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Oxidation reactions catalyzed by Vanadium peroxidases.

ten Brink, H.B.

Publication date2000

Link to publication

Citation for published version (APA):ten Brink, H. B. (2000). Oxidation reactions catalyzed by Vanadium peroxidases.

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Download date:19 Aug 2021

ChapterChapter 5

Oxidationn Reactions Catalyzed by Vanadium Chloroperoxidases from CurvulariaCurvularia inaequalis

J.J. Inorg. Biochem. 2000 accepted

Hildaa B. ten Brink,1 Henk L. Dekker,1 Hans E. Schoemaker2 and Ron Wever1

11 E. C. Slater Institute, Biocentrum, University of Amsterdam, Plantage Muidergracht 12, 10188 TV Amsterdam, The Netherlands 22 DSM Research, Bio-organic Chemistry, P.O. Box 18, 6160 MD Geleen, The Netherlands

CfiapterS CfiapterS

Oxidationn Reactions Catalyzed by Vanadium Chloroperoxidase from CurvulariaCurvularia inaequalis

Hildaa B. ten Brink,3 Henk L. Dekker/ Hans E. Schoemakerb and Ron Wever3

11 E.C. Slater Institute, University of Amsterdam, Plantage Muidergracht 12,1018 TV Amsterdam, Thee Netherlands. bDSMM Research, Bio-Organic Chemistry, P.O. box 18, 6160 MD Geleen, The Netherlands

Abstract t

Vanadiumm haloperoxidases have been reported to mediate the oxidation of

halidess to hypohalous acid and the sulfoxidation of organic sulfides to the

correspondingg sulfoxides in the presence of hydrogen peroxide. However,

traditionall heme peroxidase substrates were reported not to be oxidized by

vanadiumm haloperoxidases. Surprisingly, we have now found that the recombinant

vanadiumm chloroperoxidase from the fungus Curvularia inaequalis catalyzes the

oxidationn of ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)], a

classicall chromogenic heme peroxidase substrate. The enzyme mediates the

oxidationn of ABTS in the presence of hydrogen peroxide with a turnover frequency

off 11 s"1 at its pH optimum of 4.0. The Km of the recombinant enzyme for ABTS

wass observed to be approximately 35 \iM at this pH value. In addition, the

bleachingg of an industrial sulfonated azo dye, Chicago Sky Blue 6B, catalyzed by

thee recombinant vanadium chloroperoxidase in the presence of hydrogen peroxide

iss reported.

Introduction n

Vanadiumm haloperoxidases catalyze the oxidation of halides in the presence of

hydrogenn peroxide to a highly reactive intermediate, the corresponding hypohalous

acid,, which may either react with a suitable nucleophilic acceptor, if present,

formingg a halogenated compound [1, 2] or with hydrogen peroxide, yielding !02 [3,

4].. A variety of halogenated organic compounds, ranging from simple volatile

halohydrocarbonss (pollutants of the atmosphere) [5, 6] to relatively complicated

chirall structures (antibiotic activity) [7], are believed to be the natural products of

thee vanadium haloperoxidases.

Thee vanadium haloperoxidases are named according to their oxidation ability;

vanadiumm iodoperoxidases only oxidize iodide, while vanadium bromoperoxidases

82 2

aree also able of oxidizing bromide and vanadium chloroperoxidases mediate the

oxidationn of chloride, in addition to bromide and iodide. The vanadium iodo-and

bromoperoxidasess are predominantly found in marine organisms [8] and vanadium

chloroperoxidasess mainly originate from terrestrial fungi [9]. The active site of the

vanadiumm haloperoxidases harbors a vanadium metal, which is present as vanadate

andd resides in the highest oxidation state as vanadium(V), also during catalysis. A

well-knownn feature of these enzymes is their remarkable stability towards oxidative

inactivation,, the presence of high concentrations of organic solvents and elevated

temperaturess [10-12].

Thee vanadium chloroperoxidase (VCPO) from the fungus Curvularia

inaequalisinaequalis has been studied in great detail [10, 13]. Several extensive kinetic studies

havee been carried out [13-15] and both the primary structure [16] and the X-ray

structuree of the native enzyme [17] and the peroxo-intermediate have been

determinedd [18]. The vanadium chloroperoxidase can now be obtained in large

quantitiess from a developed Saccharomyces cerevisiae expression system [15]. As

thee recombinant VCPO (r-VCPO) behaves kinetically very similar to the native

enzymee from C. inaequalis, after activation with vanadate, the recombinant enzyme

iss used in most of our present research [15, 19]. Site-directed mutagenesis of highly

conservedd active site residues has been conducted and the effect on catalytic activity

hass been studied in great detail in order to elucidate the role of these amino acids in

halidee oxidation catalysis [15, 20].

Recently,, it has been established that vanadium haloperoxidases are also able

too mediate the oxidation of organic sulfides to the corresponding sulfoxide in the

presencee of hydrogen peroxide [21-23]. Vanadium bromoperoxidases (VBPO's)

weree shown to catalyze sulfoxidation reactions in a highly selective manner. The

vanadiumm bromoperoxidase (VBPO) from the brown seaweed Ascophyllum

nodosumnodosum converts methyl phenyl sulfide to the corresponding (i?)-sulfoxide with up

too 96% enantiomeric excess (ee) [21]. The VBPO from the red seaweed Corallina

officinalisofficinalis was observed to convert organic sulfides structurally resembling indenes

andd small sulfides, possessing a cis-positioned carboxyl group with respect to the

sulfurr atom, to the (5)-enantiomer of the corresponding sulfoxide showing high

selectivitiess exceeding 95% ee [22, 23]. As expected the presence of halides was

observedd to cancel the selectivity completely. In contrast, the r-VCPO was observed

too mediate the formation of only racemic sulfoxides [21].

Anotherr class of peroxidases, harboring a heme group as the prosthetic group

inn the active site, has been shown to catalyze not only the oxidation of halides and

83 3

sulfides,, but also the oxidation of various other organic compounds [24-26]. The

oxidationn of substrates like halides and sulfides is in general described as two-

electronn transfer mechanism, whereas the heme peroxidases also catalyze oxidation

reactionss through a 1-electron transfer mechanism. The heme peroxidases are

knownn to catalyze the oxidation of several organic compounds, including o-

methoxyphenoll (gaiacol), o-dianisidine and 2,2'-azino-bis(-3-ethylbenzthiazoline-6-

sulfonicc acid) (ABTS), Scheme la, in the presence of hydrogen peroxide through a

radicall oxidation mechanism. The oxidation of these chromogenic compounds is

frequentlyy used to assay for peroxidase activity [27-29]. In particular the latter due

too the high solubility and stability of both the substrate and product (ABTS+) in

waterr and because it is neither toxic nor carcinogenic [30, 31].

Thee vanadium bromoperoxidases did not show activity in the traditionally

usedd assay methods for peroxidase activity [32]. Therefore the peroxidative

halogenationn of monochlorodimedone [33, 34], has been used for the vanadium

enzymess to determine activity. Until now the vanadium peroxidases were thought to

bee unable to mediate radical oxidation reactions. In contrast, it has been shown that

severall vanadium(V) peroxo-complexes, which structurally resemble the peroxo-

intermediatee of the VCPO [18], are able to mediate the oxidation of a variety of

organicc compounds through the radical oxidation mechanism [35].

\\ J >=N-N=<

1a a

NH2OHH / = / = OH NH2

- O 3 S ^ X N = N H H

Kf^^Kf^^ H3CO SO3--

Now,, we demonstrate that ABTS can be oxidized by the r-VCPO in the

presencee of hydrogen peroxide. It was observed that only highly purified enzyme

preparationss exhibit ABTS oxidation activity. In some experiments lactoperoxidase

wass used for comparison. In addition, the r-VCPO catalyzed oxidation of an

industriall sulfonated azo dye [36], Chicago Sky Blue 6B (Scheme lb), is

demonstrated. .

84 4

Materialss and Methods Thee vanadium chloroperoxidase is obtained from the developed S. cerevisiae

expressionn system and after activation with vanadate the r-VCPO behaves

kineticallyy very similar to the native enzyme from C. inaequalis. The isolation and

purificationn of the enzyme was conducted as described [15].

Thee enzymatic activity of the r-VCPO was determined spectrophotometrically

byy measuring the formation of ABTS+ from ABTS at 414 nm (s = 36.0 rnM^cm"1)

onn a Varian Gary-17 spectrophotometer or on a Hewlett Packard 8452A diode array

spectrophotometerr supported by PC. The ABTS oxidation activity was measured in

1000 mM sodium acetate buffer (pH 4.0) with r-VCPO (150 nM) and ABTS (1.7

mM).. Hydrogen peroxide (2.5 mM) was added to start the reaction, unless

otherwisee specified.

Forr the oxidation of Chicago Sky Blue 6b r-VCPO (150 nM) was incubated in

1000 mM of sodium acetate buffer (pH 4.0) with the dye (10 uM) and H202 (2 mM)

forr 24 hours at room temperature. The oxidation activity was measured

spectrophotometricallyy by following the degradation of the azo dye at 610 nm [36].

Results s OxidationOxidation of ABTS

Thee one-electron oxidation reaction of ABTS (la) by r-VCPO from the fungus

C.C. inaequalis in the presence of hydrogen peroxide was assayed by measuring the

increasee in absorbance at 414 nm owing the formation of the positively charged

ABTSS radical. The initial rate of the reaction was used for kinetic analysis as it was

observedd that the rate of formation of ABTS+ gradually decreases in time. No

increasee in absorbance could be observed when apo-recombinant enzyme was used,

howeverr after the addition of an excess of vanadate (10 uM) the formation of

ABTS++ could clearly be monitored at 414 nm (results not shown).

Thee ABTS oxidation activity of vanadium haloperoxidases using the VBPO

fromfrom A. nodosum and the native VCPO from C. inaequalis was also previously

measured,, however enzymatic oxidation activity was not observed. During our

studiess using the r-VCPO we observed that some enzyme preparations exhibited

ABTSS oxidation activity, whereas others were unable to mediate the formation of

thee ABTS radical or at a very low level. However, on SDS gels after staining for

protein,, these preparations looked very similar. Therefore non-protein

contaminationss may be responsible for the observed difference and an additional

purificationn step was included in the purification procedure of r-VCPO using a

85 5

Poross HQ column (a strong anion-exchanger) on a FPLC system after the final

DEAEE ion-exchange. The resulting enzyme preparations were observed to exhibit

increasedd ABTS oxidation activity. However, the chloride oxidation activity of

thesee preparations is not affected by the presence of additional components, since

thee specific chlorination activity before and after the additional purification step

remainedd the same.

Too determine whether an intrinsic factor was responsible for the suppressed

ABTSS oxidation activity of r-VCPO three separate ABTS oxidation reactions were

investigatedd using a r-VCPO preparation only purified by a DEAE column (2 " "^ o

nmm of 1.4), the same preparation after the additional Poros HQ column purification

(2600 nm/28o nm of 0.5) and an enzyme preparation consisting of an equimolar mixture

off both the contaminated and highly purified preparation. The same concentration

off r-VCPO was used in these three experiments. Hardly any formation of ABTS+

wass observed in the oxidation reaction catalyzed by the enzyme preparation, which

wass purified only on a DEAE column. The r-VCPO preparation after the final Poros

HQQ column step, however, mediates the conversion of ABTS with 0.4 s" using 10

pJVII ABTS and 0.5 mM of hydrogen peroxide at pH 4.0 (25 s"1 when

lactoperoxidasee is used under these reaction conditions). When the combined

enzymee preparation was used the ABTS oxidation was found to be cancelled. These

findingss establish that there is a factor in the enzyme preparation that prevents the

oxidationn of ABTS by r-VCPO. Highly purified enzyme preparations were used for

thee further studies.

V) )

< < o o

t j j ra ra b b

VCPOO (nM)

Figuree 1. The dependence of ABTS oxidation activity of r-VCPO on the enzyme concentration at pHH 4.0. The r-VCPO oxidation activity was determined by following the increase of absorbance at 4144 nm due to the formation of ABTS+ in time. In this particular experiment 1 mM hydrogen peroxidee was used.

Thee oxidation activity was found to be dependent on the concentration of r-

VCPOO present as can be inferred from Figure 1. The pH dependence of the r-VCPO

200 0

86 6

catalyzedd oxidation of ABTS is presented in Figure 2 and shows that the one-

electronn oxidation of ABTS is only mediated by the vanadium enzyme under acidic

reactionn conditions with an optimal pH of approximately 4.0 under these reaction

conditionss (see Materials & Methods). At a higher concentrations of ABTS the pH

optimumm shifts to a slightly higher pH of approximately 5.0 (results not shown).

However,, it was observed that the direct reaction between ABTS and hydrogen

peroxidee also significantly contributes to the conversion of ABTS at this pH. A

turnoverr frequency for r-VCPO of approximately 11 s~' for the radical oxidation of

ABTSS at the pH optimum of 4.0 was calculated from Figure 2 using an extinction

coefficientt of 36.0 mM~' cm"1 at 414 nm. By comparison, the maximal turnover

frequencyy for chloride oxidation by r-VCPO at pH 5.0 was observed to be 22 s"1

(unpublishedd observations). Prior studies have shown that a pH of 5.0 is optimal for

thee oxidation of halides to hypohalous acid by VCPO in the presence of H202 [13].

Apparentlyy the r-VCPO catalyzes the oxidation of ABTS under more acidic

conditionss and a steady-state kinetic analysis was conducted.

Figuree 2. The pH-dependence of the r-VCPO ABTS oxidation activity. For details see Materials andd Methods.

Thee Km of r-VCPO for ABTS was found to be approximately 35 uM at a pH

off 4.0 (not shown) and the Km for hydrogen peroxide was approximately 120 uM

(dataa not shown), a value close to that obtained from steady-state kinetics of the

enzymaticc chlorination reaction [14]. The results of the steady-state experiments

showw that the r-VCPO catalyzed ABTS oxidation follows classical Michaelis-

Mentenn kinetics. Due to an increased contribution of the non-enzymatic reaction

betweenn ABTS and H202 at higher pH values and a strongly decreased ABTS

oxidationn rate at lower pH values (Figure 2), it was not possible to determine the

kineticc parameters in detail.

87 7

Sincee a specific binding site for organic compounds in the active site of the

vanadiumm bromoperoxidase from A. nodosum was suggested to be present [37] we

havee tried to identity such a site for ABTS in the r-VCPO. Increasing amounts of

concentratedd r-VCPO (from 0.5 uM to 20 uM final concentration) were added to a

solutionn containing 20 pM of ABTS in 100 mM acetate buffer (pH 4.0) and the

absorbancee at 340 nm (absorbance maximum of ABTS, 8340 = 36 mM"1 cm"1) was

monitored.. Indeed, the absorbance of ABTS at 340 nm decreased at higher enzyme

concentrationss indicating that ABTS binds to r-VCPO. No ABTS+ was formed

underr these conditions, as no H202 was present. When BSA was used, however,

insteadd of r-VCPO similar quenching of the absorbance of ABTS was observed

(resultss not shown). Therefore the presence of a specific binding site for organic

compoundss in r-VCPO could not be assessed. Unfortunately, it is not possible to

studyy the r-VCPO directly using optical spectrophotometry.

~~ 0.5 5 5 <. . 11 0.25 n n t_ _ o o n n

X> X>

<< 0

Figuree 3. The r-VCPO assisted formation of ABTS+ in time at pH 4.0. The oxidation of ABTS catalyzedd by r-VCPO was followed in time at 414 nm. In this particular experiment 350 nM of r-VCPO,, 10 uM of ABTS and 100 uM of H202 was used. After 720 s 10 uM of ABTS was added andd after 840 s 350 nM of r-VCPO was added.

Thee stoichiometry between H202 consumption and ABTS+ formation was

examinedd to obtain a better understanding of the nature of the r-VCPO catalyzed

ABTSS oxidation. Hydrogen peroxide harbors two oxidizing equivalents, which

impliess that at the most two ABTS molecules can be converted by one molecule of

H202.. Therefore, a substoichiometric amount of hydrogen peroxide (50 uM) was

addedd to ABTS (100 pM) in the presence of 350 nM r-VCPO at pH 4.0. The

reactionn was monitored by following the increase in absorbance at 414 nm and 640

nmm due to the formation of ABTS+. Under these reaction conditions 43 pM of

ABTS++ is formed (results not shown). However, when equimolar amounts of ABTS

andd H202 (100 pM) are used also merely 45 pM of ABTS is oxidized by the

enzymee (results not shown). In both reactions a gradual decrease in the rate of

3000 600 900 1200 timee (s)

88 8

ABTSS conversion was observed before reaching the final absorbance corresponding

too approximately 45 pM of ABTS+. By contrast, when lactoperoxidase (2 nM) is

presentt instead of r-VCPO in these reactions it was observed that, as expected,

approximatelyy two equivalents of ABTS+ are formed at the expense of one

equivalentt of H2O2.

AA study on the gradual inhibition of the enzymatic oxidation of ABTS reveals

thatt the enzyme is slowly inactivated during catalysis. The increase in absorbance

duee to the formation of ABTS+ gradually levels off in time as can be seen in Figure

3.. The addition of hydrogen peroxide and ABTS at this stage does not influence the

ratee of ABTS+ formation and also the addition of small amounts of chloride during

thee reaction does not effect the rate of ABTS oxidation (results not shown).

However,, upon addition of r-VCPO the absorbance increases again with about the

samee rate as the initial rate of oxidation induced by the first enzyme aliquot.

00 20 40 0 50 100

1'[H202]] 1/[ABTS]

00 20 40 0 50 100

activityy (A A414 nm/s)/[H202] activity (A A414 nm/s)/[ABTS]

Figuree 4. The determination of the binding order of ABTS and hydrogen peroxide in the r-VCPO catalyzedd oxidation of ABTS. Double-reciprocal plots of a) fixed concentrations of ABTS (10 uM

,, 20 uM , 40 uM (A) and 80 uM ) against varying concentrations of H202 and b) fixed concentrationss of H202 (25 uM , 50uM , 100 uM (A) and 200 uM ) against varying concentrationss of ABTS. The same data are plotted as Eady-Hofstee plots in Figure 4c and 4d, respectively. .

AA more detailed investigation was conducted in order to elucidate the binding

orderr in the ABTS oxidation mediated by r-VCPO in the presence of hydrogen

89 9

peroxide.. The steady-state oxidation activity of r-VCPO was studied by varying the

concentrationn of ABTS at a hydrogen peroxide concentration, which was fixed at

differentt concentrations in the proximity of the Km value, and vice versa. The

primaryy plots in the form of double-reciprocal plots are shown in Figure 4. Clearly

thee lines are not parallel but intercept in the second or third quadrant (Figure 4a and

4b,, respectively). Also the Eady-Hofstee plots are not parallel or intercept at the X-

axiss (Figure 4c and 4d). Unfortunately, it is therefore difficult to establish a binding

orderr for the r-VCPO catalyzed oxidation of ABTS.

Duringg the investigations it was discovered that the r-VCPO oxidation of

ABTSS is easily and completely inhibited. Low concentrations of several detergents,

includingg SDS (0.05%), Tween 80 (0.1%) and Triton (0.1%), were observed to

cancell the catalytic formation of ABTS+ . Also the choice of buffer was observed to

bee essential as experiments conducted in either citrate or Tris containing buffers

weree observed to yield irreproducible results, probably due to a secondary reaction

betweenn ABTS+ and the buffer [38].

1/1 1

3 3 <a a CM M O O

' —

'u 'u a a

o o

1bb r I I I

10 0

55 -

00 50 100 150 200 250

azidee cone. (nM)

Figuree 5. Azide inhibition of the ABTS oxidation activity of r-VCPO at pH 4.0. In this particular experimentt 30 nM of r-VCPO was used.

Azidee is known to bind irreversible to the vanadium metal consequently

inhibitingg vanadium peroxidase catalyzed halide oxidation [19]. Indeed, the r-

VCPOO catalyzed oxidation of ABTS is inhibited strongly at low concentrations of

azidee as shown in Figure 5. Equimolar amounts of azide and r-VCPO in the nM

rangee reduce the rate of ABTS oxidation to approximately 25% of the initial

uninhibitedd rate. It is clear that azide in a nearly stoichiometric manner is able to

preventt the enzymatic formation of ABTS+.

OxidationOxidation of Chicago Sky Blue 6B

Inn addition to the studies on the r-VCPO catalyzed oxidation of ABTS in the

presencee of H202, we studied the oxidation of a comparable, but more complex

90 0

structure,, Chicago Sky Blue 6b [Direct Blue 1] ( lb) [36]. The oxidation activity

wass followed by measuring the decrease in absorbance at 610 nm due to the

disproportionationn of the blue sulfonated azo compound. Figure 6 shows the gradual

bleachingg of Chicago Sky Blue by r-VCPO in the presence of H202. As has been

observedd for the enzyme-catalyzed oxidation of ABTS, the rate of bleaching of the

bluee dye by r-VCPO slowly decreases in time. However, it is clear that the r-VCPO

catalyzedd oxidation of Chicago Sky Blue is a much slower process than the enzyme-

assistedd oxidation of ABTS. No oxidation of the organic structure was observed in

thee absence of the enzyme or in the presence of an equal amount of unactivated

enzyme,, indicating that the r-VCPO is involved in the oxidation.

__ 1 5 5 ££ 0.75 o o Ü Ü

|| 0.5

88 0.25

Figuree 6. Oxidation of Chicago Sky Blue 6B by r-VCPO. Optical absorption spectra of 10 uM of thee dye after 24 hours incubation in: a) sodium acetate buffer (pH 4.0), b) with 2 mM H202 and c) inn the presence of 2 mM of H202 and 150 nM r-VCPO.

Althoughh the unactivated CPO is not able to catalyze the oxidation reaction,

ann initial decrease in absorbance (quenching) of the dye could be noticed. As

observedd for ABTS the dye probably binds on the surface of the enzyme. This is

supportedd by the fact that a blue precipitation was formed when the r-VCPO

concentrationn was increased to 0.5 uM. We also tried to study the relation between

enzymee concentration and oxidation of the dye, however due to the extensive

bindingg of the dye on the enzyme surface it was difficult to obtain accurate data

relatingg the oxidation activity and enzyme concentration.

Becausee halides are known to be present in trace amounts the following

experimentt was conducted in order to prove that Chicago Sky Blue is oxidized

withoutt the involvement of halides. The blue dye (10 uM) was incubated for 3 days

inn the presence of active enzyme (150 nM) and H202 (2 mM) in buffered solution

(pHH 4.0). After 3 days the decrease in absorbance at 610 nm showed that the blue

dyee was bleached (not shown). Simultaneously, phenol red (40 uM) was incubated

5500 650 750

Wavelengthh (nm)

91 1

underr identical reaction conditions (the bromination of phenol red can be

determinedd from the increase in absorbance at 590 nm). However, phenol red was

nott halogenated under these conditions as the absorbance at 590 nm did not

increase,, indicating that Chicago Sky Blue is oxidized by r-VCPO directly.

Discussion n

Sincee the discovery of a new class of peroxidases, bearing vanadate in the

activee site as the prosthetic group, it was believed that these enzymes, in contrast to

thee earlier known heme peroxidases, were not able to catalyze the oxidation of

traditionall peroxidase substrates. However, we have found that highly purified

preparationss of r-VCPO catalyze the oxidation of ABTS to ABTS+ in the presence

off hydrogen peroxide with a turnover frequency of approximately 11 s"1 at the

optimall pH of 4.0. For comparison, we observed that the turnover frequency of

lactoperoxidasee exceeds that of r-VCPO by a factor of at least 100 under these

reactionn conditions (not shown).

Inn addition, the enzymatic oxidation of the somewhat structurally related

industriall dye Chicago Sky Blue 6b by r-VCPO was observed. The bleaching of a

commerciall dye by r-VCPO with hydrogen peroxide as oxidant has not been

observedd before. Lignin peroxidase has been known to catalyze the oxidation of

azoo dyes in the presence of hydrogen peroxide [39] and even a polymeric dye in the

presencepresence of the natural mediator, veratryl alcohol [40]. Several laccases are known

too oxidize azo dyes [36] and more complicated structures in the presence of a

mediatorr [41, 42].

Alsoo o-dianisidine was oxidized by r-VCPO and absorption spectra were

obtainedd of the products, which were different when halides were present or absent

duringg enzymatic oxidation (results not shown) indicating different oxidation

mechanisms.. Guaiacol and veratryl alcohol are not oxidized by r-VCPO (not

shown),, probably due to the high oxidation potential of these substrates, and also

ferrouss cyanide could not be oxidized, presumably due to the strong negative

charge. .

Itt is conceivable that halides in some way or the other are involved in the

enzymaticc ABTS oxidation considering the high affinity of the enzyme for halides

[14,, 43] and the fact that hypochlorous acid reacts with ABTS to form the positively

chargedd ABTS radical (results not shown). However, addition of chloride in

concentrationss possibly present in the ABTS assay did not influence the rate of

92 2

enzymaticc ABTS+ formation. In addition, the r-VCPO preparations were dialyzed

extensivelyy against buffered millipore solutions and did no longer contain halides.

Thee native VCPO from the fungus C. inaequalis was also used to study the

oxidationn of ABTS and the data were compared with those obtained from the

experimentss conducted with the recombinant system. The specific activity of the

nativee enzyme was observed to be a factor of 4 lower compared to the recombinant

system.. The native enzyme is colored due to a dye present in the growth medium

[13],, which is strongly bound to the enzyme. It may well be that the presence of this

dyee strongly affects the ABTS oxidation activity of the native enzyme. In this

contextt it should be noted that the sulfoxidation activity of the VBPO from A.

nodosumnodosum was also affected by the presence of a brown component present in the

enzymee preparation [21]. The nature of the component inhibiting the various

recombinantt enzyme preparations is not clear at present, though it can be removed

byy a more extensive purification of the preparation.

Itt was observed that the r-VCPO catalyzed formation of the ABTS radical

graduallyy decreases in time. The gradual suppression of ABTS oxidation appears to

bee a consequence of a slow inactivation of the enzyme during turnover, since the

ratee remains unaffected upon addition of hydrogen peroxide or ABTS during

catalysis.. It may well be that, as the first ABTS molecules are converted, the formed

ABTSS radicals inactivate the r-VCPO. However, the ABTS radical represents a

stablee structure [38] and inactivation of heme peroxidases due to an interaction with

ABTS++ has not been reported. We believe that inactivation of r-VCPO due to direct

interactionn with ABTS+ can be excluded as the initial rate of enzymatic ABTS

oxidationn can be completely restored by adding r-VCPO again to the assay in which

ABTS++ is already present (Figure 3). We assume that the inactivation of the enzyme

duringg turnover is an intrinsic process induced by the formation of ABTS and is,

therefore,, part of a presumably complex radical oxidation mechanism.

Forr haloperoxidases in general "ping pong mechanisms" are observed with an

orderedd binding of substrates [44]. Also for the VCPO a "bi bi ping pong

mechanism"" has been established in the catalyzed halide oxidation by vanadium

haloperoxidasess [14] and for the primary plots parallel-line kinetics are found [14,

46,, 45]. In the reaction cycle of the enzyme an enzyme peroxo-intermediate is

formedd first [18], which subsequently reacts with chloride. However, for the r-

VCPOO catalyzed oxidation of ABTS the lines in the primary plots are clearly not

parallell (Figure 4) suggesting a more random mechanism. Unfortunately, the

93 3

patternss observed in Figure 4 are also not consistent with any of the patterns known

forr other traditional catalyzed bisubstrate reactions, including random or

compulsoryy order mechanisms [45, 46]. Probably the nature of the binding order

andd the actual ABTS oxidation mechanism of VCPO are more complex and consist

off a mixture of the described mechanisms.

Theree are two likely possibilities to be considered: a vanadium peroxo-

intermediatee is formed, which oxidizes ABTS in two successive one-electron

oxidationn steps, or alternatively ABTS may reduce the vanadium in the enzyme to a

lowerr valence state, which is re-oxidized by hydrogen peroxide. This possibility has

beenn suggested for the bromide oxidation catalyzed by the VBPO in the presence of

H2O22 [47]. EPR spectra taken of samples in turnover failed to show the formation of

thee vanadium(IV) state [48], although its presence may have been obscured by the

strongg intensity of the EPR signal of ABTS+ (not shown). Also in the presence of

equimolarr amounts of enzyme and ABTS (20 uM) only quenching of the ABTS

absorbancee is observed and no formation of ABTS+. Therefore, both substrates are

neededd to observe ABTS+ formation. To propose a mechanism for the ABTS

oxidationn catalyzed by r-VCPO is difficult, however a scheme is presented in Figure

7.. Also, a more complex mechanism is found for the oxidation of ABTS by

horseradishh peroxidase and a random mechanism is favored for horseradish

peroxidasee under ABTS saturation conditions [28].

pABTS S

ABTSS J? \ H 2 O 2

pp ». pH202 ». pH 2 0 2

'' H 2 0 2 ABTS. ABTS

/ \ \ rr v ABTS+

X X ABTS** J \

ABTS S :<V V

pp inactive

Figuree 7. Schematic representation of the r-VCPO catalyzed oxidation of ABTS in the presence of hydrogenn peroxide. The enzyme, represented as E, may react with both H202 and ABTS.

Presently,, the available data do not preclude either of the two possibilities.

However,, it is clear that one-electron transfer steps are involved. It is therefore

feasiblee that a radical, which resides on the r-VCPO peroxo-intermediate after the

firstt 1 -electron transfer step, is responsible for the inactivation of the enzyme. This

inactivationn may be somewhat analogous to the radical inactivation occurring in

94 4

Compoundd III (Fe3+C>22") of heme peroxidases [49], In the r-VCPO the electron may

residee either on the bound peroxide or on an amino acid in the near vicinity [50],

followedd by the oxidation of another ABTS molecule or the inactivation of the

enzymee (Figure 7).

Inn line with our findings it is interesting to note that r-VCPO also oxidizes a

smalll aromatic sulfide, methyl phenyl sulfide, via a one-electron oxidation step

yieldingg a sulfide radical [51]. Presumably due to the lack of a binding site, the

sulfidee radical leaves the enzyme active site to form racemic sulfoxide [21]. In

contrast,, VBPO from A. nodosum catalyzes sulfoxidation reactions with very high

selectivityy [21] probably due to the presence of a specific binding site [37], which

facilitatess the direct transfer of oxygen from the peroxide to the sulfide and sulfide

radicalss are not formed [51]. In line with this ABTS is not converted to the

positivelyy charged ABTS radical by a high concentration (0.5 \xM) of VBPO.

Althoughh r-VCPO has been shown to mediate the conversion of ABTS in the

presencee of hydrogen peroxide, it is not possible to use this traditional peroxidase

substratee to screen for vanadium peroxidase activity, as only highly purified enzyme

preparationss exhibit this oxidation activity. However, the catalytic system may be of

somee interest from another point of view. The VCPO may be used in (mediator-

based)) oxidation of polymeric structures and dyes [39, 41, 42] and different

colorimetricc methods for the determination of various compounds, ranging from

anti-oxidantss to flavonoids [52-54].

Acknowledgments s

Lactoperoxidasee was a gift from A. Tuynman for which the authors like to

thankk him. This work was supported by the Innovation Oriented Research Programs

Catalysiss (IOP Catalysis). We also received financial support from the Council for

Chemicall Sciences (CW) of the Netherlands Organization for Scientific Research

(NWO)) and the Netherlands Technology Foundation (STW). This work has been

performedd under the auspices of NIOK, The Netherlands Institute for Catalysis

Research,, Laboratory Report, No. 00-1-01.

95 5

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