The activation of molecular oxygen by horseradish peroxidase with sodium sulfite

Article abstract of DOI:10.1039/b104529f

Horseradish peroxidase (HRP) utilizes molecular oxygen (O₂) with sodium sulfite (Na₂SO₃) to oxidize thioanisole and styrene at the exterior of the heine pocket.

Full text of DOI:10.1039/b104529f

The activation of molecular oxygen by horseradish peroxidase with
sodium sulfite†
Shin-ichi Ozaki,* Seiko Watanabe, Sachiko Hayasaka and Megumi Konuma
Faculty of Education, Yamagata University, Kojirakawa, Yamagata 990-8560, Japan.
E-mail: ozaki@ke-sci.kj.yamagata-u.ac.jp
Received (in Cambridge, UK) 22nd May 2001, Accepted 23rd July 2001
First published as an Advance Article on the web 16th August 2001
Horseradish peroxidase (HRP) utilizes molecular oxygen
(O2) with sodium sulfite (Na2SO3) to oxidize thioanisole and
styrene at the exterior of the heme pocket.
amount of the sulfoxide product is detected when the reaction is
performed under anaerobic conditions. The results clearly
indicate that HRP together with sulfite can activate molecular
oxygen to produce a potential oxidant for the monooxygenation
22
Horseradish peroxidase (HRP) bearing iron protoporphyrin
(IX) as the prosthetic group normally utilizes hydrogen
peroxide (H2O2) as an oxidant to generate an oxoferryl species
(ONFeIV) paired with porphyrin radical cation, so-called
compound I.1 Compound I is reduced back to the ferric state by
either sequential one-electron transfer from typical phenolic
substrates such as guaiacol or by ferryl oxygen transfer to
thioethers.2–4 Since HRP is known to react with racemic
hydroperoxides to afford chiral hydroperoxides and alcohols in
high enantioselectivity, the enzyme is also used for kinetic
resolutions.5–9 In contrast to P450 monooxygenases, HRP does
not efficiently activate O2 with a reductase and NADH or
NADPH because the peroxidase does not mediate the electron
transfer from external electron sources on the protein surface to
the heme iron buried inside.10 However, the direct electron
transfer from sulfite (SO322) to the ferric heme iron would
reaction. Interestingly, sulfoxidation with SO3 and O2 does
not proceed enantioselectively as observed in the reaction with
H2O2 (Table 1); therefore, compound I does not seem to be a
catalytic species in the HRP–SO322–O2 system (i.e. pathway
step 3 ? 4 ? 5 in Scheme 1 is not dominant). Since the
oxidation of thioanisole with monoperoxysulfate (HSO52) has
been found to afford a racemic mixture of sulfoxide in the
presence or absence of HRP, we speculated that monoperoxy-
sulfate released from the heme pocket would be involved in
sulfoxidation with sulfite and molecular oxygen.
In order to investigate the mechanism further, we have
performed the sulfoxidation reaction in the presence of
superoxide dismutase (SOD) and catalase. The reaction is
subject to significant inhibition (97%) although SOD and
catalase do not decelerate the chemical sulfoxidation by
monoperoxysulfate (Table 2). By contrast, the rate of sulfoxida-
tion is reduced by < 18% in the presence of OH· radical
·
produce SO3 (step 1 in Scheme 1), which could initiate the
activation of O2 to oxidize the substrates.11,12 In order to explore
transition metal promoted oxidative reactions with sulfite, we
have investigated one- and two-electron oxidation by HRP with
SO322 and O2.
scavengers such as methanol or tert-butyl alcohol and SO4
·2
radical quenchers like ethanol (Table 2). The results imply that
(a) superoxide (O2·2) is involved in the production of
monoperoxysulfate and (b) the lack of inhibition observed in the
presence of ethanol, methanol or tert-butyl alcohol argues
Thioanisole is oxidized to methyl phenyl sulfoxide by HRP in
the presence of sodium sulfite and ambient O2 at a rate more
than four times faster than the value obtained in the incubation
with H2O2 as an oxidant (Table 1). Not more than a trace
·2
against the involvement of SO4 or OH· radicals in the
catalysis. The ferrous–dioxygen and ferric–superoxide complex
of HRP are in equilibrium (step 8 in Scheme 1). Thus, the
·2
·2
recombination of O2 and SO3 generated in step 9 and 1
(Scheme 1), respectively, could produce SO522, which is
subsequently protonated to produce monoperoxysulfate
(HSO52). The release of HSO52 from the intermediate by Fe–O
Table 1 Oxidation of thioanisolea
Initial rate/
Protein
Oxidant
turnover min21
%ee
HRP
HRP
H2O2
sulfite and O2
1.1
1.2
77b
0
a The reaction mixture containing HRP (5 mM) and thioanisole (2 mM) was
incubated with either H2O2 (0.6 mM) or sodium sulfite (0.6 mM) at 25 °C
in sodium phosphate buffer (50 mM, pH 7.0). The sulfoxide product
extracted with CH2Cl2 was analyzed by HPLC equipped with a Dicel OD
chiral column as reported in ref. 4. The reported values are the average of
two independent experiments. For anaerobic experiments, the vessel
containing the reaction mixture was frozen, evacuated and then filled with
nitrogen. The procedure was repeated three times to remove dissolved
oxygen. Sulfoxidation was then performed under a nitrogen atmosphere.
b The S isomer is the major product.
Scheme 1 Reaction scheme for the HRP–SO322–O2 system. The catalytic
cycle postulated by our results is indicated inside the square; SO3 and
O2·2 generated by step 1 and 9, respectively, would mediate the oxidation
reaction outside of the active site under the conditions described here.
·2
† Electronic supplementary information (ESI) available: plots of pH vs. rate
of sulfoxidation. See http://www.rsc.org/suppdata/cc/b1/b104529f/
1654
Chem. Commun., 2001, 1654–1655
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b104529f

Table 2 Inhibition of thioanisole oxidation by additivesa
(step 3 in Scheme 1) or/and the heterolytic cleavage of the O–O
bond in Fe(III)–SO5 species (step 5 in Scheme 1) is not an
22
Additive
Relative activity (%)
efficient process since guaiacol can not be oxidized by
monoperoxysulfate but only by compound I. The addition of an
excess of monoperoxysulfate to ferric HRP (step 7 in Scheme 1)
somewhat facilitates compound I formation to improve the one-
electron oxidation activity (4.1 turnover min21); however, the
rate is 6500-fold slower than the value obtained in guaiacol
oxidation with H2O2. It was previously reported that high valent
metal–oxo species generated from water-soluble porphyrins
and oxygen atom donors (KHSO5, H2O2, … etc.) mediate the
oxidation of alcohols, olefins or DNA,19–23 but the active site of
b
—
100
3
96
82
96
SOD and catalasec
Methanold
Ethanold
tert-Butyl alcohold
a Reactions were conducted with HRP (5 mM), thioanisole (2 mM), sodium
sulfite (0.6 mM) and additive(s) in sodium phosphate buffer (50 mM, pH
7.0) at 25 °C for 10 min. The reported values are the average of two
independent experiments. SOD and catalase did not inhibit chemical
oxidation of thioanisole by monoperoxysulfate (0.6 mM). b No additive.
c SOD (10 units) and catalase (11 units) added to the reaction mixture. d The
concentration of additive is 25 mM.
2
HRP may not be large enough to accommodate HSO5 as a
good oxidant.
In summary, we report that HRP can utilize SO322 and O2 to
oxidize thioanisole and styrene, but the catalytic species is not
compound I as established in the oxidation with H2O2. Our
bond cleavage (step 6 in Scheme 1) is excluded because
superoxide is not involved in the catalytic cycle (step 1 ? 2 ?
3 ? 6 in Scheme 1) and the observed inhibition in the presence
of SOD and catalase can not be rationalized.
Plots of sulfoxidation rate vs. pH examined in the range pH
5–10 reveal that the reaction with sulfite does not proceed below
pH 5, and the optimum pH is found to be 7 (see ESI†). The trend
here is similar to that observed for nickel-catalyzed oxidative
deamination with sulfite under aerobic conditions13 but differs
from the pH profile for the HRP–H2O2 system, which can
produce sulfoxide even below pH 6. The result is consistent
with our hypothesis that the oxidation mechanism is altered
when SO322–O2 instead of H2O2 is utilized.
2
mechanistic studies imply that monoperoxysulfate (HSO5
)
generated from O2·2 and SO3·2 mediates the oxidation reaction
outside of the heme pocket. A similar mechanism might be
involved in the metalloprotein associated biological toxicity of
sulfite inhaled from industrial emissions or ingested as a
preservative in foods.24
We thank Drs Naohiko Masuda, Minoru Ishii and Yoshihito
Watanabe for their technical support. Financial support of this
work by Grant-in-Aid for Scientific Research (No. 10680575)
and Mitsubishi Chemical Corporation Fund for S. O. is
gratefully acknowledged.
Styrene and guaiacol oxidations by HRP with sulfite and
oxygen provide further support for the proposed reaction
scheme (Scheme 1). In contrast to sulfoxidation, the epoxida-
tion reaction by compound I requires interactions of the two
vinyl carbons with a ferryl oxygen atom. Since the active site of
HRP is sterically hindered, it was previously reported that
compound I of HRP could not efficiently oxidize styrene.2,4
However, styrene oxide is detected in the mixture of HRP–
styrene–SO322–O2 (Table 3) although the rate of oxidation is
slow. In addition, phenylacetaldehyde, which is normally
observed as a side product of compound I mediated epoxidation
Notes and references
1 H. B. Dunford, Heme Peroxidases, Wiley-VCH, New York, 1999.
2 R. Z. Harris, S. L. Newmyer and P. R. Ortiz de Montellano, J. Biol.
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3 S. Ozaki and P. R. Ortiz de Montellano, J. Am. Chem. Soc., 1994, 116,
4487.
4 S. Ozaki and P. R. Ortiz de Montellano, J. Am. Chem. Soc., 1995, 117,
7056.
5 E. Hoft, H. J. Hamann, A. Kunath, W. Adam, U. Hoch, C. R. Sahamoller
and P. Schreier, Tetrahedron: Asymmetry, 1995, 6, 603.
6 W. Adam, U. Hoch, M. Lazarus, C. R. Sahamoller and P. Schreier,
J. Am. Chem. Soc., 1995, 117, 11 898.
7 W. Adam, R. T. Fell, U. Hoch, C. R. Sahamoller and P. Schreier,
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8 W. Adam, U. Hoch, H. U. Humpf, C. R. SahaMoller and P. Schreier,
Chem. Commun., 1996, 2701.
9 W. Adam, C. R. Saha-Moller and K. S. Schmid, J. Org. Chem., 2000,
65, 1431.
by other hemoproteins, is not produced.14–18 The results
22
indicate that monooxygenation reactions in the HRP–SO3
–
O2 system do not proceed via compound I but occur outside of
the heme pocket by monoperoxysulfate. Significantly, slow
one-electron oxidation of guaiacol exhibited by HRP with
22
SO3 and O2 also indicates that the intermediate formation
10 P. R. Ortiz de Montellano, Cytochrome P450, Plenum Press, New York,
2nd edn., 1995.
Table 3 Epoxidation of styrene
11 J. F. Perez-Benito and C. Arias, Collect. Czech. Chem. Commun., 1991,
56, 1552.
Experiment
Protein
Oxidant
Rate
12 Y. Song, C.-M. Yang and R. Kluger, J. Am. Chem. Soc., 1993, 115,
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13 J. Levine, J. Etther and I. Apostol, J. Biol. Chem., 1999, 274, 4848.
14 C. E. Catalano and P. R. Ortiz de Montellano, Biochemistry, 1987, 26,
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1a
2a
3d
4e
HRP
HRP
—
H2O2
NDc
200b
250b
NDc
Sulfite and O2
Monoperoxysulfate
Sulfite and O2
—
15 V. P. Miller, G. DePillis, D. J. C. Ferrer, A. G. Mauk and P. R. Ortiz de
Montellano, J. Biol. Chem., 1992, 267, 8936.
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19 S. VilainDeshayes, A. Robert, P. Maillard, B. Meunier and M.
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a Styrene (1 mL) was added to HRP (5 mM) in 0.5 mL of sodium phosphate
buffer (50 mM, pH 7.0). The concentration of styrene was expected to be 17
mM, but the reaction mixture appeared to be slightly turbid. To the styrene-
saturated solution, either H2O2 (0.6 mM) or sodium sulfite (0.6 mM) was
added to initiate the reaction at 25 °C. Products were extracted with
CH2Cl2 and analyzed by GC on a Shimadzu CBP1 capillary column. The
reaction time varied from 20 to 120 min to obtain the time vs. epoxide
formation plot. A linear relationship was observed for 120 min, and the rate
was determined as the slope of the plot. The quoted values are the average
of two independent experiments. b pmol of epoxide min21 c Not de-
.
tected.d The reaction was performed with monoperoxysulfate (0.6 mM) in
the absence of HRP. Since the exact concentration of monoperoxysulfate in
the HRP–sulfite–O2 solution can not be determined, direct comparison of
the rates in experiments 2 and 3 would not be appropriate, however, the
results indicated that the epoxide could be produced from styrene with
monoperoxysulfate. e Oxidation was also performed with sodium sulfite
(0.6 mM) with styrene aerobically in the absence of HRP, but the epoxide
product was not detected.
Chem. Commun., 2001, 1654–1655
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