Aniline-hydroxylation activity of a flavin-linked βαβα-type polypeptide packing an iron porphyrin

Article abstract of DOI:10.1246/cl.2003.6

Polypeptide containing iron porphyrin and flavin showed aniline-hydroxylation activity in the presence of 1-benzyl-1,4-dihydronicotinamide and O₂. The increased activity at pH 5.5 compared with that at pH 7.0 suggests that the dissociation of His from the iron porphyrin favored the hydroxylation.

Full text of DOI:10.1246/cl.2003.6

6
Chemistry Letters Vol.32, No.1 (2003)
Aniline-Hydroxylation Activity of a Flavin-linked ꢀꢁꢀꢁ-Type Polypeptide Packing an Iron
Porphyrin
y
yy
Ãyy
Kin-ya Tomizaki, Hidekazu Nishino, Toru Arai, Tamaki Kato, and Norikazu Nishino
Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu 804-8550
y
Institute for the Fundamental Research of Organic Chemistry, Kyushu University, Fukuoka 812-8581
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196
yy
(Received September 12, 2002; CL-020783)
Polypeptide containing iron porphyrin and flavin showed
globular structure.
Polypeptide 1 and the control compounds (2, 3) were
synthesized via the condensations of the peptide fragments as
aniline-hydroxylation activity in the presence of 1-benzyl-1,4-
dihydronicotinamide and O2. The increased activity at pH 5.5
compared with that at pH 7.0suggests that the dissociation of His
from the iron porphyrin favored the hydroxylation.
4
reported. Flavin (7a-bromoacetyl-10-methylisoalloxazine) was
attached to Cys side chain of the deprotected 49-mer polypeptide
6
before iron insertion. After the final purification with HPLC using
a Superdex Peptide HR 10/30 column, polypeptides 1–3 were
characterized by MALDI-TOF-MS. Unfortunately, 1H NMR
signals of the polypeptides were too broad to assign even before
iron insertion.
One of the key steps in the oxidation with cytochrome P-450
is the electron transfer to this enzyme from cytochrome P-450
1
reductase. Flavin derivatives reduce nearby hemes during the
2
oxidation reaction catalyzed by enzymes or artificial 4a-helix
UV/vis spectrum of 1 at pH 7.0was characteristic for the low-
spin Fe(III) complex with a narrow Soret band at 420nm and
3
bundle polypeptides. Unlike 4a-helix bundle, which seems to
7
surround porphyrin too closely, we recently synthesized a baba-
type polypeptide loosely packing porphyrin and reported its
broad Q bands at 550and 575 nm (sh) (Figure 2a), which indicate
the bis-His coordination as fifth and sixth ligands. The UV/vis
4
peroxide-dependent oxidation catalysis. In order to utilize O2 as
spectra of 1 was concentration-independent from 0.6 mM (M =
À3
oxidant, we have attached flavin to this baba-type polypeptide
containing iron porphyrin, which could successfully hydroxylate
aniline under the aerobic conditions (Figure 1).
mol dm ) to 15 mM, indicate its monomeric structure. At pH 2.7,
the Soret band shifted to 416 nm with the Q bands appeared at 570
and 615 nm, which indicate the formation of high-spin Fe(III)
7
species. The His side chains were protonated at pH 2.7 and did
(
a)
β
α
(b)
flavin
not coordinate to iron porphyrin. However, these spectral changes
showed no clear isosbestic points and showed a bimodal profile in
the Abs420 vs pH plot (Figure 2b), which suggests some
intermediate species at around pH 5.5. The CD spectra of 1 at
pH 7.0showed negative bands at 222 and 208 nm, characteristic
of the polypeptide containing a-helices. The CD spectrum is
indeed ambiguous in the polypeptides containing b-strand due to
the overlapping of the small Cotton effect of b-strand. However,
AaPA-X EVKV-AaPA-ELLKAHAELLK-
1
AaPA-VEVKX -AaPA-ELLKAHAELLKG
2
β
α
1
2
3
: X =Orn(PorFe), X =Cys(Fla)
1
2
iron porphyrin
: X =Orn(PorFe), X =Val
1
2
3
: X =Val
, X =Cys(Fla)
1
2
N
N
N
O
O
-
NH-CHR-CO-
Cys(Fla): R=
N
2
2
2
À1
O
when the ½ꢀŠ
value of 1 (À12500 deg cm dmol ) was
2
22
Orn(PorFe): R=-(CH ) NHCO-p-C H -tritolylporphyrinato iron
4
2
3
6
4
employed to analyze the a-helicity, 1 was evaluated to be 34%
a-helix, which coincided with the 37% (18/49) value assuming
the a-helical structure for the nine residues of each a-segment.
We have no direct evidences for the conformation of b-strands at
present, therefore, we wish to denominate 1 as the baba-‘type’
polypeptide. However, CD spectra showed that the b-strand are
not helical, consequently, it is at least sure that the porphyrin was
loosely surrounded by the polypeptide at pH 7.0. The peptide 2
without flavin showed a CD spectrum (½ꢀŠ222 ¼ À14000) similar
Figure 1. (a) Polypeptides 1–3 with the iron porphyrin and/or flavin.
A, Ala; a, D-Ala; E, Glu; G, Gly; H, His; K, Lys; L, Leu; P, Pro, and
V, Val. (b) Supposed baba-structure of 1.
Polypeptides 1 consist of b-strand segments and a-helix
segments linked by spacers (Figure 1a). The b-strand segment
(
ꢀ1:2 nm) are of alternative hydrophobic (X1, Val, and X2)/
5
hydrophilic (Glu/Lys ion pair) sequences and the a-helix
segments (ꢀ1:5 nm) are of hydrophobic (four Leu)/hydrophilic
5
(
two pairs of Glu/Lys) residues on the opposite side of the helix,
0.45
0.40
0.35
both designed to cover the porphyrin (ꢀ1:0 nm). When the
hydrophobic faces of the helices come close to the porphyrin to
coordinate with iron by His, the hydrophobic Val in the b-strands
would also come close to the porphyrin and the whole molecule
would arranged to give a structure with hydrophobic interior
0.04
(
a)
(b)
pH 7.0
pH 4.0
0.02
pH 2.7
(
(
porphyrin, Val, and Leu) and hydrophilic (Glu/Lys) exterior
Figure 1b). The flavin moiety exists at the molecular surface (see
0
0.30
500
600
Wavelength / nm
700
2
3
4
5
6
7
pH
below), therefore, the reductant in the solution is expected to
reduce flavin, and then the intramolecular electron transfer from
the reduced flavin to iron porphyrin takes place efficiently in the
Figure 2. UV/vis spectra of 1 (3.5 mM) (a) at pH 7.0, 4.0, and 2.7. (b)
Absorbance at 420nm of 1 vs pH.
Copyright ꢀ 2003 The Chemical Society of Japan

Chemistry Letters Vol.32, No.1 (2003)
7
to 1, indicating that the flavin attachment induce only slight
changes tothe conformation of the polypeptide. This factsupports
the hypothesis that the flavin moiety is at the surface of the
molecule (Figure 1b) due to the hydrophilic nature of the
isoalloxazine ring. The bis-His coordination to Fe(III) in 1 and 2
stabilize their folded structures. However, polypeptide 3 without
a porphyrin (½ꢀŠ ¼ À9500) and the polypeptide 1 at pH 2.7
1.0
0.8
.6
0.4
(
a)
(b)
0.8
0.6
0.4
0
1
1
0.2
0.2
0
2
2
2
22
0
0
10 20 30 40 50
[aniline] / mM
4
5
6
pH
7
8
with protonated His (½ꢀŠ ¼ À10200) show smaller helicities.
222
0
Next, we examined aniline hydroxylation in the presence of
O2 (air) catalyzed by 1–3 with 30equivalents of the reductant (1-
Figure 3. p-Aminophenol formation ([1 or 2] = 7.0 mM, [BNAH] =
ꢂ
8
210 mM, 37 C). (a) 1 or 2 vs [aniline] at pH 7.0, (b) 1 or 2 vs pH,
benzyl-1,4-dihydronicotinamide, BNAH) at pH 7.0(Eq 1). The
amount of the product p-aminophenol formed after the 15 min
[
aniline] = 10mM).
reaction, the only one product detected by HPLC, was determined
8
access of aniline. In natural hemes, the distal side is exchangeable
or vacant and the substrate is easily accessible showing the large
by the literature method. The formation of p-aminophenol with 1
was six times greater than that with 2 at pH 7.0(Table 1), which
showed efficient electron relay via the attached flavin in 1. To
verify the electron relay through flavin we monitored the UV/vis
spectra of the solution of 1 and 2 after addition of BNAH under Ar
atmosphere. For polypeptide 1, the absorptions at 432, 536, and
kcat and small KM, for instance in myoglobin, kcat ¼ 0:14 and
À3 2b
Thus, the aniline hydroxylation is carried
KM ¼ 8:4 ꢁ 10
.
out in acidic buffer solutions in which the protonated His
dissociated from the iron porphyrin (Figure 3b). The maximal
activity of 1 was at pH 5.5, where p-aminophenol formation was
5
68 nm increased after the addition of BNAH, which correspond
1
75% compared to that at pH 7.0(Table 1). It is interesting to note
that the maximal absorption of the Soret band was obtained at pH
.5 (Figure 2b). Although the pKa of His is around 6.0, His might
7
to the peaks of Fe(II) porphyrin. However, the Fe(II) species
formation was significantly slow for 2 in the presence of BNAH.
This clearly illustrates the importance of the flavin moiety in the
polypeptide for the efficient electron transfer. Aniline hydro-
xylation hardly occurred with 3, indicating that the hydroxylation
was actually catalyzed by the iron porphyrin. Although hydro-
5
be in the mid of the deprotonation/protonation and coordination/
dissociation at pH 5.5, which implied the formation of some
catalytic active species (for instance, the 5-coordinated iron
porphyrin). At high pH, two His coordinated tothe iron porphyrin,
therefore, the substrate was not easily accessible. At low pH such
as pH 4, His was protonated and dissociated from the iron
porphyrin to collapse the molecular structure and then the
porphyrin and flavin might be separated. Thus, 1 showed a bell-
shaped activity depending on the pH and its maximum activity
was observed at pH 5.5. The addition of a denaturant, guanidine
HCl, caused a decrease in the hydroxylation activity of 1 to
9
xylation of aniline occurs with Fe(III) porphyrin/H2O2, the
reactivity of 1 was not changed by the addition of an H2O2
À3
À3
À
scavenger, catalase (350unit cm ), or an O2 scavengers,
superoxide dismutase (100 unit cm ). These results showed that
1
utilize O2 and electrons derived from BNAH via flavin and not
À
using H2O2 and O2 generated from the reducing system.
Although excess amounts of BNAH was added, ꢀ20 equivalents
of BNAH was consumed after 15 min in the reactions including 1
and 3. However, BNAH was less consumed in the reaction with 2
ꢀ
10%. This is probably because the conformational change by
the denaturant increased the distance between the porphyrin and
flavin. Further spectroscopic studies of the catalytic active species
are underway.
(
ꢀ5 equivalents after 15 min). Although BNAH reduces flavin at
the surface of 1, aerobic reoxidation of the reduced flavin may
occur to consume excess BNAH (Eq 2). This may be the reason of
the low conversion of aniline and the low yield of p-aminophenol.
This work was partly supported for N. N. by a Grant-in-Aid
for Scientific Research (10480153) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan.
BNAH
BNA
flavin
reduced flavin
aniline
p-aminophenol
Fe porphyrin
(1)
(2)
+
+
O , H
2
BNAH
flavin
reduced flavin
O2
References
+
BNA
1
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ꢂ
Table 1. Aniline hydroxylation catalyzed by 1–3 at 37 Ca
2
Peptide
361
pH
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.
7.00
3
4
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.5
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2
5
3
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5
6
7
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a
[
Peptide] = 7.0 mM, [aniline] = 10mM, [BNAH] = 210 mM.
Saturation kinetics for aniline hydroxylation was observed
for 1 at the ꢀ40 mM aniline concentration (Figure 3a). The
8
9
O. Takikawa, R. Yoshida, and O. Hayaishi, J. Biol. Chem., 258, 6808
(
À2
À1
Michaelis-Menten parameters of 1 are, kcat ¼ 1:2 ꢁ 10 min
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À2
and KM ¼ 2:6 ꢁ 10 M. Such small kcat and large KM values of 1
are attributed to bis-His coordination in 1, which prevents the