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THE JOURNAL OF BIOLOGICAL CHEMISTRY
63 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265. No. 26, Issue of September 15. pp. 15671-15679.1990
Printed
in U.S.
A.
Mechanism of Dioxygen Formation Catalyzed by
Vanadium Bromoperoxidase
STEADY STATE KINETIC ANALYSIS AND COMPARISON TO THE MECHANISM OF BROMINATION*
(Received for publication, April 23, 1990)
Richard R. Everett, Helena S. Soedjak, and Alison Butlers
From the Department of Chemistry, University of California, Santa Barbara, California 93106
The steady state kinetic mechanism of the bromide-
assisted disproportionation of hydrogen peroxide,
forming dioxygen, catalyzed by vanadium bromo-
peroxidase has been investigated and compared to the
mechanism of monochlorodimedone (MCD) bromina-
tion under conditions of 0.0125-6 mM HzOz, l-500
Il’IM Br-, and pH 4.55-6.52. Under these conditions, 50
PM MCD was sufficient to inhibit at least 90% of the
dioxygen formation during MCD bromination. The
rate data is consistent with a substrate-inhibited Bi Bi
Ping Pong mechanism, in which the substrate bromide,
is also an inhibitor at pH 4.55 and 5.25, hut not at pH
5.91 and 6.52. The kinetic parameters
KmB’, KmH~02,
Kis*‘,
and
KiiB’
determined for the reactions of hro-
mide-assisted disproportionation of hydrogen peroxide
and MCD bromination are similar, indicating that the
mechanisms of both reactions occur via the formation
of a common intermediate, the formation of which is
rate-limiting. Fluoride is a competitive inhibitor with
respect to hydrogen peroxide in both reactions at pH
6.5. At high concentrations of hydrogen peroxide, the
bromide-assisted disproportionation of hydrogen per-
oxide occurs during the bromination of MCD. The sum
of the rates of MCD bromination and dioxygen forma-
tion during MCD bromination is equal to the rate of
dioxygen formation in the absence of MCD. The appor-
tionment of the reaction through the MCD bromination
and dioxygen formation pathways depends on pH, with
much lower hydrogen peroxide concentrations causing
significant dioxygen formation at higher pH.
Vanadium bromoperoxidase (V-BrPO)’ is a relatively newly
discovered enzyme found in marine algae (1). V-BrPO is also
the first naturally occurring vanadium enzyme to be isolated,
although the second vanadium enzyme, a nitrogenase has
been purified recently (2, 3). V-BrPO has been isolated from
many species of marine algae (4-7) and vanadium-dependent
bromoperoxidase activity has been found in many other spe-
cies of marine algae (5, 8). Vanadium bromoperoxidase is a
member of a new class of non-heme containing haloperoxi-
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
soiely to indicate this fact.
$ Supported by National Science Foundation Grant DMB87-16229,
the Committee on Research of the University of California, Santa
Barbara, Academic Senate, and the American Cancer Society, Junior
Faculty Research Award JFRA-216. To whom correspondence should
be addressed.
’ The abbreviations used are: BrPO, bromoperoxidase; MCD, mon-
ochlorodimedone; HEPES, /i-(Z-hydroxyethyl)-l-piperazineethane-
sulfonic acid.
dases distinct from the well-known FeHeme haloperoxidases.
In addition to FeHeme haloperoxidases in terrestrial systems,
FeHeme bromoperoxidase is known in marine organisms,
including the alga Penicilh cupitatw (9).
V-BrPO is an acidic (i.e. p1 = 4), glycoprotein (10) which
binds about 1 equivalent of vanadium per subunit (iUr 65,000)
(5, 11). Vanadium can be removed from V-BrPO by complex-
ation with EDTA, producing the inactive apoenzyme deriva-
tive (1). The activity can be fully restored by the addition of
vanadate (H,VO;/HVO:-) to apo-(V)-BrPO (12). As isolated,
V-BrPO contains a substoichiometric vanadium/subunit ratio
(i.e. about 0.4), however, the specific activity increases upon
addition of vanadate, up to a vanadium/subunit stoichiometry
of l/l (12). The resting oxidation state of V-BrPO is consid-
ered to be vanadium (V) since reduction of native V-BrPO by
dithionite produces an ESR signal consistent with the for-
mation of the vanadyl derivative, VO*+-BrPO; this derivative
reportedly lacks bromoperoxidase activity (12).
V-BrPO catalyzes the bromination of a variety of organic
substrates, including monochlorodimedone (2-chloro-5,5-di-
methyl-1,3-dimedone, MCD), a cyclic @-diketone. With the
exception of MCD and possibly other @-diketone and p-keto
acid moieties, very few organic substrates are efficiently bro-
minated. For example, V-BrPO can catalyze the formation of
1,3,5-tribromophenol, 5-bromocytosine, and 5-bromouracil,
however, the reaction is not stoichiometric with hydrogen
peroxide consumption, since much more than 1 equivalent of
hydrogen peroxide is consumed per equivalent of brominated
product produced (11). In addition to the bromination reac-
tion, V-BrPO catalyzes the formation of dioxygen in a reac-
tion best described as a “bromide-assisted disproportionation
of hydrogen peroxide” or bromide-assisted catalatic activity
(11). The rate of dioxygen formation in the absence of MCD
equals the rate of MCD bromination (at pH 6.5,47 pM MCD,
2 mM hydrogen peroxide), indicating that both MCD bromi-
nation and dioxygen formation occur via the generation of a
common intermediate, whose formation is rate-limiting
(Scheme 1; Ref. 11). The identity of the intermediate has not
been established unambiguously.
HLO- + Br- V-BrPO a.
> n
- mtermediate” (ie. HOBr, Br3, Enz-OBr or Enz-Br)
k,
MCD
Br-MCD //
k? HZO,
O1 + Br- + H?O
SCHEME
1
Recently we have shown that the dioxygen produced in the
bromide-assisted disproportionation of hydrogen peroxide is
in the singlet excited state (13). The singlet dioxygen was
15671
This is an Open Access article under the CC BY license.
15672
Steady State Kinetic Mechanism
of
Vanadium Bromoperoxidase
identified by the characteristic emission at 1268 nm resulting
from the decay of ‘O&A,) to 302(“2;), the effect of specific
singlet oxygen quenchers and the solvent isotope effect on the
singlet oxygen intensity and lifetime (Ref. 13; for reviews, see
Refs. 14 and 15). Singlet oxygen is produced in near stoichi-
ometric yield (i.e. 80%) by V-BrPO. The striking feature of
V-BrPO is its exceptional stability; V-BrPO is not inactivated
by singlet oxygen or oxidized bromine intermediates, contrary
to the Fe-Heme haloperoxidases, lactoperoxidase, and chlo-
roperoxidase (13).
A steady state kinetic analysis of the mechanism of MCD
bromination has been reported (16). It is consistent with a
“bromide-inhibited Bi Bi Ping Pong” mechanism. Given our
initial results demonstrating the similar rates of MCD bro-
mination and dioxygen formation (ll), we have undertaken
the steady state kinetic analysis of dioxygen formation cata-
lyzed by V-BrPO. Indeed, the mechanisms of dioxygen for-
mation and MCD bromination are similar, and these are
competitive processes. We have also investigated the kinetic
mechanism of inhibition by fluoride.
MATERIALS AND METHODS
Bromoperoxidase
Preparation-Vanadium bromoperoxidase was
isolated from
Ascophyllum nodosum
collected at Kornwerderzand,
Holland, in April 1986 and 1989. A modified isolation and purification
procedure of Wever
et
al. (4) was used: an additional final purification
step consisting of a concanavalin A (Bio-Rad) column was used to
separate two possibly glycosylated forms of the enzyme. The bro-
moperoxidase not retained by the concanavalin A column was used
throughout this study. This-enzyme is reported to be glycosylated
(10). The snecific activitv for the bromination of MCD is about 132
units/mg in a
0.1
M phosphate assay buffer, pH 6.5, containing 0.1 M
KBr, 2 mM H202, 50 pM MCD, 0.2 M Na2S04. The specific activity
for V-BrPO is usually given at pH 6.5 even though the pH optimum
(under the given conditions) is less than pH 6.5 (see-below). The
enzyme stock solution was stored in 50
mM
HEPES, pH 7.0, or water
at 0 or 4 “C.
Dioxygen Measurements-Rates of dioxygen formation were meas-
ured with a Yellow Snrintrs Instrument (YSI) oxygen probe (YSI
5331) and monitor (YSI 5300). The reaction medium was sparged
with nitrogen gas to reduce the dioxygen concentration so that the
oxygen probe would not become saturated during the course of the
reaction. The reaction was initiated by the addition of V-BrPO. The
rate of dioxygen formation was calculated directly from a plot of the
percent dioxygen in solution
versus
time using an oxygen concentra-
tion of 0.247 mM in air-saturated water as the standard (17). The
reported rates have also been corrected for an apparent background
rate of dioxygen formation
or drift in the electrode of 0.2-0.6%/min.
Bromoperoxidase Activity Measurements-The standard assay for
determining the specific bromoperoxidase activity is the conversion
of 2-chloro-5,5-dimethyl-1,3-dimedone (MCD) to 2-bromo-2-chloro-
5,5-dimethyl-1,3-dimedone (Br-MCD) (18). The reaction is followed
spectrophotometrically at 290 nm, i.e. the X.,,, of MCD. The specific
activity is expressed in units/mg which is defined as the micromole
of MCD brominated per min/mg of bromoperoxidase. The difference
in extinction coefficients, ac, of MCD and Br-MCD at 290 nm is
19,900
M-l
cm-’ for pH > 4 and 18,200
Mm’
cm-’ for pH 4. Under the
conditions described below, aqueous vanadate and adventitiously
bound vanadium(V) do not have BrPO activity (11).
Conditions for Steady State Kinetic Analysis of the Mechanism of
Dioxygen Formation and MCI) Bromination-The reaction medium
for the steady state dioxygen formation and MCD bromination kinetic
studies consisted of 50
mM
sodium citrate buffered at pH 4.55, 5.25,
and 5.91 or 50
mM
sodium phosphate at pH 6.52 with l-500
mM
potassium bromide and sufficient sodium sulfate to give a final ionic
strength of 0.8
M,
unless otherwise specified. The hydrogen peroxide
concentration was varied from 12.5
pM
to 6
mM. In
some experiments
the pH was measured before and after the reaction to check that the
pH remained constant during the reaction.
The reaction mixture for the study of fluoride inhibition contained
0.1
M
sodium phosphate, pH 6.5, and sufficient sodium sulfate to
bring the ionic strength to 0.4 M. The competition between fluoride
and hydrogen peroxide was investigated under conditions of 75
mM
potassium bromide, 0.2-2
mM
hydrogen peroxide and O-20
mM
po-
tassium fluoride. The competition between fluoride and bromide was
investigated in reactions containing 5-50 mM bromide and O-40 mM
fluoride, at 1
mM
hydrogen peroxide.
The
spectrophotometric determination of the MCD bromination
rate was performed concomitantly with the dioxygen formation rate
measurements using the same concentrations and stock solutions of
buffer, hydrogen peroxide, and V-BrPO, except that the total volume
of the experiments measuring dioxygen formation were twice the
volume of the MCD bromination experiments (i.e. 3 and 1.5 ml,
respectively) and 50
pM
MCD was included in the MCD bromination
reactions. The V-BrPO concentration was varied from 0.62 to 6.2 nM.
The V-BrPO concentration was sufficient to obtain a dioxygen evo-
lution rate at least twice and usually 5-10 times greater than that
observed for the background rate of dioxygen formation. All kinetic
experiments were determined at 25 ? 0.5 “C.
Steady State Kinetic Analyses-The initial rates, u, as a function
of hydrogen peroxide or bromide concentration were fit to the Mi-
chaelis-Menton expression (i.e. Y = V[H~O,]/(K + [H202]1) by an
iterative process (19), where V and K are functions of K,,,B’, K_“z02,
V
mBr, and
K,”
(see “Appendix”). The kinetic parameters,
KmBr,
K “202, KIsB’ and K. LLBr were obtained from appropriate fits of initial
rate data as a function of hydrogen peroxide and bromide concentra-
tions using Cleland’s programs “PINGPONG,” “COMP,” and “NON-
COMP” (19). All measurements were performed in duplicate or
triplicate. The figures show the least square fits to the data and not
the best fit by the PINGPONG,
NONCOMP, or COMP programs.
Conditions to Investigate Competition between Dioxygen Formation
and
MCD Bromination-The reaction medium used to investigate
the
competition between dioxygen formation and MCD bromination
consisted of 0.1
M
sodium citrate for pH 4.0-5.5 or 0.1 M sodium
phosphate for pH 6.0-8.0, 0.1
M
potassium bromide, 0.5-400
mM
hydrogen peroxide, 3-12
nM
V-BrPO at an ionic strength of 0.97
M
adjusted with sodium sulfate. The volume of the reaction mixture for
the
dioxygen formation measurements was three times the volume of
the MCD bromination experiments (i.e. 3 and 1 ml, respectively). 50
FM
MCD was included in the MCD bromination reactions. All glass-
ware was
soaked with NoChromix (Godax) and the buffers were
passed through a Bio-Rex Chelex Ion Exchange Membrane before
use.
If these precautions were not followed, high rates of oxygen
formation could occur in the absence of enzyme.
General Reagents and
Procedures-The
concentration of Hz02
(30% aqueous solution, Fisher Scientific) was determined spectropho-
tometrically by the formation of triiodide (I;) as described (20).
Protein concentrations were determined by the bicinchoninic acid
assay (21), with reagents purchased from Pierce Chemical Co. MCD
was prepared as reported in the literature (22) or purchased from
Sigma. All other chemicals were reagent grade.
RESULTS AND INTERPRETATION
Determination of Kinetic Parameters for the Bromide-as-
sisted Disproportionation of Hydrogen Peroxide and Bromi-
nation of Monochlorodimedone-The steady state rates of
dioxygen formation
and MCD bromination were investigated
as a function of hydrogen peroxide concentration (12.5 FM to
6 mM), bromide concentration (l-500 mM), and pH (4.55,
5.25,5.91, and 6.52). Plots of the steady state rates of dioxygen
formation and MCD bromination uersus hydrogen peroxide
concentration fit a rectangular hyperbolic function (data not
shown) satisfying the rate law for the Michaelis-Menton
kinetic scheme.
In the presence of 50 pM MCD, l-500 mM bromide, and
50-800 PM hydrogen peroxide at pH 5.25, V-BrPO catalyzes
the bromination of MCD preferentially over the bromide-
assisted disproportionation of hydrogen peroxide. Double-
reciprocal plots of the initial steady state rates of dioxygen
formation and MCD bromination versus hydrogen peroxide
concentration at fixed, low bromide concentrations (l-5 mM),
produce a set of approximately parallel lines, as shown in Fig.
1 for pH 5.25. Parallel plots are also observed at pH 4.55,
5.91, and 6.52 for both MCD bromination (see Figs.
A-C in
Steady State Kinetic Mechanism of Vanadium Bromoperoxidase
15673
0.00 ! 0.00
0.0
5.0 10.0
0.0 10.0
2dQ
l/ t-1202
(mM-‘)
1 / H202
(mM-‘)
0.06
FIG. 1. Primary double-reciprocal plot of the rate of dioxygen formation and MCD bromination as
a function of hydrogen peroxide concentration at fixed, low bromide concentrations at pH 5.25. A,
steady state rate of dioxygen formation in the absence of MCD: A, 1.25
mM
Br-;
n
, 2
mM
Br-; 0, 5 mM Br-. B,
steady state rate of bromination of 50
pM
MCD: +, 1
mM Br-; A, 1.25 mM Br-;
n
, 2 mM Br-; 0, 5 mM Br-. e is the
total enzyme concentration. The rate data at 1
mM
bromide in
A
were omitted because of extreme scatter. Each
line is the linear least squares fit to data.
TABLE I
Kinetic parameters obtained from the rate of dioxygen formation in the bromide-assisted disproportionation of
hydrogen peroxide and the rate of MCD bromination at various pH values
Km*’ was obtained from the PINGPONG program
(19). The
values of KmH202, LB’, and I(,,” were obtained from
the NONCOMP program
(19), except at pH 6.5 where KmHZoZ
was determined by the fit to the PINGPONG
program. K Hz%
m
K_R’ KLqB’ K,B’
PH MCD 02 MCD 02 MCD 02 MCD 02
PM
??LM
mh4 mM
4.55 662 1296 4.0 7.2 332 316 730 262
5.25 284 230 10.5 15.2 319 193 1040 700
5.91 183 244 20.0 36.6 836 1187 905 1198
6.52 113 94 25.8 19.2 s-2000” >2000”
0 Due to a combination of the scatter inherent in the dioxygen formation measurements and the lack of much
inhibition at pH 6.5, the NONCOMP program was not able to fit this data.
FIG. 2. Primary double-recipro-
cal plot of the rate of dioxygen for-
mation and MCD bromination as a
function of hydrogen peroxide con-
centration at fixed, high bromide
concentrations at pH 5.25. A,
steady
state rate of dioxygen formation in the
absence of MCD. B, steady state rate of
bromination of 50
pM
MCD. In the order
of increasing slope: *, 25
mM Br-; +,
100 mM Br-; A, 200 mM Br-;
n
, 350 mM
Br-;
0,500
mM Br-.
e is the total enzyme
concentration. Each line is the linear
least squares fit to data.
l/ Hz02
(mtd-‘) l/ Hz02
(ml&)
Miniprint Section)’ and the bromide-assisted disproportion-
ation of hydrogen peroxide. The parallel plots are consistent
with a
Ping Pong mechanism at all pH values investigated,
although this mechanism cannot be rigorously
tested because
neither the MCD bromination nor dioxygen formation reac-
tions are reversible. The value of
K,,,Br
at each pH (Table I)
was determined from the best fit using Cleland’s PINGPONG
program (19) (Equation 1, “Appendix”).
z Figs. A-E are presented in miniprint at the end of this paper.
Miniprint is easily read with the aid of a standard magnifying glass.
Full size photocopies are included in the microfilm edition of the
Journal that is available from Waverly Press.
Bromide can also inhibit the bromide-assisted dispropor-
tionation of hydrogen peroxide and the MCD bromination
reactions. At higher fixed bromide concentrations
(i.e. 25-500
mM)
the double-reciprocal plots for data obtained at pH 5.25
produce a set of roughly intersecting lines (Fig. 2), showing
that bromide inhibits both the bromide-assisted dispropor-
tionation of hydrogen peroxide (Fig. 2A) and the bromination
of MCD (Fig. 2B). Steady state kinetic data at high bromide
concentrations were also obtained at pH 4.55 (see Figs.
D
and
E in Miniprint), 5.91, and 6.52 for the bromide-assisted dis-
15674
Steady State Kinetic Mechanism
of
Vanadium Bromoperoxidase
FIG.
3. Secondary plot of K/V ver- Yi+
t ‘;‘
sus bromide concentration at se- 0.0 - z 00-
lected pH values.
A, the values of K & . .
and V were obtained from the dioxygen 2 =F
formation measurements. B, the values
of K and V were obtained from MCD <
bromination measurements. 0, pH 4.55; y 4.0- z 4.0-
& pH 5.25; A, pH 5.91; +, pH 6.52. Each
line is the linear least squares fit to data.
206.0 406.0
Br- (mM)
206.0 406.0
Br- (mM)
proportionation of hydrogen peroxide and the bromination of
monochlorodimedone. The intersection point of these double-
reciprocal plots is not exactly on the y axis. Thus, the “fit” of
the rate data at high bromide concentration to competitive
and noncompetitive equations using Cleland’s “COMP” and
“NONCOMP” iterative fit computer programs (Equations 6
and 7, “Appendix”) were examined (19). The data fit the
noncompetitive inhibition rate equation better as judged by
the about 2-fold lower sigma value. Moreover the plot ob-
tained from an overlay of the data and the “best fit” lines of
l/v uers~.~ 1/[H202] clearly appear to fit better to the noncom-
petitive equation (not shown). The value of Kz,Br is also
comparable to K,,‘I, indicating a significant noncompetitive
inhibition contribution by bromide (Table I).
A previous report indicates that the bromide inhibition of
MCD bromination is competitive, based on the y axis inter-
section of a primary plot of e/v uersuS 1/[H202] (see Fig. 1 in
Ref. 16). In that study, rate data were obtained over a 20-400
FM range of hydrogen peroxide concentration, with very few
points around the hydrogen peroxide concentration equal to
KmH2’2
(16). If more data points are taken in the concentration
range of 0.5-5 times
KmHpoz
(23), the effect of bromide on the
intercept term is evident since the set of lines clearly do not
intersect on the y axis (Fig. 2).
A secondary replot of the slopes in Fig. 2
(i.e. K/v) versus
[Br-] is linear as shown in Fig. 3 and predicted by Equation
12 (Appendix).3 The linearity and positive slopes in the plots
of
K/V uersus
[Br-] at pH 4.55 and 5.25 (Fig. 3) demonstrate
that bromide is an inhibitor of the bromide-assisted dispro-
portionation of hydrogen peroxide and MCD bromination at
high bromide concentrations. At pH 5.91 and 6.52, the slope
of the plot of
K/V uersus
[Br-] is nearly 0, indicating that
bromide does not form a significant concentration of the
inhibitory complex at these pH values. Of the pH values
investigated, the maximum inhibitory effect of bromide occurs
at pH 5.25. Bromide does not inhibit the MCD bromination
at higher pH (i.e. 6.6-7.9) or lower pH (i.e. 4) than the pH
values investigated in this study (16).
The steady state kinetic results for dioxygen formation
(Figs. 1A and 2A) are very similar to those of MCD bromi-
nation (Figs. 1B and 2B). Table I shows a comparison of the
values of KmB’,
KmHzo2,
KisBr, and
KiiBr
characterizing the
bromide-assisted disproportionation of hydrogen peroxide
and the MCD bromination reactions, as a function of pH.
The values of
KmB’, KmHzoz, KisB’,
and
KilB’
obtained from the
bromide-assisted disproportionation of hydrogen peroxide re-
‘I Below 10
mM
Br-, the plots of K/V deviate about 10% from
linearity (data not shown).
0.00 0.0
2.b 4.il 6.b 1
o~ooo.o 23 4.0 6.0
1/ HSO. (i-M’)
l/
l&O2 (mU’)
FIG. 4.
Primary double-reciprocal plot of the rate of diox-
yen formation and MCD bromination as a function of hydro-
gen peroxide concentration at fixed fluoride concentrations,
pH 6.5, showing inhibition by fluoride.
A, steady rate of dioxygen
formation in the absence of MCD. B, steady state rate of bromination
of 50
pM
MCD. In the order of increasing slope: 0, 0
mM
F-;
n
, 2
mM
F-; A, 5 mM F-; e, 10 mM F-; *, 20
mM
F-; e is the total enzyme
concentration. Each line is the linear least squares fit to data.
action agree with the values obtained from the MCD bro-
mination reaction within a factor of about 2, indicating that
the rate-limiting step of both reactions is the same. The values
of
KmH2’2
obtained from the dioxygen formation and MCD
bromination reactions depend on pH. At higher pH, V-BrPO
has a much higher affinity for hydrogen peroxide than at
lower pH. de Boer and Wever (16) have reported that
K,,,H20z
determined for the MCD bromination reaction is almost
invariant between pH 6 and 8.
Inhibition by Fluoride-The
effect of fluoride on the inhi-
bition of MCD bromination and dioxygen formation was
investigated at pH 6.5. Double-reciprocal plots of e/v
versus
1/[H202] for dioxygen formation and MCD bromination are
shown in Fig. 4,
A
and
B,
respectively, at defined fluoride
concentrations (O-20 mM) and 75 mM bromide. The y axis
intersection in Fig. 4 (and predicted by equation below) sig-
nifies that fluoride is a competitive inhibitor of hydrogen
peroxide.
Iel=
K”ZB’
1
” V,,.lH,O,I +V,.,[Br-l+V,.,
The inhibition constant,
KiF,
was determined from the best
fit to the competitive COMP program.
KiF
is 1.75 (k0.15) mM
determined from the dioxygen formation data;
KzF
is 1.11 (k
0.13) mM determined from the MCD bromination data. The
agreement of these values indicate that the mechanism of
fluoride inhibition of the bromide-assisted disproportionation
of hydrogen peroxide and MCD bromination is the same.
Consistent with the equation above, double-reciprocal plots
Steady State Kinetic Mechanism
of
Vanadium Bromoperoxidase
15675
of e/u versus l/[Br-] at 1 mM hydrogen peroxide and varied
fluoride concentrations (O-40 mM), produce a set of parallel
lines for MCD bromination (see Fig. F, Miniprint). Itoh et al.
reported that fluoride was an uncompetitive inhibitor in stud-
ies using the non-heme bromoperoxidase isolated from Cor-
allina pilulifera (24). However, the competition was only in-
vestigated with respect to bromide. Equation 7 indicates that
for a Bi Bi Ping Pong system, a plot characteristic of uncom-
petitive inhibition (i.e. parallel lines) will be observed with
respect to bromide, even though fluoride is a competitive
inhibitor with respect to hydrogen peroxide. The competitive
inhibitor behavior of fluoride observed for V-BrPO further
supports the Bi Bi Ping Pong type mechanism for V-BrPO,
as compared to other ordered “Bi Bi” mechanisms.
Competition between the MCD Bromination Pathway and
the Bromide-assisted Disproportionation
of
the Hydrogen Per-
oxide Pathway-Under the conditions of the MCD bromina-
tion experiments described above, 50 PM MCD was sufficient
to inhibit at least 90% of the dioxygen formation pathway
during MCD bromination. We have previously shown that
the rate of bromination of 50 PM MCD is approximately equal
to the rate of dioxygen formation (in the absence of MCD),
at 2
mM
H202, 0.1 M Br- at pH 6.5, which suggests that both
reactions occur via the formation of a common intermediate,
whose formation is rate-limiting (Scheme 1; Ref. 11). Scheme
1 suggests that the fate of the intermediate should be con-
TABLE II
Comparison of the rates of dioxygen formation
and
MCD bromination
versus hydrogen peroxide concentration
and
pH
Reaction conditions:
0.1 M
phosphate buffer containing Na,SO, (p
= 0.97
M),
0.1
M
Br- with or without 50
pM
MCD. To limit anoma-
lously high rates of dioxygen formation which were not V-BrPO
catalyzed but which were possibly catalyzed by metal ion contami-
nents, the glass chambers used with the YSI oxygen probe were
soaked in NoChromix and the buffers were filtered through a Bio-
Rex ion exchange membrane (Bio-Rad).
PH No MCD
50.0
JIM MCD
HzO, [Enz] ~ dlO,l/dt” -dlMCDl/dt dlO,l/dt”
?7lM
4.0 2.0
400.0
RM
3
3
5.0 2.0
100.0 3
3
5.5 2.0
40.0 3
3
12.5 + 0.5 12.4 + 0.3
23.2 + 0.2 22.5 + 1.5
32.3 t 0.9 30.7 k 0.9
38.1 + 0.6 33.9 + 1.1
29.7 f 0.9 30.2 + 1.0
31.7 f 0.3 28.4 + 0.1
6.0 2.0 3 30.8 f 1.5 30.0 + 0.4
20.0 3 30.8 + 0.2 21.6 + 0.2
6.5 0.5 3
1.0 3
2.0 3
4.0 3
10.0 3
20.0 3
40.0 3
100.0 3
23.9 k 1.2 20.8 + 0.5
26.0 + 0.3 21.2 + 0.8
26.7 + 0.1 21.8 + 0.5
26.4 f 0.3 20.8 f 0.4
26.2 f 0.2 16.7 + 0.8
23.4 + 0.5 11.2 + 0.1
20.9 * 0.2 7.2 f 0.2
15.8 f 0.2 3.0 + 0.1
7.0 0.5 6 32.7 f 0.5 30.8 f 0.8
1.0 6 34.6 + 0.1 30.0 f 0.5
2.0 6 34.1 + 0.3 27.6 + 0.3
8.0 0.5 12 29.9 + 0.7 25.8 f 1.8
1.0 12 28.9 -c 0.2 23.0 + 1.6
2.0 12 30.4 + 0.4 22.3 + 0.5
ND*
ND
0.3 + 0.2
2.4 + 0.2
0.1 k 0.0
2.3 + 0.2
2.0 f 0.3
9.7 + 0.8
1.2 + 0.9
2.2 f 0.1
2.7 k 0.2
3.3 + 0.1
8.5 f 0.3
10.9 -c 0.2
11.3 + 0.1
11.5 + 0.5
2.5 + 0.4
5.8 f 0.5
8.1 + 0.3
2.0 + 0.1
4.2 + 0.3
6.2 + 0.1
’ Dioxygen formation does not occur in the absence of bromide,
iodide, or V-BrPO.
* ND, not detectable.
trolled by the ratio of Izi[MCD] to kS[H201]. Indeed, as the
hydrogen peroxide concentration is raised, dioxygen forma-
tion does occur concomitant with bromination of 50 ~.LM MCD
(e.g. see pH 6.5 data, Table II). The sum of the rate of dioxygen
formation that occurs during MCD bromination (column 6)
and the rate of MCD bromination (column 5) is equal to the
rate of dioxygen formation in the absence of MCD (column
4), indicating that the fate of the intermediate is accounted
for by the MCD bromination and dioxygen formation path-
ways, at all pH values investigated.
The apportionment of the reaction through the MCD bro-
mination and dioxygen formation pathways depends on pH.
The concentration of hydrogen peroxide which effects a sig-
nificant (i.e. defined as X-10%) rate of dioxygen formation
concomitant with MCD bromination decreases dramatically
with an increase in pH (Table II). At pH 4, the bromide-
assisted disproportionation of hydrogen peroxide is not com-
petitive with MCD bromination (50 ELM) even at hydrogen
peroxide concentrations as high as 0.4 M. At pH 5.5, 40 mM
Hz02 induces the dioxygen formation reaction to occur at 8%
of the rate of MCD bromination. On the other hand at pH 8,
only 0.5 mM H202 is required to induce a significant appor-
tionment of the reaction through the dioxygen formation
pathway. Clearly at higher pH, dioxygen formation competes
very effectively with MCD bromination.
One consequence of the competition between dioxygen for-
mation and MCD bromination is manifested in pH profiles
of MCD bromination; previous investigations of the pH de-
pendence of V-BrPO were reported at 50 PM MCD and 5 mM
hydrogen peroxide (4). Under these conditions, the rate of
MCD bromination at pH 6.5 is 20% lower than the rate of
dioxygen formation, since k2[H202] competes effectively with
ki[MCD]. At higher hydrogen peroxide concentration, this
effect is even more pronounced (Fig. 5). Clearly, the bromide-
assisted disporportionation of hydrogen peroxide is a better
measure of the pH dependence of V-BrPO. A plot of the pH
dependence of oxygen formation rate, at 0.1 M bromide and
0.5-4.0 mM hydrogen peroxide, is shown in Fig. 6. The pH
optimum is similar to that reported by the MCD bromination
reaction (4), although the profile at higher pH does not drop
off as fast.
It is also apparent from the rate data in Table II at pH 6.5
that high concentrations of hydrogen peroxide inhibit the rate
of the bromide-assisted catalatic reaction. Above 10 mM hy-
14.0 -
000 02-evolution
W MCD-bromination
FIG.
5. pH profile of the MCD bromination reaction (m) and
bromide-assisted disproportionation of hydrogen peroxide (0)
at 10 mM hydrogen peroxide, 100 mM bromide.
Buffers used
are listed under “Materials and Methods.” Note the much lower rate
of MCD bromination compared with dioxygen formation for pH 2 6.
15676
Steady State Kinetic Mechanism of Vanadium Bromoperoxidase
ow 0.5 mM H202
mu 1 .O mM H202
tti 2.0 mM H202
W 4.0 mM H202
0.0 I , , , , , , , , ,
3.0 4.0 5.0 6.0 7.0 8.0 9.0
PH
FIG. 6. pH dependence of the rate of the bromide-assisted
disproportionation of hydrogen peroxide as a function of pH
and hydrogen peroxide concentration.
Reaction conditions are
0.1 M bromide, 3-12 nM V-BrPO deoending on the uH and 0.5-4.0
mM hydrogen peroxide. 0, 0.5 mM H;Os; &-LO mM &Oz; A,
2.0
mM
H202; +, 4.0 mM H,O,. The buffers used are listed under “Materials
and Methods.” The steady state rate of dioxygen formation was
normalized to the enzyme concentration.
drogen peroxide, the steady state rate of dioxygen formation
decreases. The rates reported in Table II were determined
within the first 3 min after initiating the reaction by the
addition of V-BrPO. We have compared the specific activity
of V-BrPO before and after turnover under the high hydrogen
peroxide concentrations to ascertain whether V-BrPO is ir-
reversibly inhibited. After complete turnover of 100
mM
hy-
drogen peroxide, the enzyme is completely inactivated, al-
though incubation of V-BrPO with 100
mM
hydrogen peroxide
under nonturnover conditions (i.e. without bromide) for 3 min
did not reduce the bromoperoxidase activity. However if V-
BrPO is allowed to react for only 3 min with 100
mM
hydrogen
peroxide and 100
mM
bromide, the specific activity decreases
only slightly. Since the hydrogen peroxide has not been com-
pletely consumed within 3 min, 10 pM ferrous ammonium
sulfate or ferric chloride was added to decompose the hydrogen
peroxide. The iron was removed by ultrafiltration (Centricon)
and the specific activity of V-BrPO re-determined. 7581%
of the bromoperoxidase activity was retained depending on
the pH (i.e. 6.5-8). However, in these experiments, in contrast
to the ones in which all the hydrogen peroxide was allowed to
turnover by V-BrPO, the bromoperoxidase activity could be
fully
restored by the addition of vanadate. Thus it is clear
that the inhibition is not simply due to irreversible inactiva-
tion of V-BrPO. The mechanism of the apparent inhibition
by hydrogen peroxide and the mechanism of the possible
vanadate loss in the presence of high concentrations of hy-
drogen peroxide are under investigation.
DISCUSSION AND CONCLUSIONS
In this study, we have compared the steady state kinetic
mechanism of the bromide-assisted disproportionation of hy-
drogen peroxide to the steady state kinetic mechanism of
MCD bromination, both catalyzed by V-BrPO. The kinetic
analysis of dioxygen formation (Figs. 1A and 2A) and MCD
bromination (Figs. 1B and 2B) is consistent with a “substrate-
inhibited Bi Bi Ping Pang” mechanism, in which bromide
functions as a substrate and as a noncompetitive inhibitor of
hydrogen peroxide binding. The kinetic parameters (Table I)
obtained from the bromide-assisted disproportionation of hy-
drogen peroxide and the MCD bromination reactions agree
within a factor of about 2, which indicates that the rate-
limiting step for both reactions is the same. The inhibitory
complex formed with bromide occurs between pH 4 and 6 at
relatively high bromide concentrations. In addition, fluoride
is a competitive inhibitor of hydrogen peroxide binding,
thereby inhibiting the rate of bromination of MCD and the
bromide-assisted disproportionation of hydrogen peroxide
catalyzed by V-BrPO (Fig. 4).
The equality of the maximum rate of dioxygen formation
(i.e. in the absence of MCD) and the sum of the rates of MCD
bromination and dioxygen formation during MCD bromina-
tion (see Table II) is further support that both the bromide-
assisted catalatic reaction and the MCD bromination reaction
occur via the formation of a common intermediate and that
the rate of formation of this intermediate is limiting (11).
Clearly, the fate of the intermediate is fully accounted for by
the k,[MCD] and k2[H202] pathways, independent of pH,
within pH range 4-8, and hydrogen peroxide concentration.
The competing dioxygen formation reaction is enhanced at
higher pH, since much lower concentrations of hydrogen
peroxide are required to effect a significant apportionment of
the reaction through the pathway of bromide-assisted dispro-
portionation of hydrogen peroxide. The mechanism of pH-
induced partitioning is still under investigation.
Scheme 2 presents a mechanism consistent with the kinetic
data, in which “Inh” represents the inhibitor, bromide, or
fluoride. Bromide or fluoride forms an abortive complex with
native V-BrPO and this reaction is competitive with H202
binding (i.e. described by Ki,lnh). In addition, bromide also
binds to the enzyme-peroxo complex to form a ternary com-
plex (i.e. described by KiiInh) leading to uncompetitive inhibi-
tion by bromide. Although Scheme 2 depicts binding of hy-
drogen peroxide before the substrate bromide, in actuality,
the order of binding of hydrogen peroxide and bromide cannot
be established.
The binding sites of hydrogen peroxide and bromide are
not known. By analogy to the coordination reactivity of
hydrogen peroxide and vanadate (V) in aqueous solution (25
28), it seems likely that hydrogen peroxide coordinates to
vanadium (V) at the active site. The question of bromide
binding to vanadium is more difficult to address. 51V NMR
studies show that bromide does not coordinate to vanadate or
other simple vanadium (V) complexes in aqueous solution.*
The catalytic role of vanadium in V-BrPO is also not known
yet. Vanadium (V) could function as an electron transfer
catalyst or a Lewis acid catalyst. Vanadium (V)-peroxide
complexes are strong oxidants (29) which could oxidize halide
ions without inducing vanadium oxidation state changes (i.e.
a Lewis acid role). On the other hand, bromide can reduce
vanadium (V) as demonstrated recently by the formation of
dibromotetraethyleneglycolvanadium(III) from V(V)-diglyme
and hypobromous acid (30).
Recently a series of vanadyl dicarboxylate complexes have
been reported to be V-BrPO mimics (31). The proposed
mechanism involves reduction of hydrogen peroxide by V(IV),
forming hydroxyl radical, subsequent reaction of V(V) with
hydrogen peroxide and bromide forming [V(V)(OH)(02)Brl-,
and finally reaction of 2 equivalents of hydroxyl radical with
[V(V)(OH)(O,)Br]- to produce HOBr. Several steps in this
proposed mechanism are energetically unfavorable; for ex-
ample, excess bromide in solution could react with hydroxyl
radical, instead of invoking reactions with the proposed bro-
moperoxo-V(V) complex. Another problem is that vanadyl
bromoperoxidase apparently is not oxidized by hydrogen per-
oxide and VO’+-BrPO does not catalyze the bromination of
’ R. I. de la Rosa, and A. Butler, work in progress.
Steady State Kinetic Mechanism
of
Vanadium Bromoperoxidase
15677
SCHEME 2
Br-MCD + V-BrPO
V-BrPO(lnh) V-BrPOQi,O,Nlnh)
InhllK?: •H20~ InhpF + Br-
,,-BrPO . * V-BrPO(H,P) -
MCD
(12).
Moreover, the reaction is not catalytic. Thus it is
highly unlikely that the vanadyl complexes are true vanadium
bromoperoxidase mimics.
de Boer and Wever (16) have proposed that tribromide is
the active brominating species produced by V-BrPO. They
reported observation of tribromide formed by V-BrPO under
turnover conditions at low pH (pH 5), low hydrogen peroxide
concentration (0.3 mM) 0.1 M bromide, and very high enzyme
concentration (i.e. 130 nM) (16). Low concentrations of hy-
drogen peroxide, acidic pH, and excess bromide are conditions
that favor the formation of tribromide and stabilize it against
reaction with hydrogen peroxide. Under acidic pH, HOBr and
bromamines (e.g. N-bromosuccinamide, N-bromotris) react
rapidly with excess bromide, giving tribromide (32); thus it
may not be possible to establish the identity of the brominat-
ing intermediate unambiguously. At higher pH, tribromide or
hypobromous acid cannot be detected in the V-BrPO cata-
lyzed reactions because a second equivalent of hydrogen per-
oxide reacts very rapidly with the oxidized bromine interme-
diate, producing singlet oxygen (13). Since reaction of hydro-
gen peroxide with oxidized bromine species (e.g. hypobromous
acid, bromine, brominated amines, etc) is very fast, the nature
of the active brominating intermediate in V-BrPO at neutral
pH, has not yet been determined.
In conclusion we have established that the steady state
kinetics of the bromide-assisted disproportionation of hydro-
gen peroxide is the same as for MCD bromination. Both
reactions proceed through the formation of a common inter-
mediate and partitioning of the intermediate is accounted for
by the reaction with MCD or a second equivalent of hydrogen
peroxide. We are continuing our investigations of the pH
dependence of the competition between dioxygen formation
and MCD bromination and the mechanism of the apparent
inhibition by hydrogen peroxide,
Acknowledgments-We thank Dr. Robert Petty of the Marine
Science Institute at the University of California, Santa Barbara, for
use of the atomic absorption spectrometer. We thank Profs. W. W.
Cleland (University of Wisconsin), Charles B. Grissom (University
of Utah), Daniel Purich (University of Florida), and Ming Tien
(Pennsylvania State University) for helpful discussions on the steady
state kinetic analyses.
APPENDIX
The rate equation for an enzyme-catalyzed Ping Pong re-
action with two substrate, H,O, and Br- is
v=
K_“Z’h[Br] + K,,,a’[H20~] +
[H2021FW (1)
Inh
II
K ‘Fib ‘02 + V-BrPO
which can be cast in the form of a rectangular hyperbola for
fixed values of [Br-1:
(2)
where
V
V=L K Hz02
KmB’ K=A K_R’ (3,4)
l+[Brl l+[Brl
Thus plots of
l/u
versus 1/[H202] are linear and parallel at
varying [Br-] since:
1 1 K
-=-+- (5)
” V V [H,Oz]
Competitive bromide inhibition was fit to
(6)
Noncompetitive bromide inhibition was fit to
Vnmx [Hdhl
“=K,H+‘++~)+[HzO++&$ f7)
KmBr
is not present in Equations 5 and 6, because the high
bromide concentrations required for bromide-inhibition sat-
urate the
K,,,BT
equilibrium. The full rate equation including
K,*’
for competitive and noncompetitive inhibition is given
below.
For competitive inhibition,
K
and
V
are defined by
Thus plots of l/v uersus 1/[H202] at fixed [Br-]
and should intersect on the y axis.
(69)
are linear
For noncompetitive inhibition,
K
and
V
are defined by
(10, 11)
Thus plots of
l/u
uersus 1/[H202] at fixed [Br-] are linear
and intersect to the left of the .y axis.
15678
Steady State Kinetic Mechanism
of
Vanadium Bromoperoxidase
Plots of K/V uersus [BY] should be linear:
15. Kanofsky, J. R. (1989) in Oxygen Radicals in Biology and Medicine
(Simic, M. G., Taylor, K. A., Ward, J. F., and von Sonntag, C.,
K K H?On KmHz%[Br-]
m
v=v,,,+ eds) pp. 211-218, Plenum Publishing Corp. New York
( 1
(12)
KS’ Vm., 16. de Boer, E., and Wever, R. (1988) J. Btil. Chem. 263, 12326-
12332
17. Thomas, J. A., Morris, D. R., and Hager, L. P. (1970) J. Biol.
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Figure A
Figure B
0.00 ! I I I I I I 0 1 u 1
0 10 20
l/ H202
(mM-I)*’
30 50
0.00 1 . I 9 , I , - I - 1
0.0
0.5 1.0 1.5 2.0 2.5
l/ H202 (mM-‘)
Steady State Kinetic Mechanism
of
Vanadium Bromoperoxidase
15679
0.20
0.16
0.04
Figure C
o.oo! I , I , I , I I
0 , ,
20 40 60 60 100
l/ H202 (mM-‘)
0.03
0.02
A
s
2.
<
0, 0.01
0.00 I I 1 I I I , I , I I
0.0
0.5 1.0 1.5 2.0 2.5
1/ H202 (mM-‘)
Figure D
