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.     

Preparation and characterization of alkaline phosphatase,horseradish peroxidase,and glucose oxidase conjugates with carboxylated carbon nano-onions

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO–enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ～0.5?mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.     

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).     

Effects of 2,4-substituents of deuteroheme upon peroxidase functions. Reactions of peroxidase and myoglobin with oxygen, carbon monoxide and alkylisocyanides

The oxygen affinity of myoglobins increased in the order diacetyl- < chlorocruoro- < proto- < meso-myoglobins, which was a decreasing order of electron-withdrawing capacities of 2,4-substituents of deuteroheme. The results led to a conclusion that the π bonding predominates over the σ bonding in the myoglobin-oxygen interaction.On the contrary, the affinities of myoglobins and reduced peroxidases for ethylisocyanide decreased as the electron-withdrawing capacities of the substituents were decreased and it was therefore concluded that the σ bonding plays a dominant role in the ethylisocyanide binding to these hemoproteins.No simple relationship was observed in the cases of reduced peroxidase-CO complexes and myoglobin-isopropylisocyanide and -tertiarybutylisocyanide complexes. Among the hemoproteins tested, protohemoproteins appeared to have the lowest affinities for these ligands.As was the case with ligands such as azide and cyanide, the affinities of a deuterohemo-protein for oxygen, CO, and isocyanides were invariably much higher than the value expected from the Hammett-type relationship. The linear relationship between enthalpy and entropy was found to be applied for the reactions of ligands and hemoproteins including deutero derivatives and the irregularly high affinities of deuterohemoproteins for ligands could not be explained on the basis of thermodynamic analyses.     

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.     

辣根过氧化物酶的热稳定剂

保持酶的天然状态和高催化特性具有重要的意义。本研究筛选了辣根过氧化物酶(HRP)的稳定剂并研究了其作用机制。结果发现硫酸镁和明胶能够显著提高HRP的热稳定性,并且两者具有协同作用。在硫酸镁和明胶组成的酶稳定剂存在的条件下,HRP在50oC保温80h后仍能保持89%的活性,常温下存放90d后可保持57%的活性,而未加稳定剂的对照样品中HRP的残留活性分别为6%和小于1%。通过对HRP的Soret带吸收光谱,色氨酸内源荧光,ANS荧光进行分析,揭示酶稳定剂可以明显降低在加热条件下HRP的变性程度,从而维持较为稳定的天然构象。     

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).     

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.     

Interaction of monoclonal antibodies with horseradish peroxidase

Properties of four types of monoclonal antibodies to horse-radish peroxidase were investigated. The dissociation constants and molecular-weight composition of the immune complexes were determined. The antibodies are shown to be directed to different epitopes on the polypeptide chain. Results of the theoretical prediction of the epitope localisation are presented. The interaction between the antibodies and peroxidase isozymes were studied.     

Activity, stability, and unfolding of reconstituted horseradish peroxidase with modified heme

Heme-propionates of horseradish peroxidase (HRP) were esterified by p-nitrophenol, phenol and p-methylphenol to change its electron character and to increase its hydrophobicity. These synthetic hemes were inserted apo-HRP to give a novel HRP, respectively. Of the three reconstituted HRPs, reconstituted HRP with p-nitrophenol-modified heme derivative had a larger initial rate, affinity, catalytic efficiency and substrate-binding efficiency than native HRP in aqueous buffer and some solvents. The reconstituted HRPs showed higher thermostability and tolerance of DMF because of the increase of the hydrophobicity of the active site. Changing the electron character of the aromatic moieties linked at each terminal of the two heme-propionates can control activity and stability of HRP. The initial rate, affinity, catalytic efficiency and substrate-binding efficiency increased with the increases of electron-withdrawing efficiency of substituents at 4-position of the phenolic used to synthesize the heme derivatives, contrariwise, the stability decreased. The modifications resulted in the increase in the temperature (T_m) at the midpoint of thermal denaturation and the decreases in both enthalpy and entropy change at T_m. The changes of catalytic properties and stabilities are related to the changes of the conformation of HRP. The modification changed the environment of heme and tryptophan, increased α-helix content of HRP. The present work demonstrates that enhancement of the hydrophobicity and the electron-withdrawing efficiency of heme improves the activity and stability of HRP.     

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).     

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.     

Interaction of propylthiouracil and its precursors with horseradish peroxidase

Interactions of horseradish peroxidase with propylthiouracil, thiouracil, propyluracil and uracil lead to the formation of complexes that exhibit different absorption spectra which can be attributed to the perturbation of peroxidase as the result of the drug-binding on a polar site in the protein. In this paper, by dilatometry and viscometry structural alterations in horseradish peroxidase were detected from its interaction with propylthiouracil and thiouracil only, and the physiological inhibition of peroxidase for these antithyroid drugs seems to be through structural alterations in the protein.     

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