oscillating peroxidase-oxidase reaction in an open system. Analysis of the reaction mechanism.

1. The oscillations in the peroxidase-oxidase reaction in an open system with NADH as the hydrogen donor are caused by the reaction starting and stopping at critical concentrations of the substrates O2 and NADH. The existence of such critical concentrations is typical of branched chain reactions. 2. The critical concentrations of O2 and NADH that determine the initiation of the reaction are mutually dependent. 3. The branching reactions that determine these critical concentrations involve compounds I and II. 4. Superoxide may be involved in the branching reactions by reacting with NADH and ferriperoxidase. At pH 5.1 the rate constant for the latter reaction is determined as 1.5 . 10(5) M-1 . s-1, whereas for the former reaction only an upper limit for the rate constant of 3.5 . 10(4) M-1 . s-1 could be estimated. These relatively low rate constants suggest that alternative branching reactions may also be involved.     

Optical detection of a red-shifted Michaelis complex in the reaction of HCN with ferromyeloperoxidase. Evidence for conformational stabilization of high spin iron (II).

The reaction of HCN with ferromyeloperoxidase involves the sequential formation of two monocyanide complexes. The first complex, which forms immediately on mixing, is characterized by a red shift in the Soret band of the ferroperoxidase, and a dissociation constant (measured as a Michaelis constant) of 0.67 mM. The second complex arises from the first via a first order process, whose maximal rate is 0.095 s-1 at 25 degrees C, pH 7.0. This more stable complex is characterized by a blue shift in the Soret and alpha bands and by an overall dissociation constant in the region of 4.5 microM. This gives a free energy difference between the two complexes of around 3.0 kcal mol-1 and a difference in optical absorption of 15 nm (Soret). The measured Arrhenius activation energy for the conversion of the high energy, long wavelength complex to the low energy, short wavelength complex is 16.3 kcal mol-1. A larger blue shift is observed on protein denaturation (34 nm), after which the two-step binding reaction is not observed. This, and the magnitude of the activation energy in the spontaneous complex interconversion process, shows that the latter is a conformational process. In addition, it can be concluded that the unknown structural feature of the heme site which is responsible for the anomalous red shift in the optical spectrum of native ferromyeloperoxidase, is also the link between the ligand state of the iron and the protein conformation.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Benzylhydrazine as a pseudo-substrate of bovine serum amine oxidase.

Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed.     

Energy transfer and distribution in the red alga Porphyra perforata studied using picosecond fluorescence spectroscopy

The detailed process of excitation transfer among the antenna pigments of the red alga Porphyra perforata was investigated by measuring time-resolved fluorescence emission spectra using a single-photon timing system with picosecond resolution. The fluorescence decay kinetics of intact thalli at room temperature revealed wavelength-dependent multi-component chlorophyll a fluorescence emission. Our analysis attributes the majority of chlorophyll a fluorescence to excitation originating in the antennae of PS II reaction centers and emitted with maximum intensities at 680 and 740 nm. Each of these fluorescence bands was characterized by two kinetic decay components, with lifetimes of 340-380 and 1700-2000 ps and amplitudes varying with wavelength and the photochemical state of the PS II reaction centers. In addition, a small contribution to the long-wavelength fluorescence band is proposed to arise from chlorophyll a antennae coupled to PS I. This component displays fast decay kinetics with a lifetime of approx. 150 ps. Desiccation of the thalli dramatically increases the contribution of this fast decay component.     

Reactions of reducing radicals with ribonuclease

Radiation-induced reactions of hydrated electrons, formate- and ethanol radicals with ribonuclease were studied by pulse radiolysis and by electrophoresis. Initially formate radicals react rapidly and very specifically with the disulphide bonds of ribonuclease. This reaction leads to aggregation by intermolecular S-S-interchange, the process being more effective at pH 4, since formation and decay of S-S-.-radical anions increases with decreasing pH. With high doses additional unreducible aggregates are formed. Radical formation at the positively charged histidine residues seems to be involved. Hydrated electrons do not react as selectively as the formate radicals, but with several sites in native ribonuclease. Thus with low doses unreducible aggregates are formed. Electrophoresis shows that reaction of the electrons causes fragmentation of the peptide chain, when OH-radicals are scavenged. Very weak transient spectra and very little degradation result on reaction of ethanol radicals with ribonuclease.     

KINETICS OF THE BIOLUMINESCENT REACTION IN CYPRIDINA. II

1. The decay curve of the light produced in the course of the luminescent reaction in Cypridina is, after the first second, in complete agreement with the theoretical expectation for a monomolecular reaction, if light intensity at any instant is assumed to be proportional to reaction velocity at that instant. It is shown that for such a reaction log I = - kt + log Ak and that the experimental values satisfy this equation. 2. The first second or two of the reaction is characterized by a brilliant initial flash, whose value is much too high to accord with the succeeding intensities and with the above formula. It is suggested that this initial high reaction velocity is an indication of a heterogeneous system. 3. Identical solutions run simultaneously give decay curves which check within the limits of the photographic error. 4. Stirring does not affect the reaction velocity or the form of the decay curve. 5. Reaction velocity is proportional to enzyme concentration, over the range of concentrations used in the study. 6. Changes in the concentration of the substrate do not affect the value of k, when all other factors are held constant. A diminution of luciferin concentration results only in a decrease in the value of the y-intercept, Log Ak, the two straight line plottings for two different concentrations being parallel. 7. The temperature coefficient is high, being about 4.5 for the 15–25° interval, and 3.0 for the 25–35° interval.     

A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2'-azido-2'-deoxynucleoside diphosphates

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a tyrosine radical which is essential for enzyme activity. In the reaction between ribonucleotide reductase and the substrate analogue 2'-azido-2'-deoxycytidine 5'-diphosphate a new transient radical is formed. The EPR characteristics of this new radical species are consistent with a localization of the unpaired electron at the sugar moiety of the nucleotide. The radical shows hyperfine couplings to a hydrogen and a nitrogen nucleus, the latter probably being part of the azide substituent. The formation of the nucleotide radical in this suicidal reaction is concomitant with the decay of the tyrosine radical of the B2 subunit. Kinetic data argue for a first (pseudosecond) order decay of the B2 radical via generation of the nucleotide radical followed by a slower first order decay of the nucleotide radical. End products in the reaction are cytosine and radical-free protein B2. In the reaction between bacteriophage T4 ribonucleotide reductase and 2'-azido-2'-deoxycytidine 5'-diphosphate an identical nucleotide radical is formed. The present results are consistent with the hypothesis that the appearance and structure of the transient radical mimic stages in the normal reaction pathway of ribonucleotide reductase, postulated to proceed via 3'-hydrogen abstraction and cation radical formation of the substrate nucleotide (Stubbe, J., and Ackles, D. (1980) J. Biol. Chem. 255, 8027-8030). The nucleotide radical described here might be equivalent to such a cation radical intermediate.     

Fluorescence decay kinetics of chlorophyll in photosynthetic membranes

The absorption of light by the pigments of photosynthetic organisms results in electronic excitation that provides the energy to drive the energy-storing light reactions. A small fraction of this excitation gives rise to fluorescence emission, which serves as a sensitive probe of the energetics and kinetics of the excited states. The wavelength dependence of the excitation and emission spectra can be used to characterize the nature of the absorbing and fluorescing molecules and to monitor the process of sensitization of the excitation transfer from one pigment to another. This excitation transfer process can also be followed by the progressive depolarization of the emitted radiation. Using time-resolved fluorescence rise and decay kinetics, measurements of these processes can now be characterized to as short as a few picoseconds. Typically, excitation transfer among the antenna or light harvesting pigments occurs within 100 psec, whereupon the excitation has reached a photosynthetic reaction center capable of initiating electron transport. When this trap is functional and capable of charge separation, the fluorescence intensity is quenched and only rapidly decaying kinetic components resulting from the loss of excitation in transit in the antenna pigment bed are observed. When the reaction centers are blocked or saturated by high light intensities, the photochemical quenching is relieved, the fluorescence intensity rises severalfold, and an additional slower decay component appears and eventually dominates the decay kinetics. This slower (1-2 nsec) decay results from initial charge separation followed by recombination in the blocked reaction centers and repopulation of the excited electronic state, leading to a rapid delayed fluorescence component that is the origin of variable fluorescence. Recent growth in the literature in this area is reviewed here, with an emphasis on new information obtained on excitation transfer, trapping, and communication between different portions of the photosynthetic membranes.     

Bioluminescence decay kinetics in the reaction of bacterial luciferase with different aldehydes

At 22°C the bioluminescence decay kinetics in the in vitro reaction catalysed by Vibrio harveyi luciferase in the presence of different aldehydes–-nonanal, decanal, tridecanal and tetradecanal did not follow the simple exponential pattern and could be fitted to a two-exponential process. One more principal distinction from the first-order kinetics is the dependence of the parameters on aldehyde concentration. The complex bioluminescence decay kinetics are interpreted in terms of a scheme, where bacterial luciferase is able to perform multiple turnovers using different flavin species to produce light. The initial phase of the bioluminescent reaction appears to proceed mainly with fully reduced flavin as the substrate while the final one results from the involvement of flavin semiquinone in the catalytic cycle.     

Spin trapping study on the kinetics of Fe2+ autoxidation: formation of spin adducts and their destruction by superoxide.

The oxidation of Fe2+ was investigated by electron spin resonance spin trapping techniques with N-t-butyl-alpha-phenylnitrone (PBN) and dimethyl sulfoxide. Under pure oxygen, the spin adduct PBN/.OCH3 was rapidly generated by the addition of Fe2+ (0.2-1.2 mM) into phosphate buffer containing ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide, and PBN at pH 7.4, but it decayed. The decay process of PBN/.OCH3 consists of two components. The fast decay was dependent on Fe2+ concentration. Another was due to destruction of the spin adduct by superoxide anion (.O2-), because superoxide dismutase (SOD) markedly prevented the decay. Catalase decreased the yield of PBN/.OCH3. When EDTA was replaced by diethylenetriaminepentaacetic acid (DTPA), both the generation and decay process of PBN/.OCH3 were slow. SOD and catalase effects were similar to those in EDTA. Fe2+ produced PBN/.OCH3 even in the absence of chelators. We could estimate the kinetic parameters by computer simulation, comparing the Fe2+ oxidation in EDTA with that in DTPA. These results demonstrate that Fe2+ reacts with O2 to generate .O2- and then H2O2, which produces .CH3 by reaction with Fe2+ and dimethyl sulfoxide.(.)OCH3 results from the reaction between .CH3 and O2. The adduct PBN/.OCH3 decays by reaction with Fe2+ and .O2-.     

Effect of limited modification of amino groups on the reactivity of human factor Xa

The basis of the specificity of human coagulation factor Xa has been probed with a reagent that reacts with nucleophiles, N-succinimidylpropionate. At pH 8.0 and 0.25 mM N-succinimidylpropionate, 0.4 microM factor Xa lost approx. 90% of its activity toward prothrombin in 4 min. The decay was first-order, k = 0.64 min-1, which increased to 0.98 min-1 in 1 mM Ca2+, and the dependence of k upon pH was consistent with primary amines being the target. The rate of modification was unaffected by the presence of a tetrapeptide substrate during modification; likewise, activity toward a tripeptide p-nitroanilide was unaltered during exposure of factor Xa to N-succinimidylpropionate with or without Ca2+. In addition, inhibition by antithrombin III was retained with a somewhat enhanced rate after modification; however, the acceleration of this by heparin was significantly less. Kinetic determination of the number of residues modified gave a reaction order of 2.0, while reaction with N-succinimidyl[3H]propionate yielded labeled factor Xa containing 1.0 mol N-succinimidylpropionate/mol factor Xa and 50% normal clotting activity, or 2.0 mol N-succinimidylpropionate/mol and 1% activity, respectively. Thus, one nucleophilic group is required for the reaction of factor Xa with prothrombin but not for the hydrolysis of peptides or recognition of antithrombin III. The decay of clotting activity of the factor X zymogen in N-succinimidylpropionate was much slower though still Ca2+-dependent. Conversely, the reaction of a related compound--N-succinimidyl(4-hydroxyphenyl)propionate or Bolton-Hunter reagent--with factor Xa broadly resembled that of N-succinimidylpropionate but the decay curves indicated more complex kinetics. Therefore, the target groups vary in their accessibility to modification according to the structural characteristics of both the protein and the reagent.