Co(II) is not oxidized during turnover in the copper amine oxidase from Hansenula polymorpha

Article abstract of DOI:10.1007/s00775-018-1624-y

Co(II) substitution into the copper amine oxidases (CAOs) has been an effective tool for evaluating the mechanism of oxygen reduction in these enzymes. However, formation of hydrogen peroxide during turnover raises questions about the relevant oxidation state of the cobalt in these enzymes and, therefore, the interpretation of the activity of the metal-substituted enzyme with respect to its mechanism of action. In this study, Co(II) was incorporated into the CAO from Hansenula polymorpha (HPAO). The effect of hydrogen peroxide on the catalytic activity of cobalt-substituted HPAO was evaluated. Hydrogen peroxide, either generated during turnover or added exogenously, caused a decrease in the activity of the enzyme but did not oxidize Co(II) to Co(III). These results are in strong contrast with results from the CAO from Arthrobacter globiformis (AGAO), where hydrogen peroxide causes an increase in the activity of the enzyme as the Co(II) is oxidized to Co(III). The results of this study with HPAO support previous reports that have shown that this enzyme acts by transferring an electron directly from the reduced TPQ cofactor to dioxygen rather than passing the electron through the bound metal ion. Furthermore, these results provide additional evidence to support the idea that different CAOs use different mechanisms for catalysis.

Full text of DOI:10.1007/s00775-018-1624-y

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-018-1624-y
ORIGINAL PAPER
Co(II) is not oxidized during turnover in the copper amine oxidase
from Hansenula polymorpha
Stephen A. Mills1 · Kiera E. Gazica1 · David L. Tierney2
Received: 4 June 2018 / Accepted: 8 October 2018
© SBIC 2018
Abstract
Co(II) substitution into the copper amine oxidases (CAOs) has been an efective tool for evaluating the mechanism of
oxygen reduction in these enzymes. However, formation of hydrogen peroxide during turnover raises questions about
the relevant oxidation state of the cobalt in these enzymes and, therefore, the interpretation of the activity of the metal-
substituted enzyme with respect to its mechanism of action. In this study, Co(II) was incorporated into the CAO from
Hansenula polymorpha (HPAO). The efect of hydrogen peroxide on the catalytic activity of cobalt-substituted HPAO was
evaluated. Hydrogen peroxide, either generated during turnover or added exogenously, caused a decrease in the activity
of the enzyme but did not oxidize Co(II) to Co(III). These results are in strong contrast with results from the CAO from
Arthrobacter globiformis (AGAO), where hydrogen peroxide causes an increase in the activity of the enzyme as the Co(II)
is oxidized to Co(III). The results of this study with HPAO support previous reports that have shown that this enzyme acts
by transferring an electron directly from the reduced TPQ cofactor to dioxygen rather than passing the electron through
the bound metal ion. Furthermore, these results provide additional evidence to support the idea that diferent CAOs use
diferent mechanisms for catalysis.
Keywords Amine oxidase (copper containing) · TOPA quinone · Metal substitution · Kinetics · Catalysis
Abbreviations
AGAO
PSAO
TPQ
Pea seedling amine oxidase
Arthrobacter globiformis amine oxidase
Copper amine oxidase
2,4,5-Trihydroxyphenylalanyl quinone
CAO
CoHPAO
Cobalt-substituted HPAO
CuDepHPAO Copper-depleted HPAO
Introduction
EPR
Electron paramagnetic resonance
HPAO
ICP-AES
Hansenula polymorpha amine oxidase
Inductively coupled plasma-atomic emis-
sion spectroscopy
The copper amine oxidases (CAOs) are a large family of
enzymes, found in most organisms ranging from bacte-
ria and fungi to plants and animals [1, 2]. These enzymes
catalyze the oxidation of primary amines to aldehydes and
ammonia, with the concurrent reduction of molecular oxy-
gen to hydrogen peroxide. These enzymes use two cofactors
for this reaction: a Cu(II) ion and 2,4,5-trihydroxypheny-
lalanyl quinone (TPQ). Catalysis by CAOs occurs in two
half-reactions. The amine is converted to an aldehyde in
the frst half-reaction (Scheme 1, clockwise from TPQox to
TPQred, purple arrows). The mechanism of this half-reac-
tion is well understood and the roles of the two cofactors
fairly well established [3, 4]. Oxygen is reduced to hydrogen
peroxide in the second half-reaction and ammonia is pro-
duced (Scheme 1, clockwise from TPQred to TPQox, black
arrows). The mechanism for this half-reaction is less well
understood and more controversial. Overall, two electrons
MOPS
PHZ
3-(N-Morpholino)-propanesulfonic acid
Phenylhydrazine
This work is dedicated to Prof. Justine P. Roth, who was a
brilliant scientist and great sounding board for ideas.
* Stephen A. Mills
millss4@xavier.edu
David L. Tierney
dtierney@miamioh.edu
1
Department of Chemistry, Xavier University, 3800 Victory
Parkway, Cincinnati, OH 45207, USA
2
Department of Chemistry and Biochemistry, Miami
University, 651 E. High St, Oxford, OH 45056, USA
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
Scheme 1 Reaction scheme for copper amine oxidases (CAOs) showing methylamine as the substrate. Reductive half-reaction (purple arrows)
clockwise from TPQox to TPQred. Oxidative half-reaction (black arrows) clockwise from TPQred to TPQox
Scheme 2 Two proposed mechanisms for electron transfer from
TPQred to O2. Direct electron transfer (top path): TPQred transfers an
electron directly to O2 initially. This pathway has also been referred to
as an outer-sphere electron transfer pathway. Indirect electron trans-
fer (bottom path): TPQred transfers an electron to Cu(II) initially. This
pathway has also been referred to as an inner-sphere electron transfer
pathway
and two protons must be transferred from TPQred to O2 in
this half-reaction. The question lies in how those electrons
are transferred.
Two mechanisms have been proposed for this reaction. In
one proposal (Scheme 2, top pathway), the frst electron is
transferred directly from TPQred to pre-bound O2, resulting in
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JBIC Journal of Biological Inorganic Chemistry
the semiquinone form of TPQ (TPQsq) and superoxide. In this
proposal, the Cu(II) acts to stabilize the superoxide until the
second electron and two protons are transferred, forming H2O2
[5, 6]. This mechanism can be called the direct electron transfer
mechanism, or the outer-sphere electron transfer mechanism.
The alternative mechanism that has been proposed begins with
an electron being transferred from TPQred to Cu(II), forming
TPQsq and Cu(I) (Scheme 2, bottom pathway). The resulting
Cu(I) transfers the electron to O2, in an inner-sphere electron
transfer process, again forming Cu(II) and superoxide [7–10].
This mechanism can be called the indirect electron transfer
mechanism, or the inner-sphere electron transfer mechanism.
Various reports have provided support for each mechanism
using various kinetic studies [6, 10–12], O-18 kinetic isotope
efects [6, 8], spectroscopic studies [7, 11–13], model studies [5],
and computational treatments [8, 14]. However, one of the clear-
est ways to distinguish between the two mechanisms is to replace
the Cu(II) with Co(II) [5, 9]. This substitution is expected to pre-
vent the indirect electron transfer mechanism since Co(II) cannot
be reduced to Co(I) under these conditions. If the Co-substituted
CAO is catalytically active, then it likely proceeds through the
direct electron transfer mechanism. If the Co-substituted CAO is
not catalytically active, then it is likely that the enzyme normally
uses the indirect electron transfer mechanism and cannot use the
direct electron transfer mechanism [5]. This methodology has
been used to show that Hansenula polymorpha amine oxidase
(HPAO) uses the direct electron transfer mechanism [5, 15],
while the pea seedling amine oxidase (PSAO) uses the indirect
electron transfer mechanism [9].
analysis was done on a Perkin Elmer Optima 7300 DV ICP-
AES using commercially available metal standard solutions.
Protein concentration was determined by the Micro Brad-
ford Protein Assay (Bio-Rad) using BSA as the standard.
HPAO was purifed according to published procedures [5].
CoHPAO was prepared as described previously [5].
General protein characterization
Enzyme activity was measured by monitoring the rate
of oxygen consumption using a Clark electrode (Yel-
low Springs 5300A voltmeter) at 25 °C after introducing
1 µL of enzyme into 1 mL of bufer containing 100 mM
potassium phosphate bufer at pH 7.2, containing 1.5 mM
methylamine. Phenylhydrazine derivatization was per-
formed at 37 °C as described previously [5]. Spectral
changes were recorded using an Agilent 8453 diode array
spectrophotometer.
Activity over time
The activity of native CoHPAO (240 nM) was moni-
tored over time by measuring oxygen consumption in the
Clark electrode. Separately, H2O2 (23 mM) was added to
CoHPAO (215 µM) and was allowed to react with H2O2
for approximately an hour prior to assaying the activity
of the enzyme. This experiment was repeated with WT
HPAO using similar concentrations of enzyme and H2O2.
A study of the mechanism of Arthrobacter globiformis
amine oxidase (AGAO) using Co-substitution identifed a pos-
sible problem with using cobalt substitution to determining the
mechanism of the CAOs [14]. This study demonstrated that the
Co(II) in AGAO could be oxidized to Co(III), either during
turnover or by adding exogenous H2O2. Once the cobalt is oxi-
dized, then the possibility exists that Co(III)CAOs could use the
indirect electron transfer mechanism, with initial reduction of
Co(III) to Co(II) prior to an inner-sphere electron transfer from
Co(II) to O2. If this can happen generally in the CAOs, then it
will be important to know the oxidation state of the cobalt both
initially and after catalysis to interpret the catalytic activity of
CoCAOs. In this study, we examined the possibility of Co(II)
oxidation in HPAO and how that would afect the interpretation
of the resulting catalytic activity of the substituted enzyme.
Cobalt EPR
CoHPAO (200 µL at 17 mg/mL, 230 µM) was placed in a
quartz EPR tube and frozen in liquid N2. The sample was
thawed and H2O2 was added to the sample (1 µL of 1 M
H2O2). The protein was allowed to react with H2O2 for
approximately 2 h prior to freezing the sample and col-
lecting another spectrum. The sample was thawed again
and treated extensively with substrate as described below
prior to collecting another spectrum. The spectra were
collected at 5 K using a Bruker EMX EPR spectrometer,
equipped with an ER4116-DM dual mode resonator oper-
ating at 9.62 GHz. Temperature control was accomplished
using an Oxford ESR900 cryostat and temperature con-
troller. Other spectral conditions included are microwave
power = 2.0 mW; field modulation = 10 G (100 kHz);
receiver gain=105; time constant/conversion time=20 ms.
Materials and methods
Materials and general methods
Extensive reaction with substrates
All chemicals were purchased from commercial sources and
used without further purifcation, except as noted. Glass-
ware was rinsed with nitric acid prior to use. Trace metal
The efect of extensive reaction with substrate was accom-
plished by bubbling oxygen through a bufer containing
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JBIC Journal of Biological Inorganic Chemistry
300
250
200
150
100
50
20 mM MOPS (pH 7.2), 100 mM sodium chloride, 1.7 mM
methylamine to form an oxygen-saturated bufer containing
approximately 1.2 mM oxygen. CoHPAO (200 µL, 17 mg/
mL) was diluted with 5 mL of this bufer in a centrifugal
concentrator (Vivaspin 6, 10,000 molecular weight cutof).
The enzyme was allowed to react for several minutes and
was concentrated to approximately 200 µL. This process was
repeated three additional times to provide the enzyme with
excess substrate to react with and to remove the products of
the reaction. After being diluted four times with substrate-
containing bufer, the substrates were removed by diluting
the enzyme with bufer that did not contain methylamine,
followed by concentrating the enzyme. This process was
repeated a second time to reduce the methylamine concen-
tration to below 2 µM. The resulting enzyme was evaluated
for activity on the oxygen electrode, as described above and
the EPR spectrum of the resulting protein was taken.
+ H O
2
2
+ H2O2
10
0
0
20
30
40
50
Time (min)
Fig. 1 Time traces for oxygen consumption by WT HPAO (green
traces) and CoHPAO (pink traces) over long times. The arrow indi-
cates when the enzyme was introduced. In both cases, the darker trace
is for the untreated enzyme and the lighter trace is for enzyme that
has been treated with H2O2. The concentration of WT HPAO was
210 nM before treatment and 200 nM after treatment with H2O2. The
concentration of CoHPAO was 240 nM before treatment and 215 nM
after treatment with H2O2. Conditions: 100 mM potassium phosphate,
pH 7.2, 1.5 mM methylamine, 25 °C
Spectroscopic evaluation of TPQ in CoHPAO
CoHPAO (8.5 mg/mL, 110 µM), that had been extensively
treated with substrate as described above, was placed in a
quartz cuvette and a spectrum was collected. Methylamine
(420 µM) was added aerobically to the enzyme and another
spectrum was collected after approximately 15 s. Additional
spectra were collected over time. The initial spectrum of the
enzyme was subtracted from the spectra collected at each
time point to produce the diference spectra.
Oxidation state of cobalt in HPAO
The oxidation state of cobalt in HPAO was determined
before and after the enzyme was treated with H2O2 and after
the enzyme was treated extensively with substrate. Cobalt
was incorporated into HPAO as Co(II). As seen previously
[5], the oxidation state of the metal is not changed as it is
incorporated into the enzyme (Fig. 2, top), producing a typ-
ical broad spectrum, with poorly resolved 59Co hyperfne
coupling (~77 G) observed at the lowest feld g value (~6.5).
Reacting the enzyme with 5 mM H2O2 (20 equivalents) has
little efect on the oxidation state the metal, as indicated by
the continued presence of the EPR signal from the Co(II)
(Fig. 2, center).
Results
Activity of CoHPAO
The activity of CoHPAO was followed by observing the con-
sumption of oxygen over time. In contrast with CoAGAO
[14], the activity of CoHPAO decreases over time, as shown
in Fig. 1. Incubating the CoHPAO with approximately 100
equivalents of H2O2 prior to measuring its activity does
not signifcantly change the activity of the enzyme. This is
also in contrast with results with AGAO, where incubating
CoAGAO with H2O2 activates the enzyme [14]. However,
the loss of activity over time is consistent with previous
results with CoHPAO, where low fractional conversions
were observed when measuring the O-18 KIE with this
enzyme [15]. In previous studies, no attempts were made
to understand the loss of activity with CoHPAO under long
turnover conditions.
One possibility for the diferences observed between
CoHPAO and CoAGAO is that it takes more H2O2 to oxi-
dize cobalt in HPAO. Another possibility is that H2O2
cannot access the metal in CoHPAO. To address both of
these possibilities, we allowed CoHPAO to react exten-
sively with substrate by introducing it to O2-saturated
bufer that also contained saturating methylamine. The
enzyme was allowed to react under these conditions and
the bufer removed with a centrifugal concentrator. The
enzyme was again introduced to the same buffer and
allowed to react again. This was repeated for a total of
four times. The EPR spectrum of the resulting enzyme
showed that even this extensive treatment with substrate
did not signifcantly afect the oxidation state of cobalt in
HPAO (Fig. 2, recycled compared to as prepared). These
The activity of WT HPAO does not seem to change under
long turnover conditions (Fig. 1). The enzyme only seems
to slow its activity as the oxygen is completely consumed.
As with CoHPAO, pretreating WT HPAO with H2O2 does
not seem to have much efect on the activity of the enzyme.
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JBIC Journal of Biological Inorganic Chemistry
30
25
20
15
10
5
0
0
5
10
15
20
Time (min)
Fig. 3 Reaction of phenylhydrazine with CoHPAO before (black cir-
cles) and after (blue squares) extensive treatment with substrates at
37 °C. Spectra are normalized for the amount of enzyme (mM) in
each reaction
Fig. 2 X-band EPR spectra of CoHPAO as prepared (top), after
reacting with H2O2 for about 2 h (center) and after extensive treat-
ment with substrates (bottom). To facilitate comparison, the 2-h and
recycled spectra are overlaid with the as-prepared spectrum in gray.
All data are presented on the same scale. The asterisk indicates the
signal from a small Cu(II) contaminant; the brackets above the as-
prepared spectrum indicate the 59Co hyperfne pattern and the posi-
tion of the low-feld g value. Conditions: 9.62 GHz, 5 K, micro-
wave power=2.0 mW; feld modulation=10 G (100 kHz); receiver
gain=105; time constant/conversion time=20 ms
spectroscopically and by allowing the enzyme to react with
phenylhydrazine.
CoHPAO was reacted with phenylhydrazine using the
standard conditions for determining the amount of cofactor
present in the enzyme [5]. After extensive treatment with
substrate, the reactivity of the TPQ seems to reduce by about
half (Fig. 3). Prior to reaction with substrates, CoHPAO
was found to have 68% reactive TPQ (Table 1 and Fig. 3,
black line). After extensive treatment with substrates, the
enzyme was found to have 36% reactive TPQ (Fig. 3, blue
line). The overall activity of the recovered enzyme was also
determined. Prior to extensive treatment, CoHPAO had a
specifc activity of 0.31 µmol/min/mg. After treatment, the
specifc activity was reduced by about half to 0.13 µmol/min/
mg (Table 1). A reduction in reactive TPQ and reduction in
overall activity were also seen with WT HPAO (Table 1);
however, the efect was not as strong. The amount of reac-
tive TPQ was reduced from 86 to 67% upon treatment with
substrates. The overall activity after treatment was about
60% of the initial specifc activity.
results are in strong contrast with what was reported for
CoAGAO, where either treatment with H2O2, or turnover
of the enzyme resulted in complete oxidation of Co(II) and
loss of the EPR signal from cobalt [14].
Origin of enzyme inactivation
To evaluate the cause of enzyme inactivation after extensive
turnover, the reactivity of the cofactor and the metal content
of the enzyme were determined (Table 1). No signifcant
change in the amount of metal was observed before and
after treatment with substrates, as determined by ICP-AES
metal analysis. Prior to treatment with substrate, the protein
had 0.54 equivalents of cobalt bound. After treatment with
substrate, the protein had 0.57 equivalents of cobalt bound.
The reactivity and presence of the cofactor were evaluated
Table 1 Characteristics of
Enzyme form
Specifc activity
Relative
TPQ/monomer Cu/monomer Co/monomer
diferent forms of Hansenula
polymorpha amine oxidase
(HPAO)
(µmol/min/mg)b
4.48
activity (%)
WT HPAO
100
59
7
7
3
0.86
0.67
0.68
0.73
WT HPAO (MeAm)a 2.66
Co HPAO
Co HPAO (H2O2)
Co HPAO (MeAm)
0.31
0.33
0.13
<0.05
0.54
0.57
0.36
aEnzyme extensively treated with substrate
bConditions: 100 mM potassium phosphate, pH 7.2, 1.5 mM methylamine, 25 °C, 258 µM O2
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JBIC Journal of Biological Inorganic Chemistry
0.2
0.15
0.1
1
0.8
0.6
0.4
0.2
0
B
A
0.05
0
-0.05
-0.1
-0.15
-0.2
300
400
500
600
700
800
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Fig. 4 a Spectral changes associated with aerobic addition of meth-
ylamine (420 µM) to CoHPAO (111 µM) after the enzyme had been
treated extensively with substrate. Spectra were collected at 15 s
(red), 3 min (orange), 25 min (green) and 60 min (blue) after sub-
strate was introduced to the resting enzyme (black). b Diference
spectra. The spectrum of the resting enzyme was subtracted from the
spectrum at each time point to observe the spectral changes
The reactivity of the TPQ in the treated enzyme was also
examined spectroscopically (Fig. 4). The protein sample
was initially pink to the eye due to the presence of oxidized
TPQ. The absorbance due to oxidized TPQ (around 500 nm)
bleached out immediately upon addition of methylamine to
the enzyme. After approximately 30 min, the absorbance
due to oxidized TPQ was restored, as the substrate was
consumed.
conditions has been observed previously. In the initial
characterizations of CoHPAO, it was difcult to determine
the O-18 kinetic isotope efect for this enzyme due to low
substrate conversions due to loss of enzyme activity under
turnover conditions [15]. This study confrms that observa-
tion and shows that this inactivation is not due to Co(II)
oxidation, loss of metal or destruction of the cofactor, Fig. 3.
However, the cause of the decrease in activity of CoHPAO
remains unknown.
Since the Co(II) in CoHPAO remains Co(II) under turno-
ver conditions, CoHPAO cannot use the indirect electron
transfer mechanism for catalysis. This observation and the
other results in this study support previous studies that
show that HPAO uses a direct electron transfer mechanism
in oxidative half-reaction of catalysis [5, 15]. These results,
along with those for other CAOs, add to the growing body
of evidence that the mechanism of oxygen reduction in the
CAOs depends on the particular CAO that is being studied.
The question is not “Which of the two mechanisms do the
Copper Amine Oxidases use?” but rather “Which Copper
Amine Oxidase uses which mechanism?” and following on
that, the next question is “What about the active site of each
CAO determines which mechanism will occur?”
Discussion
The observation that Co(II) inserted into AGAO could be
oxidized under turnover conditions and that the enzyme
was more active with Co(III) in the active site than with
Co(II) [14] calls into question the interpretation of all
results of Co-substituted CAOs. Co(II) has been used
to help elucidate the mechanism of CAOs based on the
assumption that it would not be reduced to Co(I) and,
therefore, blocks the indirect electron transfer mechanism
[5]. With Co(III) in the active site, an indirect electron
transfer mechanism is possible with an initial reduction of
Co(III) to Co(II), as proposed for AGAO [14]. This study
precludes this possible mechanism for HPAO and supports
previous studies that point to a mechanism where an elec-
tron is directly transferred from TPQred to O2.
Acknowledgements SAM acknowledges the other Xavier students who
work in the lab. SAM and KEG acknowledge the Xavier University
Ofce of Undergraduate Research for support.
The primary observation in this study is that CoHPAO
does not become more active under turnover conditions, as
was observed with CoAGAO. In contrast with CoAGAO, the
activity of CoHPAO decreases under long turnover condi-
tions, Fig. 1. Additionally, neither turnover conditions nor
addition of H2O2 to CoHPAO causes signifcant oxidation of
Co(II) in this enzyme, Fig. 2, as was reported for CoAGAO
[14]. The loss of activity of CoHPAO under long turnover
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