pH dependence of carbon monoxide binding to ferrous horseradish peroxidase

The kinetic parameters of the reaction of horseradish peroxidase with CO have been determined at pH values between 10 and 3. At pH 7.0 the CO binding equilibrium constant L was measured using submicromolar concentrations of horseradish peroxidase; the value obtained corresponds to the ratio of the association and dissociation kinetic constants as expected for a simple binding mechanism to a monomeric hemeprotein. The CO association rate constant is pH-independent below pH 7, whereas in going from pH 7 to pH 11 a 2-fold increase can be detected, as previously reported (Kertesz, D., Antonini, E., Brunori, M., Wyman, J., and Zito, R. (1965) Biochemistry 4, 2672-2676). On the other hand, CO dissociation displays a peculiar pH rate profile characterized by a progressive decrease from pH 10 to pH 5 and by a very marked increase as the pH is further lowered to pH congruent to 3. Furthermore, the rate of CO dissociation is markedly enhanced in peroxidase reconstituted with protoheme dimethyl ester, suggesting a role of the propionates in the regulation of this process.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

Activation thermodynamics of the binding of carbon monoxide to horseradish peroxidase. Role of pressure, temperature and solvent

The kinetics at 423 nm of the binding of carbon monoxide to ferrous horseradish peroxidase were studied as a function of three parameters: pressure (1-1200 bar), temperature (34 to -20 degrees C) and solvent (water, 40% ethylene glycol, 50% methanol) using a high-pressure stopped-flow apparatus. By using transition state theory the thermodynamic quantities delta V, delta S and delta H were determined under these different experimental conditions and were found to be greatly modulated by the physico-chemical parameters of the media. The results suggest that the macroscopic thermodynamic response is mainly controlled by the solvent. By adjusting two variables (among T, P, solvent), it is possible either to amplify or to cancel out the effect of the third.     

Interactions of photosystem II with bicarbonate, formate and acetate

In this study, we probe the effects of bicarbonate (hydrogencarbonate), BC, removal from photosystem II in spinach thylakoids by measuring flash-induced oxygen evolution patterns (FIOPs) with a Joliot-type electrode. For this we compared three commonly employed methods: (1) washing in BC-free medium, (2) formate addition, and (3) acetate addition. Washing of the samples with buffers depleted of BC and CO₂ by bubbling with argon (Method 1) under our conditions leads to an increase in the double hit parameter of the first flash (β₁), while the miss parameter and the overall activity remain unchanged. In contrast, addition of 40–50 mM formate or acetate results in a significant increase in the miss parameter and to an ∼50% (formate) and ∼10% (acetate) inhibition of the overall oxygen evolution activity, but not to an increased β₁ parameter. All described effects could be reversed by washing with formate/acetate free buffer and/or addition of 2–10 mM bicarbonate. The redox potential of the water-oxidizing complex (WOC) in samples treated by Method 1 is compared to samples containing 2 mM bicarbonate in two ways: (1) The lifetimes of the S₀, S₂, and S₃ states were measured, and no differences were found between the two sample types. (2) The S₁, S₀, S₋₁, and S₋₂ states were probed by incubation with small concentrations of NH₂OH. These experiments displayed a subtle, yet highly reproducible difference in the apparent S_i/S_−i state distribution which is shown to arise from the interaction of BC with PSII in the already reduced states of the WOC. These data are discussed in detail by also taking into account the CO₂ concentrations present in the buffers after argon bubbling and during the measurements. These values were measured by membrane-inlet mass spectrometry (MIMS).     

Transient Raman study of horseradish peroxidase. Evidence for a lack of extensive heme pocket relaxation subsequent to carbon monoxide photolysis

Time-resolved resonance Raman spectroscopy is a valuable tool for the study of the dynamics of heme-protein interactions. In particular, the photolysis of a ligand by short laser pulses allows for the examination of the dynamic evolution of heme-protein interactions subsequent to ligand dissociation. To date, such studies have been confined largely to hemoglobins and myoglobins. Here we present the results of the first transient Raman study of a peroxidase. Resonance Raman spectra of horseradish peroxidase were obtained within 10 ns of ligand (CO) photolysis at a variety of pH values. We find that there is only minimal relaxation of the heme pocket of horseradish peroxidase in response to ligand photolysis. This relaxation is pH-dependent and most probably involves the heme vinyl substituents. Such behavior is in sharp contrast to the transient behavior of most hemoglobins and beef heart cytochrome oxidase.     

The reaction of coumarins with horseradish peroxidase

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.     

Stereospecificity of horseradish peroxidase

We report here on the stereospecificity observed in the action of horseradish peroxidase (HRPC) on monophenol and diphenol substrates. Several enantiomers of monophenols and o-diphenols were assayed: L-tyrosinol, D-tyrosinol, L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, DL-alpha-methyldopa, DL-adrenaline, D-adrenaline, L-isoproterenol, DL-isoproterenol and D-isoproterenol. The electronic density at the carbon atoms in the C-1 and C-2 positions of the benzene ring were determined by NMR assays (delta1 and delta2). This value is related to the nucleophilic power of the oxygen atom of the hydroxyl groups and to its oxidation-reduction capacity. The spatial orientation of the ring substituents resulted in lower Km values for L- than for D-isomers. The kcat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same delta1 and delta2 NMR values for carbons C-1 and C-2, and therefore the same oxidation-reduction potential. In the case of substrates that cannot saturate the enzyme, the values of the binding constant for compound II (an intermediate in the catalytic cycle) followed the order: L-isomer>DL-isomer>D-isomer. Therefore, horseradish peroxidase showed stereospecificity in its affinity toward its substrates (K m) and in their transformation reaction rates (k cat).     

Heme-linked proton dissociation of carbon monoxide complexes of myoglobin and peroxidase.

It was found from spectrophotometric titration and proton balance measurement that the pKa value of a heme-linked protonation group of horseradish ferro-peroxidase C (donor:H2O2 oxidoreductase, EC 1.11.1.7) shifted from 7.25 to 8.25 upon combination with CO. The spectrophotometric titration experiment with myoglobin also revealed the presence of a heme-linked protonation group, the pKa value being 5.57 in myoglobin and 5.67 in the CO-myoglobin complex. It was concluded that the distinct shift of the pKa value in the case of peroxidase was attributable to the presence of a hydrogen bond between the sixth ligand and the distal base. The difference in the strength of such hydrogen bonding between peroxidase and myoglobin was discussed.     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase

The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Synthesis and properties of horseradish peroxidase copolymers

Conditions for copolymerization of native and sodium periodate-oxidized horseradish peroxidase (HTP; EC 1.11.1.7) have been optimized. Copolymerization products have been characterized electrophoretically, spectrally, and kinetically. Copolymers containing 2-3, 4, 5-7, and 9-10 molecules of the enzyme were found among the products of polymerization. The copolymers had lower values of D403/D280 than HRP. The copolymers had more ordered structures than the original HRP. Comparison of the thermal stability and kinetic characteristics of the fractions differing in the ratio of copolymers to the monomeric enzyme demonstrated that the polymeric products were more stable than HRP (in terms of resistance to high temperature or inhibitory effects of H202), but their kinetic activity was, on the whole, lower than that of the original enzyme.     

Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 A. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.     

Production of trace levels of carbon monoxide during methanogenesis on acetate and methanol

Summary Production of trace levels of carbon monoxide was consistently observed in the off-gas of a laboratory anaerobic digester fed Waste Activated Sludge. Inocula from this digester was enriched for acetate and methanol utilizing methanogenic populations. These enriched inocula were then monitored in batch assays for carbon monoxide and hydrogen production. Results demonstrated that carbon monoxide is produced during methanogenesis on both substrates. Subsequent utilization of CO was observed to occur after methane production was essentially complete for the assays conducted with methanol. Carbon monoxide evolution during methanogenesis on acetate displayed a markedly different trend from that observed from methanol.     

Synthesis and properties of horseradish peroxidase copolymers

Conditions for copolymerization of native and sodium periodate-oxidized horseradish peroxidase (HTP; EC 1.11.1.7) have been optimized. Copolymerization products have been characterized electrophoretically, spectrally, and kinetically. Copolymers containing 2–3, 4, 5–7, and 9–10 molecules of the enzyme were found among the products of polymerization. The copolymers had lower values of D ₄₀₃/D ₂₈₀ than HRP. The copolymers had more ordered structures than the original HRP. Comparison of the thermal stability and kinetic characteristics of the fractions differing in the ratio of copolymers to the monomeric enzyme demonstrated that the polymeric products were more stable than HRP (in terms of resistance to high temperature or inhibitory effects of H₂O₂), but their kinetic activity was, on the whole, lower than that of the original enzyme.