(Received for publication, October 2, 1995)
From the
A free radical-coupled copper complex has been identified as the
catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of
spectroscopic and biochemical studies. The native (inactive) enzyme is
activated by oxidants leading to the elimination of the cupric EPR
signal consistent with formation of an antiferromagnetically coupled
radical-copper complex. Oxidation also leads to the appearance of a
substoichiometric free radical EPR signal with an average g value (g = 2.0055) characteristic of
phenoxyl
-radicals arising from a minority apoenzyme fraction.
Optical absorption, CD, and spectroelectrochemical measurements on the
active enzyme reveal complex spectra extending into the near IR and
define the redox potential for radical formation (E
= 0.64 V versus NHE, pH 7.0). Resonance Raman
spectra have identified the signature of a modified
(cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active
complex. This radical-copper motif has previously been found only in
galactose oxidase, with which glyoxal oxidase shares many properties
despite lacking obvious sequence identity, and catalyzing a distinct
reaction. The enzymes thus represent members of a growing class of free
radical metalloenzymes based on the radical-copper catalytic motif and
appear to represent functional variants that have evolved to distinct
catalytic roles.
The white-rot wood-metabolizing basidiomycete fungi are major
degraders of lignin contributing essential chemistry to the global
carbon cycle. Phanerochaete chrysosporium, the organism most
extensively studied for its lignin-degrading ability, produces three
classes of extracellular enzyme under ligninolytic conditions: lignin
peroxidase, manganese peroxidase, and glyoxal
oxidase(1, 2) . In the presence of
HO
(3) , lignin peroxidases oxidize and
partially depolymerize lignin or lignin model
compounds(4, 5, 6, 7, 8, 9) .
The oxidizing peroxide cosubstrate for this reaction must be generated in situ for efficient turnover of extracellular lignin
peroxidase, a function performed by glyoxal oxidase, which catalyzes
the oxidation of a number of aldehyde and
-hydroxy carbonyl
compounds, reducing O
to H
O
in the
process. The enzyme exhibits a broad substrate specificity for
oxidation of simple aldehydes to the corresponding carboxylic acids, as
shown by (10) .
Two of the substrates for glyoxal oxidase (glyoxal (OHCCHO) and
methylglyoxal (CHCOCHO)) are found in extracellular fluid
of ligninolytic cultures (10) and are likely to represent
physiological substrates for the enzyme in a complex extracellular metabolic scheme(9, 10) . Purified glyoxal
oxidase is catalytically inactive but can be activated by lignin
peroxidase(11, 12) , suggesting a possible
extracellular regulatory circuit for the control of
H
O
production by glyoxal oxidase, and lignin
peroxidase activity by H
O
.
P. chrysosporium glyoxal oxidase has been purified to homogeneity(11) , cloned for high level expression in Aspergillus nidulans(13) , and its biochemical properties have been determined (11) . Glyoxal oxidase is an acidic monomeric glycoprotein with a naked molecular mass of 57 kDa (calculated from the nucleotide sequence; (14) ) requiring one g-atom of copper for full activity. In its catalytic reaction and copper requirement, glyoxal oxidase resembles another extracellular fungal enzyme, galactose oxidase (15, 16) which efficiently catalyzes the conversion of alcohols to aldehydes with formation of hydrogen peroxide. Galactose oxidase (from Dactylium dendroides) contains copper and an essential protein free radical that is required for catalysis(16) . The active site structure of galactose oxidase as revealed by crystallography (17) is illustrated in Fig. S1, showing the coordination of the copper ion by 2 histidine residues (His-496 and His-581), a simple tyrosinate (Tyr-495), and a covalently modified tyrosine (Tyr-272), cross-linked to a cysteinyl residue (Cys-228) to form a new, dimeric amino acid (cysteinyl-tyrosine). This feature has been identified by spectroscopic and modelling studies as the radical-forming site in galactose oxidase (18, 19, 20) .
Figure S1: Scheme 1.
Comparison of the nucleotide sequences for glyoxal oxidase and galactose oxidase shows no obvious homology in primary structure(14, 21) , with less than 20% amino acid sequence similarity based on standard sequence alignment algorithms. However, the present spectroscopic studies show a remarkable degree of similarity between the two enzymes at the level of active site structure and chemistry, identifying a free radical-coupled copper catalytic motif in glyoxal oxidase. Spectroscopic and biochemical comparison of the two proteins suggests that glyoxal oxidase and galactose oxidase are functional variants catalyzing distinct reactions at nearly identical active sites.
Recombinant P. chrysosporium glyoxal oxidase was
isolated from growth medium of a stable transformant of A. nidulans(13) grown in Aspergillus transformant minimal
medium containing 1% maltose as growth substrate, 70 mM NaNO, 50 mM KH
PO
, 7
mM KCl, 2 mM MgSO
, 100 µg/ml
methionine, 1 µg/ml biotin, and trace elements (22) at pH
4.6. Glyoxal oxidase was purified as described previously (11, 14) except that the protein was first
concentrated from culture media by ammonium sulfate
precipitation(23) . Enzyme activity was measured
polarographically with an oxygen-sensitive electrode using
methylglyoxal as substrate in the presence of the oxidant
Na
IrCl
. For phosphate inhibition studies,
glyoxal oxidase activity was measured using the oxygen electrode with
dimethylsuccinate buffer (pH 4.5) or MES (
)buffer (pH 6.0).
Redox-activated glyoxal oxidase was prepared by addition of a slight
excess of Na
IrCl
to the enzyme solution.
Protein concentration was determined by the method of Lowry (24) .
Absorption spectra were recorded on a Varian Cary 5E
UV-vis-NIR spectrometer interfaced to a microcomputer for data
acquisition. EPR spectra were acquired on a ER300 EPR spectrometer
equipped with X-band microwave bridge and a nitrogen flow system.
Quantitative EPR measurements were performed as described
previously(25) . Circular dichroism (CD) spectra were recorded
on an Aviv model 41DS UV-vis-NIR CD/MCD spectrometer(26) .
Copper analyses were performed on a Varian SpectrAA 20B atomic
absorption spectrometer equipped with a GTA-96 graphite furnace.
Spectrophotometric titrations were performed on a Hitachi U2000 UV-vis
absorption spectrometer. Spectroelectrochemical studies on glyoxal
oxidase used a special thin layer optical cell ()based on
modification of a commercial demountable IR cell (Spectra-Tech
demountable liquid sampling cell) similar in plan to a cell recently
described for IR spectroelectrochemistry applications(27) . The
cell is constructed by supporting a 1000 lines/inch gold grid working
electrode (Buckbee-Mears Co., St. Paul, MN) between quartz disks
separated by a 0.2 mm polyvinylchloride gasket, using silver/saturated
AgCl reference electrode (0.22 V versus NHE; (28) )
and a platinum wire counter electrode. Electroanalytical measurements
were performed with an EG& model 263 potentiostat interfaced with
a microcomputer for data acquisition and analysis. Resonance Raman
spectra were collected with a custom McPherson 2061/207 spectrograph
(0.67-m focal length, 600-groove grating, 7 cm
spectral resolution) using a Coherent Innova 302 krypton (647 nm)
laser, a Kaiser optical super-notch filter, and a Princeton Instruments
(LN-1100PB) liquid N
-cooled CCD detector. Spectra were
obtained on samples in glass capillaries at 278 K using a 90°
scattering geometry and a 10-min accumulation time. Sample integrity
was verified by the observation of the same absorption spectrum
(typical of active enzyme) before and after laser irradiation.
Figure 1:
EPR
spectra for glyoxal oxidase complexes. Spectra for native glyoxal
oxidase (1.0 mM enzyme in 50 mM sodium phosphate
buffer, pH 6.5) and redox-activated enzyme (same conditions, in
presence of 1 equivalent of NaIrCl
).
Instrumental conditions: microwave frequency, 9.39 GHz; microwave
power, 10 milliwatts; modulation frequency, 100 kHz; modulation
amplitude, 10 G; temperature 120 K. Inset, expansion of the
m
= +1/2 copper hyperfine feature (solid
line) and gaussian fit to five components having a 10.5 G
separation (dotted line).
Figure 2:
Glyoxal oxidase free radical EPR signal.
Spectrum of glyoxal oxidase (1 mM protein) oxidized with
excess NaIrCl
in 50 mM sodium
phosphate buffer, pH 6.5. Instrumental conditions: microwave frequency,
9.55 GHz; microwave power, 2 milliwatts; modulation frequency, 100 kHz;
modulation amplitude, 2 G; temperature 120
K.
Spectroelectrochemical measurements on glyoxal oxidase (Fig. 3, left) provide an estimate of the redox potential of the radical-forming site. In these studies, the growth of intense absorption features associated with the radical-containing protein (see below) are used to monitor the progressive redox conversion of the sample, and the absorption/potential data are analyzed in terms of the Nernst equation ().
Figure 3:
Spectroelectrochemical oxidation of
glyoxal oxidase. Left, native enzyme (2.2 mM protein)
in 10 mM sodium phosphate buffer, pH 7.0, containing 2
mM KMo(CN)
was progressively oxidized
in a thin layer cell under applied potentials ranging from 0.34 to 0.5
V (versus Ag/AgCl) in 20-mV steps. Right, Nernst plot
of potential/absorption data (
) for redox conversion of glyoxal
oxidase and theoretical line for E
= 0.64
V versus NHE, n =
1.2.
A is the sample absorbance, A and A
are the limiting absorbances for
fully reduced and fully oxidized enzyme, respectively, R is
the gas constant (8.314 J/K
mol), T is the absolute
temperature, n is the number of electrons involved in the
redox couple, and F is the Faraday constant (96.48 kC/mol). A
Nernst plot of the absorption intensities through the oxidation step (Fig. 3, right) averaged between increasing and
decreasing potential scans is linear (r
=
0.995) with a slope of 0.048 mV consistent with a single-electron step
for the oxidation (n = 1.2) and a midpoint potential (E
= 0.42 V versus Ag/AgCl; 0.64
V versus NHE) significantly higher than that found for
galactose oxidase (0.4 V versus NHE at pH 7.3; ref. 31). This
is consistent with the requirement for a relatively high potential
oxidant for activation of glyoxal oxidase
(Na
IrCl
(0.892 V; (32) ) or
K
Mo(CN)
(0.798 V; (33) )) compared to
galactose oxidase, which is completely converted to the active,
radical-containing form by milder oxidants like ferricyanide (0.424 V; (34) ). Activation of glyoxal oxidase by diffusible high
potential manganese chelates (illustrated here by Mn
EDTA oxidation) may have a physiological role in regulation of
the extracellular activity of glyoxal oxidase through the metabolic
networking of the ligninolytic enzymes(1, 2) . The
biological significance of this interaction is presently under further
study. The stability of the redox-activated proteins (reflecting the
stability of the free radical sites) differs between glyoxal oxidase
and galactose oxidase. While the latter is stable for days at 4
°C(25) , the stability of the active glyoxal oxidase is
markedly lower, with a half-life of 4 h for the radical under similar
conditions (50 mM sodium phosphate buffer, pH 6.5).
Figure 4: Optical absorption spectra. Solid line, active (top) and native (bottom) glyoxal oxidase (0.15 mM enzyme, in 50 mM sodium phosphate buffer, pH 6.5 (active) or 8.34 (native)). Dotted line, active (top) and reductively inactivated (bottom) galactose oxidase shown for comparison.
The
sensitivity of the active site of glyoxal oxidase to protons is
reflected in spectrophotometric titrations. While the spectrum of the
redox activated glyoxal oxidase is nearly unchanged over a wide pH
range (4.5-8.5), more dramatic pH-dependent changes occur in
spectra of the inactive enzyme (Fig. 5). Titration of inactive
glyoxal oxidase in 100 mM potassium phosphate buffer converts
the enzyme to a purple form at low pH ( =
1365 M
cm
), maintaining
two isosbestic points (at 504 and 568 nm) that identify the reaction as
a simple two-state transition. More detailed analysis by
Henderson-Hasselbach fitting of the titration data gives an estimate
for the pK
of the protein group involved in the
protonation step (pK
= 7.7) (Fig. 5, inset). The spectral changes associated with the pH-dependent
structure change (loss of 451 nm phenolate-to-Cu
LMCT
absorption at low pH) suggest that the titratable group is a
coordinated tyrosinate, ionizing more readily in the metal complex than
is typical of the free amino acid (pK
=
10.1; (37) ) and dissociating from the metal on protonation.
The assignment to the unmodified tyrosinate (corresponding to Tyr-495
in the active site of galactose oxidase, Fig. S1) is supported
by the observation that resonance Raman profiles at low pH reveal only
vibrational modes of a Cys-Tyr group, whereas at high pH contributions
from both tyrosinates are observed (see below).
Figure 5:
pH-dependent spectral changes for native
glyoxal oxidase. Glyoxal oxidase (0.15 mM protein in 100
mM sodium phosphate buffer (1-12): pH 8.34,
8.15, 7.98, 7.86, 7.71, 7.50, 7.39, 7.16, 7.10, 6.92, 6.44, 4.53). Inset, absorption data fit to a protonation equilibrium model
with pK=
7.7.
In glyoxal oxidase,
the acid titration of the coordinated tyrosinate reflects a
perturbation of the phenolate basicity by approximately 2.5 pH units in
the metal complex. The lack of sensitivity to protons for the
corresponding low and high pH spectra of the redox activated enzyme
implies a more dramatic perturbation of phenolate basicity in the
radical-copper complex. This difference can be understood in terms of
the decrease in overall charge of the ligand set associated with
oxidation of the coordinated cysteinyl-tyrosinate anion to generate the
neutral (Cys-TyrO) phenoxyl, making the copper a more
strongly acidic metal center(38) . The modulation of ligand
acidity by metal interactions is an essential feature of ligand
participation in catalysis in galactose oxidase (16) (see
below). In that enzyme, the spectra of both active and inactive forms
are relatively insensitive to pH, but there is evidence for a
temperature-dependent proton transfer equilibrium in the active site
and for proton-coupled anion binding(38) .
Azide acts as an analog of peroxide in
oxygen-metabolizing enzymes, forming stable complexes that mimic
reactive oxygen species. Addition of azide to both native and redox
activated forms of glyoxal oxidase leads to the appearance of new
spectral features (Fig. 6, Table 1), that are again
similar to those observed for the azide complexes of galactose oxidase
and other copper proteins (39, 40, 41, 42) . By analogy, the
intense absorption near 380 nm is assigned to azide-to-Cu LMCT. The lower energy absorption bands have more metal-centered d
d character, as indicated by their
relatively large CD anisotropy (see below). As previously observed for
galactose oxidase(25, 38) , there is a significant
difference in affinity for azide between active and inactive forms;
active glyoxal oxidase binds azide much tighter (K
= 0.5 mM) than the native enzyme (K
= 25 mM). Although the trend in
affinity parallels the results for galactose oxidase, the dissociation
constants for both forms are approximately 25 times (active) and 130
times (native) larger than the corresponding values found for that
enzyme.
Figure 6:
Optical
spectra of azide complexes. Spectra of active (top) and native (bottom) glyoxal oxidase (0.15 mM enzyme, 50 mM sodium phosphate buffer, pH 6.5) in presence of 10 mM (active) or 250 mM (native)
NaN.
The similarity in the shifts in absorption bands between the two enzymes on binding exogenous ligands is particularly interesting in view of the evidence for pseudorotation of the copper complex and an intrinsic proton transfer coordinate in the active site of galactose oxidase, relating to substrate activation in catalysis(16, 38) . Previous studies on galactose oxidase have demonstrated that coordination of an exogenous ligand results in a displacement of the unmodified tyrosinate (Tyr-495) through a fluxional interchange of strong and weak interactions in the metal complex that can be regarded as a pseudorotation distortion of the active site(16, 38) . Displacement of the tyrosinate ligand increases its basicity, allowing it to serve as a general base for a proton abstraction step in catalysis(38) . In galactose oxidase, the pseudorotation mechanism leads to a thermodynamic linkage between anion binding and proton uptake(16) . The close correspondence in the spectroscopic behavior of the two proteins under ligand perturbations indicates that not only are the two active sites structurally related but that these dynamical motifs are reproduced as well.
Figure 7:
Circular dichroism spectra for glyoxal
oxidase complexes. Comparison of CD spectra for active (A) and
native (B) glyoxal oxidase in absence (solid lines)
and presence (broken lines) of 10 mM (active) or 250
mM (native) NaN.
Figure 8:
Resonance Raman spectra obtained with 647
nm excitation. -, glyoxal oxidase (1.5 mM enzyme,
in 25 mM sodium phosphate buffer pH 7) in presence of 1.2
equivalents of NaIrCl
(A) and on
addition of 10 mM NaN
(C). - - -,
corresponding spectra for active galactose oxidase in absence (B) or presence (D) of NaN
for
comparison.
The copper-coordinated
phenoxyl radical (Cys-Tyr 272) in active galactose oxidase is
associated with another set of vibrational modes at 1382, 1487, and
1595 cm (Fig. 8B), which persist in
the azide adduct (Fig. 8D) (36, 45) .
These three modes are characteristic of the Raman spectra of phenoxyl
radicals, with the 1487 cm
feature being assigned to
the C-O stretch on the basis of its isotope dependence in model
compounds(36, 46) . The residual features at 1185,
1246, 1312, 1417, and 1439 cm
in the azide complex (Fig. 8D) are also assigned to vibrations of the
phenoxyl radical. Active glyoxal oxidase exhibits a similar set of
major features at 1375, 1486, and 1591 cm
with 647
nm excitation (Fig. 8A). Both these and the minor
features at 1250, 1309, 1421, and 1444 cm
are again
found in the azide adduct (Fig. 8C). All of these modes
have diminished intensity with 458 nm excitation (data not shown),
indicating that they are all due to the same chromophore. The
similarity of the vibrational frequencies and intensities to those of
galactose oxidase allows us to conclude that active glyoxal oxidase has
a similar (Cys-Tyr) phenoxyl radical as a copper ligand.
The native
(inactive) form of glyoxal oxidase exhibits distinctive resonance Raman
features for both the unmodified tyrosinate and Cys-Tyr ligands (Table 2). At pH 8.1 where both ligands are coordinated, 458 nm
excitation selectively enhances the vibrations of the unmodified
tyrosinate at 1171, 1260, and 1609 cm, whereas 647
nm excitation selectively enhances the vibrations of the Cys-Tyr ligand
at 1267, 1481, and 1596 cm
. At pH 6.1, only
vibrational modes of the Cys-Tyr are observed at 1240, 1486, and 1596
cm
. The decrease in energy of the C-O stretch from
1267 to 1240 cm
is explained by an increase in the
Cu-O bond strength of the Cys-Tyr ligand on protonation and
dissociation of the other Tyr ligand. Similarly, in the presence of
azide, only Cys-Tyr modes are observed at 1272, 1483, and 1599
cm
. Thus, as in the case of the oxidized enzyme, the
resonance Raman profiles again indicate that the unmodified tyrosinate
ligand no longer binds to copper in the presence of azide and also does
not bind at low pH. The enhancement of the Cys-Tyr modes with 647 nm
excitation demonstrates that the unusually intense absorption bands in
the 600-650 nm region of the reduced enzyme ( Fig. 4and Fig. 6) have substantial Cys-tyrosinate
Cu
charge transfer character in addition to the Cu
ligand field character indicated by CD spectroscopy (Fig. 7).
The distinct redox potentials for the radicals in the two enzymes may be important in the context of the catalytic reactions, the higher potential redox site being favorable for aldehyde oxidation chemistry. The tuning of the redox potentials of the metal and radical may be accomplished by varying the strength of ligand interactions in the active site complex. Inorganic modelling studies on a chelate that reproduces the complex functionality of the active site of galactose oxidase shows that metal interactions can shift the redox potential of a thioether substituted phenolate by more than 0.5 V from the midpoint potential of the free ligand(51) .