(Received for publication, November 21, 1996, and in revised form, January 22, 1997)
From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606
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 M1 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 s1, 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.
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 O
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 s1, 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 O
2 and 2 mol of
H2O2 per mol of XO, with the O
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 O
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 M1 s
1. From 1.7-1.9 mol of
O
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 O
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 s1, 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.
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 M1 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.
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 EnzymeXDH 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 IntermediatesSpectra 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.
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 DetectionSuperoxide 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
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 were performed
by the method of initial rates. Xanthine/oxygen turnover was measured
by monitoring the increase in absorbance of urate at 295 nm
(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
(
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 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.
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 min1. 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.
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 s1,
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.
|
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 s1, 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 O
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.
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 M1 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.
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 s1 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.
To assess the ability of XDH to form O2 relative
to XO, and to elaborate on the mechanism in Scheme 1, the amount of
O
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 O
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 O
2 per mol of
XDH. Of the 1.7 total eq O
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 O
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.
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 × 103 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
O
2-dependent reduction, and the measured 1.7 eq
O
2 per XDH probably represents a lower limit of the true
value.
To measure O2 formation in a system more relevant to turnover,
and to determine from which redox states O
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 O
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
O
2 per XDH2e
or to 0.93 O
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 O
2
is formed per XDH molecule. As XDH is reduced to an oxidation state
below XDH2e
, little additional O
2 is formed. Due
to interference from the direct reduction of cyt c by XDH, 1.9 eq
O
2 per XDH is also considered a minimum value. This
measurement of 1.9 mol of O
2 per mol of XDH with xanthine as
the electron donor corresponds well with the 1.7 eq O
2
determined with photo-reduced XDH. A more conservative lower end of
0.93 to 1.1 eq represents that O
2 detected at a rate
comparable with oxidation in the absence of cyt c. As much as 0.97 eq
O
2 is formed at approximately 1.2 × 10
3
s
1. This O
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 O
2
formed. These experiments are in agreement with a model in which
O
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 O
2 being
formed than that detected by the SOD-dependent reduction of
cyt c.
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
XDH4e
XDH2e
XDH1e
XDHox. The first two steps would each produce an equivalent
of H2O2, and the last two steps would each form
an equivalent of O
2. This stoichiometry is not inconsistent
with the observed O
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.
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
O
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 O
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 O
2. Scheme 2 predicts that
XDH2e
reacts with oxygen to form 1 mol of O
2 and
0.5 mol of H2O2. This is not inconsistent with
experiments in which 0.93 to 1.9 eq O
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.
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 O
2 per XDH, and those starting with
XDH7e
predict 1.8 mol of O
2. This range of
predicted O
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 O
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.
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 s1,
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 s1 (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.
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.
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 O2, at least 1.9 mol of O
2 are
formed in the conversion of XDH2e
to XDHox. Thus,
O
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 O
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 O
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
XDH4e
XDH2e
XDH1e
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 O
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 O
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 M1 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 s1,
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
O
2 via XDH2e
. The ratio of
k8 to k9, 0.38, is
consistent with the fraction of electrons from xanthine that form
O
2 during xanthine/oxygen turnover, 0.42 (1). Although XDH
produces no more O
2 than XO during the oxidative half-reaction with oxygen, much more O
2 is produced during XDH-catalyzed
xanthine/oxygen turnover because it cycles through the higher oxidation
states where O
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 O2 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.
We thank Dr. David P. Ballou for the use of instrumentation and for helpful discussion.