Horseradish peroxidase. XXXIV. Oxidation of compound II to I by periodate and inorganic anion radicals.

The one-electron oxidation of horseradish peroxidase compound II to compound I by sodium periodate was observed. The bimolecular rate constant for the NaIO4--compound II interaction is equal to 9.5 +/- 1 x 10(-3) M-1s-1 at room temperature. Irradiation, using ultraviolet light, of the solution containing compound II and persulfate in the presence of bicarbonate, chloride, or bromide, leads ot the fast accumulation of compound I due to the oxidative action of SO4, CO3, Cl2, and Br2 anion radicals, which are products of the photolysis.     

Enhancement of the rate of the beta-elimination of phosphate from radicals derived from glycerol-2-phosphate by Cu(I)-phenanthroline. A pulse radiolysis study

Hydroxyl radicals abstract hydrogen atoms from glycerol-2-phosphate with a specific rate constant of (7.0 +/- 1.5) x 10(8) M-1s-1 forming the beta-phospho radical as the major product. At physiological pH this radical undergoes a beta-phosphate elimination with a rate constant less than or equal to 1 x 10(3) s-1. The beta-phospho radical reacts with Cu(I)-phenanthroline to produce an unstable transient with a metal-carbon sigma-bond which has an absorbance similar to that of the cuprous phenanthroline complex in the visible region. This intermediate decomposes via a beta-elimination of phosphate with a rate constant of (1.0 +/- 1.5) x 10(4) s-1, which was independent of the acidity in the pH range 4-9.     

Studies of electron migration in DNA in aqueous solution using intercalating dyes.

The reactions of the hydrated electron (e-aq) and of the hydroxyl radical (OH) with double-stranded DNA in aqueous solution at room temperature have been studied through the use of the intercalating dyes, proflavine and ethidium. These dyes react with e-aq with rate constants of (2.5 +/- 0.2) - 10(10) M-1 - s-1 and (3.0 +/- 0.3) - 10(10) M-1 - s-1, respectively; the rate constant for the reaction of OH with proflavine is (1.0 +/- 0.2) - 10(10) M-1 - s-1. When these molecules are bound within the DNA structure both the yields and the rate constants of reaction with e-aq are reduced in a manner entirely consistent with a simple competition between the DNA bases and restricted dye molecules reacting with a bimolecular rate constant of about 2 - 10(9) M-1 - s-1. No evidence of free electron migration in the DNA was obtained, and an upper limit of five base pairs for the range of such migration was derived. Reactions of the hydroxyl radical with DNA-bound proflavine also lead to a rate constant of about 2 - 10(9) M-1 - s-1. These rate constants are in good agreement with rate predictions (per base unit) for a diffusion-controlled reaction with the DNA structure.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.     

The reaction of superoxide radical with iron complexes of EDTA studied by pulse radiolysis.

The reactions of Fe3+-EDTA and Fe2+-EDTA with O2- and CO2- were investigated in the pH range 3.8--11.8. Around neutral pH O2- reduces Fe3+-EDTA with a rate constant which is pH dependent kpH 5.8--8.1 = 2 - 10(6)--5 - 10(5) M-1 - s-1. At higher pH values this reaction becomes much slower. The CO2- radical reduces Fe3+-EDTA with kpH 3.8--1- = 5 +/- 1 - 10(7) M-1 - s-1 independent of pH. At pH 9--11.8, Fe2+-EDTA forms a complex with O2- with kFe2+-EDTA + O2 = 2 - 10(6)--4 - 10(6) M-1 - s-1 which is pH dependent. We measured the spectrum of Fe2+-EDTA-O2- and calculated epsilon 290 over max = 6400 +/- 800 M-1 - cm-1 in air-saturated solutions. In O2-saturated solutions another species is formed with a rate constant of 7 +/- 2 s-1. This intermediate absorbs around 300 nm but we were not able to identify it.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV

Compared to other monomeric heme proteins and the heme peroxidases, the Glycera dibranchiata monomer hemoglobin components III and IV exhibit very slow cyanide binding kinetics. This is agreement with the previously reported behavior of component II. Similar to component II, components III and IV have been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0 by using a 100-250-fold excess of potassium cyanide at each pH. At 20 degrees C with micromolar protein concentrations, kobs for component III varies between 7.08 x 10(-5) s-1 at pH 6.0 and 100-fold cyanide excess and 1.06 x 10(-2) s-1 at pH 9.0 and 250-fold cyanide excess. For component IV, the values are 2.03 x 10(-4) s-1 for 100-fold cyanide excess at pH 6.0 and 4.13 x 10(-2) s-1 for 250-fold cyanide excess at pH 9.0. In comparison to other heme proteins, our analysis shows that the bimolecular rate constant (klapp) is small. For example, at pH 7.0, it is 3.02 x 10(-1) M-1 s-1 for component III and 1.82 M-1 s-1 for component IV, compared to 400 M-1 s-1 for sperm whale metmyoglobin, 692 M-1 s-1 for soybean metleghemoglobin a, 111 M-1 s-1 for guinea pig methemoglobin, and 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase. Our results also show that the dissociation rates (k-lapp) are extremely slow and no larger than 10(-6) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin.

Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex.     

Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content--order restored

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234-240; 2005) the authors argued that the catalytic rate constant, k(cat), for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sample that already contained excess copper did not remove spurious activity, which is incompatible with well-known stability constants of copper complexes and contradicts previous observations. In the present study we verified that the addition of EDTA eliminates completely the effect of excess copper on the decomposition rate of O2*- in the presence of Cu,Zn-SOD. We determined that k(cat) = (2.82 +/- 0.02) x 10(9) M(-1) s(-1) at low ionic strength (2 < I < 15 mM) and (1.30 +/- 0.02) x 10(9) M(-1) s(-1) in the presence of 50 mM phosphate at pH 7.8 (I = approximately 150 mM), which are about twice higher than those reported by Michel et al. We also determined k(cat) by the cytochrome c assay and demonstrated the correlation between these direct and indirect assays. The phenotypic deficits imposed by deletion of SODs, and the oxygen dependence of these deficits, have repeatedly demonstrated that the several SODs do in fact, as well as is theory, provide an important protection against that facet of oxidative stress imposed by O2*-.     

Kinetics of bimolecular decay of alpha-tocopheroxyl free radicals studied by ESR

The kinetics of bimolecular decay of alpha-tocopheroxyl free radicals (T) was studied by ESR mainly in ethanol and heptanol solvents. A second-order kinetic law was observed during the whole course of reaction (-d[T]/dt = 2k[T]2) and the following rate constants were determined with good accuracy in the temperature range 281-321 K: ethanol: log(2k) = 8.2 +/- 0.5--(6.6 +/- 0.7 kcal/mol)/(2.3RT) M-1.s-1; heptanol: log(2k) = 6.1 +/- 0.4--(4.3 +/- 0.6 kcal/mol)/(2.3RT) M-1.s-1. The global rate constant clearly increases with solvent polarity.     

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.     

Steady-state kinetics of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical.

Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 +/- 0.13 x 10(4) M-1s-1 and 9.26 +/- 0.58 x 10(4) M-1s-1 at 25 degrees C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated.     

O2-mediated oxidation of hemopexin-heme(II)-NO

Hemopexin (HPX), serving as scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. Here, kinetics of HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO are reported. NO reacts reversibly with HPX-heme(II) yielding HPX-heme(II)-NO, according to the minimum reaction scheme: HPX-heme(II)+NO kon<-->koff HPX-heme(II)-NO values of kon, koff, and K (=kon/koff) are (6.3+/-0.3)x10(3)M-1s-1, (9.1+/-0.4)x10(-4)s-1, and (6.9+/-0.6)x10(6)M-1, respectively, at pH 7.0 and 10.0 degrees C. O2 reacts with HPX-heme(II)-NO yielding HPX-heme(III) and NO3-, by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme: HPX-heme(II)-NO+O2 hon<--> HPX-heme(III)-N(O)OO l-->HPX-heme(III)+NO3- the backward reaction rate is negligible. Values of hon and l are (2.4+/-0.3)x10(1)M-1s-1 and (1.4+/-0.2)x10(-3)s-1, respectively, at pH 7.0 and 10.0 degrees C. The decay of HPX-heme(III)-N(O)OO (i.e., l) is rate limiting. The HPX-heme(III)-N(O)OO intermediate has been characterized by optical absorption spectroscopy in the Soret region (lambdamax=409 nm and epsilon409=1.51x10(5)M-1cm-1). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers.     

Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.     

Kinetic study on chemical modification of taka-amylase A. I. Location and role of tryptophan residues

1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose. Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, Ki) of the enzyme modified with K-IWS and K-IIWS were determined. The k0 value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3 +/- 0.5) x 10(5) M-1 . s-1, (2.1 +/- 0.3) x 10(3) M-1 . s-1 and (1.7 +/- 0.2) x 10(2) M-1 . s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6 +/- 0.6) x 10(6) M-1 . s-1 and (6.9 +/- 1.1) x 10(4) M-1 . s-1, respectively. On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.     

Superoxide dismutase activity of Cu(Tyr)2 and Cu, Co-erythrocuprein.

Crystalline Cu(Tyr)2 and homogeneous Cu2Co2-erythrocuprein were prepared. The reactivity of each chelated Cu2 compound with superoxide was studied by pulse radiolysis at pH 7.6 +/- 0.1 and compared with the reactivity of native erythrocuprein (superoxide dismutase). Superoxide anions were generated by a 40-ns pulse of 1.81-MeV electrons. The yield of O2 ranged between 6 - 60 muM. The kinetics of the spontaneous O2 decay were second order; in the presence of Cu2 complexes the reaction was first order with respect to O2. Taking into account the effect of the different Cu2 concentrations on the O2 decay, second-order rate constants for the reaction of chelated Cu2 with O2 were obtained. For an equivalent of Cu2 in either erythrocuprein or Cu, Co-erythrocuprein, a numerical value of 1.3 +/- 0.1 x 10(9) M-1S-1 was calculated. Surprisingly, the same value was obtained employing Cu(Tyr)2. The highest rate constant was measured for the hydrated Cu2 (2.7 x 10(9) M-1S-1). In the presence of a biologically significant chelating agent such as serum albumin, a marked decrease in the Cu2aq-induced superoxide dismutation was observed. This was not the case when the dismutation in the presence of either the Cu2 of native erythrocuprein or Cu, Co-erythrocuprein, or those Cu2 ions chelated with tyrosine or certain di- and tripeptides was measured.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Nature of oxygen activation in glucose oxidase from Aspergillus niger: the importance of electrostatic stabilization in superoxide formation.

Glucose oxidase catalyzes the oxidation of glucose by molecular dioxygen, forming gluconolactone and hydrogen peroxide. A series of probes have been applied to investigate the activation of dioxygen in the oxidative half-reaction, including pH dependence, viscosity effects, 18O isotope effects, and solvent isotope effects on the kinetic parameter Vmax/Km(O2). The pH profile of Vmax/Km(O2) exhibits a pKa of 7.9 +/- 0.1, with the protonated enzyme form more reactive by 2 orders of magnitude. The effect of viscosogen on Vmax/Km(O2) reveals the surprising fact that the faster reaction at low pH (1.6 x 10(6) M-1 s-1) is actually less diffusion-controlled than the slow reaction at high pH (1.4 x 10(4) M-1 s-1); dioxygen reduction is almost fully diffusion-controlled at pH 9.8, while the extent of diffusion control decreases to 88% at pH 9.0 and 32% at pH 5.0, suggesting a transition of the first irreversible step from dioxygen binding at high pH to a later step at low pH. The puzzle is resolved by 18O isotope effects. 18(Vmax/Km) has been determined to be 1.028 +/- 0.002 at pH 5.0 and 1.027 +/- 0.001 at pH 9.0, indicating that a significant O-O bond order decrease accompanies the steps from dioxygen binding up to the first irreversible step at either pH. The results at high pH lead to an unequivocal mechanism; the rate-limiting step in Vmax/Km(O2) for the deprotonated enzyme is the first electron transfer from the reduced flavin to dioxygen, and this step accompanies binding of molecular dioxygen to the active site. In combination with the published structural data, a model is presented in which a protonated active site histidine at low pH accelerates the second-order rate constant for one electron transfer to dioxygen through electrostatic stabilization of the superoxide anion intermediate. Consistent with the proposed mechanisms for both high and low pH, solvent isotope effects indicate that proton transfer steps occur after the rate-limiting step(s). Kinetic simulations show that the model that is presented, although apparently in conflict with previous models for glucose oxidase, is in good agreement with previously published kinetic data for glucose oxidase. A role for electrostatic stabilization of the superoxide anion intermediate, as a general catalytic strategy in dioxygen-utilizing enzymes, is discussed.