Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide

Article abstract of DOI:10.1271/bbb.69.293

Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H ₂O₂, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H₂O ₂-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H₂O₂, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.

Full text of DOI:10.1271/bbb.69.293

Biosci. Biotechnol. Biochem., 69 (2), 293–300, 2005
Indole Hydroxylation by Bacterial Cytochrome P450 BM-3
and Modulation of Activity by Cumene Hydroperoxide
y
1;
Qing-Shan LI,1 Jun OGAWA,1 Rolf D. SCHMID,2 and Sakayu SHIMIZU
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
2Institut fur Technische Biochemie, Universitat Stuttgart, Allmandring 31, D-70459 Stuttgart, Germany
¨
¨
Received July 28, 2004; Accepted November 12, 2004
Cytochrome P450 BM-3 from Bacillus megaterium
catalyzed NADPH-supported indole hydroxylation un-
der alkaline conditions with homotropic cooperativity
toward indole. The activity was also found with the
support of H2O2, tert-butyl hydroperoxide (tBuOOH),
or cumene hydroperoxide (CuOOH). Enhanced activity
and heterotropic cooperativity were observed in
CuOOH-supported hydroxylation, and both the Hill
coefficient and substrate concentration required for
half-maximal activity in the CuOOH-supported reac-
tion were much lower than those in the H2O2-,
tBuOOH-, or NADPH-supported reactions. CuOOH
greatly enhanced NADPH consumption and indole
hydroxylation in the NADPH-supported reaction. How-
ever, when CuOOH was replaced by tBuOOH or H2O2,
heterotropic cooperativity was not observed. Spectral
studies also confirmed that CuOOH stimulated indole
binding to P450 BM-3. Interestingly, a mutant enzyme
with enhanced indole-hydroxylation activity, F87V
(Phe87 was replaced by Val), lost homotropic cooper-
ativity towards indole and heterotropic cooperativity
towards CuOOH, indicating that the active-site struc-
ture affects the cooperativities.
one,5) 17ꢀ-estradiol,5) aflatoxin B1,5) and amitriptyline,5)
the enzyme activity is influenced by several effectors.
ꢁ-Naphthoflavone (ANF) stimulates P450 3A4 activity
on the oxidation of progesterone,2–4) testosterone,4)
estradiol,6) aflatoxins,7,8) polycyclic aromatic hydro-
carbons,9,10) carbamazepine,11) and acetaminophen.12)
Kinetic analysis of 7,8-benzoflavone and phenanthrene
oxidation by P450 3A4 indicated that they increase each
other’s kcat values without changes in their Km values,
suggesting that the two substrates are simultaneously
present in the same active site.10) The simultaneous
binding of two substrates in one active site has also been
observed in the crystal structures of P450 eryF/9-
aminophenanthrene and P450 ery F/androstenedione.13)
Based on these results, it is proposed that a P450 enzyme
with an active site large enough to accommodate
multiple ligands will exhibit this type of cooperativity.13)
A well studied bacterial P450, P450 BM-3 from
Bacillus megaterium, catalyzes the subterminal hy-
droxylation of long-chain saturated fatty acids, and the
corresponding amides and alcohols, or produces the
epoxides of medium and long-chain unsaturated fatty
acids.14–17) It catalyzes the oxidation of saturated or
unsaturated fatty acids without cooperativity, and only
one substrate has been observed in the active site in the
structure of the P450 BM-3 heme domain complexed
with palmitoleic acid.18)
Key words: cytochrome P450; P450 BM-3; cumene
hydroperoxide; cooperativity; peroxide
shunt pathway
Here, we report that homotropic cooperativity is
involved in indole hydroxylation catalyzed by P450
BM-3 under alkaline conditions. The activity was
greatly stimulated by cumene hydroperoxide (CuOOH)
through heterotropic cooperativity. In addition, we
created a mutant enzyme that shows high activity and
affinity for indole.19) But, we found in this study that the
mutant F87V (Phe87 was replaced by Val) does not
show homotropic cooperativity towards indole and also
loses heterotropic cooperativity towards CuOOH. These
results suggest that the environment of the binding site
influences cooperativity.
Cytochrome P450 monooxygenases (P450s) have vast
potential for environmental treatment, drug design, and
pharmaceutical and chemical syntheses.1) In this regard,
the active site structures and interactions with substrates
of P450s have been the focus of much interest. Several
mammalian P450s with a large active site exhibit
homotropic cooperativity towards a number of sub-
strates, and their activities are also influenced by
effectors. P450 3A4, the most important and abundant
P450 in the liver, is one of the most extensively studied.
In addition to its homotropic cooperativity towards
several substrates, including progesterone,2–4) testoster-
y
To whom correspondence should be addressed. Tel: +81-75-753-6115; Fax: +81-75-753-6128; E-mail: sim@kais.kyoto-u.ac.jp
Abbreviations: P450, cytochrome P450; WT, wild type of P450 BM-3; F87V, Phe87Val mutant of P450 BM-3; CuOOH, cumene hydroperoxide;
tBuOOH, tert-butyl hydroperoxide

294
Q.-S. LI et al.
Materials and Methods
supported indole hydroxylation with 1 mM CuOOH
was calculated by subtracting the values of the control
without NADPH from the measured data.
Preparation of WT and F87V enzymes. WT and F87V
enzymes were obtained as described previously.19) The
expression and purification of P450, and determination
of the concentration of P450 were carried out as
described previously.20)
Spectral binding studies. Binding spectra were re-
corded with a Shimadzu MultiSpec-1500 spectropho-
tometer. For analysis of indole binding to WT or F87V,
various concentrations of indole in 0.1 M Tris/HCl
(pH 8.6) were prepared (see the legend to Fig. 3), and
120 ml of a 20 mM P450 sample was added to 480 ml of
the indole solutions. The difference spectra were
recorded from 340 to 500 nm, using the same solution
without indole as the reference. For CuOOH stimulation
of indole binding to WT, 400 ml of 0.45 mM indole in
0.1 M Tris/HCl (pH 8.6) was mixed with 100 ml of a
24 mM P450 sample and 100 ml of 6 mM CuOOH in 0.1 M
Tris/HCl. The spectra were recorded 30 s after all the
components had been mixed together, using the same
solution without indole and CuOOH as a reference at
25 ꢂC. The change in absorbance (A) was determined by
subtracting the absorbance at 425 nm (the trough) from
that at 390 nm (the peak). The concentration of indole
added for binding was used as the concentration of free
Enzyme activity assay. Indole hydroxylation activity
was evaluated by monitoring blue color development
due to indigo formation through P450-catalyzing hy-
droxylation and successive chemical dimerization.19)
Kinetic analysis was performed as described previous-
ly.21) Nonlinear regression was used to determine Vmax
n
and S50, using the equation v ¼ ðVmaxSnÞ=ðS50 þ SnÞ.
The Hill coefficient (n) was determined by plotting
log½v=ðVmax ꢀ vÞꢁ vs. log S.
Hydroperoxide-supported reaction: To measure in-
dole hydroxylation supported by H2O2, tert-butyl hydro-
peroxide (tBuOOH; Katayama Kougyo, Osaka, Japan),
or CuOOH (Aldrich, Milwaukee, WI, U.S.A.), 60 ml of a
45 mM P450 sample, was mixed with 480 ml of 0.1 M
Tris/HCl buffer (pH 8.6) containing 0.03 to 30 mM
indole. After 2 min, 60 ml of a hydroperoxide solution
was added to start the reaction. The concentrations of
hydroperoxides in the reaction mixtures are indicated in
Fig. 2, Table 2, and Table 3. The reaction was carried
out at 25 ꢂC and indigo formation was monitored at
670 nm for 10 min. The amount of indigo was calculated
using "M ¼ 13400 Mꢀ1 cmꢀ1, which was obtained with
pure indigo. Three types of controls were carried out
under the same conditions except for the absence of one
of these compositions, P450, indole, or hydroperoxides.
NADPH-supported reaction: To measure the NADPH
consumption rate in indole hydroxylation, 60 ml of a
10 mM P450 sample was mixed with 480 ml of 0.1 M Tris/
HCl buffer (pH 8.6) containing 0.03 to 30 mM indole,
and after 2 min standing, 60 ml of 3.5 mM NADPH was
added to start the reaction. NADPH consumption was
monitored at 340 nm for 30 s at 25 ꢂC and the NADPH
indole for data analysis, i.e., ligandfree ¼ ligandtotal
.
Calculation was carried out according to the method
described previously.21,22) ꢀAmax was determined by
nonlinear regression of the plots of ꢀA vs. S using the
n
equation ꢀA ¼ ꢀAmaxSn=Ks þ Sn.
HPLC analysis. HPLC analysis was performed to
determine the amounts of degradation products of
CuOOH. The reaction samples were extracted twice
with chloroform, 6 ml in total, evaporated to dryness,
and then dissolved in 5% dimethyl sulfoxide for HPLC
analysis. HPLC analysis was carried out on a C18
reverse phase column (Cosmosil 5C18 AR-II, 4.6 mm
I.D. ꢃ 250 mm; Nacalai Tesque, Kyoto, Japan), with
elution at 1.0 ml/min with a methanol/water gradient
containing 1% acetic acid. The gradient of methanol was
as follows: 0–5 min, 0% (v/v); 5–50 min, 0–80%; and
50–60 min, 80%. The eluent was monitored at 254 nm.
ꢀ1
concentration was calculated using "M ¼ 6200 M
cmꢀ1 22) To measure the indole hydroxylation supported
.
by NADPH, 60 ml of a 45 mM P450 sample was mixed
with 480 ml of 0.1 M Tris/HCl buffer (pH 8.6) containing
0.3 to 30 mM indole, and after 2 min standing, 60 ml of
10.5 mM NADPH was added to start the reaction,
followed by standing for 1 h at 25 ꢂC. The total amount
of indigo produced was determined at 670 nm. To
monitor the effects of CuOOH on NADPH consumption
or on NADPH-supported indole hydroxylation, the
procedures were the same as those without the addition
of CuOOH, except that 1 mM CuOOH was added 1 min
before the addition of NADPH and the measurement of
indigo was carried out after the reaction mixture had
stood for 15 min at 25 ꢂC. Four kinds of control
experiments were carried out under the same conditions,
except for the absence of one of these compositions:
P450, indole, CuOOH, or NADPH. The NADPH-
Results
Detection of indole hydroxylation activity of WT P450
BM-3
Several mutants of P450BM-3, such as F87V, have
been reported to be able to hydroxylate indole, while
WT could not.19) After extensive tests under various
conditions, we found that WT also catalyzes NADPH-
supported indole hydroxylation under high pH condi-
tions. However, it is not practical to determine the indole
hydroxylation activity of WT by measuring indigo
formation, because the amount of indigo formed with
NADPH is too small. Therefore the activity was
measured by monitoring NADPH consumption. The
kinetic data are summarized in Table 1. With the
support of NADPH, WT exhibited a typical ‘‘S’’ curve

Indole Hydroxylation by P450 BM-3
295
Table 1. Kinetic Data for Indole Hydroxylation Catalyzed by WT and F87V
WT
F87V
a
b
a
b
kcat
S50 (mM)
n
kcat
Km (mM)
2:1 ꢄ 0:3
n
NADPH
323 ꢄ 31
13:2 ꢄ 2:1
1:78 ꢄ 0:24
383 ꢄ 42
1:01 ꢄ 0:02
aThe kcat unit for NADPH is nmol NADPH/min/nmol P450.
bS50 and Km are all for indole.
Table 2. Kinetic Analysis of Indole Hydroxylation Catalyzed by WT and F87V
WT F87V
kcat of indole oxidation
(nmol indigo/min/nmol P450)
Km for hydroperoxide
(mM)
kcat of indole oxidation
(nmol indigo/min/nmol P450)
Km for hydroperoxide
(mM)
H2O2
tBuOOH
CuOOH
0:32 ꢄ 0:05
0:24 ꢄ 0:03
30:7 ꢄ 3:2
20:4 ꢄ 2:7
26:3 ꢄ 3:5
14:4 ꢄ 1:1
29:4 ꢄ 2:2
10:9 ꢄ 1:4
45:5 ꢄ 5:1
25:6 ꢄ 4:3
23:9 ꢄ 3:9
22:3 ꢄ 3:1
The hydroperoxide concentrations in the reaction mixtures were in the range of 1 to 30 mM. The indole concentrations were fixed at 11.2, 11.2, 6.4, 0.9, 6.4, and
6.4 mM in the reactions with WT and H2O2, WT and tBuOOH, WT and CuOOH, F87V and H2O2, F87V and tBuOOH, and F87V and CuOOH respectively.
Fig. 2. Turnover Numbers of WT in Indole Hydroxylation Supported
by 8 mM H2O2, 8 mM tBuOOH and 8 mM CuOOH with a Series of
Concentrations of Indole.
Each point represents the average value for three independent
measurements.
WT and n ¼ 1:01 ꢄ 0:02 for F87V (Fig. 1B). In sum,
WT exhibited homotropic cooperativity toward indole,
but F87V did not.
Hydroperoxide-supported indole hydroxylation by WT
and F87V
Indole hydroxylation by WT was further investigated
with the support of different hydroperoxides. We
observed that CuOOH strongly supported the reaction
with a kcat value nearly 100-fold of that obtained in the
Fig. 1. NADPH Consumption by WT and F87V in Indole Hydroxy-
tBuOOH or H2O2-supported reaction (Table 2 and
lation with a Series of Concentrations of Indole (A), and Linear
Fig. 2). In the absence of P450 BM-3 enzymes, no
Regression of Plots of log½v=ðVmax ꢀ vÞꢁ vs. log S (B).
indigo was formed with any of these hydroperoxides
(data not shown), suggesting that the hydroxylation was
actually an enzyme-catalyzed reaction. Kinetic analysis
indicated that the Km values of CuOOH, tBuOOH, and
H2O2 for WT were close to each other (Table 2). The
small differences in affinities of these hydroperoxides to
WT appear not to be responsible for the large differences
Each point presents the average value for three independent
measurements.
for NADPH consumption with different concentrations
of indole, but F87V did not (Fig. 1A). The Km value of
F87V was less than 16% of the S50 for indole of WT
(Table 1). The Hill coefficient was n ¼ 1:78 ꢄ 0:24 for

296
Q.-S. LI et al.
Table 3. Kinetic Analysis of Hydroperoxide-Supported Indole Hydroxylation by WT
H2O2 (mM)
16
tBuOOH (mM)
16
CuOOH (mM)
2
1
4
kcat of indole oxidation
(nmol indigo/min/nmol P450)
0:26 ꢄ 0:04
7:71 ꢄ 0:54
1:96 ꢄ 0:34
0:27 ꢄ 0:04
7:54 ꢄ 0:67
1:98 ꢄ 0:29
2:20 ꢄ 0:20
1:76 ꢄ 0:19
1:37 ꢄ 0:10
3:56 ꢄ 0:24
1:75 ꢄ 0:22
1:16 ꢄ 0:07
7:70 ꢄ 0:52
3:34 ꢄ 0:27
1:02 ꢄ 0:03
S50 for indole
(mM)
Hill coefficient
(n)
in indole hydroxylation activities. These results suggest
that, rather than as oxygen donor, CuOOH may play
another important role(s) in WT-catalyzing indole
hydroxylation. As described below, CuOOH enhanced
the activity through increasing the binding affinity of
indole to WT.
On the other hand, as shown in Table 2, all three
hydroperoxides effectively supported indole hydroxyla-
tion catalyzed by a mutant enzyme F87V, which has a
substitution of Phe87 by Val and shows increased indole
hydroxylation activity supported by NADPH.19)
gradually changed to a Michaelis–Menten type curve
when higher concentrations (2 or 4 mM) of CuOOH
were used (data not shown). The Hill coefficients (n),
indicators of the degree of cooperativity, were 1:96 ꢄ
0:34 and 1:98 ꢄ 0:29 for the H2O2- and tBuOOH-
supported reactions (Table 3) respectively, but decreas-
ed greatly to 1:37 ꢄ 0:10 and further to 1:02 ꢄ 0:03
when the reactions were supported by 1 and 4 mM
CuOOH respectively. The S50 values of indole for WT
were 7:71 ꢄ 0:54 and 7:54 ꢄ 0:67 mM for the H2O2- and
tBuOOH-supported reactions respectively, but also
decreased greatly to 1:76 ꢄ 0:19 and 1:75 ꢄ 0:22 mM
for the reactions supported by 1 and 2 mM CuOOH
(Table 3) respectively. These results indicate that indole
binds to WT with homotropic cooperativity and that
CuOOH can significantly stimulate indole binding to
WT and reduce the homotropic cooperativity of indole.
CuOOH supports WT-catalyzing indole hydroxylation
through a P450-type reaction
Since the CuOOH-supported hydroxylation activity of
WT to its native substrates is very low,22) it should be
clarified whether CuOOH supports indole hydroxylation
through a P450-type reaction. The degradation products
of CuOOH produced through indole hydroxylation by
WT were analyzed by HPLC. The results indicated that
13 and 87% of the CuOOH was converted to acetophe-
none and cumyl alcohol respectively (these compounds
were not contained in the original CuOOH solution).
The latter was produced through heterolytical cleavage
of the o–o bond of CuOOH, i.e., a P450-type reaction.23)
The molar amount of cumyl alcohol was 2.85-fold that
of indigo, for one mol of which at least two mols of
CuOOH were required. The small amount of acetophe-
none and a little more of cumyl alcohol might be
products derived from the CuOOH decomposed by WT
without any contribution to the indole hydroxylation,
because P450 can catalyze the transformation of
CuOOH into the two compounds without the participa-
tion of any other substrate.23) These results suggest that
WT-catalyzing indole hydroxylation involves heterolyt-
ical cleavage of the o–o bond of CuOOH, i.e., through a
P450-type reaction, not a peroxidase-type reaction.
Spectral observation of indole binding to WT and
F87V in the absence of CuOOH
The homotropic cooperativity of indole binding to
WT was further confirmed by spectral binding assays in
the absence of CuOOH. The results indicate that indole
bound to WT with half-maximal binding at 7:4 ꢄ
0:7 mM and a Hill coefficient of 1:73 ꢄ 0:15 (Fig. 3A
and C). On the other hand, F87V did not show any
cooperativity for indole binding. F87V showed high
affinity to indole with half-maximal binding at
0:75 ꢄ 0:06 mM and a Hill coefficent of 1:00 ꢄ 0:12,
i.e., no cooperativity (Fig. 3B and C).
Spectral observation of indole binding to WT in the
presence of CuOOH
It is difficult to carry out kinetic analysis of indole
binding to WT with CuOOH addition, because CuOOH
oxidizes WT and also mediates WT-catalyzed indole
hydroxylation, and a steady substrate-binding spectrum
is hard to obtain. However, it is still possible to observe
the stimulation of indole binding to WT caused by
CuOOH addition. Both indole and CuOOH can induce a
type I binding spectrum with a peak at 390 nm and a
trough at 425 nm. For comparison, spectra were record-
ed under the same conditions except that the enzyme
was mixed with the substrate solution containing only
indole, or CuOOH, or indole and CuOOH together. The
concentrations of the substrate-binding complex were
2:4 ꢄ 0:8, 15 ꢄ 5 and 30 ꢄ 9% of the total enzyme
Kinetic analysis of CuOOH-supported indole hydrox-
ylation by WT
Kinetic analysis of WT-catalyzing indole hydroxyl-
ation was performed with the support of 16 mM H2O2,
16 mM tBuOOH, or 1, 2 or 4 mM CuOOH. With 16 mM
tBuOOH or 16 mM H2O2, WT exhibited a typical ‘‘S’’
curve for indigo formation with different concentrations
of indole. With 1 mM of CuOOH, an ‘‘S’’ curve for
indigo formation was also observed. The ‘‘S’’ curve

Indole Hydroxylation by P450 BM-3
297
Fig. 3. Indole-Induced Spectral Shifts of P450 BM-3.
(A) Difference spectra from a single assay of 4 mM of WT to a series of concentrations of indole: 1.92, 2.4, 3.6, 4.8, 6.0, 7.2, 9.6, 12, and
14.4 mM. (B) Difference spectra from a single assay of 4 mM of F87V to a series of concentrations of indole: 0.24, 0.48, 0.72, 0.96, 1.44, 1.92, 2.4,
3.6, 4.8, 6.0, and 7.2 mM. The arrows in (A) and (B) indicate the direction of spectral changes following low to high concentrations of indole. (C)
Kinetic analysis of indole binding. Hill coefficients (n) are the averages of the individual values determined from the slopes of the linear
regression lines of three independent titrations for WT and two titrations for F87V.
concentration with the addition of 0.3 mM indole, 1 mM
CuOOH, and both, respectively. Therefore, CuOOH
stimulated indole binding to WT.
CuOOH enhances NADPH consumption by WT with
low concentrations of indole
The NADPH consumption rate of WT with the
addition of 1 mM CuOOH and a series of concentrations
of indole was investigated (Fig. 4). The kinetic data are
presented in Table 4. The addition of CuOOH greatly
enhanced the consumption of NADPH by WT in the
presence of low concentrations of indole. There was no
NADPH consumption observed without P450 BM-3
enzymes, suggesting that NADPH consumption was
actually a P450-catalyzing reaction. The S50 value for
indole decreased more than 80-fold, from 13.2 to
Fig. 4. Effect of 1 mM CuOOH on NADPH Consumption by WT in
the Presence of Indole.
0.16 mM, without any significant difference in the kcat
value with the addition of 1 mM CuOOH. The Hill
coefficient also largely decreased with the addition of
1 mM CuOOH (Table 4). However, the S50 value for
indole did not change obviously with the addition of
H2O2, tBuOOH, acetophenone, or cumyl alcohol instead
of CuOOH (data not shown). These results indicate that
the molecular structure of the whole CuOOH molecule,
rather than the properties of a hydroperoxide or its
Each point represents the average value for three independent
measurements.
degradation products, played a role in the heterotropic
stimulation of indole binding to WT. On the other hand,
the addition of CuOOH did not significantly affect the
NADPH consumption rate of F87V.

298
Q.-S. LI et al.
Table 4. Effect of 1 mM CuOOH on Kinetics of NADPH Consumption by WT in the Presence of Indole
kcat of NADPH consumption
(nmol NADPH/min/nmol P450)
S50 for indole
(mM)
Hill coefficient
(n)
Without CuOOH
With 1 mM CuOOH
323 ꢄ 31
256 ꢄ 29
13:2 ꢄ 2:1
1:78 ꢄ 0:24
1:03 ꢄ 0:04
0:16 ꢄ 0:02
The concentrations of indole used were the same as indicated in Fig. 3.
another role(s) in the reaction in addition to that of
oxygen doner.
The very close Km values of the three hydroperoxides
for WT show that the affinities of the three hydro-
peroxides to WT are similar. Kinetic analysis showed
that CuOOH reduces the Hill coefficient and S50 of
indole hydroxylation by WT, as a P450 3A4 activator
does.2–4,21) These results clearly support the view that
CuOOH acts as a heterotropic stimulator in indole
hydroxylation by WT, in addition to its role as an
oxygen donor. NADPH consumption and NADPH-
supported indole hydroxylation by WT under low indole
concentrations can significantly increase with the addi-
tion of CuOOH, but not with the addition of the products
derived from CuOOH, viz., acetophenone or cumyl
alcohol. These results indicate that the whole molecule
acts as a stimulator, i.e., the stimulation might be caused
by CuOOH binding. But the coupling efficiency was still
low, approximately 3%, as found with several aromatic
substrates in WT-catalyzing hydroxylation.25,26)
Fig. 5. Effect of 1 mM CuOOH on NADPH-Supported Indole
Hydroxylation by WT.
Each point represents the average value for three independent
measurements.
CuOOH enhances NADPH-supported indole hydroxy-
lation by WT
Cooperativity has been observed for P450 3A4 and
other mammalian P450s with a big active site. The large
active site is probably related to the ability to recognize
a wide range of substrates, but it reduces the affinity of
substrate binding. An effector is supposed to reduce the
effective size of the active site and to change the binding
environment, which is expected to result in an increase
in the binding affinity of the first substrate on the binding
of the second, thereby yielding homotropic or hetero-
tropic cooperativity.21) P450 BM-3 has a small active
site and catalyzes the hydroxylations of a limited
number of native substrates without cooperativity.14–17)
These characteristics of P450 BM-3 are in significant
contrast with those of P450 3A4. We created various
mutant enzymes.19) By analysis of these mutants, we
found that removing the aromatic ring of Phe87 from the
heme pocket of P450 BM-3 by replacement of Phe87
with Val greatly increased the activities of this enzyme
towards a variety of different compounds such as
indole, phenol derivatives, and polycyclic aromatic
compounds.19,25,26) Phe87 is an important barrier that
prevents these aromatic compounds from accessing the
active site of P450 BM-3. The P450 BM-3 structure also
indicates that the aromatic ring of Phe87 extends into the
heme pocket and is located above the porphyrin.18)
CuOOH binding in the active site could move Phe87 to a
new position and thus create a space for indole binding,
which would result in high affinity for indole. Replace-
ment of Phe87 by Val resulted in F87V losing
homotropic cooperativity as well as heterotropic stim-
Since CuOOH can stimulate NADPH consumption by
WT with low concentrations of indole, whether CuOOH
stimulates NADPH-supported indole hydroxylation by
WT was investigated. The NADPH-supported hydroxy-
lation of indole by WT with and without the addition of
1 mM CuOOH is shown in Fig. 5. With the addition of
1 mM CuOOH, production of indigo, the end product of
indole hydroxylation, was observed even with a con-
centration of 0.2 mM indole. On the other hand, without
CuOOH it occurred only when the concentration of
indole was higher than 3 mM. Acetophenone and cumyl
alcohol did not stimulate indole hydroxylation by WT
(data not shown).
Discussion
In this study, we found that WT P450 BM-3 catalyzes
indole hydroxylation with homotropic cooperativity
toward indole under alkaline conditions. Furthermore,
we found that the activity was modulated by CuOOH.
CuOOH has been used extensively to support P450
activity as an oxygen donor through the peroxide shunt
pathway,23) and its effect on the inactivation of P450
activity has also been reported,24) but no work on its role
as a stimulator of P450 activity has been reported. The
activity of WT in indole hydroxylation supported by
CuOOH is much higher than that supported by tBuOOH,
H2O2, or NADPH, suggesting that CuOOH might play

Indole Hydroxylation by P450 BM-3
299
8) Shimada, T., and Guengerich, F. P., Evidence for
cytochrome P-450NF, the nifedipine oxidase, being the
principal enzyme involved in the bioactivation of
aflatoxins in human liver. Proc. Natl. Acad. Sci.
U.S.A., 86, 462–465 (1989).
9) Koley, A. P., Buters, J. T. M., Robinson, R. C.,
Markowitz, A., and Friedman, F. K., Differential
mechanisms of cytochrome P450 inhibition and activa-
tion by ꢁ-naphthoflavone. J. Biol. Chem., 272, 3149–
3152 (1997).
10) Shou, M., Grogan, J., Mancewicz, J. A., Krausz, K. W.,
Gonzalez, F. J., Gelboin, H. V., and Korzekwa, K. R.,
Activation of CYP3A4: evidence for the simultaneous
binding of two substrates in a cytochrome P450 active
site. Biochemistry, 33, 6450–6455 (1994).
ulation by CuOOH, but greatly increased its affinity to
indole with a Km value of 0.75 mM and also the coupling
efficiency of NADPH-consumption and indole hydroxy-
lation.
Studies on substrate interactions with P450 BM-3,
combined with structural analysis, will provide infor-
mation to clarify the mechanisms of the substrate
binding and hydroxylation specificity in a P450 reaction.
Such information will be very useful for the redesign of
novel substrate specificity of a P450 and the P450
related drug design.
Acknowledgments
11) Kerr, B. M., Thummel, K. E., Wurden, C. J., Klein, S.
M., Kroetz, D. L., Gonzalez, F. J., and Levy, R. H.,
Human liver carbamazepine metabolism. Role of
CYP3A4 and CYP2C8 in 10,11-epoxide formation.
Biochem. Pharmacol., 47, 1969–1979 (1994).
12) Li, Y., Wang, E., Patten, C. J., Chen, L., and Yang, C.,
Effects of flavonoids on cytochrome P450-dependent
acetaminophen metabolism in rats and human liver
microsomes. Drug Metab. Dispos., 22, 566–571 (1994).
13) Cupp-Vickery, J., Anderson, R., and Hatziris, Z., Crystal
structures of ligand complexes of P450eryF exhibiting
homotropic cooperativity. Proc. Natl. Acad. Sci. U.S.A.,
97, 3050–3055 (2000).
14) Ruettinger, R. T., Wen, L. P., and Fulco, A. J., Coding
nucleotide, 50 regulatory, and deduced amino acid
sequences of P-450BM-3, a single peptide cytochrome
P-450: NADPH-P-450 reductase from Bacillus mega-
terium. J. Biol. Chem., 264, 10987–10995 (1989).
15) Narhi, L. O., and Fulco, A. J., Characterization of a
catalytically self-sufficient 119,000-Dalton cytochrome
P450 monoxygenase induced by barbiturates in Bacillus
megaterium. J. Biol. Chem., 261, 7160–7169 (1986).
16) Boddupalli, S. S., Estabrook, R. W., and Peterson, J. A.,
Fatty acid monooxygenation by cytochrome P450 BM-3.
J. Biol. Chem., 265, 4233–4239 (1990).
17) Graham-Lorence, S. E., Truan, G., Peterson, J. A., Falck,
J. R., Wei, S., Helvig, C., and Capdevila, J. H., An active
site substitution, F87V, converts cytochrome P450BM-3
into a regio- and stereoselective (14S,15R)-arachidonic
acid epoxygenase. J. Biol. Chem., 272, 1127–1135
(1997).
The authors wish to thank Professor T. Sakaki of
Toyama Prefectural University for helpful discussions.
Q. S. Li is a post-doctoral fellow (no. P99115) supported
by the Japan Society for the Promotion of Science
(JSPS). This work was supported in part by the Project
for the Development of a Technological Infrastructure
for Industrial Bioprocesses on R&D of New Industrial
Science and Technology Frontiers (to SS) of the New
Energy and Industrial Technology Development Organ-
ization (NEDO) of Japan, and by COE for Microbial-
Process Development Pioneering Future Production
Systems (the COE program of the Ministry of Educa-
tion, Culture, Sports, Science, and Technology of
Japan).
References
1) Joo, H., Lin, Z., and Arnold, F. H., Laboratory evolution
of peroxide-mediated cytochrome P450 hydroxylation.
Nature, 339, 670–673 (1999).
2) Schwab, G. E., Raucy, J. L., and Johnson, E. F.,
Modulation of rabbit and human hepatic cytochrome P-
450-catalyzed steroid hydroxylations by alpha-naphtho-
flavone. Mol. Pharmacol., 33, 493–499 (1988).
3) He, Y. A., He, Y. Q., Szklarz, G. D., and Halpert, J. R.,
Identification of three key residues in substrate recog-
nition site 5 of human cytochrome P450 3A4 by cassette
and site-directed mutagenesis. Biochemistry, 36, 8831–
8839 (1997).
4) Harlow, G. R., and Halpert, J. R., Alanine-scanning
mutagenesis of a putative substrate recognition site in
human cytochrome P450 3A4. Role of residues 210 and
211 in flavonoid activation and the substrate specificity.
J. Biol. Chem., 272, 5396–5402 (1997).
5) Ueng, Y. F., Kuwabara, T., Chun, Y. J., and Guengerich,
F. P., Cooperativity in oxidations catalyzed by cyto-
chrome P450 3A4. Biochemistry, 36, 370–381 (1997).
6) Kerlan, V., Dreano, Y., Bercovici, J. P., Beaune, P. H.,
Floch, H. H., and Berthou, F., Nature of cytochromes
P450 involved in the 2-/4-hydroxylations of estradiol in
human liver microsomes. Biochem. Pharmacol., 44,
1745–1756 (1992).
18) Li, H., and Poulos, T. L., The structure of the
cytochrome p450BM-3 haem domain complexed with
the fatty acid structure, palmitoleic acid. Nat. Struct.
Biol., 4, 140–146 (1997).
19) Li, Q. S., Schwaneberg, U., Fischer, F., and Schmid, R.
D., Directed evolution of the fatty acid hydroxylase
P450BM-3 into an indole-hydroxylating catalyst. Chem.
Eur. J., 6, 1531–1535 (2000).
20) Li, Q. S., Ogawa, J., and Shimizu, S., Critical role of the
residue size at position 87 in H2O2-dependent substrate
hydroxylation activity and H2O2 inactivation of cyto-
chrome P450 BM-3. Biochem. Biophys. Res. Commun.,
280, 1258–1261 (2001).
7) Ueng, Y. F., Shimada, T., Yamazaki, H., and Guenger-
ich, F. P., Oxidation of aflatoxin B1 by bacterial
recombinant human cytochrome P450 enzymes. Chem.
Res. Toxicol., 8, 218–225 (1995).
21) Harlow, G. R., and Halpert, J. R., Analysis of human
cytochrome P450 3A4 cooperativity: construction and
characterization of a site-directed mutant that displays
hyperbolic steroid hydroxylation kinetics. Proc. Natl.

300
Q.-S. LI et al.
Acad. Sci. U.S.A., 95, 6636–6641 (1998).
chrome P450 2B1: identification of an active site heme-
modified peptide. J. Biol. Chem., 268, 59–65 (1993).
25) Li, Q. S., Ogawa, J., Schmid, R. D., and Shimizu, S.,
Residue size at position 87 of cytchrome P450 BM-3
determines its stereoselectivity in propylbenzene and 3-
chlorostyrene oxidation. FEBS Lett., 508, 249–252
(2001).
26) Li, Q. S., Ogawa, J., Schmid, R. D., and Shimizu, S.,
Engineering cytchrome P450 BM-3 for oxidation of
polycyclic aromatic hydrocarbons. Appl. Environ. Mi-
crobiol., 67, 5735–5739 (2001).
22) Truan, G., and Peterson, J. A., Thr268 in substrate
binding and catalysis in P4350BM-3. Arch. Biochem.
Biophys., 349, 53–64 (1998).
23) Shimizu, T., Murakami, Y., and Hatano, M., Glu318 and
Thr319 mutations of cytochrome P450 1A2 remarkably
enhance homolytic O–O cleavage of alkyl hydroper-
oxides: an optical absorption spectral study. J. Biol.
Chem., 269, 13296–13304 (1994).
24) Yao, K., Falick, A. M., Patel, N., and Correia, M. A.,
Cumene hydroperoxide-mediated inactivation of cyto-