The Reaction of Reduced Xanthine Dehydrogenase with Molecular Oxygen
REACTION KINETICS AND MEASUREMENT OF SUPEROXIDE RADICAL*

(Received for publication, November 21, 1996, and in revised form, January 22, 1997)

Christopher M. Harris and Vincent Massey Dagger

From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Xanthine dehydrogenase (XDH) from bovine milk contains significant activity in xanthine/oxygen turnover assays. The oxidative half-reaction of XDH with molecular oxygen has been studied in detail, at 25 °C, pH 7.5, to determine the basis of the preference of XDH for NAD over oxygen as oxidizing substrate. Spectral changes of XDH accompanying oxidation were followed by stopped-flow spectrophotometry. The amount of superoxide radicals formed during oxidation was investigated to assess the ability of XDH to catalyze production of oxygen radicals. Reduced XDH reacts with oxygen in at least 4 bi-molecular steps, with 1.7-1.9 mol of superoxide per mol of XDH formed from the last 2 electrons oxidized. A model is discussed in which the flavin hydroquinone transfers electrons to oxygen to produce hydrogen peroxide at a rate constant of at least 72,000 M-1 s-1, whereas flavin semiquinone reduces oxygen to form superoxide as slow as 16 M-1 s-1.

Steady-state kinetics of xanthine/oxygen and NADH/oxygen turnover of XDH were determined to have kcat values of 2.1 ± 0.1 and 2.5 ± 0.9 s-1, respectively, at 25 °C, pH 7.5. XDH is therefore capable of catalyzing the formation of reduced oxygen species at one-third the rate of xanthine/NAD turnover, 6.3 s-1 (Hunt, J., and Massey, V. (1992) J. Biol. Chem. 267, 21479-21485), in the absence of NAD. As XDH contains a significant and intrinsic xanthine oxidase activity, estimates of relative amounts of XO and XDH based solely upon turnover assays must be made with caution. Initial-rate assays containing varying amounts of xanthine, NAD, and oxygen indicate that at 100% oxygen saturation, NADH formation is only inhibited at concentrations of xanthine and NAD below Km for each substrate.


INTRODUCTION

Purine catabolism in primates ends with the xanthine oxidoreductase-catalyzed oxidation of xanthine to urate with concomitant reduction of either molecular oxygen or NAD. This enzyme can be isolated from bovine milk in two different forms, xanthine oxidase (XO)1 and xanthine dehydrogenase (XDH). The enzyme type depends on the oxidation state of protein thiols, and the two forms are interconvertible (1, 2). Preincubation with thiol-reducing agents produces XDH, which contains high dehydrogenase activity and low oxidase activity. When the enzyme is isolated from milk without precaution to prevent disulfide formation, it is obtained as XO form and has high oxidase activity and very little dehydrogenase activity. Xanthine oxidoreductase represents a unique system in which two fundamentally different types of reactions, electron transfer to NAD or to molecular oxygen, can be catalyzed by the same protein, differing only as a result of a conformational change.

Xanthine oxidoreductase exists as a homodimer of 145-kDa subunits. Each subunit contains one FAD, one molybdopterin, and two 2Fe/2S clusters of the spinach ferredoxin type (3, 4). Reducing substrates of the purine type, such as xanthine, react at the molybdopterin site of the enzyme (5). Oxidizing substrates, such as NAD or oxygen, react at the flavin center (6, 7). These cofactors can accept a total of six electrons from 3 mol of xanthine. When reduced by artificial electron donors, an additional pair of electrons can be reversibly accepted by a cystine which is not involved in the XO/XDH interconversion (8). Both the 2Fe/2S and FAD moieties undergo distinct spectral changes on reduction, providing a sensitive means for monitoring the redox state of the enzyme.

Xanthine oxidoreductase catalysis can be separated into a reductive half-reaction in which 2 electrons at a time are transferred from xanthine to the enzyme and an oxidative half-reaction in which electrons are conveyed from the enzyme to oxygen or NAD. The reaction of reduced XDH with oxygen was investigated to determine the basis for the predisposition of XDH toward NAD, instead of oxygen, as an oxidizing substrate. XDH is known to contain an NAD binding site (1), adjacent to the flavin moiety, which is absent in XO. Also, the flavin midpoint potential of XDH, -340 mV, is sufficiently low for reduction of NAD, -335 mV at pH 7.5 (9). In contrast, the flavin midpoint potential of XO, -255 mV (10), is too high to significantly reduce NAD. These properties demonstrate that XDH, and not XO, is equipped to react with NAD. The current study addresses how these changes influence oxidation by oxygen, with particular regard to rate and products of reaction. Rates of reaction were measured by mixing pre-reduced XDH with oxygen in a stopped-flow spectrophotometer, monitoring the oxidation of iron-sulfur and flavin centers. Reduction of O2 by reduced flavins can result in the formation of H2O2 or Obardot 2. These activated oxygen species are thought to be the agents of oxidative stress acting in ischemia and the aging process. Superoxide produced was quantitated by superoxide-dependent reduction of cyt c.

The oxidative half-reaction with oxygen has been well-studied for two closely related enzymes, bovine milk XO and chicken liver XDH. In the case of XO, oxidation is triphasic with the first phase a lag, the second phase saturating at a rate of 125 s-1, and the third phase showing bi-molecular kinetics at 10,000 M-1 s-1, at 25 °C, pH 8.5 (11-13). Product analysis yielded 2 mol of Obardot 2 and 2 mol of H2O2 per mol of XO, with the Obardot 2 produced from the last 2 electrons to leave XO. In the case of chicken liver XDH, four reaction phases were measured with the first two having rate constants of 1,900 M-1 s-1 and 260 M-1 s-1, and the last two being a complex combination of rates (14). These experiments were done at 4 °C, pH 7.8. With chicken liver XDH, 3 mol of Obardot 2 and 1.5 mol of H2O2 were produced on oxidation. Superoxide was produced from the last electrons to leave chicken liver XDH as well.

In the current study with the mammalian bovine milk XDH, the oxidative half-reaction with oxygen proceeds via at least 4 bi-molecular reactions with observed rate constants of 72,000 ± 16,000, 3,500 ± 1,300, 120 ± 21, and 16 ± 3.5 M-1 s-1. From 1.7-1.9 mol of Obardot 2 per mol of XDH is formed from the last 2 electrons oxidized. A mechanism is proposed in which FADH2 reacts with oxygen to form H2O2 and FAD at a microscopic rate constant of at least 72,000 M-1 s-1, while FADH· reacts with oxygen to form Obardot 2 and FAD as slow as 16 M-1 s-1. Observed rate constants are thought to progressively decrease with the fraction of the relevant reduced flavin species, FADH2 or FADH·, as well as with the increasing redox potential of XDH.

In addition, the competence of XDH to catalyze xanthine/oxygen and NADH/oxygen turnover was determined by measuring the steady-state kinetics of these two activities by the method of initial rates. Both activities gave parallel or near-parallel lines on Lineweaver-Burk plots, consistent with ping-pong mechanisms. Xanthine/oxygen and NADH/oxygen turnover have kcat values of 2.1 ± 0.1 and 2.5 ± 0.9 s-1, respectively, at 25 °C, pH 7.5. These rates are 33 and 40% that of xanthine/NAD turnover, kcat of 6.3 s-1 (1), indicating that XDH-catalyzed reduction of oxygen in vitro is significant in the absence of sufficient competing NAD.

The ability of oxygen to compete with NAD as a substrate was determined by measuring the inhibition of xanthine/NAD turnover as a function of oxygen concentration. Significant inhibition of NADH formation under initial-rate conditions was only observed at 100% oxygen saturation (1.2 mM at 25 °C) and at concentrations of xanthine and NAD near their Km values, <= 1 and 7 µM (1), respectively. Oxygen competes very poorly with NAD as an alternative substrate for XDH. By monitoring the entire reaction with an oxygen electrode, however, oxygen consumption occurs slowly, even in the presence of 1 mM NAD. Once the xanthine/NAD reaction has gone to completion, the NADH produced can be consumed by the NADH oxidase activity of XDH.


MATERIALS AND METHODS

Xanthine dehydrogenase was purified by the method of Hunt and Massey (1). Concentrations expressed are per monomer, determined with an extinction coefficient at 450 nm of 37,800 M-1 cm-1. The percent functional enzyme was measured prior to each usage by determining the fraction of absorbance lost at 450 nm on anaerobic reduction by 200 µM xanthine relative to the total absorbance change after further reduction with an excess of sodium dithionite. Samples used were typically between 65 and 75% active. All reactions were performed in 50 mM potassium phosphate, pH 7.5, 0.3 mM EDTA, at 25 °C. XDH activity was also routinely checked by measuring the formation of NADH at 340 nm in an air-saturated assay containing 100 µM xanthine, 500 µM NAD, and a catalytic (10-50 nM) amount of XDH. Identical rates are obtained if the assay is anaerobic. Reactions were performed in temperature-equilibrated cuvettes at 25 °C. Oxidase activity was also determined to detect any spontaneous conversion to the oxidase form. Oxidase assays measure the formation of urate at 295 nm in a mixture as above, minus NAD. Xanthine dehydrogenase samples were incubated with 2.5 mM dithiothreitol for 1 h at 25 °C prior to use. Dithiothreitol was then removed by desalting over a Sephadex G-25 column. Xanthine, NADH, bovine heart cytochrome c, superoxide dismutase, urate, sodium dithionite, methylmethane thiosulfonate, and dithiothreitol were purchased from Sigma. Catalase was purchased from Calbiochem. Analytical mixtures of oxygen, nitrogen, and argon (Matheson purity) were purchased from Matheson.

Anaerobiosis

Enzyme samples were made anaerobic by 10 cycles of evacuation and equilibration with high purity argon. Stopped-flow instruments were purged of oxygen by overnight equilibration with an anaerobic solution containing 250 µM protocatechuate and 10-50 nM protocatechuate-3,4-dioxygenase (a generous gift from Dr. D. P. Ballou, University of Michigan) in 50 mM potassium phosphate, pH 7.5.

Reduction of Enzyme

XDH for stopped-flow experiments was made anaerobic, as above, in a tonometer with an attached side arm cuvette. XDH was photo-reduced until the visible spectrum indicated complete bleaching in a system containing 20 mM potassium oxalate and a catalytic amount of 5-deazaflavin (15) equal to 10% of the XDH concentration, typically about 0.5 µM. Spectra were obtained with a Hewlett-Packard model 8452A diode array spectrophotometer. Intermediate levels of reduction were obtained by careful irradiation until the visible spectrum of the sample matched that from the literature (9) for the desired species. Samples of XDH were also reduced by titrating from a syringe with an anaerobic solution of 10 mg/ml sodium dithionite. Photo-reduction leaves no excess of reducing equivalents in the sample; therefore, partial re-oxidation of starting material was frequently observed due to contaminating oxygen. Stable formation of fully reduced XDH (8 electrons) was only accomplished through titration with sodium dithionite.

Rapid-reaction Kinetics

-Data from pre-steady-state experiments were collected with a Hi-Tech Instruments model SF-61 stopped-flow instrument interfaced to a Dell model 325D computer. All stopped-flow experiments were carefully temperature controlled at 25 °C. Reactant concentrations given are after mixing in the stopped-flow apparatus. Data acquisition and analysis were performed with Program A, developed in the laboratory of Dr. David P. Ballou at the University of Michigan. Data were analyzed with Program A to exponential fits based on the Marquardt-Levenberg algorithm. Program A also allows simulation of kinetic data. Given a model complete with rate constants, initial concentrations, and extinction coefficients for all species, an absorbance trace is calculated. The shape of these curves can then be compared directly with actual data. Also, the simulated traces can be fitted to test how well the results from analysis of simulated data correlate with analysis of actual data. The diode array stopped flow was made available through the generosity of Dr. David P. Ballou. Diode array data were collected with a program supplied by Hi-Tech Scientific and analyzed using the Specfit program from Spectrum Software Associates.

Analysis of Spectral Intermediates

Spectra derived from the diode array data were analyzed to determine the oxidation state and electron distribution of each reaction species. The total extinction change at 450 nm was found to be the most accurate way to calculate the redox state of reaction intermediates due to the large signal change. At 450 nm, extinction coefficients used are as follows: 37,800 for XDHox, 11,600 for XDH8e-, with extinction increases of 7,000 per 2Fe/2S center, 12,200 for FAD, and 2,440 for FADH·, in units of M-1 cm-1. Using these values and the redox potentials of the centers (9, 10), extinction coefficients at 450 nm for redox states of XDH were calculated to be 12,400 for XDH7e-, 12,800 for XDH6e-, 14,500 for XDH5e-, 16,800 for XDH4e-, 19,400 for XDH3e-, 23,000 for XDH2e-, and 29,600 for XDH1e-, in units of M-1 cm-1.

Preparation of Desulfo XDH

The very closely related xanthine oxidase is known to be inactivated by cyanolysis of the labile molybdenum site sulfur (16). Desulfo XDH was prepared by incubating a 200 µM solution of XDH with 35 mM potassium cyanide at 25 °C, pH 7.5, until no residual activity remained. The sample was passed over a Sephadex G-25 column to remove excess potassium cyanide. Inactive XDH was then prepared for an oxidative half-reaction experiment as described above.

Superoxide Detection

Superoxide formed in the reaction of reduced XDH with oxygen was measured as the superoxide dismutase (SOD) inhibited reduction of cytochrome c (cyt c) (17). Samples containing 6.5 µM XDH, 20 mM potassium oxalate, 0.65 µM 5-deazaflavin, 10 µg/ml catalase, 500 µM methylmethane thiosulfonate (MMTS), and 10 µg/ml SOD (when used) in a total volume of 1 ml were held at 25 °C for 10 min. MMTS was included to derivatize cysteines on XDH. This modification inhibited reduction of cyt c which was observed on mixing oxidized XDH with cyt c. Following preincubation, samples of XDH were made anaerobic in an anaerobic cuvette and then photo-reduced. The cuvette was then opened to air, and 25 µl of 2 mM cyt c was added (50 µM final), followed by immediate mixing with air. Reduction of cyt c was followed spectrophotometrically, with the Hewlett-Packard Diode Array instrument, until completion (about 30 min). This reaction was also performed in the presence of SOD. The total absorbance change at 550 nm in the presence of SOD was subtracted from the total change in the absence of SOD to yield the absorbance change due to superoxide-dependent reduction of cyt c, with Delta E550 = 21,000 M-1 cm-1 (18). Values were multiplied by 1.12 as a correction factor for the relatively poor (2 nm) spectral resolution of the diode array instrument. This value was obtained by comparing the reduction of cytochrome c with excess dithionite measured with the diode array instrument to that measured with a Cary 219 double-beam spectrophotometer. Superoxide was also measured in the stopped-flow instrument, allowing greater kinetic resolution. A sample of 8 µM active XDH was incubated at 25 °C for 15 min with 500 µM MMTS. The sample was transferred to a tonometer and made anaerobic but was left in the oxidized state. XDH samples were mixed (1:1) in the stopped-flow apparatus with solutions containing xanthine, 100 µM cyt c, 20 µg/ml catalase, and plus or minus 40 µg/ml SOD. These cyt c solutions were pre-equilibrated in tonometers with 100% oxygen, which at 25 °C results in 1.2 mM oxygen solutions.

Steady-state Kinetics

Steady-state kinetics were performed by the method of initial rates. Xanthine/oxygen turnover was measured by monitoring the increase in absorbance of urate at 295 nm (Delta E = 9,600 M-1 cm-1). NADH/oxygen turnover was determined by measuring the decrease in absorbance at 340 nm as NADH is converted to NAD (Delta E = 6, 200 M-1 cm-1). The stopped-flow instrument was used to accurately control oxygen concentration. The concentration of oxygen was varied by bubbling syringes containing buffer and substrate at least 15 min with different mixtures of oxygen and nitrogen.

Oxygen Electrode

Oxygen consumption was measured with a Yellow Springs Instruments electrode. The electrode assembly was connected to a water bath set at 25 °C. Samples containing 500 µM xanthine, air-saturated buffer (260 µM oxygen), and varying concentrations of NAD were incubated in the sample compartment until a stable reading was obtained, about 10 min. Reactions were initiated by the addition of XDH (0.5 µM final). The sample compartment was closed to air for the remainder of the measurement.


RESULTS

Quantitation of Fraction of Xanthine Oxidase

The amount of residual XO-type enzyme in preparations of XDH was measured to distinguish the inherent reactivity of XDH toward oxygen from that of contaminating XO. The difference in reactivity of XO and XDH toward NADH was used. A sample of 3.8 µM XDH was reacted anaerobically with an NADH-generating system containing 1 µM NAD, 1 mM glucose 6-phosphate, and 5 units/ml of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. A strong thermodynamic driving force is supplied by spontaneous hydrolysis of the 6-phosphoglucono-1,5-lactone produced. The reaction was initiated by tipping in NAD from the side arm of an anaerobic cuvette. The XDH sample was reduced biphasically by the NADH-generating system at observed rates of 1.4 and 0.04 min-1. At the end of 3 h, 95% of the absorbance at 450 nm was bleached, relative to reduction with excess sodium dithionite (not shown). In contrast, 3.8 µM XO reacted with the NADH-generating system was reduced at approximately 7 × 10-4 min-1. This reaction was not followed to completion; however, at 26 h the XO sample was only 61% reduced (not shown). As XO is reduced by NADH much more slowly than XDH, these experiments are consistent with the conclusion that the 95% bleaching of the XDH sample is due to reduction of the dehydrogenase form enzyme and that the oxidase form constitutes no more than 5% of the total sample.

Spectral Intermediates in the Reaction with Oxygen

To determine the number and type of species in the reaction with oxygen, fully reduced XDH was reacted with oxygen in the diode array stopped-flow instrument. It should be noted that only the iron-sulfur and FAD centers undergo significant changes in the visible spectrum; oxidation of the molybdenum center is not directly observed in these experiments. A solution of dithionite-reduced XDH (17 µM after mixing) was reacted with 610 µM oxygen at 25 °C, pH 7.5. Spectra were recorded from 1.25 ms to 30 min, with a logarithmic bias to the distribution of data points. Spectra were analyzed with the Specfit program from 400 to 650 nm, as inclusion of the entire data set contained more data points than the analysis program could accommodate. Evolving factor analysis of the data by singular value decomposition indicated that four species were present in the reaction, with the possibility of up to two additional species. Spectra were fit to a five-species consecutive model due to the observation of four reaction phases in single-wavelength stopped-flow data (see below). Spectra of enzyme species derived from a four-exponential free fit to the diode array data are shown in Fig. 1. Observed rates from this free fit are k1 obs = 22 s-1, k2 obs = 2.5 s-1, k3 obs = 0.15 s-1, and k4 obs = 0.011 s-1. To determine the oxidation state of each reaction species, spectra were analyzed by comparing the extinction at 450 nm to that calculated for XDH at discrete reduction states (see "Materials and Methods"). From this analysis (Table I) it is clear that intermediates 1 and 3 correspond to mixtures of enzyme species. With an E450 of 26,300 M-1 cm-1, intermediate 1 appears to be a mixture of 50% XDH2e- and 50% XDH1e- which have calculated extinction coefficients of 23,000 and 29,600 M-1 cm-1, respectively. Intermediate 2 has an E450 of 29,400 M-1 cm-1 which corresponds closely to that of XDH1e-. At E450 of 33,400 M-1 cm-1, intermediate 3 appears to be composed of 45% XDH1e- and 55% XDHox. This analysis indicated that the number of electrons remaining in XDH at the end of the first through fourth phases is 1.50, 0.97, and 0.45, and 0.00 electrons, respectively (Table I). Diode array data were also analyzed by a fixed fit with kobs values of 44, 2.1, 0.070, and 0.0095 s-1 for the first through fourth phases, respectively, as calculated from the experimentally determined rate constants (see below). Spectra derived from this fixed fit were indistinguishable from those shown in Fig. 1. The oxidation of XDH8e- with molecular oxygen can be described as proceeding through the species described in Scheme 1.


Fig. 1. Spectral intermediates in the reaction of fully reduced XDH with molecular oxygen. Reduced XDH (17.3 µM) was reacted with 610 µM oxygen at 25 °C, pH 7.5. Spectra were recorded from 1.25 ms to 30 min in a diode array stopped-flow instrument. Spectra are derived from a free fit to the data. Spectrum A, fully reduced XDH; spectrum B, intermediate 1; spectrum C, intermediate 2; spectrum D, intermediate 3; spectrum E, oxidized XDH.
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Table I.

Kinetic and spectral properties of intermediates in the reaction of reduced XDH with molecular oxygen

Rate constants were calculated from the slopes in Fig. 3, as described in the text. Extinction coefficients were normalized such that the E450 of oxidized XDH was equal to 37,800 M-1 cm-1 to correct for base-line shifts. Extinctions were calculated from the amplitudes of exponential fits. The number of remaining electrons in each intermediate was calculated from the E450 of the species and the extinctions calculated for each redox state (see "Materials and Methods").


Enzyme species Observed rate constant of formation Extinction 450 nm Remaining electrons

M-1 s-1 M-1 cm-1
Reduced XDH 12,900  ± 1,900 5.93  ± 0.87
Intermediate 1 k1,ave   = 72,000  ± 16,000 26,300  ± 1,200 1.50  ± 0.07
Intermediate 2 k2,ave   = 3,500    ± 1,300 29,400  ± 1,400 0.97  ± 0.05
Intermediate 3 k3,ave   = 120       ± 21  34,000  ± 700 0.45  ± 0.01
Oxidized XDH  k4,ave   =   16        ± 3.5 37,800 0.00


Scheme 1. Summary of the oxidation of reduced XDH with molecular oxygen. Rate constants and redox states are those calculated from the data. Each step involves at least one second-order reaction with oxygen.
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Kinetics of Oxidation by Molecular Oxygen

XDH (15 µM after mixing) was reduced by dithionite and then reacted in the stopped-flow instrument with solutions of phosphate buffer pre-equilibrated with oxygen, at 25 °C, pH 7.5. Data were recorded at individual wavelengths for greater kinetic resolution. Reaction traces at 610 µM oxygen are presented in Fig. 2, along with fits to the data at 450, 550, and 620 nm. There are four reaction phases apparent in the data in Fig. 2. Data at 450, 550, and 620 nm were fit (see "Materials and Methods") with kobs values of 24, 1.9, 0.14, and 0.013 s-1, in order of appearance. As the dithionite couple (-460 mV) in near that of the reducible disulfide and FADH·/FADH2 couples of XDH (-420 and -410 mV, respectively (9)), an excess of dithionite was typically necessary for full reduction. At low oxygen concentrations, this excess dithionite resulted in deviation from pseudo first-order kinetics as well as incomplete oxidation of XDH. While stable formation of XDH8e- was only observed with dithionite as reductant, these deviations complicated analysis. A reaction trace with dithionite-reduced XDH (8 electron-reduced) is presented because it represents the entire oxidation reaction. To obtain reliable rates over a wide range of oxygen concentrations, photo-reduced XDH was used. A sample of XDH (4.7 µM after mixing) was photo-reduced to approximately XDH6e- and reacted with oxygen over the range 31-610 µM. Observed rates from a four-exponential fit to a trace at 610 µM oxygen (Fig. 4B) are 44, 1.4, 0.070, and 0.0098 s-1 in order of appearance. The rate of the first phase in photo-reduced samples was typically twice that of dithionite-reduced samples. It is proposed that the lower redox state of the dithionite-reduced enzyme (XDH8e-) results in a contribution from an additional step of oxidation than is seen with the less-reduced (XDH6e-) sample from photo-reduction. The observed rate as a function of oxygen concentration is presented in Fig. 3 for all four kinetic phases. Estimates of kobs for each phase except the fourth (not shown for fully reduced XDH) were linearly dependent on oxygen concentration (Fig. 3) with y intercepts close to or at the origin. This kinetic behavior is diagnostic of irreversible second-order reactions with oxygen. Rate constants determined from the slope of each line are k1 = 68,000 ± 17,000 M-1 s-1, k2 = 2400 ± 310 M-1 s-1, and k3 = 110 ± 18 M-1 s-1. Values of kobs for phase 4 varied chaotically between 0.0031 and 0.072 s-1 (not shown). The simplest interpretation of these data is that at least 4 eq of oxygen react with reduced XDH, each in a second-order fashion, to yield XDHox (Scheme 1). This could result in the formation of a mixture of H2O2 and Obardot 2. As reactions were very slow, the possibility of contributions from photochemistry was tested by covering the lamp source for the majority of a trace. The lamp was uncovered occasionally to check for correspondence with normal traces. Agreement was precise (not shown); therefore, photochemistry was considered to be insignificant.


Fig. 2. Plot of absorbance versus time for the oxidation of fully reduced XDH with molecular oxygen. Dithionite-reduced XDH (15 µM) was reacted with 610 µM oxygen at 25 °C, pH 7.5, in a single-wavelength stopped-flow instrument. Reaction traces, bold curves, are overlaid with fits to the data, narrow curves. Data are presented at 450, 550, and 620 nm. Note log time scale.
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Fig. 4. Reaction of partially reduced XDH with molecular oxygen. XDH was photo-reduced to various levels and then reacted with 610 µM oxygen in the stopped-flow instrument at 25 °C, pH 7.5. XDH at the 6- and 2-electron-reduced states were 4.69 µM, whereas XDH at the 4-electron-reduced state was 4.47 µM. A, spectra of the initial species taken in the stopped-flow instrument on mixing with anaerobic buffer. Spectra were normalized by subtraction at 700 nm to correct for base-line shifts but were not corrected for concentration differences. Spectrum a (- - -), 6-electron-reduced XDH; spectrum b, 4-electron-reduced XDH; spectrum c, 2-electron-reduced XDH; spectrum d, oxidized XDH from the experiment with 2-electron-reduced XDH. B, reaction traces at 450 nm, bold curves, overlaid with fits to the data, narrow curves. Traces were normalized by subtraction to the same end point to correct for base-line shifts. Solid curve, 6-electron-reduced XDH; (- - -) 4-electron-reduced XDH; dotted curve, 2-electron-reduced XDH. Note log time scale.
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Fig. 3. Dependence of observed rate on oxygen concentration for the oxidation of XDH at various reduction states. XDH was photo-reduced to differing levels and then reacted with varying oxygen concentrations in a stopped-flow instrument at 25 °C, pH 7.5. Solid circles, fully reduced XDH; solid squares, 4-electron-reduced XDH; solid triangles, 2-electron-reduced XDH. Data are fit to a linear equation using the Kaleidograph program. Solid lines, fully reduced XDH; dashed lines, 4-electron-reduced XDH; (- - -), 2-electron-reduced XDH. A, phase 1; B, phase 2; C, phase 3; D, phase 4.
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As fitting reaction traces with four exponentials can lead to non-unique solutions, the oxygen reaction was repeated with samples of XDH that had been reduced to approximately the 6-, 4-, and 2-electron stages. Samples of XDH (4.5 µM for XDH4e- and 4.7 µM for XDH2e-, after mixing) were carefully photo-reduced (Fig. 4A) until the visible spectra closely resembled that of the desired species. Samples were then reacted in the stopped-flow instrument with buffer at various oxygen concentrations at 25 °C, pH 7.5. Traces of the reactions at 450 nm and 610 µM oxygen are shown in Fig. 4B. There is almost no absorbance change at 550 or 620 nm in the oxidation of XDH4e- and XDH2e-. The reaction of XDH4e- with oxygen required four exponentials to adequately fit, with k obs values at 610 µM oxygen of 48, 2.8, 0.073, and 0.0083 s-1 for the first through fourth phases. Each phase is linearly dependent on oxygen concentration (Fig. 3) with calculated rate constants k1 = 76,000 ± 26,000 M-1 s-1, k2 = 4,500 ± 2,500 M-1 s-1, k3 = 110 ± 43 M-1 s-1, and k4 = 12 ± 5.6 M-1 s-1. The reaction of XDH2e- with oxygen required three phases for an acceptable fit. At 610 µM oxygen, kobs values 8.5, 0.080, and 0.011 s-1 were determined. Each of these three phases is also linearly dependent on oxygen concentration (Fig. 3) with rate constants of k2 = 13,000 ± 2,500 M-1 s-1, k3 = 130 ± 43 M-1 s-1, and k4 = 19 ± 4.3 M-1 s-1. Note that the starting points for these reactions (XDH4e- and XDH2e-) are only approximate. Also, initial spectra represent the sum of a population of enzyme species. The observation of these kinetic phases in the oxidation of XDH2e- supports the assignment of intermediate 1 as a significantly oxidized species.

To obtain a set of rate constants consistent with all three of these kinetic experiments, rate constants corresponding to the same reaction were averaged (Table I). Values for k4 of 12 and 19 M-1 s-1 from the XDH4e- and XDH2e- experiments, respectively, were averaged to give k4 ave = 16 ± 3.5 M-1 s-1. Excellent agreement was obtained when kobs values for each oxygen concentration were calculated from k4 ave and used to fit data from the XDH6e- experiment, whose k4 obs values varied inconsistently in free fits. Values for k3 of 110, 110, and 130 M-1 s-1 were averaged to give k3 ave = 120 ± 21 M-1 s-1. Values for k2 of 2,400, 4,500, and 13,000 M-1 s-1 were first averaged to give k2 ave = 6,700 ± 5,700 M-1 s-1, but kobs values calculated from this rate constant could not be used to fit data from experiments starting with XDH6e- and XDH4e-. Instead, the values of k2 from these latter two experiments were averaged to give k2 ave = 3,500 ± 1,300 M-1 s-1, which could be used to fit data from all three experiments. As can be seen in Fig. 4B, the amplitude of the first phase is extremely small for the reaction of XDH2e- with oxygen, thus explaining the poor agreement with the other experiments. Values of k1 of 68,000 and 76,000 M-1 s-1 were averaged to give k1 ave = 72,000 ± 16,000 M-1 s-1. These four average rate constants can be used together to fit all of the experimental data at all wavelengths studied.

Influence of Desulfo Enzyme, Ligands, and Method of Reduction on the Kinetics of Oxidation

Samples of XDH used in these studies contained approximately 25-35% inactive desulfo form. To assess the influence of this inactive XDH on the observed kinetics, desulfo XDH was prepared by prior incubation with potassium cyanide, until no measurable activity remained. Desulfo XDH (7.5 µM after mixing) was photo-reduced and reacted with 610 µM oxygen. Reaction traces (not shown) were identical in form to those of native samples. Four exponential fits with observed rates of 40, 3.2, 0.17, and 0.021 s-1 were required to account for the reaction traces at 450 nm. XDH was also reacted with the mechanism-based inhibitor allopurinol (19). A solution of 8.8 µM XDH was incubated with 1 mM allopurinol and 2.5 mM DTT for 1 h at 25 °C, pH 7.5. No detectable activity remained. The sample was desalted on Sephadex G-25 and prepared for the stopped-flow as above. Allopurinol-inhibited XDH (6.1 µM after mixing) was photo-reduced and reacted with 610 µM oxygen at 25 °C, pH 7.5. Resulting traces (not shown) were similar to that of native XDH and fit freely to kobs values of 42, 3.7, 0.13, and 0.014 s-1. XDH (7.5 µM after mixing) photo-reduced in the presence of urate (250 µM) also reacted with 610 µM oxygen as normal XDH. Values of kobs from the four exponential fits are 31, 2.0, 0.098, and 0.011 s-1. As the oxidation kinetics of dithionite- and photo-reduced XDH are nearly identical (except for the extent of reaction), it is also apparent that neither the method of reduction nor sulfite derived from dithionite influences oxidation. We conclude from these experiments that alterations at the molybdenum center such as cyanolysis or binding of allopurinol, urate, or sulfite do not affect the kinetics of oxidation by molecular oxygen.

Quantitation of Superoxide Formed in the Reaction with Molecular Oxygen

To assess the ability of XDH to form Obardot 2 relative to XO, and to elaborate on the mechanism in Scheme 1, the amount of Obardot 2 formed during XDH oxidation was measured. The superoxide dismutase (SOD) -inhibited reduction of cytochrome c (cyt c) (17) has been used successfully to quantitate the amount of Obardot 2 produced in the reaction of bovine milk XO with oxygen (12, 13). To minimize complications arising from partial re-oxidation of reduced XDH samples prior to reaction with O2 and cyt c, and because of the slowness of the reaction, experiments were performed in anaerobic cuvettes. A solution of 6.5 µM XDH was photo-reduced anaerobically. The cuvette was opened, 50 µM cyt c was added, and the sample was then quickly equilibrated with air. Reduction of cyt c was measured at 550 nm in a diode array spectrophotometer (Fig. 5). Only about 20% of the total absorbance change occurred in the dead time of these experiments, about 30 s. The reaction exhibited biphasic kinetics with rates of 0.0074 and 0.0012 s-1 (Fig. 5, inset) At air saturation and 25 °C, these correspond to second-order rate constants of 29 and 4.7 M-1 s-1, respectively. Prior to anaerobiosis, XDH samples were preincubated with 500 µM methylmethane thiosulfonate (MMTS) to derivatize any reactive cysteines. In the absence of MMTS treatment, reduction of cyt c on mixing with oxidized XDH was observed. Presumably these reactive cysteines can reduce cyt c. XDH assayed after MMTS treatment was not altered in xanthine/NAD or xanthine/oxygen turnover, indicating that MMTS does not inactivate XDH or convert XDH to XO. The final spectrum after re-oxidation in the absence of SOD indicated that a total of 4.7 ± 0.16 eq cyt cred per XDH were formed. Identical reactions in the presence of 10 µg/ml SOD (Fig. 5) showed that 3.0 ± 0.14 eq cyt cred per XDH are formed in a superoxide-independent manner. The difference between these values, 1.7 ± 0.15, is the total number of mol of Obardot 2 per mol of XDH. Of the 1.7 total eq Obardot 2 detected, 1.1 ± 0.03 are observed in the faster phase of cyt c reduction, which is consistent with the oxidation kinetics in the absence of cyt c. One explanation for the slow formation of the remaining 0.6 eq Obardot 2 is that cyt c may be forming a complex with XDH and thus be retarding the reaction with oxygen. Note that a large amount of cyt c is reduced in the presence of SOD. Increasing the concentration of SOD from 10 to 50 µg/ml had no effect on the quantity of cyt c reduced.


Fig. 5. Detection of superoxide in reaction of reduced XDH with oxygen. Samples of XDH were prepared as described under "Materials and Methods." - - -, oxidized XDH, 6.5 µM; - - -, fully reduced XDH; · · ·, 30 min after addition of 50 µM cyt c and oxygen at air saturation; solid spectrum, repeat experiment, 30 min after addition of 50 µM cyt c and oxygen in the presence of 10 µg/ml SOD; - - -, spectrum of 50 µM oxidized cyt c mathematically added to spectrum of 6.5 µM oxidized XDH. Inset, time course of superoxide-dependent reduction of cyt c. The rate of cyt c reduction at 550 nm was measured in the absence and in the presence of SOD. Absorbance values measured in the presence of SOD were subtracted from those measured in the absence of SOD to give the time course of superoxide-dependent cyt c reduction. Data were analyzed by exponential stripping procedures. The faster phase occurs at 0.0074 s-1 and involves formation of 1.1 eq Obardot 2. The slower phase occurs at 0.0012 s-1 and corresponds to formation of 0.6 eq Obardot 2.
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The anaerobic reaction between photo-reduced XDH and cyt c was measured by mixing cyt c from a side arm of an anaerobic cuvette. At 2 µM XDH, the concentration of cyt c was varied from 10 to 100 µM. Monophasic reduction of cyt c was observed with essentially no concentration dependence on the observed rate, 1.7 × 10-3 s-1 at 10 µM cyt c and 1.4 × 10-3 s-1 at 100 µM cyt c. This indicates that cyt c binds tightly to XDH. This tight binding is proposed to impede the reaction of XDH with oxygen, thus explaining the slow kinetics of superoxide-dependent cyt c reduction. Also, this binding may favor cyt c as an electron acceptor over oxygen. Clearly, the direct reduction of cyt c by XDH significantly complicates the determination of Obardot 2-dependent reduction, and the measured 1.7 eq Obardot 2 per XDH probably represents a lower limit of the true value.

To measure Obardot 2 formation in a system more relevant to turnover, and to determine from which redox states Obardot 2 is formed, oxidized XDH (4.0 µM active enzyme after mixing) was pretreated with MMTS and reacted in the stopped-flow apparatus with solutions of xanthine, 10 µg/ml catalase, 610 µM oxygen, and 50 µM cyt c with and without 20 µg/ml SOD (not shown). Experiments with 2 µM xanthine (0.5 equivalents xanthine per active XDH) yielded total absorbance changes equal to 3.9 µM cyt cred, after subtraction of the SOD-independent change. This is equivalent to a total of 1.9 mol of Obardot 2 per mol of xanthine. Traces were multiphasic, with 0.97 eq cyt c formed at the slowest rate of 0.0011 s-1, significantly slower than the value of 0.0095 s-1 predicted from k4 ave of 16 M-1 s-1. Assuming the enzyme is reduced to only XDH2e-, this corresponds to 1.9 total Obardot 2 per XDH2e- or to 0.93 Obardot 2 per XDH2e- counting only that portion consistent with the rate of oxidation in the absence of cyt c. The reaction of 4 µM active XDH with 2 µM xanthine and 610 µM oxygen was also monitored in the absence of cyt c (not shown). After an initial decrease in A450 corresponding to reduction at 8.6 s-1, a biphasic increase in A450 at 0.17 and 0.018 s-1 restored XDHox. These oxidation rates are reasonably consistent with those determined with pre-reduced XDH. Experiments with 8 µM xanthine (2.0 eq xanthine per active XDH) yielded 7.5 µM cyt cred after subtraction of the total SOD-independent change. 6.2 µM cyt cred was formed in that portion occurring during the time scale of XDH oxidation in the absence of cyt c. A total of 1.6 to 1.9 eq Obardot 2 is formed per XDH molecule. As XDH is reduced to an oxidation state below XDH2e-, little additional Obardot 2 is formed. Due to interference from the direct reduction of cyt c by XDH, 1.9 eq Obardot 2 per XDH is also considered a minimum value. This measurement of 1.9 mol of Obardot 2 per mol of XDH with xanthine as the electron donor corresponds well with the 1.7 eq Obardot 2 determined with photo-reduced XDH. A more conservative lower end of 0.93 to 1.1 eq represents that Obardot 2 detected at a rate comparable with oxidation in the absence of cyt c. As much as 0.97 eq Obardot 2 is formed at approximately 1.2 × 10-3 s-1. This Obardot 2 production is proposed to be slow due to inhibition by cyt c binding to XDH. Binding and subsequent reduction of the cyt c may also perturb the amount of Obardot 2 formed. These experiments are in agreement with a model in which Obardot 2 is formed from the last 2 electrons oxidized from XDH. But, because of the complications arising from binding of cyt c, these experiments do not rule out the possibility of more Obardot 2 being formed than that detected by the SOD-dependent reduction of cyt c.

Mechanism and Simulation of Pre-steady-state Kinetic Data

Models discussed were simulated in their entirety with Program A (see "Materials and Methods"). All reactions were simulated as second order with respect to oxygen. Analysis of curves simulated at different oxygen concentrations recapitulated this bi-molecular behavior.

One interpretation of the data is that the four phases observed represent four discrete reactions as follows: XDH6e- right-arrow XDH4e- right-arrow XDH2e- right-arrow XDH1e- right-arrow XDHox. The first two steps would each produce an equivalent of H2O2, and the last two steps would each form an equivalent of Obardot 2. This stoichiometry is not inconsistent with the observed Obardot 2 formation. Such a mechanism has been proposed for bovine milk XO (12, 13). The extinction coefficients for these species can easily be calculated. As there is no evidence for oxygen binding, the spectrum of each intermediate should in no manner be perturbed from that predicted thermodynamically. Using the rate constants given in Table I, and extinction coefficients calculated for the discrete species above (see "Materials and Methods"), a simulation of this simple mechanism is given (Fig. 6, curve A). While the rates compare favorably to those of the actual data, the nature of the intermediate species is clearly inconsistent. Thus the mechanism of XDH oxidation by oxygen is quite different from that of XO.


Fig. 6. Simulation of kinetic data from reaction of reduced XDH with molecular oxygen. A reaction trace of 15 µM dithionite-reduced XDH with 610 µM O2 is presented (solid curve). The trace was normalized by subtraction to correct for a base-line shift. Mechanisms were simulated in their entirety with program A. In all cases presented, total XDH concentration was 15 µM, and initial oxygen concentration was 610 µM. Curve A, simulation of model XDH6e- right-arrow XDH4e- right-arrow XDH2e- right-arrow XDH1e- right-arrow XDHox. Extinction coefficients used are those predicted by the redox potentials of the various centers (see "Materials and Methods"). Rate constants used are those in Table I. Curve B, simulation of the mechanism in Scheme 1. Extinction coefficients used are 11,600 M-1 cm-1 for XDHred and those in Table I for remaining species. Rate constants used are those in Table I. Curve C, simulation of the mechanism in Scheme 2. Extinction coefficients are predicted from the redox potentials of the centers. Rate constants used are those given in Scheme 2.
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A more accurate but simplistic model is given in Scheme 1, which includes only the intermediates observed. Use of the average extinction coefficients derived from the actual data and the apparent rate constants (Table I) yielded a simulation in good agreement with the data (Fig. 6, curve B). Although an accurate model requires bifurcations to explain the mixture of redox states observed, the four-step model in Scheme 1, along with the empirical extinction coefficients, is still useful for predicting the behavior toward oxygen of XDH samples at particular average redox states.

A more complete model, using the theoretical extinction coefficients, requires bifurcations to account for the mixture of intermediate states (Scheme 2). A combination of XDH1e- and XDH2e- has been observed to be formed in a single phase at 72,000 M-1 s-1 when starting with XDH6e- and at approximately 30,000 M-1 s-1 when starting with XDH8e-. This requires up to 6.5 electron oxidation exhibiting overall monophasic kinetics. Attempting to unravel the elementary steps that comprise this phase would amount to speculation. Starting with XDH8e-, the model in Scheme 2 predicts that rate constant k1 has a value of a least 70,000 M-1 s-1. Rate constants k2 and k3 are suggested to occur with an overall rate constant of approximately 70,000 M-1 s-1. The 2-fold apparent discrepancy of rates between dithionite- and photo-reduced XDH in the first step of oxidation is consistent with the different starting points, XDH8e- and XDH6e-, respectively, and the rate constants listed. Altogether, steps k1, k2, and k3 are proposed to produce XDH2e- and 3 eq H2O2. The XDH2e- formed can react in another 2-electron process, k4, to yield XDHox and 1 mol of H2O2, or it can react in two consecutive 1-electron processes, k5 and k10, to give XDHox and 2 mol of Obardot 2. The values of k4 and k5 must be similar in magnitude to account for the oxidation state of intermediate 3. Starting with XDH7e-, two consecutive 2-electron oxidations, k6 and k7, can occur with an overall rate constant of 70,000 M-1 s-1, resulting in XDH3e- and 2 mol of H2O2. XDH3e- is predicted to react in either a 1-electron process through k8 to form XDH2e- and an equivalent of Obardot 2 or in a 2-electron process through k9 to yield XDH1e- and an equivalent of H2O2. The rate constant k8 was assigned a value of 3,000 M-1 s-1 to correspond with steady-state data (see "Discussion"), although simulations indicate this rate constant can be varied over a factor of 2 with little effect. The 2-electron oxidation of XDH3e- to XDH1e-, k9, was modeled at 8,000 M-1 s-1 to give good correspondence with the data. From XDH1e- a single 1-electron oxidation, k10, would result in XDHox and 1 mol of Obardot 2. Scheme 2 predicts that XDH2e- reacts with oxygen to form 1 mol of Obardot 2 and 0.5 mol of H2O2. This is not inconsistent with experiments in which 0.93 to 1.9 eq Obardot 2 are detected when XDH is reduced with [one-half] eq xanthine, forming mostly XDH2e-. The binding of cyt c to XDH bas been shown to impede the rate of oxygen oxidation; this binding may also change the product distribution.


Scheme 2. Predicted model of the oxidation of reduced XDH with molecular oxygen. Reactions described are predicted from simulations to the data. Bifurcations were included to account for observed mixtures of redox states. Individual rate constants are given for the bifurcations. The rate at which XDH3e- is predicted to decay is k8 + k9 = 11,000 M-1 s-1, and the rate at which XDH2e- is estimated to decay is k4 + k5 = 400 M-1 s-1.
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Simulating the mechanism in Scheme 2 with 67% XDH8e- and 33% XDH7e- as initial species and with the theoretically predicted extinctions yields a reaction trace in reasonable correspondence with the actual data (Fig. 6, curve C). Note that reactions starting with 100% XDH8e- predict formation of 1 eq Obardot 2 per XDH, and those starting with XDH7e- predict 1.8 mol of Obardot 2. This range of predicted Obardot 2 stoichiometries is consistent with that observed experimentally. A mixture of starting material oxidation states is reasonable as the extinction coefficients at 450 nm of XDH8e-, XDH7e-, and XDH6e- vary over only 1,200 M-1 cm-1. Modeling with only XDH8e- as the initial species results in simulations in which the extinction of intermediate 1 is far too low (not shown). This can only be rectified by adding a bifurcation at XDH4e- involving 1-electron oxidation to form XDH3e- and Obardot 2. Such a step is certainly possible. Decreasing rates of 2-electron oxidation, >= 70,000, 8,000, and 200 M-1 s-1, are proposed to correspond with the decreasing fraction of FADH2 as the oxidation state of XDH increases. While the fraction of FADH2 varies at least 100-fold over the various redox states, the fraction of FADH· only varies approximately 3-fold. The model in Scheme 2 is only approximate and is severely limited by the data available. This mechanism is presented to demonstrate the reactions proposed to be significant but should only be considered in a qualitative manner.

Steady-state Kinetics of Xanthine/Oxygen and NADH/Oxygen Turnover

Since XDH can use molecular oxygen as an electron acceptor, appreciable xanthine oxidase activity is expected. Experiments presented above estimate XO contamination to be no more than 5%, indicating this xanthine/oxygen activity is inherent to XDH. The xanthine/oxygen turnover of XDH was measured by the method of initial rates at 25 °C, pH 7.5. The sample of XDH used in this experiment was determined to be 76% active (see "Materials and Methods"). As samples contained 0.50 µM total XDH after mixing, the concentration of active XDH used is (0.50 µM) (0.76) = 0.38 µM. A Lineweaver-Burk plot of the data shows near-parallel lines, suggestive of a ping-pong mechanism (not shown). For xanthine/oxygen turnover of XDH, kcat is 2.1 ± 0.1 s-1, Km for xanthine is 1.9 ± 0.4 µM, and Km for oxygen is 65 ± 9.0 µM. For comparison, kcat for xanthine/NAD turnover of XDH is 6.3 s-1 (1), 3-fold faster. Also, at pH 7.5, 25 °C, XO has a kcat of 13 s-1 (20).

As NADH can reduce XDH at a rate of 18 s-1 (21) XDH is predicted to contain NADH oxidase activity. NADH oxidase activity of XDH was measured by the method of initial rates, at 25 °C, pH 7.5. A Lineweaver-Burk plot of the data shows a set of parallel lines, consistent with a ping-pong mechanism (not shown). NADH oxidase activity was determined to be independent of the fraction of desulfo XDH by assaying before and after treatment with potassium cyanide. For NADH oxidase activity, XDH has a kcat of 2.5 ± 0.9 s-1, a Km for NADH of 2.8 ± 0.5 µM, and a Km for oxygen >= 2 mM. A replot of Vmax (app), from the primary velocity versus NAD concentration plot, versus oxygen concentration yielded a straight line out to 610 µM oxygen with no evidence of saturation. Therefore, determination of kcat involves a very large extrapolation and is thus less accurate.

Competition of NAD and Molecular Oxygen for Reducing Equivalents

The ability of oxygen to compete with NAD as an electron acceptor was tested by measuring the rate of NADH formation in initial-rate assays while systematically varying the concentrations of xanthine, NAD, and oxygen (not shown). Detectable inhibition of the rate of NADH formation was only observed at high oxygen and very low xanthine or NAD concentrations. At 100% oxygen saturation, 1.2 mM at 25 °C, and at 2 µM xanthine and 14 µM NAD, 77% of the anaerobic rate was observed. Km values for xanthine and NAD are <= 1 and 7 µM, respectively (1). These xanthine and NAD concentrations are at the lower limit of our spectrophotometric detection. At xanthine and NAD concentrations equal to 8 and 56 µM, respectively, 91% of the anaerobic rate is observed. Under initial-rate conditions, oxygen can only successfully compete with NAD as a substrate for XDH at NAD or xanthine concentrations significantly below each Km.

These experiments with xanthine, NAD, and oxygen were also performed with an oxygen electrode, monitoring the disappearance of molecular oxygen (not shown). The oxygen electrode is not sensitive enough to demonstrate inhibition of oxygen consumption by NAD during the initial-rate portion of the reaction; therefore, the entire reaction was followed. Experiments were performed at 500 µM xanthine. At concentrations of NAD from 0 to 1 mM, all of the oxygen (260 µM) was eventually consumed over a time course that ranged from 15 to 50 min. The time required for complete oxygen depletion was proportional to the NAD concentration. This result can be simply explained as consumption of oxygen by the NADH oxidase activity of XDH once the xanthine/NAD reaction has gone to completion. This conclusion is supported by the observation of a plateau and a decrease in 340 nm absorbance when aerobic xanthine/NAD assays are followed at longer times. The significance of this observation is that electron equivalents from xanthine can ultimately be transferred to oxygen in the presence of NAD and NADH. Although oxygen competes poorly for electron equivalents from XDH when there is an excess of NAD present, the NAD is eventually consumed. Although disfavored kinetically, presumably oxygen is the ultimate electron acceptor due to the high redox potential of the O2/H2O2 couple, +300 mV at pH 7.0 (22), much higher than that of the NAD/NADH couple, -320 mV at the same pH.


DISCUSSION

A simplified model for the oxidative half-reaction of bovine milk XDH with molecular oxygen is presented in Scheme 1. This mechanism describes oxidation as proceeding by four irreversible second-order reactions with oxygen, although these are certainly complex combinations of rate constants. Using the reduction of cyt c as an indicator for Obardot 2, at least 1.9 mol of Obardot 2 are formed in the conversion of XDH2e- to XDHox. Thus, Obardot 2 is produced from the last electrons to be oxidized. The rate constants calculated from the data decrease by over 3 orders of magnitude from the start to the end of the reaction. It is proposed that these changes in rate may be due to a decrease in electron density at the FAD center, the site at which oxygen reacts. The inherent reactivity of reduced XDH toward oxygen may also decrease as its oxidation state changes, and the redox potential of the enzyme becomes more positive. Two different types of flavin reactivity can be distinguished. The reaction of FADH2 with oxygen to produce FAD and H2O2 is thought to be fast, while the reaction of FADH· with oxygen to produce FAD and Obardot 2 is slow. The redox potentials predict that FADH2 is present significantly only at XDH3e- and lower, while a large fraction of FADH· persists from XDH6e- to complete oxidation. Superoxide is not formed until the higher oxidation states because the reaction of oxygen with FADH2 is proposed to be much faster than with FADH·, even when only a small fraction of FADH2 is present. At XDH2e- and XDH1e-, very little FADH2 is predicted to be present, thus favoring the slower reaction to make Obardot 2. There is substantial precedent for altered reactivity of flavin hydroquinone and semiquinone species toward molecular oxygen. Such behavior has been shown in the oxidation of the simpler flavoproteins glucose oxidase from Aspergillus niger (25) and flavodoxin from Megasphaera elsdenii (26, 27). Experiments with chicken liver XDH were consistent with such selectivity of reaction rates with oxygen, with hydroquinone reacting at 1,900 M-1 s-1 and semiquinone at 260 M-1 s-1, at pH 7.8, 4 °C (14). The overall oxidative half-reaction of bovine milk XDH is suggested to proceed by at least four second-order reactions, controlled by the electron distribution within XDH and by the redox potentials of the enzyme. Equilibria controlling electron distribution are expected to be maintained quickly from studies on intramolecular electron transfer (23, 24) and the rapid equilibrium hypothesis of Olson et al. (28).

The current results share some similarities to those determined in the oxidative half-reaction of bovine milk XO. Those experiments were carried out at 25 °C, pH 8.5, and were proposed to follow a four-step reaction sequence: XDH6e- right-arrow XDH4e- right-arrow XDH2e- right-arrow XDH1e- right-arrow XDHox (12, 13). Three kinetic phases at 450 nm were observed, a short lag phase, a saturating phase with a limiting rate of 125 s-1, and an apparent Kd of 500 µM, and a phase directly proportional to oxygen concentration with a second-order rate constant of 10,000 M-1 s-1. At the end of the second phase, spectral changes indicated that XO had been oxidized by 5 electron equivalents. Each research group that has studied the reaction of XO with oxygen has proposed the formation of a binary complex between XOred and oxygen based on the observation of saturation kinetics (11-13). The reaction of most known reduced flavoproteins with oxygen proceeds via an irreversible second-order reaction (29), and the current data certainly support this in the case of bovine milk XDH. It is possible that the reaction of XO with oxygen also proceeds via purely bi-molecular reactions. Saturation kinetics could be observed due to complications arising from a series of chemical reactions that are exhibited in a single spectrophotometric phase.

The oxidation of chicken liver XDH is quite different from that of bovine XDH. The chicken liver enzyme reacts with oxygen according to a model similar to that of XO, only with a bifurcation at XDH4e- to form XDH2e- in either a single 2-electron step or in two consecutive 1-electron steps (14). Between 2.8 and 3.0 mol of Obardot 2 were detected with the chicken liver enzyme. With bovine milk XDH, the rate at which FADH· reacts with oxygen is much slower than in the chicken liver enzyme (260 M-1 s-1 (14)) and cannot compete with the 2-electron rate until the fraction of FADH2 is less than 0.01, which is at XDH3e- or higher. This also explains why more Obardot 2 is detected in the oxidation of the chicken liver enzyme. For all three of the enzymes discussed, the reaction with oxygen would appear to involve a series of bi-molecular collisions, with the reaction rate and products at least partially a function of the type of reduced flavin encountered.

If reduced bovine milk XDH can react with oxygen at least as fast as 72,000 M-1 s-1, then how can NAD effectively compete as an electron acceptor? It has been proposed previously that during xanthine/NAD catalysis, XDH cycles between the 2- and 4-electron-reduced states (21). XDH4e- would thus be the species for which NAD and oxygen compete. Reduced XDH reacts with NAD at a limiting rate of 170 s-1, subsequent to rapid binding.2 According to Scheme 1, XDH4e- reacts with oxygen with a rate constant of at least 72,000 M-1 s-1, which at air saturation would give a kobs of 18 s-1, much less than 170 s-1 for the NAD reaction. In addition, NAD appears to bind very rapidly, with an estimated association rate constant of at least 1 × 107 M-1 s-1,2 140-fold greater than the fastest observed oxygen reaction. For comparison, at 200 µM oxygen XO is estimated to react at 35 s-1, at pH 8.5 (12). At its lowest oxidation states, XDH reacts with oxygen at rates comparable with those of XO. At the higher oxidation states the rates are much slower than the slow phase of the XO reaction, 16 M-1 s-1 versus 1 × 104 M-1 s-1 (12). This difference is proposed to be due in part to the greater stabilization of FADH· with respect to FADH2 in XDH; the semiquinone form appears to react much slower than the hydroquinone. In addition, there must be a significant (630-fold) reduction in the rate at which the semiquinone of XDH reacts with respect to that of XO. Not only is the oxygen reaction retarded by the distribution of electrons in XDH, but the inherent reactivity of the flavin semiquinone is much smaller.

XDH contains appreciable xanthine/oxygen and NADH/oxygen activities. The kcat values for these reactions are very close, 2.1 ± 0.1 and 2.5 ± 0.9 s-1, respectively, and constitute 33 and 40% of the kcat value for xanthine/NAD catalysis, respectively. Since the reductive half-reaction in both cases has been shown to be faster than 2.5 s-1 (21), the reaction with oxygen must greatly limit turnover for both. The spectrum of XDH during xanthine/oxygen catalysis approximates XDH3e- (1); the rate-limiting step is oxidation, and the spectrum represents the more reduced form. XDH is therefore thought to cycle between XDH1e- and XDH3e- during xanthine/oxygen turnover. According to Scheme 2, XDH3e- decays at k8 + k9 = 11,000 M-1 s-1. Using the concentration of oxygen at air saturation, 260 µM, this would give an oxidation rate of 2.8 s-1. During xanthine/oxygen turnover, xanthine is proposed to reduce XDH1e- to XDH3e- at approximately 7.0 s-1, the same rate at which xanthine reduces XDHox to XDH2e- (21). From kred of 7.0 s-1 and kox of 2.8 s-1, a kcat of 2.0 s-1 can be calculated which agrees quite well with the experimental value of 2.1 s-1. XDH3e- can either be oxidized directly to XDH1e- by k9 to form 1 mol of H2O2 or it can proceed through k8 and k5 to form 2 eq Obardot 2 via XDH2e-. The ratio of k8 to k9, 0.38, is consistent with the fraction of electrons from xanthine that form Obardot 2 during xanthine/oxygen turnover, 0.42 (1). Although XDH produces no more Obardot 2 than XO during the oxidative half-reaction with oxygen, much more Obardot 2 is produced during XDH-catalyzed xanthine/oxygen turnover because it cycles through the higher oxidation states where Obardot 2 is formed. NADH/oxygen turnover is proposed to cycle through XDH1e- and XDH3e- as well. Using kred of 18 s-1 (21) and kox of 2.8 s-1, the calculated kcat of 2.3 s-1 agrees well with the experimental value of 2.5 s-1. Under initial-rate conditions, steady-state experiments performed in the presence of xanthine, NAD, and oxygen indicate that oxygen can only effectively compete at very high concentrations (100%), and only at concentrations of xanthine and NAD below their Km values, <= 1 and 7 µM, respectively (1).

McCord (30) has suggested that conversion of XDH to XO in vivo may be responsible for production of H2O2 and Obardot 2 in postischemic reperfusion injury. Results from the current work suggest that the mammalian bovine milk XDH is itself capable of producing such reactive oxygen species, albeit at 16% the rate of XO (calculated as the ratio of kcat values 2.1 and 13 s-1 (20) at 25 °C, pH 7.5). Estimates of relative amounts of XO and XDH based solely upon turnover assays must therefore be made with caution, as XDH contains an intrinsic xanthine oxidase activity. Monitoring the reaction with an oxygen electrode, oxygen was consumed in aerobic xanthine/NAD assays over a 15-50-min time scale from 0 to 1 mM NAD. Once xanthine/NAD turnover has gone to completion, XDH can function as an NADH oxidase, even in the presence of high NAD levels. Depletion of the amount of NAD available to XDH, relative to oxygen, should be sufficient for XDH-catalyzed production of oxygen radicals.


FOOTNOTES

*   This work was supported by National Science Foundation Grant DMB-8500291 (to V. M.) and by National Institutes of Health Training Grant in Molecular Biophysics GM08270 (to C. M. H.). This material was presented in preliminary form at the 12th Symposium on Flavins and Flavoproteins, Calgary, Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan, M3441, Medical Science I, Ann Arbor, MI 48109-0606.
1   The abbreviations used are: XO, xanthine oxidase; XDH, xanthine dehydrogenase; XDHne-, xanthine dehydrogenase reduced by n electrons; cyt c, cytochrome c; SOD, superoxide dismutase; MMTS, methylmethane thiosulfonate.
2   C. M. Harris and V. Massey, manuscript in preparation.

ACKNOWLEDGEMENT

We thank Dr. David P. Ballou for the use of instrumentation and for helpful discussion.


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