Conversion of myoglobin into a peroxygenease: A catalytic intermediate of sulfoxidation and epoxidation by the F43H/H64L mutant

Full text of DOI:10.1021/ja970453c

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J. Am. Chem. Soc. 1997, 119, 6666-6667
Communications to the Editor
Conversion of Myoglobin into a Peroxygenase: A
Catalytic Intermediate of Sulfoxidation and
Epoxidation by the F43H/H64L Mutant
Shin-ichi Ozaki,† Toshitaka Matsui,‡ and
Yoshihito Watanabe*,†,‡
Institute for Molecular Science
Okazaki, Myodaiji, 444 Japan
Department of Structural Molecular Science
The Graduate UniVersity for AdVanced Studies
Okazaki, Myodaiji, 444 Japan
Figure 1. Superposition of the heme and some selected residues
including distal histidine (His-64 in Mb and His-52 in CcP) in crystal
structures of sperm whale myoglobin (Mb) and cytochrome c peroxidase
(CcP). Light and dark balls indicate oxygen molecules bound to heme
iron in Mb and CcP, respectively. The distances between Nꢀ of the
distal histidine and iron are 4.3 and 5.6 Å in Mb and CcP, respectively.
Only heme in Mb is shown in this figure. (a) Side view. (b) Top view.
ReceiVed February 11, 1997
Myoglobin (Mb) has been one of the most intensively
investigated hemoproteins as evident from the accumulated
biochemical, biophysical, and spectroscopic data.1 The heter-
ologous overexpression system for recombinant sperm whale
Mb in Escherichia coli has been developed,2 and high-resolution
X-ray crystal structures of the wild type as well as some Mb
mutants are available.3 Thus, superposition of the active site
structures of Mb and other hemoproteins enables us to utilize
Mb as heme enzyme models for the elucidation of structure-
function relationships.4 We have engineered sperm whale Mb
based on the comparison of crystal structures of oxymyoglobin
and an oxy form of cytochrome c peroxidase (CcP) (Figure 1).5
Although the Leu-29 f His and His-64 f Leu double
replacement of Mb seems to create a peroxidase-like active site,
the imidazole is located too far from the heme center to interact
with hydroperoxide bound to the iron. Our previous results
suggest that L29H/H64L Mb cannot efficiently cleave O-O
bond to generate a ferryl (FeIVdO) radical cation species,
equivalent to compound I of peroxidase.6 Thus, we have
mutated Phe-43 to a histidine residue because the predicted
distance between His-43 and the heme iron is approximately
equal to that of CcP. The novel F43H/H64L Mb mutant
oxidizes sulfide and styrene more efficiently than peroxidase.
More intriguingly, we have identified a compound I-like species
of the Mb mutant as the catalytic intermediate for the first time.
The replacement of Phe-43 in the wild type with a histidine
residue increases the rate of thioanisole oxidation by 14-fold,
and the mutation of His-64 f Leu in F43H Mb further enhances
the sulfoxidation rate by 13-fold (Table 1). The enantiomeric
excess is improved from 25% to 85% by the His-64 f Leu
and Phe-43 f His double mutation of Mb, and the dominant
enantiomer is R. More than 92% of 18O incorporation in the
sulfoxide from H218O2 in the oxidation by wild type, F43H,
and F43H/H64L Mb indicates that the ferryl oxygen is
transferred to thioether. In comparison with the wild type, the
F43H/H64L mutant oxidizes styrene 300 times faster with an
improvement of enantioselectivity from 9 to 68%. Incubations
of styrene and H218O2 with wild type and F43H/H64L Mb
resulted in incorporation of 20% and 94% of 18O in epoxide,
respectively. The low 18O incorporation into the epoxide in
the presence of the wild type and H218O2 could be rationalized
by the competition of the ferryl oxygen transfer and cooxidation
mechanism. The cooxidation mechanism requires protein
radical formation followed by binding of molecular oxygen to
generate a protein-peroxy radical, and His-64 was suggested
as the initial radical site.7 The replacement of His-64 with an
unoxidizable leucine residue could prevent generation of the
protein radical and decrease the cooxidation. In fact, the value
of 18O incorporation from 18O-labeled hydrogen peroxide for
the F43H mutant, bearing two histidines in the active site, is
54%, which is between the values for F43H/H64L and wild
type Mb.8
We have attempted to identify the catalytic species of F43H/
H64L Mb involved in a net two-electron oxidation of thioanisole
and styrene. The horseradish peroxidase compound I-like
spectrum is not observed by monitoring the changes in absorp-
tion spectra of the incubation mixture containing the mutant
and hydrogen peroxide.9 However, the mixing of F43H/H64L
Mb and m-chloroperbenzoic acid (mCPBA) causes the decrease
in absorbance at 406 nm, followed by the shift to longer
wavelength by 12 nm (Figure 2a). The formation of the first
intermediate proceeds at the rate of kobs1 ) 110 s-1 (standard
† Institute for Molecular Science.
‡ The Graduate University for Advanced Studies.
* Corresponding author. Phone: 81-564-55-7430. FAX: 81-564-54-2254.
E-mail: yoshi@ims.ac.jp.
(1) Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in Their
Reactions with Ligands; North-Holland: Amsterdam, 1971.
(2) Springer, B. A.; Sligar, S. G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84,
8961.
(3) (a) Phillips, G. N.; Arduini, R. A.; Springer, B. A. ; Sligar, S. G.
Proteins: Struct. Funct. Genet. 1990, 7, 381. (b) Quillin, M. L.; Arduini,
R. M.; Olson, J. S.; Phillips, G. N. J. Mol. Biol. 1993, 234, 140.
(4) (a) Adachi, S.; Nagano,S.; Ishimori, K.; Watanabe, Y.; Morishima,
I.; Egawa, T.; Kitagawa, T.; Makino, R. Biochemistry 1993, 32, 241. (b)
Matsui, T.; Nagano, S.; Ishimori, K.; Watanabe, Y.; Morishima, I.
Biochemistry 1996, 35, 13118.
error 3.8), and the Soret shifts to 418 nm at the rate of kobs2
)
(7) (a) King, N. K.; Winfield, M. E. J. Biol. Chem. 1963, 238, 1520. (b)
Catalano, C. E.; Ortiz de Montellano, P. R. Biochemistry 1987, 26, 8373.
(c) Tschirretguth, R. A.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys.
1996, 335, 93. (d) Fenwick, C. W.; English, A. M. J. Am. Chem. Soc. 1996,
118, 12236.
(8) Peroxygenase activity of HRP was previously reported in: Ozaki,
S.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1995, 117, 7056. The
rate of sulfoxidation for F43H Mb is equal to that for native HRP, and the
F43H/H64L mutant is found to oxidize thioanisole approximately 13 times
faster than HRP. Styrene is oxidized by the Phe-43 f His mutants at least
100-fold faster than HRP and its mutants.
(5) For crystal structures of Mb and CcP, see: Phillips, S. E. J. Mol.
Biol. 1980, 142, 531. Miller, M. A.; Shaw, A.; Kraut, J. Nat. Struct. Biol.
1994, 1, 524. Although the X-ray crystal structure of HRP has not been
published yet, the modeling studies in Zhao et al. (Zhao, D.; Gilfoyle, D.
J.; Smith, A. T.; Loew, G. H. Proteins: Struct. Funct. Genet. 1996, 26,
204) suggest that the alignment of distal histidine in HRP is similar to that
of CcP.
(6) Ozaki, S.; Matsui, T.; Watanabe, Y. J. Am. Chem. Soc. 1996, 118,
9784.
(9) (a) Yonetani, T.; Schleyer, H. J. Biol. Chem. 1967, 242, 1974. (b)
Dunford, H. B.; Stillmann, J. S. Coord. Chem. ReV. 1976, 19, 187.
S0002-7863(97)00453-8 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 28, 1997 6667
Table 1. Peroxygenase Activity of Mb and Its Mutantsa
a All the values are the average of at least two determinations. Incubations were carried out as described in ref 8. b The absolute stereochemistry
of the dominant isomer is R.
for other heme containing peroxidases.10 Upon the addition of
styrene or thioanisole to the compound I-like intermediate,
absorbance of the Soret band for the intermediate is recovered
back to the level of ferric state. Thus, it is now convincing
that the observed species bears two-electron oxidation equiva-
lents.11 Since the close examination of a crystal structure for
sperm whale Mb suggests that the orientation of the distal
histidine in F43H/H64L Mb could be somewhat similar to that
of bovine liver catalase,12 compound I of the mutant appears to
be readily reduced to the ferric state in the presence of hydrogen
peroxide.13 On the contrary, the wild type reacts with mCPBA
more slowly than the F43H/H64L mutant to generate compound
II, and there is no accumulation of compound I (Figure 2b),
presumably due to the rapid electron transfer from compound
I to His-64, which lies closer to the heme iron.
In summary, we have engineered the distal pocket of Mb to
mimic the active site of peroxidase, and the F43H/H64L mutant
is found to be a much better peroxygenase than wild type Mb
and even HRP. Furthermore, the utilization of mCPBA enables
us to identify a compound I-like species of F43H/H64L Mb as
a catalytic intermediate for peroxygenase activity. Our results
clearly indicate that the alignment of the distal histidine is
important for the reactivity as well as the direct observation of
the transient catalytic species for Mb.
Acknowledgment. We thank Dr. John S. Olson (Rice University)
for providing cDNA of sperm whale myoglobin and Dr. Akio Murakami
(National Institute for Basic Biology) for his help in measuring catalase
activity. This work was supported by Grant-in-Aid for Scientific
Research (No. 07458147) and for Priority Areas, Molecular Biome-
tallics, for Y.W.
Figure 2. Absorption spectra of (a) ferric F43H/H64L (5 µM) or (b)
JA970453C
wild type Mb (5 µM) with mCPBA (0.5 mM) in 50 mM sodium acetate
(10) Stillmann, J. S.; Stillmann, M. J.; Dunford, H. B. Biochem. Biophys.
buffer, pH 5.3. Spectra were recorded on a Hi-Tech SF-43 stopped-
Res. Commun. 1975, 63, 32. (b) Smith, A. T.; Sanders, S. A.; Thorneley,
flow apparatus equipped with MG 6000 diode array spectrometer at 5
°C. Insets are changes in absorbance at 415 nm. The black line spectra
represent ferric. Dashed and dotted line spectra are for compounds I
and II, respectively.
R. N. F.; Burke, J. F.; Bray, R. R. C. Eur. J. Biochem. 1992, 207, 507.
(11) The percentage of enantiomeric excess for thioanisole oxidation by
F43H/H64L Mb with mCPBA is identical to the value with H2O2. The result
implies that the same catalytic species is involved in the sulfoxidation.
(12) Fita, I.; Rossmann, M. G. Proc. Natl. Acad. Sci. U.S.A. 1985, 82,
1604.
9.2 s-1 (standard error 0.38). Decrease in the absorbance of
the Soret and intense absorbance around 500-700 nm are
characteristic for the conversion of ferric to ferryl porphyrin
radical cation, and the following Soret shift is due to the decay
of compound I (Por+•FeIVdO) to II (PorFeIVdO) as observed
(13) The dismutation of hydrogen peroxide has been determined by
monitoring oxygen evolution using an oxygen electrode, and the 50-fold
increase in catalase activity versus the wild type (1.7 ( 0.2 turnovers/min)
was observed for the F43H/H64L mutant (79 ( 2 turnovers/min). The
catalase activity of F43H/H64L Mb is still less than 1% of that for bovine
liver catalase.