Mechanistic implications of variable stoichiometries of oxygen consumption during tyrosinase catalyzed oxidation of monophenols and o-diphenols

The stoichiometry of oxygen consumption during tyrosinase-catalyzed oxidation of an o-diphenol (4-tert-butylcatechol, TBC) and a monophenol (4-tert-butylphenol, TBP) has been determined. At high [substrate]/[enzyme] ratios, in the case of o-diphenols, the stoichiometry of the enzyme-catalyzed reaction was always 1 O(2)/2 o-diphenols, although if the o-quinone product was unstable, the apparent stoichiometry could tend to 1 O(2)/1 o-diphenol due to regeneration of an o-diphenol in a side reaction. In the case of monophenols, the stoichiometry could be 1 O(2)/1 monophenol or 1.5 O(2)/1 monophenol depending if the o-quinone product was stable or unstable, respectively. However, at low [substrate]/[enzyme] ratios, the oxygen/substrate stoichiometry could, even in the case where stable products are formed, be lower than 1 O(2)/2 substrates for o-diphenols or higher than 1 O(2)/1 substrate for monophenols. These data supported the mechanism proposed by Rodríguez-López et al. [J. Biol. Chem. 267 (1992) 3801-3810], in which, during hydroxylation of monophenols, tyrosinase first transformed monophenol to o-diphenol and then either catalyzed a further oxidation to form o-quinone or released it into the reaction medium. In this second case, subsequent oxidation of the o-diphenol resulted in additional oxygen consumption.     

Generation of superoxide during the enzymatic action of tyrosinase.

Evidence for the generation of superoxide anion in an enzymatic action of tyrosinase is reported. In the dopatyrosinase reaction, 1 mol of O2 is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and 1/4 mol of O2-. Superoxide dismutase and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (a chemiluminescence probe and O2 trap) do not inhibit the rate of dopachrome formation from dopa in the presence of tyrosinase, indicating that free O2- is not utilized for metabolizing dopa. ESR studies for the accumulation of semiquinone radicals generated from tyrosine and N-acetyltyrosine in the presence of tyrosinase imply that O2- is not generated by the semiquinone + O2 reaction. Since the addition of H2O2 and dopa to tyrosinase promotes the release of O2- and formation of dopachrome, the Cu(II)O2-Cu(I) complex could be formed as a intermediate (an active form of tyrosinase); [Cu(II)]2 + H2O2 in equilibrium Cu(I)O2-Cu(II) + 2H+.     

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Calibration and use of a Clark-type oxygen electrode from 5 to 45 degrees C

A calibration procedure for a Clark-type oxygen electrode over a wide range of temperatures is described. The autoxidation of duroquinol (2,3,5,6-tetramethyl-1,4-benzenediol) was used to verify the electrode's ability to accurately sense the total amount of dissolved O2 in an aqueous buffer. Electrode response time was measured by using oxygenated ethanol to deliver a rapid increase in O2 concentration to the reaction medium. An oxygen-producing system (spinach thylakoids) was utilized to test the range of O2-evolution rates able to be sensed. It was concluded that a Clark-type oxygen electrode has the absolute sensitivity, rapidity, and range necessary to accurately track rates of O2 production or consumption from 5 to 45 degrees C.     

Tyrosinase-mediated formation of a reactive quinone from the depigmenting agents, 4-tert-butylphenol and 4-tert-butylcatechol

Exposure of the skin to certain phenols or catechols such as 4-tert-butylphenol (TBP) and 4-tert-butylcatechol (TBC) may cause leukoderma. These substances are used in the polymer industry and numerous cases have been reported. Several theories of the mechanism for chemical leukoderma have been suggested. In the present study, TBP and TBC are shown to be oxidised by tyrosinase. The oxidation of TBC yields a quinone that is further investigated on its reactions with cysteine or glutathione (GSH). The products formed are isolated and identified by mass spectrometry and nuclear magnetic resonance as being 4-tert-butyl-6-S-cysteinylcatechol (cys-TBC) and 4-tert-butyl-6-S-glutathionylcatechol (GS-TBC). The reactive quinone is a strongly electrophilic substance that rapidly reacts with GSH. A depletion of the GSH defence system may give conditions where the quinone lives long enough to effect its toxic properties. The influence of the reactive tert-butylquinone on enzymatic activities is demonstrated by the inhibition of glyceraldehyde-3-phosphate dehydrogenase.     

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.     

O2 solubility in aqueous media determined by a kinetic method

A kinetic method for the determination of O2 solubility in air-saturated aqueous solutions of widely varying composition and temperature is described. It is based on the precise molar stoichiometry between the rates of uptake of H+ and O2, measured with response-matched electrodes, in the reaction NADH + H+ + 1/2O2----NAD+ + H2O, catalyzed by an NADH oxidase preparation. To the initially anaerobic test system, which contains an excess of NADH and NADH oxidase in a buffered medium, an aliquot of the O2-containing solution to be tested is added and the rates of both O2 uptake and H+ uptake are recorded; the H+ electrode is calibrated against standard HCl. From these data the amount of O2 in the aliquot is calculated. Some representative values for O2 solubility at 25 degrees C and 760 mm in air-saturated systems are (i) distilled H2O, 516 nmol O/ml, (ii) 0.15 M KCl, 480 nmol O/ml, and (iii) 0.25 M sucrose, 458 nmol O/ml. Data and equations are also given for the solubility of O2 at 760 mm in air-saturated and lightly buffered 0.15 M KCl and 0.25 M sucrose over the range 5 to 40 degrees C. In the method described the rates of O2 and H+ uptake are precisely linear and stoichiometric when NADH is present in large excess over O2. However, when O2 is in excess and small additions of 340-nm-standardized NADH are made, as in earlier methods based on NADH oxidation, the endpoint is approached very gradually and tends to overestimate O2 solubility, owing to (i) the higher Km for NADH than for O2, (ii) the relatively slow response of the Clark O2 electrode, and (iii) the incomplete oxidation of NADH in the presence of 340-nm-absorbing inhibitory substances.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Composite electrochemical biosensors: a comparison of three different electrode matrices for the construction of amperometric tyrosinase biosensors

A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphite-Teflon; reticulated vitreous carbon (RVC)-epoxy resin; and graphite-ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite-EPD electrodes, whereas a longer useful lifetime was observed for the graphite-Teflon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphite-EPD>graphite-Teflon>RVC-epoxy resin. A correlation between sensitivity and the catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix influences the interactions in the tyrosinase catalytic cycle.     

Mechanism of Bacterial Pyrite Oxidation

The oxidation by Ferrobacillus ferrooxidans of untreated pyrite (FeS(2)) as well as HCl-pretreated pyrite (from which most of the acid-soluble iron species were removed) was studied manometrically. Oxygen uptake was linear during bacterial oxidation of untreated pyrite, whereas with HCl-pretreated pyrite both a decrease in oxygen uptake at 2 hr and nonlinear oxygen consumption were observed. Ferric sulfate added to HCl-pretreated pyrite restored approximately two-thirds of the decrease in total bacterial oxygen uptake and caused oxygen uptake to revert to nearly linear kinetics. Ferric sulfate also oxidized pyrite in the absence of bacteria and O(2); recovery of ferric and ferrous ions was in excellent agreement with the reaction Fe(2)(SO(4))(3) + FeS(2) = 3FeSO(4) + 2S, but the elemental sulfur produced was negligible. Neither H(2)S nor S(2)O(3) (2-) was a product of the reaction. It is probable that two mechanisms of bacterial pyrite oxidation operate concurrently: the direct contact mechanism which requires physical contact between bacteria and pyrite particles for biological pyrite oxidation, and the indirect contact mechanism according to which the bacteria oxidize ferrous ions to the ferric state, thereby regenerating the ferric ions required for chemical oxidation of pyrite.     

Transient phase calibration of tyrosinase-based carbaryl biosensor

The inhibition of tyrosinase, used as a selective compound in amperometric biosensor for the detection of carbaryl and the possibilities of calibration of carbaryl biosensor have been studied. The kinetic analysis of biosensor data was based on the application of the biosensor dynamic model, allowing a quick calculation of independent on each other kinetic and steady-state parameters. It was found that carbaryl acts as an inhibiting substrate of tyrosinase and at low concentrations accelerates the tyrosinase-catalyzed oxidation of tyrosine by dissolved oxygen. The reaction mechanism is analogous to that usually considered for uncompetitive inhibition and the plot of kinetic parameter as a function of carbaryl concentration has a flat asymmetric maximum. Consequently, the kinetic parameter alone is not sufficient for the calibration of carbaryl biosensor and simultaneous application of other carbaryl-dependent parameters, like steady-state parameter, is essential.     

Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols

A new disposable amperometric bi-enzyme sensor system for detecting phenols has been developed. The phenol sensor developed uses horseradish peroxidase modified screen-printed carbon electrodes (HRP-SPCEs) coupled with immobilized tyrosinase prepared using poly(carbamoylsulfonate) (PCS) hydrogels or a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) matrix. Optimization of the experimental parameters has been performed with regard to buffer composition, pH, operating potential and storage stability. A co-operative reaction involving tyrosinase and HRP occurs at a potential of -50 mV versus Ag/AgCl without the requirement for addition of extraneous H(2)O(2), thus, resulting in a very simple and efficient system. Comparison of the electrode responses with the 4-aminoantipyrine standard method for phenol sample analysis indicated the feasibility of the disposable sensor system for sensitive "in-field" determination of phenols. The most sensitive system was the tyrosinase immobilized HRP-SPCE using PCS, which displayed detection limits for phenolic compounds in the lower nanomolar range e.g. 2.5 nM phenol, 10 nM catechol and 5 nM p-cresol.     

Isoproterenol oxidation by tyrosinase: intermediates characterization and kinetic study

The present work deals with isoproterenol oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced at short reaction time were characterized by scanning repetitive spectrophotometry and the stoichiometry of the respective aminochrome appearance was established. The oxidation pathway from isoproterenol to aminochrome is parallel to the previously proposed for L-dopa oxidation by mushroom tyrosinase, whose steps are as follow: Isoproterenol----o-quinone-H+----o-quinone----leukoaminochrome---- aminochrome. The stoichiometry for the conversion of o-quinone-H+ into the aminochrome of isoproterenol followed the equation: 2 o-quinone-H+----isoproterenol + aminochrome. The kinetics of chemical reactions that take place from the o-quinone-H+ to aminochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction (EzCC: Enzymatic-Chemical-Chemical mechanism).     

Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.     

Study of the Kinetics of Oxidation of Monophenols by Tyrosinase. The Effect of Reducers

Kinetics of oxidation of monophenols by tyrosinase from the fungus Aspergillus flavipes 56003 and the effect of Fe²⁺, serine, and ascorbic acid on this reaction were studied. The effectors were shown to accelerate the oxidation of monophenols, decreasing the lag-time of the reaction. It is assumed that the activation of the tyrosinase in the presence of Fe²⁺ is due to a direct reduction of the active site copper ions. Serine and ascorbic acid are supposed to affect the reaction of quinone transformation. The activation of the enzyme in the presence of Fe²⁺ suggests that the oxidation of monophenols is an autocatalytic process.     

Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.     

Selective uptake of 2-thiouracil into melanin-producing systems depends on chemical binding to enzymically generated dopaquinone

2-Thiouracil (TU), an antithyroid drug, is receiving growing interest as a specific tumor marker for malignant melanoma, owing to its capability of being selectively accumulated into active melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the tyrosinase catalyzed oxidation of tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit melanin formation by tyrosinase catalyzed oxidation of 0.25 mM tyrosine in phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the tyrosine-tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to ninhydrin and ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on Sephadex G-10 and was formulated as the TU-dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented melanomas and other melanin-producing systems is due to the covalent binding to dopaquinone, produced by tyrosinase catalyzed oxidation of tyrosine.     

Study of alpha-methyldopa oxidation by tyrosinase

The pathway for alpha-methyldopa oxidation to alpha-methyldopachrome, by mushroom tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been undertaken. The steps for alpha-methyldopa transformation into its aminochrome would be: alpha-methyldopa----o-alpha-methyldopaquinone-H+----o-alpha- methyldopaquinone----leuko-alpha-methyldopachrome----alpha- methyldopachrome. The stoichiometry for this conversion corresponded to the equation: 2 o-alpha-methyldopaquinone-H+----alpha-methyldopa + alpha-methyldopachrome. At very acid pH values, another route implying the addition of water to the quinonic ring, competes with the first one. Two chemical pathways can be proposed from alpha-methyldopaquinone-H+, the relative importance of which is determined by the pH. A theoretical and experimental kinetic approach was applied to this oxidative reaction. Rate constants and thermodynamic activation parameters of the chemical steps, have been evaluated. The results obtained confirmed that alpha-methyldopa oxidation by tyrosinase followed a scheme similar to that established for L-dopa and alpha-methylnoradrenaline.     

Tyrosinase‐Mediated Formation of a Reactive Quinone from the Depigmenting Agents, 4‐tert‐Butylphenol and 4‐tert‐Butylcatechol

Exposure of the skin to certain phenols or catechols such as 4‐tert‐butylphenol (TBP) and 4‐tert‐butylcatechol (TBC) may cause leukoderma. These substances are used in the polymer industry and numerous cases have been reported. Several theories of the mechanism for chemical leukoderma have been suggested. In the present study, TBP and TBC are shown to be oxidised by tyrosinase. The oxidation of TBC yields a quinone that is further investigated on its reactions with cysteine or glutathione (GSH). The products formed are isolated and identified by mass spectrometry and nuclear magnetic resonance as being 4‐tert‐butyl‐6‐S‐cysteinylcatechol (cys‐TBC) and 4‐tert‐butyl‐6‐S‐glutathionylcatechol (GS‐TBC). The reactive quinone is a strongly electrophilic substance that rapidly reacts with GSH. A depletion of the GSH defence system may give conditions where the quinone lives long enough to effect its toxic properties. The influence of the reactive tert‐butylquinone on enzymatic activities is demonstrated by the inhibition of glyceraldehyde‐3‐phosphate dehydrogenase.