Use of apomyoglobin to gently remove heme from a H2O2-dependent cytochrome P450 and allow its reconstitution

Article abstract of DOI:10.1039/c6nj02882a

The heme of hydrogen peroxide-dependent cytochrome P450_BSβ (P450_BSβ) was removed by apomyoglobin under mild conditions to give apo-P450_BSβ without the need for acidic conditions and organic solvents. The circular dichroism spectrum of the apo-P450_BSβ was essentially identical to that of holo-P450_BSβ, showing a small structural change resulting from the removal of heme using apomyoglobin. The apo-P450_BSβ was reconstituted with hemin or manganese protoporphyrin IX (MnPPIX), and the resulting reconstituted P450_BSβ catalyzed the one-electron oxidation of guaiacol using hydrogen peroxide as an oxidant. A higher catalytic activity was observed for P450_BSβ reconstituted with MnPPIX when meta-chloroperoxybenzoic acid (mCPBA) was used as the oxidant.

Full text of DOI:10.1039/c6nj02882a

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Use of apomyoglobin to gently remove heme
from a H O -dependent cytochrome P450 and
2
2
Cite this:DOI: 10.1039/c6nj02882a
allow its reconstitution†
a
ab
c
c
Shih-Cheng Chien, Osami Shoji,* Yoshiko Morimoto and Yoshihito Watanabe*
The heme of hydrogen peroxide-dependent cytochrome P450BSb (P450BSb) was removed by
apomyoglobin under mild conditions to give apo-P450BSb without the need for acidic conditions and
organic solvents. The circular dichroism spectrum of the apo-P450BSb was essentially identical to that of
holo-P450BSb, showing a small structural change resulting from the removal of heme using apomyoglobin.
The apo-P450BSb was reconstituted with hemin or manganese protoporphyrin IX (MnPPIX), and the
resulting reconstituted P450BSb catalyzed the one-electron oxidation of guaiacol using hydrogen
peroxide as an oxidant. A higher catalytic activity was observed for P450BSb reconstituted with MnPPIX
when meta-chloroperoxybenzoic acid (mCPBA) was used as the oxidant.
Received (in Montpellier, France)
16th September 2016,
Accepted 21st November 2016
DOI: 10.1039/c6nj02882a
www.rsc.org/njc
6
of horse heart and skeletal muscle myoglobin (Mb). Several
methods based on a slight modification of this acid–butanol
Introduction
Heme, Fe-protoporphyrin IX, is one of the most important method have been reported for the preparation of apo forms of
metal complexes in nature, serving as the prosthetic group hemoproteins such as HRP and P450cam (see Fig. S1 and S2 for
7
,8
for hemoproteins that perform diverse functions, including these different heme enzyme structures, ESI†).
However,
oxygen storage and transport, gas sensing, electron transfer, these methods are not suitable if the hemoproteins as well as
1
,2
and catalysis. The properties of hemoproteins largely depend their apo-forms are not sufficiently stable in organic solvents
on the nature of heme; therefore, replacement of the heme with and/or under acidic conditions. Although the preparation of
heme analogues are expected to drastically change the properties apoproteins in bacterial cells has been developed as an alternative,
3
of hemoproteins. A variety of reconstituted hemoproteins their reconstitution was limited to metal complexes possessing a
containing heme analogues, and heme derivatives including protoporphyrin IX framework. To avoid the use of harsh chemical
different metal ions, chemically modified hemes, and synthetic treatments in the preparation of apoproteins, and to expand the
metal complexes have been constructed with the aim of developing range of applicable metal complexes to include various synthetic
synthetic proteins with (improved) catalytic activity or entirely metal complexes, it is important to develop a simple strategy for
4,5
different functions. To allow the reconstitution of hemoproteins removing the heme of hemoproteins without the need for organic
with synthetic metal complexes, the tightly bound heme must be solvents and acidic conditions.
removed to give apoproteins. Noncovalently bound hemes (b-type
Cytochrome P450BSb (P450BSb) is a hemoprotein that is
hemes) can be extracted from hemoproteins using organic irreversibly denatured during the preparation of apoprotein under
solvents such as butan-2-one and acetone under acidic conditions the methods described above. Therefore, it has been believed to be
(pH 2–4). Teale et al. first reported the removal of heme from impossible to prepare reconstituted P450BSb. However, P450BSb is
hemoproteins utilizing acid–butanol (pH = 2) to prepare apo forms regarded as a promising candidate for the construction of a
biocatalyst because P450BSb and its unique family, the hydrogen
peroxide-dependent P450s, efficiently utilize hydrogen peroxide to
a
Department of Chemistry, Graduate School of Science, Nagoya University,
catalyze various C–H bond hydroxylations and some other types of
Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
9,10
oxidation reactions.
If the heme of P450BSb could be replaced
E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
b
c
Core Research for Evolutional Science and Technology (CREST), Japan Science and
Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan. E-mail: p47297a@nucc.cc.nagoya-u.ac.jp
Electronic supplementary information (ESI) available: Supplementary figures
with synthetic metal complexes, the properties of the enzymatic
reaction catalyzed by P450BSb could be altered to allow the
development of a versatile biocatalyst.
Here we report a simple and useful method for the preparation
of apo-P450BSb using apo-Mb under very mild conditions
(Scheme 1). We decided to employ apo-Mb as a heme scavenger
†
including crystal structures, UV-Vis and CD spectra, the spectrum of purification and
the proposed mechanisms of Mn–P450BSb with mCPBA. See DOI: 10.1039/c6nj02882a
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À1
2
60 nm at 24 1C and 20 nm min scan speed, and an average
spectrum was produced from four independent consecutive
scans. Size distributions were recorded by dynamic light scattering
(DLS) using a Nano ZS (Malvern Instruments Ltd) with disposable
plastic cuvettes and caps. Inductively coupled plasma-optical
emission spectrometer (ICP-OES) measurements were performed
on a Varian Vista-Pro ICP Spectrometer (Varian Inc.). The
purification procedures described below were carried out using
a Bioassist eZ system (Tosoh Corporation). The protein figures
were generated using the PyMOL Molecular Graphics System
(
version 1.8; DeLano Schr ¨o dinger, LLC.).
Protein expression and purification
For the expression of recombinant P450BSb, 6Â histidine-tagged
(
6Â His-tagged) P450BSb was expressed in the transformed
Scheme 1 Schematic illustration of apo-Mb capturing the native cofactor
heme (red with the iron atom in yellow) and subsequently reconstituted
Escherichia coli strain M15. Cells were cultivated at 27 1C in
À1
with an artificial cofactor (blue with the manganese atom in magenta) to Luria–Bertani medium supplemented with 100 mg mL
of
À1
2 2
generate a functional enzyme from holo-form H O -dependent P450BSb.
ampicillin and 25 mg mL of kanamycin. Once the culture
reached an optical density at 600 nm (OD600) of 0.7–0.8, heme
precursor d-aminolevulinic acid (0.5 mM) was added, and the
culture was incubated for an additional 30 min. At OD600 = 1.0–1.2,
gene expression was induced by addition of IPTG (0.1 mM), and
cultivation was followed by culturing at 20 1C for 20 h rotating
at 80 rpm. Cells were then harvested by centrifugation and
lysed in 0.1 M potassium phosphate buffer (pH 7.0) with 0.3 M
potassium chloride, 50 mM imidazole, and 20% (v/v) glycerol.
because it binds strongly to heme and can be readily prepared
by the aforementioned acid–organic solvent-methods; the
dissociation constant of apo-Mb for heme is reported to be
À14
À1 11
1
0
M
.
We also demonstrate that the resulting apo-P450BSb
can be reconstituted with manganese protoporphyrin IX
MnPPIX) and that the resulting reconstituted P450BSb exhibits
(
improved one-electron oxidation activity when mCPBA was
employed as an oxidant.
Purification of 6Â His-tagged P450 was performed by Ni-chelate
BSb
affinity chromatography (GE Healthcare, Little Chalfont, UK). The
protein was further purified using S-200 Sephacryl (GE Healthcare)
size exclusion chromatography. An ASoret/A280 absorbance ratio Z1
indicated the high purity of the protein, which was also confirmed
by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Reduced
CO-difference spectra were used to determine the concentration of
P450 by subjecting the purified recombinant protein to sodium
Experimental methods
Materials
All chemicals were purchased from commercial sources and
used without further purification unless otherwise indicated.
12
Mn(III) protoporphyrin IX chloride was obtained from Frontier dithionite and CO gas. Protein samples were stored at À80 1C for
Scientific, Inc. (Logan, UT, USA) and used after exchanging its
further use.
À
À
Cl for BF4 by treating with AgBF . Hydrogen peroxide,
4
Preparation of apo-Mb
dithiothreitol (DTT), ethylene glycol, sodium dodecyl sulfate
(
SDS), isopropyl-b-D-1-thiogalactopyranoside (IPTG), and kanamycin
sulfate were obtained from WAKO Pure Chemical Industries, Ltd
Osaka, Japan). Glycerol, potassium chloride, urea, hydrochloric
acid, potassium phosphate, ampicillin sodium salt and phenyl-
A heme moiety was extracted from horse skeletal muscle Mb by
the butan-2-one extraction according to a published procedure
with slight modifications. The pH of a Mb solution (100 mg Mb
in 40 mL Milli-Q water) was adjusted to 2.5 in an ice-water bath
6
(
methylsulfonyl fluoride were purchased from Nacalai Tesque using chilled 0.1 M HCl(aq). The solution was transferred to a
Inc. (Kyoto, Japan). Horse skeletal muscle Mb was obtained
from Sigma-Aldrich Co. (St. Louis, MO, USA). Concentrations of
chemicals described in procedures are the final concentrations.
separation funnel, and an equal volume of 2-butanone was
added. The mixture was gently shaken for 30 s and then allowed
to stand for 10 min at 4 1C. The colorless aqueous phase was
separated and dialyzed for 24 h at 4 1C against 5 L of Milli-Q
water, and then for 24 h against PBS buffer (pH 7.3). Next, the
Measurements
UV-visible (UV-Vis) absorption spectra were recorded on a UV-2600 solution was passed through a 0.22 mm filter and stored at 4 1C
PC spectrophotometer (Shimadzu Corporation) or an Agilent 8453 for further use. The concentration of prepared apo-Mb was
equipped with an 89090A thermal controller (Agilent Technologies, spectrophotometrically determined using its extinction coefficient
À1
À1
13
Inc.), and the data were collected using screw-capped quartz of 15 700 M cm at 280 nm.
cuvettes of 1 cm path length. Circular dichroism (CD) spectra
were recorded with quartz cuvettes of 0.1 cm path length using a
Preparation of apo-P450BSb
J-720WI spectropolarimeter (Jasco Corporation) equipped with a The removal of heme from P450BSb to prepare apo-P450BSb was
temperature controller, and the data were recorded from 200 to performed by treating P450BSb with apo-Mb. The reaction was
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carried out in 0.1 M potassium phosphate buffer (pH 7.0) pH 7.0 under an argon atmosphere. The apoprotein solution was
containing 0.3 M potassium chloride and 20% (v/v) glycerol combined with urea (2 M), DTT (10 mM), histidine (10 mM), and
with P450BSb (25 mL, 8.5 mM). Solid Na S O was dissolved into Tritont X-100 (0.02%), and then left to stand for 15 min at
2
2 4
À1
the solution (1 mg mL ) at 25 1C to generate reduced P450BSb
.
room temperature. A 210 mL KOH solution (0.1 M) containing
Five equivalents of apo-Mb (25 mL, 42.5 mM) was added to the MnPPIX(BF ) (2.5 eq., 0.5 mM, the concentration determined
4
À1
À1 15
reaction buffer, and the final concentration of glycerol was using a e462nm of 25 000 M cm ), diluted tenfold with Milli-Q
adjusted to 5% using additional PBS buffer. The reaction was water, was added dropwise to the solution of apoprotein. The
maintained in a gas-tight flask for 24 h without stirring. Before whole reaction was then incubated in a gas-tight flask wrapped in
purification, 0.02% of Triton X-100 was added to aid the aluminum foil for 24 h without stirring. Before purification, an
separation of P450BSb and Mb. Purification of 6Â His-tagged Amicon ultracentrifugal filter was used to remove excess MnPPIX
apo-P450BSb was performed by Ni-chelate affinity chromatography and other chemicals. Purification of 6Â His-tagged rMn–P450
BSb
to remove holo-Mb and excess apo-Mb. This is the same was performed by Ni-chelate affinity chromatography to remove
purification procedure used for recombinant P450BSb. Sample free MnPPIX by the same procedure as that used for recombinant
purity was checked by SDS-PAGE. Pure apo-P450BSb was stored P450BSb. The final purified rMn–P450BS gave a single band on
at 4 8C for further use or stored at À80 8C for long-term preservation. SDS-PAGE, and the ASoret(381)/A280 absorbance ratio was 0.7.
Reconstitution of apo-P450BSb
Determination of P450 concentration
Reconstitution of apo-P450BSb was performed by adapting the
The concentration of the recombinant P450 was measured by
reduced CO-difference spectroscopy. In the case of rFe–P450BSb
with a higher content of P420 isoform (Fig. S4, ESI†), the
1
4
BSb
procedure for apo-P450cam at 25 1C as well as subsequent
protein purification at 4 1C. After the preparation of protopor-
phyrin complex solution, it should be added to apo-P450BSb
buffer immediately. Any delay in the addition of protopor-
phyrin complex solution to apo-P450BSb led to incomplete
12
concentration was measured by reduced pyridine-hemochrome
16
spectroscopy. The concentrations of holo- and apo-P450 for CD
spectroscopy measurements were determined by the Bradford
1
3
recombination due to its low solubility in water solution.
17
protein assay. rMn–P450BSb was quantitated from the Bradford
rFe–P450BSb. A 7 mL apo-P450BSb (6 mM) solution at pH 7.0
under an argon atmosphere was combined with urea (2 M), DTT
protein assay and multiplied by values of ASoret/A280
.
(
10 mM), histidine (10 mM), and Triton X-100 (0.02%). The solution
was then left to stand for a further 15 min at room temperature. A
0 mL KOH solution (0.1 M) containing hemin (2.5 eq., 2.1 mM) and
Guaiacol oxidation
Using H as an oxidant. For each catalytic reaction, the
5
2 2
O
myristic acid (0.2 mM), diluted tenfold with Milli-Q water, was total volume of the mixture was maintained at 1 mL. The
added dropwise to the solution of apoprotein. The whole reaction reaction was carried out in a 0.1 M potassium phosphate buffer
was performed in a gas-tight flask for 24 h without stirring. Before (pH 7.0) at 25 1C in the presence of 50 nM P450, 10 mM
purification, an Amicon ultracentrifugal filter was used to remove guaiacol (30 mL of 0.4 M ethanol solution), and 20 mM
excess hemin and other chemicals. Purification of 6Â His-tagged n-heptanoic acid (50 mL of 0.4 M ethanol solution). Reactions
rFe–P450BSb was performed by Ni-chelate affinity chromatography were initiated by the addition of 4 mM hydrogen peroxide
to remove free hemin by the same procedure used for the (300 mL of 14 mM aqueous solution). Initial oxidation rates
preparation of recombinant P450BSb. The final purified rFe–P450BSb were determined by monitoring the absorption change at
gave a single band on SDS-PAGE.
470 nm relative to the tetramer of guaiacol with an extinction
À1
À1 18
rMn–P450BSb. The insertion of MnPPIX into the apoprotein coefficient of 26 600 M cm
.
All initial turnover numbers
was carried out using a 7 mL apo-P450BSb (6 mM) solution at (TONs) are the average of at least three measurements, and are
Fig. 1 Time course of UV-Vis absorption spectra changes during the reactions at 25 1C for 24 h. (A) P450BSb with apo-Mb. Inset: spectral expansion of
4
80 B 600 nm; (B) Reduced P450BSb with apo-Mb.
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expressed as nmol product per nmol P450 per min Æ standard
deviation.
Using mCPBA as an oxidant. The reaction conditions were
similar to those described above, except that the oxidant was
changed to mCPBA and the reaction mixture does not contain
n-heptanoic acid. The reactions were started by adding 4 mM
mCPBA (70 mL of 60 mM ethanol solution) and monitored by
the absorption change at 470 nm. The oxidation products were
also determined quantitatively, as described previously.
ICP-OES
The iron and manganese contents in rP450BSb were determined
by ICP-OES. Concentrated samples of purified rFe–P450BSb and
rMn–P450BSb protein were diluted with 0.2 M HNO to 2 mL of
3
1.5, 3.0, 4.5 and 6.0 mM for the determinations. The solution
was placed in a new glass vial and subjected to ICP-OES via a
peristaltic pump. The sample was aspirated with argon, passed
over plasma, and analyzed for iron and manganese. A control
experiment was performed using Milli-Q water as a sample.
Commercial calibration standards and multi-element standards
(Wako Pure Chemical Industries, Ltd) were used.
Results and discussion
The removal of the heme from P450BSb to prepare apo-P450BSb
was performed by treating P450BSb with apo-Mb. When five
equivalents of apo-Mb were added to a solution of histidine-
tagged P450BSb at 25 1C, apparent shifts of the Soret absorption
band and Q-band were observed.‡ The absorption peak of
P450BSb at 417 nm shifted to 409 nm, which is readily attributed
to the formation of holo-Mb (Fig. 1A). After 24 h incubation, the
resulting apo-P450BSb was purified by Ni-chelate affinity chromato-
graphy, but a small amount of holo-P450BSb was still observed. To
bring the heme transportation to completion, P450BSb was reduced
2 2 4
using Na S O because the dissociation rate constant of reduced
II
heme (Fe ) coordinated with cysteine is rapid compared with that of
III 19
heme (Fe ). DFT calculations also showed a weaker binding of
cysteine to ferroheme than to ferriheme. A clear shift of the
absorption maxima of heme was again observed after the
addition of apo-Mb, and no remarkable change was observed Fig. 2 Comparisons between holo- and apo-form P450BSb. Holo-P450BSb
is shown as a blue solid line and apo-P450BSb as a red solid line. (A) UV-Vis
after 24 h (Fig. 1B).
Apo-P450BSb purified by Ni-chelate affinity chromatography
did not show any absorption assignable to heme, indicating
absorption spectra of holo- and apo-P450BSb. (B) CD spectra (0.0025 M) of
holo- and apo-P450BSb; the concentrations of protein were calculated
using the Bradford method. (C) DLS spectra of holo- and apo-P450BSb
.
that the heme of P450BSb can be removed completely (Fig. S5,
ESI†). Interestingly, the CD spectrum of apo-P450BSb was essentially
identical to that of the holo form, indicating a small structural
change upon the removal of the heme (Fig. 2B), while the CD
spectra of apoproteins produced by acid–organic solvent methods
(
Fig. 2C and Fig. S6, ESI†), which are consistent with the size of
holo-P450BSb (PDB ID: 3WSP), indicating that there is no
appreciable denaturation or aggregation. These results clearly
showed that apo-P450BSb could be prepared using apo-Mb and
the resulting apo-P450BSb retained almost the same conformation
as its holo form without denaturation.
20
were generally significantly different from those of the holo form.
For example, apo-P450cam prepared by the acid–butanone
method gave a weaker CD signal, indicative of partial denaturation.
The hydrodynamic diameters of holo- and apo-P450BSb deter-
mined by DLS analysis were 4.36 and 5.05 nm, respectively
With pure apo-P450BSb in hand, we reconstituted it according
1
4
to the method reported for preparing reconstituted P450cam
.
Initially, apo-P450BSb was reconstituted with heme to confirm
that it is possible to reinsert heme into an appropriate position
in P450BSb and to fully recover its catalytic activity. A solution of
‡
The absorption spectral change was very slow when the reaction of preparing
apo-P450BSb was under 4 1C after 24 h incubation (Fig. S3, ESI†).
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apo-P450BSb was added with 2.5 equivalents of hemin in the
presence of 10 mM DTT and 10 mM histidine, and then incubated
for 24 h under argon. Reconstituted P450BSb (rFe–P450BSb) could
be purified by Ni-chelate affinity chromatography. The UV-Vis
absorption spectrum showed a Soret peak at 417 nm. The CD
spectrum of reconstituted P450BSb remained similar to those of
recombinant P450BSb and a-helical proteins (Fig. S7, ESI†).
rFe–P450BSb exhibited catalytic activity similar to that of
native P450BSb for the one-electron oxidation of guaiacol with
the assistance of n-heptanoic acid, which accelerates the
generation of active species of P450BSb because the carboxylate
2
1
of n-heptanoic acid serves as an acid–base catalyst. The initial
TON (nmol product per nmol P450 per min) of one-electron
oxidation of guaiacol by rFe–P450BSb with heptanoic acid was
estimated to be 3150, which is almost the same as that of
P450BSb under similar conditions (Table 1). These results indicate
that apo-P450BSb prepared using apo-Mb can be reconstituted with
Fig. 3 UV-Vis absorption spectrum (black line) of rMn–P450BSb in 0.1 M
potassium phosphate buffer (pH 7.0) containing 0.3 M KCl and 20% (v/v)
glycerol. The Soret band from MnPPIX in the deep pocket of the active site
of P450BSb was at 381 nm, and there was no signal of free MnPPIX or
adherent (noncovalently bound) MnPPIX at the surface of P450BSb in the
22
heme and that the reconstituted rFe–P450BSb has a catalytic activity UV-Vis spectrum. The ASoret(381)/A280 absorbance ratio was 70%. The
CD spectrum showed that rMn–P450BSb remained in the same a-helix
comparable to that of recombinant P450BSb
.
structure as the recombinant P450BSb. Inset: CD spectrum (red line) of
rMn–P450BSb in the same potassium phosphate buffer.
Given that apo-P450BSb could be reconstituted with heme,
we have attempted to reconstitute apo-P450BSb with MnPPIX in
a similar manner, and obtained P450BSb reconstituted with
MnPPIX (rMn–P450BSb). The CD spectrum of rMn–P450BSb
confirmed that rMn–P450BSb had the same protein structure
as P450BSb (Fig. 3 and Fig. S7, ESI†). The Soret absorption band
of rMn–P450BSb was observed at 381 nm, which corresponded
We also examined the hydroxylation of cyclohexane by rMn–P450BSb
with mCPBA, but no product was detected, suggesting either the
IV
V
Mn QO, not the Mn QO, species generated by homolytic
III
V
cleavage of Mn –OOR or very rapid reduction of Mn QO under
the conditions and eventually Mn QO is responsible for one-
23,24
IV
to that of P450cam reconstituted with MnPPIX (Fig. 3).
ICP-OES
showed that 70% of the apo-P450BSb was reconstituted with
MnPPIX. The UV-Vis absorbance ratio (ASoret(381)/A280) of
rMn–P450BSb was consistent with 70% reconstitution.
electron oxidation by rMn–P450
(Scheme S2, ESI†).
BSb
in the presence of mCPBA
2
5
The catalytic activity of rMn–P450BSb for one-electron oxidation
of guaiacol using hydrogen peroxide as an oxidant was examined, Conclusions
À1
but the TON was very low at 40 min . In contrast, when mCPBA
We have demonstrated that apo-P450BSb can be prepared using
apo-Mb as a heme scavenger without subjecting the proteins to
harsh and denaturing conditions. Although the preparation of
apo-P450s and their reconstitution is generally difficult, the
heme of P450BSb was completely removed by apo-Mb to give
pure apo-P450BSb that could be reconstituted with MnPPIX.
The holo-, apo-, rFe– and rMn–P450BSb remained as analogous
a-helical structures, in contrast to other reported reconstituted
P450s and their apo-P450s prepared using the acid–butanone
method or incubation with hydrogen peroxide. Although we
focused on the preparation of apo-P450BSb, other P450s whose
apo-forms are difficult to prepare by conventional acid–organic-
solvent methods may also be potent targets for our methodology.
Furthermore, we believe that this method is useful to selectively
remove the heme of hemoproteins containing other cofactors
such as flavin derivatives or iron–sulfur clusters.
was used as the oxidant, the catalytic activity was improved
À1
(
2850 min ) compared with that of recombinant P450BSb
(Table 1). On the other hand, hydroxylation of cyclohexane was
not catalyzed by neither recombinant P450BSb nor rFe–P450BSb
.
Table 1 Catalytic activity for one-electron oxidation of guaiacol by
c
different oxidants with recombinant and reconstituted P450BSb. The units
of catalytic activity are described as the TON (all values are the average of
five measurements and are expressed in terms of nmol product per nmol
P450 per min Æ standard deviation)
2
0
26
À1
TON (min
)
a
Enzyme protein
H
2
O
2
mCPBA
b
Acknowledgements
P450BSb
rFe–P450BSb
rMn–P450BSb
3750
113 Æ 5
—
3150 Æ 50
This work was supported by Grants-in-Aid for Scientific
Research (S) to Y. W. (24225004) from the Ministry of Education,
Culture, Sports, Science, and Technology (Japan) and CREST,
40 Æ 8
2860 Æ 40
a
b
Heptanoic acid assisted these catalytic experiments. The value of
TON is adapted from Angew. Chem., Int. Ed., 2007, 46, 3656–3659. For the
c
proposed oxidation mechanism of guaiacol with P450, see Scheme S1, ESI. Japan Science and Technology Agency.
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