Identification of the rate-limiting step of the peroxygenase reactions catalyzed by the thermophilic cytochrome P450 from Sulfolobus tokodaii strain 7

Article abstract of DOI:10.1111/febs.12712

Cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st) is a thermophilic cytochrome P450 that shows high tolerance of harsh conditions and is capable of catalyzing some peroxygenase reactions. Here, we investigated t

Full text of DOI:10.1111/febs.12712

Identification of the rate-limiting step of the peroxygenase reactions catalyzed
by the thermophilic cytochrome P450 from Sulfolobus tokodaii strain 7
Shohei Hayakawa1, Hirotoshi Matsumura1,2,†, Nobuhumi Nakamura1, Masafumi Yohda1, and
Hiroyuki Ohno1
1Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology,
Koganei, Tokyo 184-8588, Japan
2Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health
and Science University, Beaverton, OR97006, USA
Correspondence
N. Nakamura, Department of Biotechnology and Life Science, Tokyo University of Agriculture and
Technology, Koganei, Tokyo 184-8588, Japan
Fax: +81-42-388-7482
Tel: +81-42-388-7482
E-mail: nobu1@cc.tuat.ac.jp
Present address
†Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon
Health and Science University, Beaverton, OR 97006, USA
Running title
The rate-limiting step of peroxygenase reactions
Article type
: Original Article
Keywords
cytochrome P450; peroxygenase reaction; pH dependence; rate-limiting step; site directed mutagenesis
ABSTRACT
Cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st) is a
thermophilic cytochrome P450 that shows high tolerance for harsh conditions and is capable of catalyzing
some peroxygenase reactions. Here, we investigated the pH dependence of the peroxygenase reactions
catalyzed by wild-type P450st and a mutant, in which the residues located close to the proximal heme
ligand are mutated. Both hydrogen peroxide-driven ethylbenzene hydroxylation and styrene epoxidation
by wild-type P450st were found to be activated in weak-acidic and weak-basic solutions. Under these
conditions, the Michaelis constant for hydrogen peroxide (KmH2O2) was decreased. The turnover rate (kcat)
of ethylbenzene hydroxylation was increased and followed an S-shaped curve with an increase in the pH
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value. The apparent acid dissociation constant (pKaapp) of the kcat was 7.0, which suggests that the
rate-limiting step of this reaction is the deprotonation of the FeIII-H2O2 complex. By introducing a double
mutation around the proximal heme ligand, the peroxygenase activity was increased over a wide pH range
and was dramatically increased at pH 5. The spectroscopic properties of this F310A/A320Q mutant
indicated that the Lewis acidity of the heme was increased by this mutation. Kinetic investigations
showed that the rise in the Lewis acidity of the heme facilitates the rate-limiting step of peroxygenase
H2O2
reactions and decreases the Km
value. Differences in the pH dependence of the kcat value between
wild-type P450st and the mutant suggest that the rate-limiting step switches to the protonation of the
ferric-hydroperoxo species (compound 0) under alkaline conditions.
INTRODUCTION
Cytochrome P450s (P450s) [1] are a family of heme-thiolate protein that are involved in various
metabolic and biosynthetic pathways in ubiquitous organisms. P450s catalyze the monooxygenation of
hydrophobic substrates by activating dioxygen for insertion into inactive hydrogen-carbon bonds. Their
ability to catalyze various oxidation reactions (e.g., hydroxylation, epoxidation, dealkylation, and C-C
bond cleavage) with high regio- and stereoselectivity potentially expands the application of P450s to
industrial chemistry.
In general, monooxygenation by P450s proceeds along a reaction cycle that requires a dioxygen, two
electrons from NAD(P)H, and two protons [1]. However, some P450s and P450 variants catalyze
peroxide driven-monooxygenation reactions via the peroxide shunt pathway [2-8]. The overall reaction
equation of this “peroxygenase reaction” is as follows:
RH + H2O2 → ROH + H2O
In this reaction, a molecule of peroxide (e.g., hydrogen peroxide, cumene hydroperoxide, t-butyl
hydroperoxide, or m-chloroperbenzoic acid (m-CPBA)) accesses the heme and directly forms active
oxidants. This reaction is especially useful for industrial applications because the P450 electron transport
system and NAD(P)H, which is expensive, are not required. However, the peroxygenase activities of
P450s are generally low. Although some P450 mutants [9, 10] and P450 derivatives (several fungal
peroxygenases [8, 11-13]) have been reported to catalyze peroxygenase reactions efficiently, there is still
not enough knowledge of the peroxygenase reaction properties.
In recent years, several thermophilic P450s from various thermophilic archaea and bacteria have been
reported [14-18]. These P450s possess a rigid structure and maintain their enzymatic activities even under
extreme conditions. For example, CYP119 from the acidothermophilic archaeon Sulfolobus
acidocaldarius was the first reported thermophilic P450. CYP119 exhibits a much higher melting
temperature (Tm = 91°C) than typical mesophilic P450s, such as P450cam (Tm = 54°C) [19, 20]. The high
structural stability of thermophilic P450s is adaptable not only for industrial applications but also for
elucidating the biochemical properties of P450s under a wide range of conditions.
We previously isolated a thermophilic P450 from the thermoacidophilic crenarchaeon Sulfolobus
tokodaii strain 7 (P450st). P450st exhibits high tolerance for heat (does not denature even at 80°C), acid,
and alkaline (maintains its local structure around the heme at pH 1.5-11) [16, 21-23]. We also revealed the
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capability of P450st to catalyze peroxygenase reactions (the kcat value of styrene epoxidation is 8.7 × 10-3
s-1 at pH 7.5) [21]. Therefore, P450st is an appropriate model for the elucidation of the peroxygenase
properties of P450s under various conditions. In this paper, we report the pH dependence of the
peroxygenase reactions catalyzed by P450st. The rate-limiting step of these reactions was identified by
kinetic investigations over the wide pH range. We also developed a more active mutant by mutating the
residues around the proximal heme ligand and determined that the Lewis acidity of the heme controls the
rate of this rate-limiting step.
RESULTS
pH dependence of the peroxygenase reactions catalyzed by wild-type P450st. The specific activities of
several peroxygenase reactions catalyzed by wild-type P450st were investigated at various pH values. The
endogenous substrates for P450st are unknown. However, long-chain saturated fatty acids (especially
lauric acid) have been experimentally shown to bind to P450st with high affinity, inducing a type I
spectral change. In this study, ethylbenzene and styrene were selected as model substrates because they
are commonly utilized and are easy to detect by gas chromatography. However, the binding of these
substrates to P450st does not induce a type I spectral change. After incubation with wild-type P450st and
hydrogen peroxide, ethylbenzene was converted into two products. The major product was identified as
1-phenylethanol by direct chromatographic comparison with an authentic standard, while the minor
product was unidentified. The peak area ratio of the minor product to the major product was 1:15 and was
relatively unchanged when the pH value was varied. Styrene was converted into a sole product, styrene
oxide, as reported previously [21]. Under all pH conditions, the identities of the products were unchanged,
and no products were detected in the absence of P450st.
The pH dependences of the specific activities of ethylbenzene hydroxylation and styrene epoxidation
by wild-type P450st via the peroxide shunt pathway are shown in Figure 1. At all pH values, the
ethylbenzene hydroxylation activity was higher than the styrene epoxidation activity. This result could be
due to a difference in the binding affinity of the two substrates. Notably, the pH dependences of these
reactions were in good agreement with each other although their substrate oxidation mechanisms are
different. This result suggests that the rate-limiting steps of ethylbenzene hydroxylation and styrene
epoxidation are the same and could be any step before the formation of the active oxidant.
To provide further insight into hydrogen peroxide-driven ethylbenzene hydroxylation at each pH value,
steady-state kinetic measurements of this reaction were made. At all pH values, plots of the initial rate
versus the hydrogen peroxide concentration gave a Michaelis-Menten-type saturation curve. The kinetic
parameters at each pH values are shown in Table 1 and Figure 2. Wild-type P450st showed the highest
Michaelis constant value (~80 mM) for hydrogen peroxide (KmH2O2) at pH 7. This value was reduced to
about one-third at pH 5 and 10, indicating that the affinity of P450st for hydrogen peroxide is increased in
H2O2
weak-acidic and weak-basic solutions. These changes in the Km
value could not be due to changes in
the protonation state of hydrogen peroxide due to its high pKa value (pKa ≈ 12 in water). Thus, some
changes in the protein structure and/or the electronic properties of heme could affect the affinity of P450st
for hydrogen peroxide.
On the other hand, the turnover rate (kcat) exhibited an S-shaped curve with increasing pH until pH 10
(Figure 2B). The apparent acid dissociation constant (pKaapp) value was 7.0, which was estimated by
fitting the curve to the Henderson-Hasselbalch equation. These findings indicate that the rate-limiting step
is any deprotonation reaction with a pKa value of 7.0.
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Modulation of the Lewis acidity of the heme by mutating the residues around the proximal heme site.
Previously, we prepared a series of P450st mutants in which the heme binding motif residues were
mutated [21]. In one of these mutants, the conserved phenylalanine (Phe310) is substituted with alanine
and Ala320 is substituted with a glutamine. This mutant (F310A/A320Q) exhibits a 30 mV positive-shift
in redox potential for the FeIII/FeII couple compared with that of wild-type P450st due to the weakening of
the push effect of the proximal thiolate in the mutant. This result indicates that the electron density around
the heme is decreased, and thus, the Lewis acidity of the heme is expected to be increased. We also
revealed that the F310A/A320Q mutant maintains the higher thermal stability than typical P450s [21].
Hence, this mutant is suitable to confirm the effects of increasing of the Lewis acidity of the heme on the
peroxygenase activity of P450st.
A previous spectroscopic study of wild-type P450st has noted that the Lewis acidity of the heme can be
evaluated by the pKa value of “the acid-alkaline transition”, which is due to the protonation equilibrium
between FeIII-H2O and FeIII-OH- (pKaAA) [23]. To investigate effects of the mutations on the Lewis acidity
of the heme, the pH dependence of the spectroscopic properties of the F310A/A320Q mutant was
investigated. The optical absorption spectra of the mutant at various pH values are shown in Figure 3A.
The mutant exhibited a typical spectrum of a ferric low-spin heme at pH 3-7, as previously demonstrated
by optical absorption spectroscopy and resonance Raman spectroscopy at pH 7.5 [21]. Below pH 2, the
mutant lost the heme due to cleavage of the Fe-S bond; heme dissociation occurs below pH 1.5 for
wild-type P450st [23]. This difference in tolerance for acid is likely due to a slight decrease in the
structural stability of the mutant [21].
In weak-basic solutions, the mutant exhibited a type II-like spectral change with no aggregation and/or
precipitation, as was previously observed for wild-type P450st [23]. This change was reversible and
showed well-defined isosbestic points at 376, 423, and 516 nm, demonstrating that only two states are
present during this transition. These findings demonstrate that the observed spectral change is the
acid-alkaline transition. The pKaAA value was calculated as 8.2
0.1 by fitting the curve to the
Henderson-Hasselbalch equation. This value is lower than that of wild-type P450st (pKaAA = 8.7 0.1)
[23], demonstrating that the Lewis acidity of the heme is increased by the mutations as expected. The
high Lewis acidity of the mutant reflects the low electron density around the heme.
pH dependence of the peroxygenase reactions catalyzed by the F310A/A320Q mutant. By mutating
F310 and A320, the peroxygenase activities were significantly increased over a broad pH range. Figure 4
shows the specific activities of the peroxygenase reactions (ethylbenzene hydroxylation and styrene
epoxidation) catalyzed by the F310A/A320Q mutant at various pH values. Similar to wild-type P450st,
the mutant converted ethylbenzene and styrene to 1-phenylethanol (with a small amount of unknown
byproduct) and styrene epoxide, respectively. The pH dependences of these reactions were changed to the
bell-shaped pH dependences with maximum activities near pH 5. At pH 5, the mutant was over 15-fold
more active than wild-type P450st. Notably, the pH dependence of styrene epoxidation was in good
agreement with that of ethylbenzene hydroxylation even for the mutant. This result suggests that, similar
to the case of wild-type P450st, the rate-limiting steps of both reactions are the same.
The kinetic parameters of hydrogen peroxide-driven ethylbenzene hydroxylation by the F310A/A320Q
mutant are shown in Table 2. Similar to wild-type P450st, plots of the initial rate versus the hydrogen
peroxide concentration gave a Michaelis-Menten-type saturation curve at all pH values investigated. The
H2O2
Km
values were significantly decreased in the mutant at all of the examined pH values, while its pH
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dependence was unchanged. The binding of hydrogen peroxide is most likely facilitated due to the
increase in the Lewis acidity of the heme iron.
Earlier, we revealed that wild-type P450st exhibited an S-shaped pH-kcat profile with a pKaapp value of
7.0 (Figure 2B). For the mutant, the pH-kcat profile was changed to a bell-shaped curve with a maximum
value at neutral pH (pH 5 ≈ pH 10 < pH 7). At pH 7, the kcat value was increased by 2.7-fold for the
mutant. This increase was likely due to the acceleration of the rate-limiting deprotonation of the
FeIII-H2O2 complex by the increase in the Lewis acidity of the heme. On the other hand, the kcat value was
decreased by 0.6-fold for the mutant at pH 10. This change indicates a change in the rate-limiting step.
DISCUSSION
We have investigated the peroxygenase property of wild-type P450st and a mutant at various pH values.
For wild-type P450st, hydrogen peroxide-driven ethylbenzene hydroxylation and styrene epoxidation
were activated in weak-acidic and weak-basic solutions, and the pH dependences of these reactions were
in good agreement with each other. The kcat value exhibited an S-shaped curve with increasing pH and the
pKaapp value of 7.0. These results indicate that the rate-limiting step is any deprotonation reaction before
the formation of the active oxidant with a pKa value of 7.0.
As shown in Scheme 1, the reaction mechanism of peroxygenase reactions by P450s involves many
protonation/deprotonation steps [1, 2, 24]. The resting state of P450s has a heme with a water ligand (or a
hydroxide ligand at high pH) [23] at the sixth coordination site. When a substrate and a hydrogen
peroxide access the active site, the ligand at the sixth coordination site is substituted by the hydrogen
peroxide and the ferric-hydroperoxide (FeIII-H2O2) complex is generated. Then, this complex is
deprotonated and converted to a ferric-hydroperoxo species (compound 0). Because compound 0 is a
good Lewis base, it undergoes protonation to the outer oxygen atom. This protonation induces heterolytic
O-O bond cleavage and forms an oxoferryl porphyrin π-cation radical species (compound I), which is
expected to be the major active oxidant of hydroxylation by P450s. During these steps, there is only one
deprotonation step, the formation of compound 0 by the deprotonation of the FeIII-H2O2 complex (Scheme
1, a). Therefore, this step should be the rate-limiting step of the peroxygenase reactions catalyzed by
wild-type P450st.
The pKa value of the FeIII-H2O2 complex of P450st and other P450s has not yet been experimentally
confirmed. Thus, we decided to estimate this value from the pKaAA value. Egawa et al. revealed that the
formation of compound 0 by several myoglobins via reaction with hydrogen peroxide is activated in
weak-basic solutions with a pKaapp value of 7.00-7.27 [25]. They also elucidated that the pKaapp values are
~1.7 units lower than the pKaAA values. The relationship between these two pKa values should be
applicable to P450st. The pKaAA value of wild-type P450st has been previously estimated as 8.7 [20],
suggesting that the pKa value of the formation of compound 0 by the deprotonation of the FeIII-H2O2
complex is 7.0. This value is completely in agreement with the pKaapp value that was calculated from the
pH dependence of the kcat value. This agreement confirms that the rate-limiting step of peroxygenase
reactions by P450st is the deprotonation of the FeIII-H2O2 complex.
This rate-limiting step is quite different from the case of typical monooxygenation reactions by P450s:
the rate-limiting steps are considered to be any step after formation of the active oxidant (e.g. C-H bond
breaking or product dissociation from P450s) as well as the electron transfer steps [26-30]. If the active
oxidant is formed rapidly enough, the rate-limiting step of peroxygenase reaction should be the substrate
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oxidation or the conformational change in the substrate-bound complex as described in recent studies of
CYP119 compound I [31-33].
Then, we attempted to improve the peroxygenase activity of P450st on the basis of the reaction
mechanism by a site-directed mutagenesis study. P450s have highly conserved residues around the
proximal cysteine ligand, which are collectively called the “heme-binding” motif. Previous studies have
revealed that several residues within this region control the redox potential of the heme. For example,
several site-directed mutagenesis studies of cytochrome P450BM3 have reported that a highly conserved
phenylalanine residue within the heme binding loop (Phe393 in P450BM3) controls electron donation
from the thiolate ligand [34-37]. In the present study, the F310A/A320Q mutant of P450st, which is also
mutated around the heme-binding motif, was utilized. We previously revealed that this mutant keeps the
high thermostability and exhibits a positive-shift in redox potential for the FeIII/FeII couple due to decrease
of the electron donation from the thiolate ligand [21].
The decreased pKaAA value of the F310A/A320Q indicated that the Lewis acidity of the heme is
increased by the mutations. The peroxygenase activity of this mutant was higher than that of wild-type
P450st over a broad pH range, and the pH-kcat profile was changed to a bell-shaped curve with a
maximum value at neutral pH. This change of the pH-kcat profile indicates a change in the rate-limiting
step in weak-basic solutions. The increase in the Lewis acidity of the heme not only accelerates the
deprotonation of the heme ligand but also inhibits its protonation. Especially in basic solutions, the
protonation of the heme ligand should be strongly inhibited. Thus, it is likely that a change in the
rate-limiting step of the peroxygenase reaction from the deprotonation of the FeIII-H2O2 complex to the
formation of compound I by the protonation of compound 0 results in the decrease in the kcat value at pH
10. This hypothesis is reasonable because the heterolytic cleavage of the O-O bond of compound 0 to
form compound I must be disturbed in the mutant due to the weak push effect of the proximal thiolate.
We previously reported that the F310A/A320Q mutant catalyzes styrene epoxidation via the peroxide
shunt pathway more efficiently than wild-type P450st in neutral solutions [21]. This report suggested that
the activation of the peroxygenase reaction in spite of the weakening push effect of the proximal heme
ligand indicates the participation of compound 0 in this reaction. However, this assumption is
contradicted by the present study because ethylbenzene hydroxylation, which is not catalyzed by
compound 0, is also activated with the mutant. Kinetic studies over a wide pH range revealed that
ethylbenzene hydroxylation and styrene epoxidation progress with the same rate-limiting step, the
deprotonation of the FeIII-H2O2 complex. As revealed in the present study, the previously reported
acceleration of styrene epoxidation via the peroxide shunt pathway for the mutant is thus attributed to the
acceleration of this step. These studies confirm that rates of the peroxygenase reactions catalyzed by
P450st are adjustable by changing the Lewis acidity of the heme.
Peroxygenase reactions catalyzed by P450s were discovered in the 1970’s, and enormous efforts to
understand this type of reaction have been undertaken. The reaction mechanism has been gradually
elucidated, and recently, highly reactive P450 mutants have been developed by protein engineering. In
addition, studies of some P450 derivatives (fungal peroxygenases) have revealed the property of
peroxygenase reaction. However, further investigation (e.g. kinetics, pH dependence, thermodynamic
analysis, and structural insight) is required to better understand this reaction. In this study, we revealed the
pH dependence of the peroxygenase reactions catalyzed by wild-type P450st and identified the
rate-limiting step of this reaction. We also succeeded to increase the peroxygenase activity of P450st on
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the basis of this mechanistic insight. These findings provide detailed insights into peroxygenase reactions
catalyzed by P450s.
The kinetic parameters determined in this study show that the low affinity for hydrogen peroxide and
the interruption of the deprotonation of the FeIII-H2O2 complex are primary problems for the efficient
progression of peroxygenase reactions. We demonstrated that an increase in the Lewis acidity of the heme
is an effective solution for these problems. This method should be applicable to other P450s because the
reaction mechanism of peroxygenase reactions is common for P450s. Our observations also revealed that
the reaction rate of the mutant is reduced in weak-alkaline solutions due to a change in the rate-limiting
step. This result indicates that the Lewis acidity of the heme must be optimized according to the reaction
conditions, taking into account the efficiency of the protonation/deprotonation steps in the reaction cycle.
To address this issue, arrangement of proton donor/accepters around the distal heme site seems to be
effective as well as modulation of the Lewis acidity of the heme only if substrate binding of substrates is
not disturbed.
EXPERIMENTAL PROCEDURE
Expression, purification, and spectroscopic observation of P450st. Wild-type P450st and the
F310A/A320Q mutant were expressed in Escherichia coli BL21 (DE3) (Merck) and purified as reported
previously [16, 21]. Optical absorption spectroscopy of the F310A/A320Q mutant was carried out with a
Shimadzu UV-2450 spectrometer at 25°C in several buffers (40 mM): glycine-HCl (pH 2-3), acetate (pH
4-5), potassium phosphate (pH 6-8), and glycine-NaOH (pH 9-12). The pKa value of the acid-alkaline
transition (FeIII H2O ↔ FeIII OH− + H+) of the mutant was determined by the shift of the Soret region with
fitting to the Henderson-Hasselbalch equation (eq. 1) as reported previously [23].
[FeIIIOH− ]
[FeIIIH2O]
pH = pKa +
(eq. 1)
Specific activities of the peroxygenase reactions. The specific activities of the peroxygenase reactions
catalyzed by wild-type P450st and the F310A/A320Q mutant were examined at 25°C in the various 40
mM buffers (pH 2-12) described above. The reaction mixtures (total volume of 200 μl) contained 40 μM
enzyme, 2 mM substrate (ethylbenzene or styrene), and 3 mM hydrogen peroxide. The substrates were
dissolved in acetonitrile, and 2 μl substrate was added to the reaction mixture. The reaction was initiated
by the addition of enzyme. Control incubations were performed without enzyme or hydrogen peroxide.
The samples were extracted with dichloromethane containing an internal standard (styrene for
ethylbenzene hydroxylation, and 2-phenylethanol for styrene epoxidation). The combined
dichloromethane layers were concentrated by evaporation and then analyzed by isothermal gas
chromatography (GC-4000, GL Science) at 80°C on an InterCap 1 column (30 m × 0.25 mm inner
diameter, GL Science). Under these conditions, the retention times for ethylbenzene, 1-phenylethanol,
styrene, and styrene oxide were 3.0, 6.9, 3.3, and 7.0 min, respectively. The products formations were
quantified using relative peak areas with respect to the internal standards.
Steady-state kinetic analysis. All steady-state kinetic measurements of ethylbenzene hydroxylation were
performed at 30°C in several 40 mM buffers (pH 4-10). The reaction mixtures (total volume of 200 μl)
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contained 10 μM enzyme, 10 mM ethylbenzene, and 1-300 mM hydrogen peroxide. The reaction,
extraction, and product detection were performed as described above (in the “Specific activities of the
peroxygenase reactions” section). The kinetic constants were calculated with Hanes-Woolf plots.
ACKNOWLEDGMENTS
This study was supported by a grant from the Japan Society for the Promotion of Science (No.
21605004).
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Table 1. Kinetic parameters of hydrogen peroxide-driven ethylbenzene hydroxylation by wild-type
P450st at various pH values.
H2O2
kcat/Km
KmH2O2 (mM)
kcat (min-1)
(× 10-2 min-1 mM-1)
pH 5
pH 7
pH 10
23.2 2.0
81.4 3.6
26.2 13.9
5.6 1.4
7.5 0.5
9.5 3.1
24.0 4.9
9.3 0.7
41.5 13.6
Table 2. Kinetic parameters of hydrogen peroxide-driven ethylbenzene hydroxylation by the
F310A/A320Q mutant at various pH values.
H2O2
kcat/Km
KmH2O2 (mM)
kcat (min-1)
(× 10-2 min-1 mM-1)
pH 5
pH 7
pH 10
7.2 2.4
30.6 1.6
9.3 1.7
4.3 0.6
20.4 5.2
5.3 0.5
62.2 14.5
66.7 17.4
57.8 5.4
SCHEME 1. The reaction mechanism of hydroxylation by P450s via the peroxide shunt pathway.
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FIGURE 1. Specific activities of the peroxygenase reactions (ethylbenzene hydroxylation (black) and
styrene epoxidation (gray)) catalyzed by wild-type P450st at pH 2-12.
FIGURE 2. The pH dependences of (A) the Km value for hydrogen peroxide (KmH2O2) and (B) the kcat
value of hydrogen peroxide-driven ethylbenzene hydroxylation by wild-type P450st. Plots of the kcat value
as a function of pH were fitted to the Henderson-Hasselbalch equation for calculating the apparent acid
dissociation constant (pKaapp).
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FIGURE 3. (A) Optical absorption spectra of the ferric state of the F310A/A320Q mutant at pH 2
(dashed line), pH 7 (solid line), and pH 10 (dotted line). (B) Shift of the Soret region as a function of pH.
The plots were fitted to the Henderson-Hasselbalch equation to calculate the pKa value.
FIGURE 4. Specific activities of the peroxygenase reactions (ethylbenzene hydroxylation (A) and
styrene epoxidation (B)) catalyzed by wild-type P450st (black) and the F310A/A320Q mutant (gray) at
pH 2-12.
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