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INTRODUCTION |
Xanthine oxidoreductase catalyzes the oxidation of hypoxanthine
and xanthine to urate and is involved in purine catabolism in mammals.
Isolated from bovine milk, the enzyme exists as a dimer, containing one
molybdopterin, one FAD, and two plant ferredoxin-type 2Fe/2S centers
per 145-kDa subunit (1-4). Reducing substrates such as xanthine react
at the molybdenum center (5), whereas oxidizing substrates such as
oxygen or NAD react at the FAD (6, 7). Like other molybdenum
hydroxylases, the oxygen incorporated into substrate is derived from
water, and electron equivalents are released in the hydroxylation
reaction. Electrons from xanthine are transferred either to NAD or to
molecular oxygen, depending on the form of the enzyme present. Xanthine
dehydrogenase-type (XDH)1
enzyme prefers NAD as an electron acceptor (8) but in the absence of
NAD will catalyze xanthine/oxygen turnover at 30% the rate of
xanthine/NAD turnover (9). Xanthine oxidase-type (XO) enzyme only
utilizes oxygen as its oxidizing substrate, to any significant extent.
XDH from bovine milk can be converted to XO irreversibly by proteolysis
(10, 11) or reversibly by oxidation of cysteines to cystines (8, 12).
Approximately eight cysteines are oxidized to four cystines on
converting milk XDH to XO (12). XDH is thought to be the major form
present in vivo (13), and there is evidence that reduced
oxygen species formed from XDH or XO may be important in several
oxidative pathologies (14-16).
There is strong evidence for a conformational change between XDH and XO
in the FAD binding region. XDH strongly destabilizes anionic forms of
the flavin relative to XO (17, 18). XDH contains an NAD-binding site
adjacent to the FAD, as addition of the NAD analog aminopyridine
adenine dinucleotide causes marked perturbations of the visible
spectrum of XDH (19, 20), and NAD causes only very small perturbations
to XDH, 
= 600 M
1 cm
1. In
contrast, no spectral changes occur on the addition of either compound
to oxidized XO. The redox potential of the FAD/FADH2 couple
in XDH,
340 mV (20), is favorable for the reduction of NAD,
335 mV
at pH 7.5 (21). However, the FAD/FADH2 couple in XO,
255
mV (22), is too high for efficient reduction of NAD.
This paper investigates the role of the low redox potential of the
FAD/FADH2 couple and the salient NAD-binding site of XDH in
conferring reactivity toward NAD as an oxidizing substrate. The
midpoint potential of the FAD/FADH2 couple was engineered by replacement of the normal FAD with FAD analogs containing different redox potentials. By reversing the redox potentials of the two enzyme
forms, it might be possible to reverse the use of NAD as an oxidizing
substrate. The low potential 1-deaza-FAD,
280 mV for the unbound
flavin, (23) was used to prepare 1-deaza-XO, an oxidase-type enzyme
with a low FAD/FADH2 midpoint potential. Previous work of
Hille and Massey (24) has shown that 1-deaza-XO has a low flavin
potential of
340 mV at the higher pH, 8.5. The recently synthesized
8-CN-FAD has a redox potential of
50 mV (25). Preparation of 8-CN-XDH
resulted in a dehydrogenase-type enzyme with an FAD/FADH2
potential predicted to be too high for efficient NAD reduction. The
flavin potentials of these substituted enzymes were determined relative
to the 2Fe/2S centers by reductive titrations or by reduction in the
presence of suitable redox dyes. Steady-state kinetics of the
xanthine/NAD and xanthine/oxygen activities were measured to assess any
changes in the preferred oxidizing substrate.
As NAD binding experiments have focused on spectral changes of the
enzyme on addition of NAD, NAD binding was also assessed by measuring
the concentration of unbound NAD in mixtures of NAD and oxidized XO or
XDH and by isothermal calorimetry. Note that NAD binding to reduced XDH
and XO is more catalytically relevant, but it is not possible to
measure in the case of XO. Differences in substrate binding and
reactivity are discussed.
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MATERIALS AND METHODS |
XDH was purified by the method of Hunt and Massey (18). XO was
purified in the same manner but with the omission of dithiothreitol. The preparation of 1-deaza-FAD has been described previously by Spencer
et al. (23). The synthesis and characterization of 8-CN-FAD was performed by Murthy and Massey and has recently been described (25). Immediately before use, XDH samples were incubated for 1 h
at 25 °C with 2.5 mM dithiothreitol. Dithiothreitol was
removed by desalting on a Sephadex G-25 column. All experiments were
performed at 25 °C in 50 mM potassium phosphate, pH 7.5, 0.3 mM EDTA.
Preparation of Artificial FAD-substituted Enzymes--
Samples
of deflavo XO were prepared by the method of Komai et al.
(6). Deflavo XDH was prepared in the same manner but with the addition
of 2.5 mM dithiothreitol at all stages. The visible spectra
and activity of deflavo enzymes indicated greater than 95% removal of
FAD. Reconstitution of holoproteins was performed by incubating deflavo
enzyme (approximately 20 µM) with a 3-fold excess of the
artificial FAD for at least 30 min on ice. Excess flavin was removed by
repeated concentration and dilution in a Centricon-100 spin
concentrator. Reconstitution of deflavo XO and XDH with normal FAD
ensured that native activity could be recovered.
Anaerobic Titrations--
Samples of 1-deaza-FAD substituted
enzyme were purged of oxygen in anaerobic cuvettes, as described
previously (9). Photo reductions (26) were performed by careful
irradiation of anaerobic enzyme in a sample containing 20 mM potassium oxalate and 5-deazaflavin equal to 10% of the
enzyme concentration. Dithionite titrations were performed by minute
additions of an anaerobic sodium dithionite solution (0.5 mg/ml) from a
titrating syringe into an anaerobic solution of enzyme.
Titrations are presented as proportionality plots in which the percent
absorbance lost at a principally 2Fe/2S wavelength is plotted as a
function of the percent absorbance lost at a mostly FAD wavelength.
Redox potentials of the enzyme-bound artificial flavins were determined
relative to the 2Fe/2S centers by fitting proportionality plots with
simulated curves calculated from the Nernst equation, the known redox
potentials of 2Fe/2S I and 2Fe/2S II, and the extinction changes of the
iron-sulfur and FAD centers. The fraction of each center reduced was
calculated at a given system potential, E. Percent spectral
changes were calculated by dividing the sum of absorbance changes for
each center by the total absorbance change reached at full
reduction,
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(Eq. 1)
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where the extinction changes (
) are at the FAD wavelength.
The fraction reduced at the primarily 2Fe/2S wavelength was calculated in the same way but with the corresponding extinction changes. Concentrations of 2Fe/2S Ired, 2Fe/2S IIred,
FADH°, and FADH2 were calculated as follows from the
system potential, E, and the known potentials,
E° (19, 21), of the various centers:
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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The system potential was varied at fixed values of
E°FAD/FADH° and
E°FADH°/FADH2. This method
relies on the assumption that the potentials of the 2Fe/2S centers are
unchanged by flavin substitution. The method is accurate insofar as the
potential of the FAD is not too far removed from the 2Fe/2S potentials
(E°2Fe/2S I =
310 mV, and
E°2Fe/2S II =
235 mV (22)).
Redox Determinations by Dye Equilibration--
Anaerobic
8-CN-FAD-substituted XO was photo-reduced stepwise as above in the
presence of the indicator dye indigotrisulfonate (20 µM,
Em at pH 7.5 =
96 mV). The extent of
reduction of the dye was estimated from its absorbance change at 600 nm, where XO shows negligible absorbance changes. The extent of
reduction of the enzyme was estimated at 492 nm, an isosbestic point
for the reduction of indigotrisulfonate. To calculate the difference between the redox potential of indigodisulfonate and that of the enzyme-bound 8-CN-FAD, the log(ox/red) of the indicator was plotted versus the log(ox/red) of the enzyme-bound 8-CN-FAD
according to the method of Minneart (27), a method previously utilized for the measurement of the redox potentials of native XDH (20).
1-ml samples of 8-CN-FAD-substituted XDH containing an appropriate
indicator dye (indigodisulfonate, indigotrisulfonate, or 1-HO-phenazine) plus 3 units of glucose-6-phosphate dehydrogenase plus
2 µM benzyl viologen were purged of oxygen in an
anaerobic cuvette with 2 side arms by gentle agitation under a constant stream of oxygen-free argon for 20 min. The visible spectrum was then
recorded. To begin reduction of the XDH by an NADPH generating system,
0.1 µM NADP and 5 mM glucose 6-phosphate were
tipped in from the side arms. The solution was thoroughly mixed in the
main cuvette, and the reaction was followed by recording visible
spectra at 25 °C with time as the NADPH generating system slowly
reduced the XDH. For indigodisulfonate, the extent of reduction of the dye was estimated by the decrease in absorbance at 612 nm, where the
enzyme gives rise to less than 5% of the total absorbance change under
the conditions used, and the extent of reduction of the flavin was
estimated at 463 nm, which is isosbestic for the dye reduction. For
indigotrisulfonate, the extent of reduction of the dye was estimated by
the extent of decrease in absorbance at 600 nm, where the enzyme gives
rise to less than 5% of the total absorbance change, and the extent of
reduction of the flavin was estimated from the change in absorbance at
460 nm after the absorbance changes because of the dye were subtracted.
For 1-hydroxyphenazine, the extent of reduction of the dye was
estimated by the decrease in absorbance at 372 nm. At this wavelength,
absorbance changes associated with formation and reduction of the
flavin semiquinone of 8-CN-XDH are negligible. Absorbance changes at
this wavelength associated with the 2Fe/2S centers of the enzyme were
subtracted. The reduction of FADH° was monitored at 620 nm, where
1-hydroxyphenazine does not absorb in either its oxidized or reduced forms.
Steady-state Kinetics--
Steady-state kinetics were measured
with a Hi-Tech SF-61 and with a Kinetics Instruments stopped-flow
spectrophotometer, wherein excellent control of oxygen concentration
can be maintained. The method of initial rates was used.
Xanthine/oxygen turnover was measured by the increase in urate
concentration at 295 nm (
= 9,600 M
1
cm
1). Xanthine/NAD turnover was measured anaerobically as
the increase in NADH concentration at 340 nm (
= 6,200 M
1 cm
1). Concentrations of
xanthine (2-80 µM) and NAD (2-80 µM) or
molecular oxygen (31-1220 µM) were varied independently.
The enzyme concentration was between 0.1 and 0.5 µM. The
enzyme concentration was corrected for inactive enzyme; the fraction of
active enzyme was measured as the fraction of absorbance lost within a
few s on anaerobic addition of 200 µM xanthine relative
to that lost on addition of excess sodium dithionite. Assays with NAD
analogs were performed at fixed concentrations: 100 µM
xanthine and 500 µM pyridine dinucleotide. The
wavelengths monitored and the extinction changes are as follows: thionicotinamide adenine dinucleotide, 
395 = 11,300 M
1 cm
1; acetylpyridine
adenine dinucleotide, 
361 = 9,100 M
1 cm
1; pyridine aldehyde
adenine dinucleotide, 
356 = 9,300 M
1 cm
1 (28).
NAD Binding--
Native XDH or XO (80 µM) was
mixed with varying concentrations of NAD at 25 °C. The sample was
put in a Centricon-30 spin concentrator, and was centrifuged at 4,000 rpm for 30 min. The concentration of free ligand was taken as the
concentration of NAD that passes through the filter. Samples run
without enzyme indicated that no NAD was retained by the Centricon
filter. NAD was measured by its UV spectrum (
260 = 17,800 M
1 cm
1). NAD biding was
also measured by isothermal titration calorimetry, as described by
Wiseman et. al. (29) using enzyme concentrations in the
range 100-220 µM. These measurements were kindly made by Dr. Bruce A. Palfey, employing a Calorimetry Sciences Corp. (Provo, Utah) 4200 instrument.
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RESULTS |
Preparation and Properties of Artificial FAD-substituted
Enzymes--
Deflavo XO and XDH were prepared by the method of Komai
et al. (6). The visible spectrum as well as activity assays
indicated that greater than 95% of the FAD had been removed (not
shown). Mixing deflavo XO with 8-CN-FAD and 1-deaza-FAD yielded enzymes with near-complete flavin reconstitution. The addition of 8-CN-FAD and
1-deaza-FAD to deflavo XDH also resulted in near-complete binding, as
shown below.
The spectrum of 8-CN-FAD is not significantly changed on binding to
deflavo XO or XDH. The extinction at 450 nm of 8-CN-FAD is 9,990 M
1 cm
1 (25). The spectrum of
8-CN-XO minus that of deflavo XO has an extinction difference of 8,800 M
1 cm
1, indicating that only
88% of the 8-CN-XO contains bound flavin. The extinction difference at
450 nm because of 8-CN-FAD binding to deflavo XDH is 9,500 M
1 cm
1, indicating 95% bound
flavin in the 8-CN-XDH sample. After reconstitution with 8-CN-FAD, the
excess flavin was removed from the enzyme samples in a Centricon-100
concentrator. The spectrum of the initial material (enzyme + excess
flavin) minus that of the free 8-CN-FAD that had passed through the
filter confirmed the extinctions reported here. Spectra of 8-CN-XO and
8-CN-XDH are shown in Figs. 1,
A and B, respectively.

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Fig. 1.
Reduction of 8-CN-XO and 8-CN-XDH.
Panel A, 7.7 µM 8-CN-XO; panel B,
14 µM 8-CN-XDH. Samples were made anaerobic and reduced
by minute additions of 0.5 mg/ml sodium dithionite. Spectra are shown
of the following enzyme species: dotted spectrum, oxidized
enzyme; solid spectrum, maximal semiquinone; dashed
spectrum, reduced enzyme. Inset to panel B,
change in absorbance at 620 nm versus the change in
absorbance at 460 nm for the reduction using an NADPH-generating system
of a 7.1 µM sample of 8-CN-XDH.
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The spectra of 1-deaza-XO and 1-deaza-XDH are identical to that
reported previously for 1-deaza-XO (24). The difference spectra of
enzyme-bound minus deflavo enzyme indicated the extinction at 550 nm of
bound 1-deaza-FAD to be 9,150 M
1
cm
1 for 1-deaza-XO and 9,310 M
1
cm
1 for 1-deaza-XDH. These values are quite close to that
of 9,700 M
1 cm
1 reported for
1-deaza-XO (24) and to that of 8,700 M
1
cm
1 for the free 1-deaza-FAD (30). These spectral data
indicate near-complete binding of 1-deaza-FAD to deflavo XO and XDH.
Again, the spectrum of enzyme with excess 1-deaza-FAD minus that of the unbound 1-deaza-FAD support these extinctions. Spectra of 1-deaza-XO and 1-deaza-XDH are shown in Figs. 5, A and B, respectively.
Reductive Titrations and Determination of Flavin Redox
Potentials--
The redox potentials of the bound artificial flavins
were determined to ensure that the FAD potential had been shifted in the desired direction and amount. Enzyme samples were reduced in very
small increments by photo-reduction, by sodium dithionite, or by an
NADPH-generating system. Sufficient time (30 to 60 s) was given
between irradiations or additions to ensure equilibration. For the
1-deaza-FAD-substituted enzymes, the 2Fe/2S centers were used as redox
indicators and are expected to be reduced before the flavin. For the
higher potential 8-CN-FAD-substituted enzymes, dye equilibration
studies were carried out, as the 2Fe/2S centers were expected to be of
too low a potential to be of use as redox indicators.
Spectra from the reduction of 7.7 µM 8-CN-XO by sodium
dithionite are shown in Fig. 1A. In addition to spectra of
the oxidized and reduced enzyme, the spectrum containing the maximally
obtained FADH° is given. The peak at 414 nm indicates this to be the
anionic form of 8-CN-FAD semiquinone (25). Only about 12% of the
semiquinone is stabilized, because this species has an extinction
increase relative to oxidized 8-CN-FAD of 26,600 M
1 cm
1 (25). It is evident from
the spectra in Fig. 1A that at the stage of maximal
semiquinone formation there is no reduction of 2Fe/2S centers, because
there is no change in the spectrum at wavelengths greater than 520 nm.
The FAD/FADH° potential is therefore much higher than that of the
2Fe/2S centers. Identical results are obtained by photo-reduction.
Extinction changes for this reduction are given in Table
I.
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Table I
Extinction changes of artificial FAD-substituted enzymes
Extinction coefficients are given in units of
M 1 cm 1 along with the wavelength
in nm at which they were measured. Negative values indicate extinction
increases relative to oxidized enzyme. Extinction changes for 2Fe/2S
centers are based on those of deflavo XO (6). Extinction changes for
oxidized and reduced flavins are from Figs. 1 and 5. Extinction changes
for flavin semiquinones are estimated from those of 8-CN-OYE (anionic)
and 8-CN-flavodoxin (neutral) from the work of Murthy and Massey (25)
and from that of 1-deaza-flavodoxin (neutral) (30).
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Spectra from the photoreduction of 9.6 µM 8-CN-XO and 20 µM indigotrisulfonate are shown in Fig.
2. The amounts of oxidized and reduced
indigotrisulfonate and the amounts of oxidized and reduced enzyme-bound
8-CN-FAD were estimated as indicated under "Materials and Methods."
The log(ox/red) for the indigotrisulfonate was plotted
versus the log(ox/red) for the flavin (Fig. 2,
inset). An initial slope of 2 corresponds to the small
amount of observed semiquinone formation, which is followed by a slope
of 0.9 as compared with a theoretical slope of 1 for the 2-electron
reduction of the flavin. The plot gives a potential of
90 mV for the
2-electron reduction of the enzyme-bound 8-CN-FAD. This figure
represents a decrease in the potential of 8-CN-FAD of 40 mV on binding
to XO, a figure in reasonable agreement with decreases of 47 mV and 50 mV on the binding of native FAD and 1-deaza-FAD, respectively, to XO
(Table II).

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Fig. 2.
Photoreduction of 8-CN-XO in the presence of
indigotrisulfonate. A mixture of 9.6 µM 8-CN-XO and
20 µM indigotrisulfonate was made anaerobic and
photoreduced in the presence of 1 µM 5-deazaflavin and 50 mM oxalate as outlined under "Materials and Methods."
Spectra are shown of the following species: solid spectrum,
anaerobic 8-CN-XO plus indigotrisulfonate; dotted spectrum,
after 3.2 min of irradiation; dashed spectrum, fully reduced
8-CN-XO plus indigotrisulfonate obtained after 13.4 min of irradiation.
Inset, data obtained at 492 nm for enzyme reduction and at
600 nm for dye reduction as outlined under "Materials and
Methods."
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Spectra from the dithionite reduction of 14 µM 8-CN-XDH
are shown in Fig. 1B. The most notable difference with
respect to the reduction of 8-CN-XO is that the semiquinone formed is
the neutral semiquinone, as seen by the extinction increases from 550 to 650 nm (25). This is entirely consistent with the destabilization of
anionic flavins in XDH (17, 18). A plot of A460
versus A620 obtained from the
reduction of a 7.1 µM sample of 8-CN-XDH using a
NADPH-generating system (inset, Fig. 1B)
indicates that, in contrast with the low yield of semiquinone observed
during the reduction of 8-CN-XO, 70% of the semiquinone is stabilized. This compares to a stabilization of a large proportion (90%) of the
semiquinone obtained during the reduction of XDH-containing native FAD
(20). A 70% stabilization of the semiquinone implies that the
FAD/FADH° and the FADH°/FADH2 potentials are separated by 79 mV.
To determine the potential of the 8-CN-FAD/FADH° couple in XDH, the
enzyme was reduced anaerobically using an NADPH-generating system (see
"Materials and Methods") in the presence of 20 µM indigodisulfonate (Em at pH 7.5 =
140 mV) and
in a separate experiment in the presence of 20 µM
indigotrisulfonate (Em at pH 7.5 =
96 mV. The
amounts of oxidized and reduced dye and the amounts of oxidized and
reduced enzyme-bound flavin were estimated as indicated under
"Materials and Methods." The data obtained from the reduction of
8-CN-XDH with indigodisulfonate are shown in Fig.
3. Plots of the log(ox/red) of
indigodisulfonate (see inset, Fig. 3) and of the log(ox/red)
of indigotrisulfonate versus the log(ox/red) for the
formation of FADH° give values of
120 mV and
116 mV,
respectively, for the FAD/FADH° potential. These plots should give a
2-unit slope as both indigodisulfonate and indigotrisulfonate are
2-electron acceptors. However, slopes of 1.0 and 0.7 were obtained.

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Fig. 3.
Reduction of 8-CN-XDH in the presence of
indigodisulfonate using a NADPH-generating system. A mixture of
4.4 µM 8-CN-XDH and 20 µM indigodisulfonate
was made anaerobic and then was reduced using an NADPH-generating
system as outlined under "Materials and Methods." Spectra are shown
of the following species: solid spectrum, anaerobic 8-CN-XDH
plus indigodisulfonate; dotted spectrum, after 27.2 min of
reduction; dashed spectrum, after 6 h of reduction.
Inset, data obtained at 462 nm for enzyme reduction and at
612 nm for dye reduction as outlined under "Materials and
Methods."
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To determine the potential of the 8-CN-FADH°/FADH2 couple
in XDH, the enzyme was reduced anaerobically using an NADPH-generating system, as above, in the presence of 25 µM
1-hydroxyphenazine (Em at pH 7.5 =
187 mV).
The amounts of oxidized and reduced dye and the amounts of oxidized and
reduced enzyme-bound flavin were estimated as indicated under
"Materials and Methods." A plot of the log(ox/red) of
1-hydroxyphenazine versus the log(ox/red) of the
FADH°/FADH2 couple (inset, Fig.
4) gives a value of
198 mV for the
FADH°/FADH2 potential. This value is in very close agreement to a theoretical value of
197 mV, assuming an FAD/FADH° potential of
118 mV and 70% stabilization of the semiquinone form.
The plot in Fig. 4 is expected to yield a 2-unit slope as 1-HO-phenazine is a 2-electron acceptor. A slope of 1.2 was obtained. Although the slopes of the plots shown are not easily explained, the
measured potentials are all in excellent agreement. A lowering of the
potential of 8-CN-FAD from
56 to
158 mV on binding to XDH is also
in reasonable agreement with results obtained with other flavins (see
Table III).

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Fig. 4.
Reduction of 8-CN-XDH in the presence of
1-hydroxyphenazine using an NADPH-generating system. A mixture of
5.3 µM 8-CN-XDH and 25 µM
1-hydroxyphenazine was made anaerobic and reduced using an
NADPH-generating system as outlined under "Materials and Methods."
Spectra are shown of the following species: solid spectrum,
anaerobic 8-CN-XDH plus 1-hydroxyphenazine; dotted spectrum,
after 13.5 min of reduction; dashed spectrum, after 2 h
and 40 min of reduction. Inset, data obtained at 620 nm for
enzyme reduction and at 372 nm for dye reduction as outlined under
"Materials and Methods."
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Table III
Activity of NAD analogs with native xanthine oxidase and xanthine
dehydrogenase
Initial-rate assays were performed as described under "Materials and
Methods." Assays were measured at 100 µM xanthine
and 260 µM oxygen for XO and at 100 µM xanthine, 500 µM NAD, and no
oxygen for XDH. Enzyme concentration was 50-100 nM.
Redox potentials were corrected for pH 7.5 by adding 15 mV ( 30
mV/pH unit) to values reported at pH 7.0 (33). Activities are expressed
as percent xanthine/oxygen rate for XO and as percent xanthine/NAD rate
for XDH.
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Spectra from a dithionite reduction of 7.4 µM 1-deaza-XO
are shown in Fig. 5A. There is
only about 10% of the 1-deazaflavin semiquinone species stabilized,
which is barely visible as an absorbance increase above 650 nm, where
the neutral semiquinone of 1-deaza-FAD has maximal absorbance (31). A
large amount of 2Fe/2S reduction occurred by the point of maximal
semiquinone, indicating a low potential for this flavin. This
observation is pictured more clearly in the proportionality plot (Fig.
6) whose upward curvature indicates the
flavin to have a lower potential than the 2Fe/2S centers. This is
consistent with the results of Hille and Massey at pH 8.5 (24). Fitting
these data to the extinctions given in Table I gave optimal
correspondence at E°FAD/FADH° =
360 mV and
E°FADH°/FADH2 =
290 mV
(Fig. 6). The midpoint potential determined here at pH 7.5 of
325 mV
is close to that of
340 mV determined at pH 8.5 (24); the more
positive potential of
295 mV would be expected for a pH decrease of
1.0 unit. The potential of
325 mV is similar to that of native XDH,
340 mV (20), indicating that substitution with 1-deaza-FAD does
indeed produce a low flavin potential form of XO.

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Fig. 5.
Reduction of 1-deaza-XO and 1-deaza-XDH.
Panel A, 7.4 µM 1-deaza-XO; panel
B, 9.8 µM 1-deaza-XDH. Samples were made anaerobic
and reduced by minute additions of 0.5 mg/ml sodium dithionite. Spectra
are shown of the following enzyme species: solid spectrum,
oxidized enzyme; dotted spectrum, maximal semiquinone;
dashed spectrum, reduced enzyme.
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Fig. 6.
Proportionality plot of the reduction of
1-deaza-XO and 1-deaza-XDH. Data are plotted as the percent
absorbance lost at 470 nm on reduction with sodium dithionite as a
function of the percent absorbance lost at 550 nm. Data points for
1-deaza-XO (circles) and 1-deaza-XDH (squares)
are shown along with simulations to the data for 1-deaza-XO
(solid curve) and 1-deaza-XDH (dashed curve).
Simulations were calculated as described under "Materials and
Methods" using the extinction changes in Table I and the following
redox potentials: 1-deaza-XO, E°FAD/FADH° = 360 mV, E°FADH°/FADH2 = 290 mV;
1-deaza-XDH, E°FAD/FADH° = 345 mV,
E°FADH°/FADH2 = 375
mV.
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The reduction of 9.8 µM 1-deaza-XDH is shown in Fig.
5B. Except for stabilizing significantly more flavin
semiquinone during reduction, 1-deaza-XDH behaves very similarly to
1-deaza-XO. The proportionality plots of the two enzymes are very
similar to each other (Fig. 6). The redox potentials determined from
fitting these data are E°FAD/FADH° =
345
mV and E°FADH°/FADH2 =
375 mV. The midpoint potential of
360 mV is 20 mV more negative
than that of native XDH.
Steady-state Kinetics of Artificial FAD-substituted XO and
XDH--
The ability of the artificial FAD-substituted enzymes to
support xanthine/oxygen and xanthine/NAD turnover was measured to assess the role of the flavin midpoint potential in controlling NAD
versus oxygen reactivity. Concentrations of both reducing and oxidizing substrates were varied independently. In all cases, Lineweaver-Burk plots displayed sets of parallel lines, consistent with
a ping-pong mechanism (not shown). Kinetic constants from steady-state
measurements are presented in Table II.
The kcat of 8-CN-XO-catalyzed xanthine/oxygen
turnover, 12 s
1, is quite close to that of native enzyme,
13 s
1 (32). This indicates that the artificial flavin is
catalytically active, and it extends the range of data used to study
the effect of the flavin midpoint potential on catalysis (24). The high error associated with the oxygen Km for this
turnover as well as the xanthine/oxygen turnover of 8-CN-XDH is because of the Km value being only slightly lower than the
solubility of oxygen at 25 °C, 1.2 mM. 8-CN-XO, like all
of the XO forms measured, catalyzes a barely detectable level of
xanthine/NAD turnover. This may possibly be because of a small amount,
0.1-2.5%, of contaminating dehydrogenase-type enzyme, because the NAD
Km values of the XO-type enzymes are near those of
XDH. The 8-CN-XDH is competent in xanthine/oxygen turnover,
kcat of 5.1 s
1. However, no amount
of xanthine/NAD turnover was detected. The neutral semiquinone species
seen on reduction of 8-CN-XDH (Fig. 1B) is evidence that the
sample is still a dehydrogenase-type enzyme. Also, 74% of the visible
spectrum (relative to dithionite reduction) was bleached on mixing
anaerobic 8-CN-XDH with 400 µM NADH (not shown). By
comparison, the similar NADH reaction with native XO results in no
detectable reduction; 2% of absorbance was lost at 15 min. These data
demonstrate that 8-CN-XDH is truly a dehydrogenase-type enzyme, despite
the lack of xanthine/NAD reductase activity. The nondetectable
xanthine/NAD activity of 8-CN-XDH establishes that a low flavin
midpoint potential is required for xanthine/NAD catalysis.
Xanthine/oxygen turnover of 1-deaza-XO has a
kcat of 2.0 s
1, quite similar to
that measured at pH 8.5, 1.7 s
1 (24). The sample is
clearly a competent enzyme form. The very small xanthine/NAD activity
of 1-deaza-XO, kcat of 0.049 s
1,
indicates that the low flavin midpoint potential of this enzyme,
325
mV, is not sufficient to bestow dehydrogenase activity. Xanthine/NAD activity of 1-deaza-XDH is 40% that of native XDH, demonstrating that
substitution with this artificial flavin has not markedly disrupted the
enzyme. The lower xanthine/NAD activity of 1-deaza-XDH is not a
surprising result. Nishino et al. (7) found that
substitution of chicken liver XDH with the low potential flavins
6-OH-FAD, 8-SH-FAD, and 8-OH-FAD resulted in enzyme containing 30-61%
xanthine/NAD activity of the native enzyme at a single set of reaction
conditions (7). The low potential of 1-deaza-XDH may result in a
greatly decreased fraction of fully reduced flavin during turnover.
Pyridine Nucleotide Analogs--
To test separately the role of
substrate binding and the need for the flavin midpoint potential to be
near or below that of its electron acceptor, the ability of native XO
and XDH to use a series of pyridine nucleotide analogs of different
potentials was measured (Table III). Xanthine/oxygen activity was
measured at 260 µM oxygen, thus explaining why for XO,
xanthine/NAD activity is a higher percent of xanthine/oxygen activity
than that in Table II. Redox potentials for pyridine nucleotides were
corrected for pH 7.5 by adding
15 mV (
30 mV/pH unit) to the values
reported at pH 7 (33). Raising the potential of the electron acceptor as high as
245 mV, more positive than the
255-mV flavin potential of XO (22), has no effect on the xanthine/pyridine nucleotide activity
of XO. This supports the conclusion from the 1-deaza-XO experiments
that favorable thermodynamics are not sufficient for dehydrogenase
activity. Raising the potential of the pyridine nucleotide acceptor had
only modest effects on the xanthine/pyridine nucleotide activity of
XDH. This is not surprising, as reduction by xanthine is known to be
the slow step in catalysis (34).
NAD Binding to XDH and XO--
The flavin spectrum of oxidized XDH
is known to be perturbed on the addition of the NAD analog
aminopyridine adenine dinucleotide (Refs. 18 and 19 and Fig.
7B), indicating the pyridine
nucleotide binds in close proximity to the FAD. Only minute changes
(
450 = 600 M
1
cm
1) are observed on the addition of 2.5 mM
NAD to 20 µM XDH, although NAD has a much larger
perturbation on the spectrum of chicken liver XDH (19). No spectral
changes were observed on the addition of either compound to oxidized XO
(Fig. 7A), indicating that NAD does not bind to XO adjacent
to the FAD. NAD binding by nonspectral methods was performed to
distinguish whether XO has a disrupted NAD-binding site or if there is
a binding site and it is not in the vicinity of the FAD. The best
comparison would be that of NAD binding to the reduced enzymes. A
Kd of NAD binding to fully reduced XDH of 25 µM has been measured kinetically (35). The addition of
NAD to photo-reduced XO results in small absorbance increases
(
340 = 1,940 M
1
cm
1, 
450 = 2,040 M
1 cm
1) throughout the visible
spectrum, consistent with oxidation of a small fraction of XO and not
consistent with any FADH2:NAD complex (not shown). As
mixing reduced XO and NAD provides no information about binding,
binding of NAD to the oxidized enzymes was measured. In initial
experiments, 80 µM solutions of oxidized XDH and XO were
each mixed with varying concentrations of NAD from equimolar to 1 mM. Measurements of the unbound NAD concentration was
obtained from the spectrum of the flow-through when the sample was
centrifuged in a Centricon-30 concentrator. Using this method, a
Kd of 280 ± 145 µM was obtained
for the binding of NAD to XDH, but no evidence could be obtained
indicating binding of NAD to XO. NAD binding to XDH was also measured
by a completely different analytical method, that of isothermal
titration calorimetry (29), yielding a Kd of
160 ± 40 µM (results not shown). Again, in a
separate experiment, no indication of NAD binding to the oxidase form
could be obtained.

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Fig. 7.
Spectral perturbations on addition of
pyridine dinucleotides to XDH and XO. Panel A: solid
spectrum, 20 µM XO; dotted spectrum, 20 µM XO + 2.5 mM NAD; dashed
spectrum, 20 µM XO + 1 mM aminopyridine
adenine dinucleotide. Panel B: solid spectrum, 20 µM XDH; dotted spectrum, 20 µM
XDH + 2.5 mM NAD; dashed spectrum, 20 µM XDH + 1 mM aminopyridine adenine
dinucleotide.
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DISCUSSION |
The goal of the present work was to assess the role of the low
flavin midpoint potential and of NAD binding in determining specificity
toward oxidizing substrates for XO and XDH. The flavin midpoint
potential was engineered by preparing enzyme samples substituted with
artificial flavins. FAD redox potentials of these enzyme forms
indicated that the desired changes in potential had indeed occurred.
Preparation of 8-CN-XDH resulted in an enzyme with a flavin midpoint
potential of
158 mV, predicted to be too high to efficiently reduce
NAD,
335 mV at pH 7.5 (21). This enzyme was completely lacking in
xanthine/NAD activity even though several experiments indicated it was
still dehydrogenase-type. A low flavin potential is certainly necessary
for NAD reactivity. Preparation of 1-deaza-XO yielded an oxidase-type
enzyme with a flavin midpoint potential close to that of native XDH.
Although as functional as an oxidase, 1-deaza-XO has no more
dehydrogenase activity than does native XO. This residual activity
could be because of a small amount, 0.1-2.5%, of contaminating
dehydrogenase-type enzyme. Measuring the activity of native XO and XDH
in assays using NAD analogs of higher redox potentials also
demonstrated that only dehydrogenase-type enzyme could utilize pyridine
nucleotides as an oxidizing substrate. These data indicate that
favorable thermodynamics are necessary, but not sufficient, for
dehydrogenase activity. Clearly NAD binding is an essential factor.
The visible spectrum of XO is not perturbed upon the addition of NAD or
of aminopyridine adenine dinucleotide, unlike that of XDH (19, 18).
Also, NAD does not inhibit xanthine/oxygen turnover of XO. At 100 µM xanthine, 260 µM oxygen, and 500 µM NAD, xanthine/oxygen turnover proceeds at 99.6% the
rate obtained in the absence of NAD (not shown). Under these
conditions, XDH-catalyzed xanthine/oxygen turnover would be completely
inhibited in favor of xanthine/NAD turnover (9). The existence of an
NAD binding site in XDH, but not in XO, was found by measuring the
concentration of unbound NAD in mixtures of NAD and XO or XDH and
independently by isothermal titration calorimetry. Values of
Kd for NAD binding to oxidized XDH 280 ± 145 µM and 160 ± 40 µM, respectively, were measured using these methods. Thus, binding of NAD to XDH is
relatively weak, with a Kd ~ 220 µM.
Nishino and Nishino (19) measured a Kd of
310 µM for NAD binding to oxidized chicken liver XDH
(19). NAD binding to the two-electron reduced state of XDH has
previously been modeled to be approximately 200 µM,
supporting the concept of weak NAD binding to more oxidized enzyme
forms (35). These binding data, along with the lack of spectral
perturbation on pyridine nucleotide addition, the lack of xanthine/NAD
activity of 1-deaza-XO, and the insensitivity of xanthine/oxygen
activity to the presence of NAD indicate that the conformational
changes that convert XDH to XO also disrupt the NAD-binding site in XO.
The binding experiments reported here are with the oxidized enzymes.
NAD binding to reduced XO and XDH is more catalytically relevant
although not possible to measure for XO. Note that oxygen binding is
not an issue in determining substrate specificity; XDH has been shown
to react with oxygen by second-order reactions (9), and there is no
good evidence for oxygen binding to XO or any other flavoprotein
oxidase (36).
NAD binding to oxidized XDH, Kd ~ 220 µM, is approximately 10-fold weaker than to fully reduced
enzyme, Kd of 25 µM (34). Using these
Kd values and
340 mV as the flavin midpoint
potential of unbound XDH (20), a flavin midpoint potential of
370 mV
can be calculated for NAD-bound XDH based on a thermodynamic box
(Scheme 1). This 30-mV lowering in flavin
potential may facilitate reduction of NAD. This might be at the expense
of decreasing electron density at the flavin and increasing it at the
iron and molybdenum centers. But, in oxidation with NAD (35) and in
reduction with NADH (34), intramolecular electron transfer into and out
of the flavin appear to only happen in the absence of bound NAD.
Intramolecular electron transfer to make FADH2 is only
slightly disfavored, Keq = 0.12, before NAD
binding. Results from the oxidative half-reaction with NAD (35)
indicate that formation of FADH2 precedes NAD binding. Thus
the energy of substrate binding is used to lower the midpoint potential
of the FADH2, shifting the difference between FAD and NAD
potentials from 5 to 35 mV. The Kd of 310 µM for NAD binding to oxidized chicken liver XDH (19) is
also much higher than that measured kinetically for the fully reduced
enzyme, 80 µM (37).