Enhancement of peroxygenase activity of horse heart myoglobin by modification of heme-propionate side chains

Article abstract of DOI:10.1246/cl.2003.496

A myoglobin reconstituted with an artificially created heme having two benzene moieties bound at the terminal of two heme-propionates shows a clear enhancement in peroxygenase activity toward thioether and styrene oxidations due to the expanding hydrophob

Full text of DOI:10.1246/cl.2003.496

496
Chemistry Letters Vol.32, No.6 (2003)
Enhancement of Peroxygenase Activity of Horse Heart Myoglobin by Modification of
Heme-propionate Side Chains
Takashi Hayashi,ꢀy;yy Takaaki Matsuda,y and Yoshio Hisaedaꢀy
yDepartment of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka 812-8581
yyPRESTO in Japan Science and Technology Corporation (JST)
(Received March 17, 2003; CL-030232)
A myoglobin reconstituted with an artificially created heme
that a neutral small substrate such as thioanisole or styrene
may be of suitable size for the artificial binding site in rMb.
Thus, we investigated the peroxygenase activity of the myoglo-
bins toward the substrate oxidation in the presence of H2O2.
The general procedure for the thioanisole oxidation catalyzed
by myoglobins is as follows: A solution of myoglobin
(5.0 mM; pH 7.0, 100 mM phosphate buffer)was incubated upon
the addition of H2O2 (5.0 mM)at 20 ꢁC.13 The product was
mainly the corresponding sulfoxide characterized by GC–MS.
The oxidation process was monitored by HPLC after the addi-
tion of thioanisole (0–1.5 mM).14 The catalytic reaction fol-
lowed the conventional Michaelis-Menten kinetics as shown
in Figure 1. From the plots, we determined the kinetic para-
meters, Km and kcat as shown in Table 1. In particular, the Km
value of the thioanisole oxidation by rMb is 6-fold smaller than
that observed for the nMb, thus, the catalytic efficiency toward
the thioanisole oxidation in rMb, which is represented by the
kcat/Km value, is significantly increased compared to that ob-
served in the nMb. In addition, the incorporation ratio of the
18-labeled oxygen atom into the product upon the addition of
H218O2 was 96%, suggesting that the activated oxygen of the
oxoferryl species as a reaction intermediate is directly trans-
ferred to the substrate in the heme pocket. These findings sup-
port the fact that the artificial substrate binding site enhances the
peroxygenase activity of myoglobin.
having two benzene moieties bound at the terminal of two
heme-propionates shows a clear enhancement in peroxygenase
activity toward thioether and styrene oxidations due to the ex-
panding hydrophobic heme pocket.
Modification of hemoproteins such as myoglobin, heme
peroxidases or cytochromes is generally carried out by site-di-
rected mutagenesis.1 Particularly, myoglobin is one of the best
scaffolds to engineer functions into the protein. It is known that
the active site modification of myoglobin by replacement of an
amino acid at the distal site of the heme pocket with an appro-
priate one enhances the peroxidase and/or peroxygenase
activity.1{6 However, there have been few examples which de-
monstrate the construction of an artificial substrate binding site
on the protein surface by mutagenesis.7;8 In contrast, we have
focused on a different approach, that is, replacement of native
heme with a modified heme, to create a new binding domain
in the myoglobin.9{11 For example, we have recently reported
that the modification of two heme-propionate side chains by in-
troducing hydrophobic benzene moieties accelerates the one-
electron oxidation of phenol derivatives such as guaiacol in
the presence of hydrogen peroxide,10 whereas native myoglo-
bin, nMb, shows little peroxidase activity. In the next stage, it
is of particular interest to study the oxygenation from oxoferryl
species by the reconstituted myoglobin having an artificially
created substrate binding site. In this communication, we wish
to report the conversion of myoglobin into peroxygenase by
chemical modification of the heme-propionate side chains.
The modified heme, which has two benzene moieties linked
at each terminal carboxylate of the two heme-propionates in the
native heme, has been prepared and incorporated into the horse
heart apomyoglobin to yield a reconstituted myoglobin, rMb.12
Based on computer modeling, the benzene moieties may form a
hydrophobic pocket on the myoglobin surface. It is expected
Next, to evaluate the selectivity of the substrate binding
0.08
0.06
0.04
0.02
protein
O
COOH
COOH
COOH
COOH
N
N
0
0.4
0.8
1.2
1.6
apomyoglobin
NH
O
[thioanisole] /mM
O
O
N
N
N
N
Figure 1. Initial rate, V, of myoglobin-cat-
alyzed oxidation as a function of thioanisole
concentration for a fixed amount of myoglo-
bin. Closed and open circles represents the
plots for rMb and nMb, respectively.
[rMb] = [nMb] = 5.0 mM, [H2O2] =
5.0 mM, pH 7.0 (100 mM phosphate buffer)
Fe
COOH
COOH
COOH
COOH
O
N
N
NH
artificial heme
O
reconstituted myoglobin, rMb
ꢁ
Scheme 1.
at 20 C.
Copyright ꢀ 2003 The Chemical Society of Japan

Chemistry Letters Vol.32, No.6 (2003)
497
Table 1. Kinetic Parameters and Stereochemistry for the
Thioanisole Oxidation Catalyzed by rMb and nMba
Ministry of Education, Culture, Sports, Science and Technol-
ogy.
Protein
rMb
nMb
References and Notes
Km
kcat
0:28 ꢃ 0:03
0:096 ꢃ 0:010
0.34
1:7 ꢃ 0:2
0:086 ꢃ 0:009
0.050
1
E. L. Raven and A. G. Mauk, in ‘‘Advances in Inorganic
Chemistry,’’ ed. by A. G. Sykes and G. Mauk, Academic
Press, San Diego (2001), Vol. 51, pp 1–51 and references
are cited therein.
Km/kcat
Initial rateb
(relative rate)
Incorporation of
18O from H218O2
6.18
1
2
3
4
5
6
7
8
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96 ꢃ 3
92 ꢃ 3
c
areaction conditions: [nMb] = [rMb] = 5.0 mM, [thioanisole]
= 0–1.5 mM, [H2O2] = 5.0 mM, at 20 C, pH 7.0 (100 mM
ꢁ
phosphate buffer). Reaction was monitored by HPLC
equipped with YMC Pack ProC4. Turnover frequency per
b
minute during initial stage. cProportionation of 18-labeled
oxygen in sulfoxide determined by GC–MS.
site, we compared two substrate oxidations catalyzed by rMb
and the native protein. The initial turnover rate of thioanisole
oxidation by rMb (vinit. ¼ 1:3 minꢂ1)is 6.2-fold faster than that
observed for the nMb, whereas the relative rate for the oxidation
of the anionic substrate, 4-carboxythioanisole, by rMb is re-
duced to approximately 60% of that observed for the nMb under
the same conditions.15 This clear difference indicates that the
neutral thioanisole can be easily bound into the artificially cre-
ated heme pocket, whereas the charged substrates such as 4-car-
boxythioanisole will not easily access the hydrophobic binding
site.
Furthermore, it is known that styrene is also one of the sui-
table substrates to monitor the peroxygenase activity.4;16;17 The
styrene oxidation catalyzed by rMb was monitored by a proce-
dure similar to the thioanisole oxidation. The GC–MS and
HPLC analyses support the formation of three products, styrene
oxide, phenylacetaldehyde and benzaldehyde, that were identi-
fied by comparison with authentic samples, where no decompo-
sition of styrene oxide occurred under the HPLC analytical con-
ditions. The product ratio between styrene oxide and
phenylacetaldehyde by rMb is 4.0 : 1.0 which is comparable
with that observed by nMb. The initial turnover rate of styrene
oxidation catalyzed by rMb (vinit. ¼ 0:28 minꢂ1)is more than
10-fold faster than that observed for the nMb.18 This finding in-
dicates that the modified heme pocket formed by the artificial
prosthetic group plays an important role in the acceleration of
styrene oxidation in the presence of H2O2.
9
T. Hayashi and Y. Hisaeda, Acc. Chem. Res., 35, 35 (2002).
10 T. Hayashi, Y. Hitomi, T. Ando, T. Mizutani, Y. Hisaeda,
and H. Ogoshi, J. Am. Chem. Soc., 121, 7747 (1999).
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13 Native horse heart myoglobin (Sigma M1882)and rMb
were purified by CM-52 and G-25 column chromatography.
14 The reaction was quenched by adding CH2Cl2 to the myo-
globin solution, and then the reaction tube was vortexed for
over 2 min. The organic layer, which was separated from the
aqueous solution, was used for the oxidation analysis.
15 [Mb] = 1.0 mM, [substrate] = 100 mM, [H2O2] = 1.0 mM, at
ꢁ
20 C, pH 7.0 phosphate buffer.
16 S. Ozaki and P. R. Ortiz de Montellano, J. Am. Chem. Soc.,
117, 7056 (1995).
17 S. Ozaki, T. Matsui, and Y. Watanabe, J. Am. Chem. Soc.,
118, 9784 (1996).
18 [Mb] = 10 mM, [substrate] = 8.7 mM, [H2O2] = 1.0 mM, at
20 C, pH 7.0 phosphate buffer.
19 T. Asakura and T. Yonetani, J. Biol. Chem., 247, 2278
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Commun., 1996, 2205.
In conclusion, the peroxygenase activity toward hydropho-
bic thioanisole and styrene oxidation is enhanced by the modi-
fication of the heme-propionate side chains.19{22 This result
strongly suggests that further fine-tuning of the artificial moi-
eties at the terminals of the propionates in myoglobin will give
us a unique oxidation catalyst with a high substrate specificity
and/or stereoselectivity.
ꢁ
22 A. D. Ryabov, V. N. Goral, L. Gorton, and E. Csoregi,
¨
Chem.–Eur. J., 5, 961 (1999).
This work was partially supported by JST, the Yazaki
Memorial Foundation for Science and Technology, and the
Published on the web (Advance View)May 6, 2003; DOI 10.1246/cl.2003.496