(Received for publication, June 2, 1995; and in revised form, June 16, 1995)
From the
The UV-visible absorbance change associated with reduction of
the molybdenum centers of xanthine oxidase and xanthine dehydrogenase
has been determined using a double-difference technique. At pH 8.5, the
Mo(VI) minus Mo(IV) difference spectrum seen with xanthine
oxidase exhibits a positive feature at 420 nm, having an extinction
change of 3,000 M
cm
as well as evidence for a negative feature below 340 nm. In
xanthine oxidase this change is found to exhibit a marked pH
dependence, implicating protonation/deprotonation events associated
with changes in the molybdenum oxidation state. Application of the
double-difference protocol to the respective circular dichroism spectra
of xanthine oxidase and xanthine dehydrogenase reveals appreciable CD
changes at 420 and 580 nm associated with the reduction of the
molybdenum center. The present results demonstrate a direct
spectroscopic handle on the molybdenum centers of both xanthine oxidase
and xanthine dehydrogenase.
Milk xanthine oxidase and chicken liver xanthine dehydrogenase
belong to the molybdenum hydroxylase class of enzymes, with each
possessing a MoOS catalytic unit in its active site. Both enzymes are
homodimers of molecular weight 300,000 with two iron-sulfur centers (of
the spinach ferredoxin variety) and one molecule of flavin adenine
dinucleotide in addition to the molybdenum center in each subunit (1, 2, 3, 4) . The oxidative
hydroxylation of a variety of aromatic heterocycles and simple
aldehydes takes place at the molybdenum centers of these enzymes, with
reducing equivalents thus introduced passed to the flavin site where
they are removed by either molecular oxygen (for the oxidase) or
NAD (for the dehydrogenase). In the case of xanthine
oxidase, it has been shown that the hydroxylation reaction passes
through an intermediate in which the nascent uric acid product is
coordinated to the (reduced) molybdenum via the newly introduced
hydroxyl group as Mo(IV)-OR. Oxidation of this species by 1 eq yields a
Mo(V) species that gives rise to the well characterized ``very
rapid'' molybdenum EPR signal(3, 5, 6) .
Xanthine dehydrogenase is thought to operate via a similar reaction
mechanism.
The kinetics of the reductive half-reaction of these
enzymes have been studied extensively by both UV-visible absorption and
EPR spectroscopy (7, 8, 9, 10) .
Studies monitoring absorbance changes associated with enzyme reduction
necessarily focus on the iron-sulfur and flavin centers, as these are
responsible for the preponderance of the spectral changes seen upon
reduction for both enzymes. Any spectral change attributable to the
molybdenum site has remained ill defined because of the much larger
absorption changes associated with these other chromophores. Strong
evidence exists, however, for transient absorption changes attributable
to the molybdenum center of xanthine oxidase in the course of the
reaction of enzyme with lumazine (2,6-dihydroxypteridine; Refs. 11 and
12), 2-hydroxy-6-methylpurine(7) , and xanthine(13) .
With lumazine, two long wavelength-absorbing species have been
identified as intermediates in the reductive half-reaction, the first
ascribed to ES (i.e. Mo(VI)
lumazine) and the second to E
P (Mo(IV)
violapterin). The latter
intermediate, having an absorbance maximum at 650 nm with an extinction
change of approximately 8000 M
cm
, is readily formed by anaerobic addition of
violapterin to dithionite-reduced enzyme(11) , while the
existence of the former has been inferred from the kinetic behavior of
the enzyme(12) . The same long wavelength-absorbing species has
been observed with xanthine dehydrogenase. (
)The reaction of
xanthine oxidase with 2-hydroxy-6-methylpurine has also been found to
possess two successive reaction intermediates detectable by UV-visible
spectrophotometry, exhibiting absorption differences (relative to
oxidized enzyme) at 470 and 540 nm, respectively, with extinction
changes of approximately 410 M
cm
. These spectral intermediates have been
attributed to the molybdenum center of the enzyme in the Mo(IV) and
Mo(V) valence states, respectively, the latter corresponding to the
species exhibiting the ``very rapid'' Mo(V) EPR signal known
to be formed in the course of the reaction(14) . A comparable
species has also been seen in the reaction of xanthine oxidase with
xanthine(13) , with an associated spectral change above 350 nm
very similar to that observed upon addition of product uric acid to
reduced enzyme(15) . It has recently been shown that analogous
intermediates are also observed in the reaction of xanthine
dehydrogenase with either xanthine or xanthopterin(16) .
Despite the above evidence for spectral intermediates attributable to the molybdenum center of xanthine oxidase in the course of its reaction with substrate, there is no unambiguous evidence to date for a spectral change associated with reduction of the molybdenum center. Using a double-difference spectroscopic technique, we report the determination of this spectral change in both xanthine oxidase and xanthine dehydrogenase and its associated circular dichroism change.
Xanthine oxidase was purified from unpasteurized cow's
milk (obtained from the dairy herd of Ohio State University) according
to the procedure of Massey et al.(17) . Sephacryl
S-300 gel filtration chromatography and CM-52 ion-exchange column
chromatography steps at the end of the procedure ensured removal of
contaminating lactoperoxidase(18) . The purified enzyme
exhibited a ratio of absorbance at 276 to 450 nm of 5.4 and was
typically
70% functional. The
30% inactive enzyme found in
all conventional preparations of xanthine oxidase lacks a catalytically
essential sulfur atom at the molybdenum center (19) and for the
purposes of the present studies can be considered to be inert. Routine
enzyme assays and the determination of the specific activity of
xanthine oxidase were performed as described by Massey et
al.(17) . Xanthine dehydrogenase was purified by a method
involving homogenization of fresh chicken livers in liquid nitrogen,
followed by centrifugation, ammonium sulfate, and butanol fractionation
and sequential chromatography on hydroxylapatite, Sephacryl S-300, and
a folate affinity column. This procedure avoids use of acetone
extraction, thereby minimizing damage to the molybdenum center of this
enzyme.
UV-visible absorption spectra were obtained with a Hewlett-Packard 8452 diode-array spectrophotometer interfaced to a Hewlett-Packard Chemstation computer. Circular dichroism spectra were obtained using an AVIV-40DS UV-visible near infrared spectrophotopolarimeter. Alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,6-diol) was purchased from Sigma. All other reagents and buffers were of the highest quality commercially available and used without further purification.
Figure S1: Scheme 1.
Fig. 1shows the
several difference spectra obtained according to Fig. S1for
both xanthine oxidase and xanthine dehydrogenase at pH 8.5. PanelA shows the difference spectra of oxidized enzyme and
enzyme that has been reduced, treated with alloxanthine, and reoxidized
for 10 min for xanthine oxidase (solidline) and
xanthine dehydrogenase (dashedline). The data
obtained using xanthine oxidase are in good agreement with the
literature(20) . PanelB shows the difference
spectrum for reduced xanthine oxidase in the absence and presence of
alloxanthine (solidline) and the same for xanthine
dehydrogenase (dashedline). In the latter spectrum,
some reoxidation of the large amount of sodium dithionite required to
achieve full formation of the alloxanthine complex results in a large
negative artifact below 360 nm, but above this wavelength the
difference spectrum is minimally perturbed. ()PanelC of Fig. 1gives the double-difference spectra
obtained by subtraction of the appropriate spectra from PanelsA and B for xanthine oxidase (solidline) and xanthine dehydrogenase (dashedline). Again, the difference spectrum obtained with
xanthine dehydrogenase is accurate only above 360 nm. It is readily
evident in the double-difference spectrum obtained with each enzyme
that a spectral change having a difference maximum at
420 nm is
observed, and on the basis of the argument summarized above (Fig. S1) this spectral change must correspond to that observed
on reduction of the molybdenum center of each enzyme. The extinction
change at 420 nm is estimated to be
3,000 M
cm
in the case of xanthine oxidase;
corrections made for uncomplexed alloxanthine resulted in a negligible
shift in the molybdenum absorption profile above 380 nm and were
considered negligible in the overall treatment. It is noteworthy that
the extinction change associated with reduction of the molybdenum
center of xanthine oxidase is of a comparable magnitude with that
observed upon formation of the Mo(IV)
alloxanthine complex (20) and the Mo(IV)
violapterin complex(11) ,
although the wavelength maxima are very different for the latter two
spectral changes.
Figure 1: Determination of spectral change associated with reduction of the molybdenum center of xanthine oxidase and xanthine dehydrogenase. PanelA (difference spectrum 1), solid line, difference spectra obtained after the addition of excess alloxanthine to dithionite-reduced xanthine oxidase under anaerobic conditions; dashed line, the same with xanthine dehydrogenase. The experimental conditions for xanthine oxidase were 68 µM functional enzyme and 125 µM alloxanthine (after mixing) in 0.1 M pyrophosphate, 0.3 mM EDTA, pH 8.5, 25 °C. Spectra were recorded in a diode-array spectrophotometer shortly after the addition of product to dithionite-reduced enzyme. Absorbance changes are given relative to reduced enzyme. Panel B (difference spectrum 2), difference spectrum obtained after introduction of air to the dithionite-reduced enzyme-alloxanthine complex. Spectra are for xanthine oxidase (solid line) and xanthine dehydrogenase (dashed line). Absorbance changes are given relative to oxidized enzyme. Panel C, double-difference spectra obtained after subtraction of difference spectrum 2 from difference spectrum 1 for xanthine oxidase (solid line) and xanthine dehydrogenase (dashed line).
Xanthine oxidase as isolated from cow's milk
is known to possess a variable amount (usually 20-30%) of
nonfunctional enzyme lacking a catalytically essential Mo=S
moiety. It has been shown, however, that alloxanthine binds only to the
reduced, functional form of the enzyme (20) (also confirmed in
the present studies, data not shown). As a result, the
double-difference spectrum for xanthine oxidase shown in Fig. 1C reflects the spectral change associated with
reduction of the functional form of xanthine oxidase only. Chicken
liver xanthine dehydrogenase, on the other hand, does not possess a
substantial amount of the desulfo-form as isolated. Differences in the
results obtained with the two enzymes appear to be associated with
differences in pKs associated with their
molybdenum centers (see below).
Figure 2: Deconvolution of absorption profiles attributable to prosthetic centers of xanthine oxidase. Solid line, difference spectrum corresponding to the subtraction of native xanthine oxidase (oxidized) from native xanthine oxidase (reduced), corresponding to the cumulative UV-visible spectral change due to reduction of the flavin, iron/sulfur, and molybdenum centers. Dashed line, difference spectrum corresponding to the subtraction of deflavoxanthine oxidase (oxidized) from deflavoxanthine oxidase (reduced), corresponding to the cumulative spectral changes attributable to the iron/sulfur and the molybdenum centers, respectively. Dotted line, difference spectrum corresponding to the spectral change associated with reduction of the molybdenum center of xanthine oxidase.
Figure 3: Effect of pH on the spectral change associated with reduction of the molybdenum center of xanthine oxidase. Solid line, double-difference spectrum obtained under the same experimental conditions as described above, that is in 0.1 M pyrophosphate, 0.3 mM EDTA, pH 8.5; dashed line, double-difference spectrum obtained as above except in 0.1 M CAPS (3-(cyclohexylamino)propanesulfonic acid), 0.1 N KCl, 0.3 mM EDTA, pH 10; dotted line, the double-difference spectrum obtained as above except in 0.1 M MES (4-morpholineethanesulfonic acid), 0.1 N KCl, 0.3 mM EDTA, pH 6.0.
Figure 4: The circular dichroism change associated with the reduction of the molybdenum center of xanthine oxidase and xanthine dehydrogenase. Solid line, the double-difference CD spectra obtained for xanthine oxidase after subtraction of its oxidized difference spectrum from its reduced difference spectrum as described above for the UV-visible absorbance spectra; dashed line, the same with xanthine dehydrogenase. The experimental conditions were the same as above, and the CD spectra were recorded in an AVIV-40DS CD spectrometer.
The results presented here clearly demonstrate a significant
but heretofore undetected spectral change associated with simple
reduction of the molybdenum centers of xanthine oxidase and xanthine
dehydrogenase. This constitutes the first determination of the
absorbance change for the MoOS centers found in these enzymes (although
as discussed in the introduction there is abundant evidence for other
types of spectral changes associated with the formation of several
types of reaction intermediates at the molybdenum center). The spectral
change appears to reflect a shift in absorbance maximum from 420 nm in
the oxidized center to below 300 nm in the reduced center of xanthine
oxidase at pH 8.5 (Fig. 1C), although the shift is
difficult to quantitate due to the deterioration in signal-to-noise in
the 260-310-nm region, where background absorption by the protein
is large. The pH dependence of the spectral change attributed to the
molybdenum center of xanthine oxidase is not surprising given the known
relationship of protonation/deprotonation events to a molybdenum
oxidation state in even the simplest inorganic complexes. The present
work indicates that at least one ionizable group is associated with the
molybdenum center of xanthine oxidase. With xanthine dehydrogenase, it
appears that the pK values associated
with the molybdenum center are somewhat lower than in the case of the
oxidase, since the dehydrogenase exhibits a spectral change for
reduction of its molybdenum center at pH 8.5 that is comparable with
that observed with the oxidase at pH 6 (compare the dashedspectrum of Fig. 1C and the dottedspectrum of Fig. 3, respectively).
Other
oxomolybdenum enzymes possessing a Mo(VI)O center in the
oxidized state rather than Mo(VI)OS (e.g. sulfite oxidase and
nitrate reductase) have very little absorption in the 200-800-nm
region attributable to the molybdenum centers, although the molybdenum
domain of sulfite oxidase exhibits an absorption band at 480 nm having
an extinction coefficient of
1600 M
cm
. Only dimethyl sulfoxide reductase exhibits
a spectral change that is clearly attributable to the molybdenum
center(25) . This indicates a major difference in the
electronic structure between the molybdenum centers of sulfite oxidase
and nitrate reductase, on the one hand, and dimethyl sulfoxide
reductase on the other. Clearly there are important differences between
the electronic structures of the molybdenum centers of these enzymes
that must ultimately be due to differences in the molybdenum
coordination sphere. The present work demonstrates for the first time a
spectral change associated with reduction of the molybdenum centers of
two MoOS-containing enzymes, xanthine oxidase and xanthine
dehydrogenase. This spectral change is pH-dependent with a maximum
extinction change at 410 nm in the high pH limit and provides a new
experimental probe of the molybdenum center that in the future should
make it amenable to techniques such as resonance Raman spectroscopy.