Studies on the pathway of methane formation from procarbazine,a 2-methylbenzylhydrazine derivative,by rat liver microsomes

The oxidative metabolism of procarbazine, its azo, hydrazone, and two azoxy derivatives, and methylhydrazine by hepatic microsomes from phenobarbital-pretreated rats was investigated to elucidate the pathway of metabolism that resulted in methane formation from procarbazine. When incubated with microsomal reaction mixtures fortified with NADPH, all of the compounds, except the azoxy isomers, were metabolized to yield methane. A lag phase in methane formation was noted for procarbazine, but not for the other compounds. Kinetic and inhibition studies utilizing methimazole and ethylhydrazine precluded methylhydrazine as an intermediate in methane formation from procarbazine. When the azo derivative was oxidatively metabolized in the presence of liver microsomes, no hydrazone tautomer was detected. Upon monitoring the production of the azo and hydrazone metabolites formed during microsomal metabolism of procarbazine, the azo derivative was formed in sufficient quantities to account for the majority of the methane produced. In addition, small amounts of hydrazone were also detected. It was concluded that both the azo and hydrazone metabolites of procarbazine contribute to methane formation from the terminal methyl group of the hydrazine with the azo derivative being the predominant source and the hydrazone derivative being a minor source of methane. Consideration of the chemical and enzymatic pathways of procarbazine oxidation and the implication of a methyl radical intermediate in methane formation are discussed.     

Role of the interactions between the active site base and the substrate Schiff base in amine oxidase catalysis. Evidence from structural and spectroscopic studies of the 2-hydrazinopyridine adduct of Escherichia coli amine oxidase

2-Hydrazinopyridine (2HP) is an irreversible inhibitor of copper amine oxidases (CAOs). 2HP reacts directly at the C5 position of the TPQ cofactor, yielding an intense chromophore with lambda(max) approximately 430 nm (adduct I) in Escherichia coli amine oxidase (ECAO). The adduct I form of wild type (WT-ECAO) was assigned as a hydrazone on the basis of the X-ray crystal structure. The hydrazone adduct appears to be stabilized by two key hydrogen-bonding interactions between the TPQ-2HP moiety and two active site residues: the catalytic base (D383) and the conserved tyrosine residue (Y369). In this work, we have synthesized a model compound (2) for adduct I from the reaction of a TPQ model compound (1) and 2HP. NMR spectroscopy and X-ray crystallography show that 2 exists predominantly as the azo form (lambda(max) at 414 nm). Comparison of the UV-vis and resonance Raman spectra of 2 with adduct I in WT, D383E, D383N, and Y369F forms of ECAO revealed that adduct I in WT and D383N is a tautomeric mixture where the hydrazone form is favored. In D383E adduct I, the equilibrium is further shifted in favor of the hydrazone form. UV-vis spectroscopic pH titrations of adduct I in WT, D383N, D383E, and 2 confirmed that D383 in WT adduct I is protonated at pH 7 and stabilizes the hydrazone tautomer by a short hydrogen-bonding interaction. The deprotonation of D383 (pKa approximately 9.7) in adduct I resulted in conversion of adduct I to the azo tautomer with a blue shift of the lambda(max) to 420 nm, close to that of 2. In contrast, adduct I in D383N and D383E is stable and did not show any pH-dependent spectral changes. In Y369F, adduct I was not stable and gradually converted into a new species with lambda(max) at approximately 530 nm (adduct II). A detailed mechanism for the adduct I formation in WT has been proposed that is consistent with the mechanism proposed for the oxidation of substrate by CAOs but addresses some key differences in the active site chemistry of hydrazine inhibitors and substrate amines.     

Oxidative metabolism of some hydrazine derivatives by rat liver and lung tissue fractions

The enzyme systems in rat liver and lung responsible for the oxidative metabolism of hydrazine derivatives were studied to determine whether these enzymes, cytochrome P-450 and monoamine oxidase, were responsible for metabolically activating hydrazines to carcinogenic/toxic metabolites. Cytochrome P-450 preferentially oxidized the nitrogen to nitrogen bond of 1,2-disubstituted hydrazines and hydrazides, while monoamine oxidase oxidized the nitrogen to nitrogen bond of all the classes of hydrazine derivatives that were tested. Oxidation of the nitrogen to nitrogen bond led to the formation of stable azo intermediates in the case of 1,2-disubstituted hydrazines and to unstable monoazo (diazene) metabolites in the case of monosubstituted hydrazines and hydrazides. In addition, cytochrome P-450 preferentially oxidized the carbon to nitrogen bond of monoalkylhydrazines; this reaction resulted in the formation of aldehyde metabolites (via hydrazone intermediates). Monosubstituted hydrazines were shown to be potent, irreversible inhibitors of mitochondrial monoamine oxidase. In contrast, the 1,2-disubstituted hydrazines appeared to be good substrates for the monoamine oxidase and served as competitive inhibitors at high concentrations. There did not appear to be any monoamine oxidase isozyme (form A or B) specificity in the metabolism of either the 1,2-disubstituted hydrazines or the monoalkylhydrazines, ethyl- and n-propylhydrazine.     

The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors

 Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic cofactor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60  °C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu²⁺ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystallineEscherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu²⁺ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co²⁺-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed. Received: 2 December 1998 / Accepted: 22 March 1999     

Investigation into the mechanism of λmax shifts and their dependence on pH for the 2-hydrazinopyridine derivatives of two copper amine oxidases

Copper amine oxidases (CuAO), from Escherichia coli (ECAO) and pea seedling (PSAO) were reacted with an excess of the hydrazine derivative 2-hydrazinopyridine (2HP) to form an initial, strongly absorbing adduct, (adduct 1; λ_max 420–430 nm) formed by the covalent binding of 2HP with the active site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). Thermal incubation of buffered solutions of adduct 1 (pH 5.65–10.7) or addition of KOH solution (giving a final pH of 13–15) led isosbestically to a dramatic λ_max shift yielding adduct 2 (λ_max 520–530 nm). For both ECAO and PSAO, an increase in pH resulted in increased formation of adduct 2 with concomitant loss of adduct 1. Maximum adduct 2 formation occurred at pH 9.84 in ECAO and at pH 10.7 in PSAO. Beyond these pH levels, adduct 2 formation occurred to a much lesser extent which was independent of pH, suggesting enzyme denaturation. It is proposed that the conversion of adduct 1 to adduct 2 occurs as a result of hydrazone to azo conversion mediated by loss of a single proton, possibly to the active site base. It is further postulated that adduct formation and subsequent deprotonation can be likened to the substrate and product Schiff base complexes in the reductive half cycle of copper/TPQ containing amine oxidases. As part of this study an extinction coefficient at 280 nm was determined for ECAO by gravimetric analysis. This yielded a value of 2.1×10⁵ M⁻¹ cm⁻¹ giving rise to the need of a correction factor when estimating the protein concentration from an absorbance reading at 280 nm. Using the estimated molecular mass of 160 kDa for the homodimeric ECAO, a correction factor of 0.76 must be applied.     

Effect of hydrazine on properties of cytochrome oxidase

The effects of hydrazine on ferrocytochrome c oxidation by cytochrome oxidase and on spectral properties of the enzyme were studied. Hydrazine was found to modify the spectral properties of lipid-depleted preparations of cytochrome oxidase dissolved in 1% cholate and to inhibit the cytochrome c oxidase activity of the enzyme, whereas the kinetic properties of lipid-enriched and Tween preparations were unchanged by hydrazine. Cytochrome oxidase was found to possess a hydrazine oxidase activity. This activity was not coupled with the specific cytochrome c oxidase activity. The effect of pH on the observed changes was studied. Hydrazine was found to yield protein bands in the optical spectra of cytochrome oxidase as 580 nm, 537 nm and 845 nm. It is concluded that hydrazine interacts with the oxygen-binding site of cytochrome oxidase. The effect of hydrazine on the formation of the "ferryl" form (Fe4+a3/Cu2+b) of the enzyme is discussed.     

Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4-dinitrophenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 1H-NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.     

Nitric oxide and mitochondrial complex IV

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.     

The haemolytic reactions of 1-acetyl-2-phenylhydrazine and hydrazine: A spin trapping study

Free radical production from the reaction of hydrazine and 1-acetyl-2-phenylhydrazine (AcPhHZ) with oxyhaemoglobin and with human red blood cells, has been observed by the electron spin resonance technique of spin trapping. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the free radical intermediates detected depended on the hydrazine derivative, oxyhaemoglobin and the oxyhaem/hydrazine derivative concentration ratio.

The reaction of hydrazine with oxyhaemoglobin in the presence of DMPO gave a nitroxide which was identified as a reduced dimer of DMPO. Whereas hydrazine-treated red blood cells, in the presence of DMPO, gave a nitroxide spin adduct which was identified as the hydroxyl radical spin adduct of DMPO, 5,5-dimethyl-1-pyrrolidino-1-oxyl (DMPO-OH).

The reaction of AcPhHZ with oxyhaemoglobin, in the presence of DMPO, gave DMPO-OH, the phenyl radical spin adduct of DMPO, 5,5-dimethyl-2-phenylpyrrolidino-1-oxyl (DMPO-Ph) and an oxidised derivative of DMPO, 5,5-dimethyl-2-pyrrolidone-1-oxyl (DMPOX). The amounts of DMPO-Ph, DMPO-OH and DMPOX observed depended on the 1-acetyl-2-phenyl-hydrazine/oxyhaemoglobin concentration ratio; DMPOX replaced DMPO-OH as the concentration of AcPhHZ was decreased. DMPOX production has been previously associated with the production of highly oxidised haem iron-oxygen intermediates. AcPhHZ treated red blood cells gave DMPO-Ph and DMPO-OH spin adducts in the presence of DMPO.

DMPO had little to no effect on the rate of oxygen consumption by oxyhaemoglobin with hydrazine and AcPhHZ. Moreover, the rate of oxyhaemoglobin oxidation induced by hydrazine, was not decreased by DMPO whereas the rate of oxyhaemoglobin oxidation induced by AcPhHZ was decreased approx. 40% by DMPO. DMPO (10 mM) gave a small decrease in haemolysis and lipid peroxidation induced by 1 mM hydrazine and AcPhHZ in a 1% suspension of red blood cells.     

Synthesis and biological evaluation of some nitrogen containing steroidal heterocycles

epi-Androsterone 1 was converted into its hydrazone derivative through the reaction with hydrazine hydrate 80%. Hydrazonoandrostane derivative 2b reacted with hydrazonoyl halides in the presence of K₂CO₃ forming the corresponding hydrazopyridazinoandrostane derivatives 6a–d. The 3β-acetyl-17-hydrazonoandrostane derivative 2b reacted with a halogen reagent, benzoyl chloride, to form the non-cyclic 16-benzoylated hydrazone 9.On the other hand, compound 2b produced the corresponding pyridazinoandrostane derivatives 11 and 12 via its reaction with phenacyl bromide and chloroacetone respectively. Reaction of the hydrazono derivative 2b with benzaldehyde in the presence of acetic acid drops led to the formation of the benzylidenehydrazonoandrostane derivative 13. The product 14, phosphinom-ethylenehydrazonoandrostane was obtained by the reaction of the derivative 13 with trisdimethylaminophosphine in the presence of dry benzene. The reaction of compound 2b with phenyl isothiocyanate followed by boiling in chloroacetic acid or thioglycolic acid produced the pyrazoloandrostane derivatives 17 and 18 respectively. The biological activity of compounds 6a, 6d, 11, 12, and 15 was evaluated as inhibitor of growth in a human liver carcinoma cell line and doxorubicine was used for comparison. Compounds 15 and 12 showed a higher potency than the other tested compounds.     

Inactivation of copper-containing amine oxidases by turnover products.

For bovine serum amine oxidase, two different mechanisms of substrate-induced inactivation have been proposed. One consists of a slow oxidation by H2O2 of a conserved residue in the reduced enzyme after the fast turnover phase [Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovì, B. & Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.267, 174-178] and the other of the oxidation by H2O2 of the dihydrobenzoxazole in equilibrium with the product Schiff base, during the catalytic cycle [Lee, Y., Shepard, E., Smith, J., Dooley, D.M. & Sayre, L.M. (2001) Biochemistry40, 822-829]. To discriminate between the two mechanisms, the inactivation was studied using Lathyrus cicera (red vetchling) amine oxidase. This, in contrast to bovine serum amine oxidase, formed the Cu+-semiquinolamine radical with a characteristic UV-vis spectrum when oxygen was exhausted by an excess of any tested amine in a closed cuvette. The inactivation, lasting about 90 min, was simultaneous with the radical decay and with the formation of a broad band (shoulder) at 350 nm. No inactivation occurred when a thousand-fold excess of amine was rapidly oxidized in an L. cicera amine oxidase solution stirred in open air. Thus, the inactivation is a slow reaction of the reduced enzyme with H2O2, following the turnover phase. Catalase protected L. cicera amine oxidase from inactivation. This effect was substrate-dependent, varying from full protection (benzylamine) to no protection (putrescine). In the absence of H2O2, a specific inactivating reaction, without formation of the 350 nm band, was induced by some aldehydes, notably putrescine. Some mechanisms of inactivation are proposed.     

Nitric oxide covalently labels a 6-hydroxydopa-derived free radical intermediate in the catalytic cycle of copper/quinone-containing amine oxidase from lentil seedlings

The reaction of NO-derivatized polyamines called "NONOates" with an amine oxidase from lentil seedlings was studied. 3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) and 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine (DPTA-NONOate) were found to be irreversible inactivators of the lentil enzyme. The spectrum of the protein was strongly affected in the course of reaction with both compounds, leading to the formation of a covalent adduct with a stable band at 334 nm. The corresponding amine compounds diethylentriamine (DETA) and norspermidine (DPTA) were substrates of the lentil enzyme that did not lead to enzyme inactivation. Diethylamine-NONOate, not containing amino groups, was found to be an irreversible inactivator of the amine oxidase only in the presence of a substrate. Since all NONOates spontaneously decompose in solution with release of NO, it seems as if the latter is responsible for the enzyme inhibition. The insensitivity of the native enzyme to NO suggested that this compound was unreactive toward both the cofactors, 6-hydroxydopa quinone (TPQ) and Cu(II), and thus a model for the irreversible inactivation could involve the attack by NO of the Cu(I)-semiquinolamine radical catalytic intermediate.     

Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase

N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Acceleration of the oxygen reaction in CuA-deficient Nitrosomonas europaea cytochrome c oxidase as revealed by the flow-flash measurement.

The oxygen reaction of Nitrosomonas europaea cytochrome c oxidase containing either 2Cu or 1Cu per two heme a molecules was investigated by the flow-flash technique at 20 degrees C. The reaction profiles of the bacterial enzyme were essentially the same as those of bovine heart cytochrome c oxidase, although the rate of the primary oxygen compound formation was much slower. The 1Cu enzyme exhibited higher rates for both primary oxygen compound formation and intramolecular electron transfer than the 2Cu enzyme. This result clearly indicates that CuA is not essential functionally for the oxidation of ferrous heme a moieties, and suggests its structural importance in maintaining the molecular integrity of N. europaea cytochrome oxidase.     

Ligand interactions with galactose oxidase: mechanistic insights.

Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.