Non-covalent modification of the heme-pocket of apomyoglobin by a 1,10-phenanthroline derivative

Article abstract of DOI:10.1016/j.bmcl.2005.10.016

To expand the repertoire of artificial enzymes that are constructed by replacing the natural prosthetic group of hemoproteins with non-natural cofactors, we examined incorporation of a non-porphyrinic ligand (1) into the heme-pocket of apomyoglobin in a n

Full text of DOI:10.1016/j.bmcl.2005.10.016

Bioorganic & Medicinal Chemistry Letters 16 (2006) 248–251
Non-covalent modification of the heme-pocket of apomyoglobin
by a 1,10-phenanthroline derivative
*
Yutaka Hitomi, Hidefumi Mukai, Hideaki Yoshimura, Tsunehiro Tanaka
and Takuzo Funabiki
Department of Molecular Engineering, Kyoto University, Kyoto Daigaku Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
Received 29 July 2005; revised 4 October 2005; accepted 6 October 2005
Available online 24 October 2005
Abstract—To expand the repertoire of artificial enzymes that are constructed by replacing the natural prosthetic group of hemopro-
teins with non-natural cofactors, we examined incorporation of a non-porphyrinic ligand (1) into the heme-pocket of apomyoglobin
in a non-covalent fashion. Ligand 1 is a highly conjugated 1,10-phenanthroline derivative, which shares some structural features
with protoporphyrin IX; for example, molecular size and arrangement of hydrophobic and anionic parts. Addition of apomyoglobin
to a solution of 1 induces clear changes in the absorption spectrum of 1, suggesting one-to-one incorporation of 1 into the heme
6
À1
cavity of apomyoglobin with an affinity of 6.3 · 10 M . We found that the hydrolytic activity of apomyoglobin toward p-nitro-
phenyl hexanoate was greatly suppressed because of the incorporation of 1 into the heme-pocket.
ꢀ
2005 Elsevier Ltd. All rights reserved.
Introduction of non-natural metal complexes into pro-
tein cavity has been recognized as a powerful method
to create new artificial metalloproteins with desirable
al similarity to the native prosthetic group has been con-
sidered to be unpromising, although it has been reported
that aromatic molecules such as 8-anilino-1-naphthalene
1
7
functions. The molecules incorporated inside protein
sulfonate bind to the heme-pocket of apomyoglobin.
cavity often exhibit significant stability and unique reac-
tivities such as enantio- or substrate-selective reactions.
For example, Distefano and co-workers have demon-
strated that a Cu(II)-1,10-phenanthroline complex that
is covalently attached to a cysteine in the protein cavity
of an adipocyte lipid-binding protein catalyzes highly
Recently, however, Watanabe and co-workers success-
fully demonstrated that Schiff base complexes can be
stably incorporated into the heme cavity of myoglobin
with assistance of protein modification by amino acid
8
mutation. Thus, expanding the range of metal complex-
es that can be incorporated into the heme cavity would
enable us to design artificial metalloproteins with func-
tions beyond those of heme proteins (Scheme 1).
1
d,2
enantioselective hydrolysis with up to 86% ee.
In
3
addition to the covalent approach, non-covalent incor-
poration of metal complexes into protein cavity also has
been carried out to create artificial metalloproteins with
Recently, we synthesized a highly conjugated phenan-
throline ligand (1), 2,9-bis(3,5-dicarboxylphenylethy-
nyl)phenanthroline, as a new water-soluble fluorescent
4–6
unique functions. For example, HayashiÕs and Casel-
laÕs groups have reported that replacement of heme in
myoglobin with protoporphyrin IX modified at the pro-
9
probe. As depicted in Figure 1, ligand 1 shares structur-
pionate group(s) can convert an O -storage protein to a
2
al similarity with protoporphyrin IX; that is, both the
molecules have a similar molecular size of ca. 1 nm
square, an aromatic plane at the upper side, and car-
boxylate groups at the bottom side. It has well known
that protoheme is tightly incorporated into the heme
cavity through the ligation of the proximal histidine to
the heme iron as well as through multiple non-covalent
interactions between the protoheme and amino acid res-
idues, which include hydrophobic interactions between
the porphyrin plane and non-polar amino acid side
chains as well as polar interactions between the propio-
nate side chains and polar side chains, for example,
4
,5
peroxidase or a peroxygenase. Since this approach
takes advantage of strong interactions between proto-
heme and heme-binding site, incorporation of any other
non-natural metal complex that bear much less structur-
Keywords: Apomyoglobin; Heme-pocket; Phenanthroline; Recons-
titution.
*
Corresponding author. Tel.: +81 75 383 2562; fax: +81 75 383
561; e-mail: hitomi@moleng.kyoto-u.ac.jp
2
Present address: Biomimetic Research Center, Doshisha University,
Kyo-Tanabe, Kyoto 610-0321, Japan.
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.016

Y. Hitomi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 248–251
249
caused by tryptophan residues of added apomyoglobin.
The molar ratio graph at 317 and 370 nm is shown in the
inset of Figure 2A. The absorption at 317 nm decreases
and the absorption at 370 nm increases almost linearly
until a ligand-to-apomyoglobin ratio of 1:1 is reached,
and further addition of apomyoglobin did not affect
the absorption spectrum, except for the absorption re-
gion increased by added apomyoglobin (250–300 nm).
The spectral change between 250 and 400 nm was ana-
lyzed using a non-linear least-squares fit algorithm
based on singular value decomposition to determine
the binding constant to be log K = 6.8 ± 0.3 (SPECFIT
native myoglobin
protoheme
1
2
software ). The binding constant of 1 for apomyoglo-
bin is rather smaller than that of the native prosthetic
artificial prosthetic group
À12
À15
10a
group hemin (K ꢀ 10 –10
M). The lower affin-
ity of 1 may be caused by lacking the metal ion that can
d
coordinate to the proximal histidine.
On the other hand, addition of holomyoglobin to 1 did
not alter the spectrum of 1. To confirm the incorpora-
tion of 1 in the heme-pocket, we prepared tetrazole-
myoglobin, whose distal histidine residue is modified
to bear a tetrazole ring according to the reported proce-
apomyoglobin
Scheme 1.
reconstituted myoglobin
1
0
13
Ser92 and Arg45 in sperm whale myoglobin. There-
fore, the above-mentioned structural similarity between
the phenanthroline 1 and protoheme suggests the possi-
bility that ligand 1 binds into the heme cavity of apo-
myoglobin in the same fashion to protoheme. Here,
we have investigated the above possibility using sperm
dure, and added its apoprotein to a solution of 1. The
resulting spectral changes were very much similar to
those for the unmodified apomyoglobin (Fig. 3). How-
ever, the estimated binding constant shows a decrease
by ca. 10-fold (log K = 5.7 ± 0.2). Thus, these results
clearly suggest that 1 is tightly incorporated into the
heme cavity of apomyoglobin due to its structural
homology with the native prosthetic group.
1
1
whale apomyoglobin as a host protein.
First, UV titration studies were carried out by addition
of sperm whale apomyoglobin to a HEPES-buffered
solution (pH 8.1) of 1. The absorption spectral changes
On the basis of the spectral analysis, spectrum for 1 inside
heme-pocket can be calculated and is shown in Figure 2B.
The spectrum shows red-shifted absorption bands com-
pared with that for 1 in the absence of apomyoglobin.
(
Fig. 2A) exhibit two isosbestic points at 305 and
48 nm, and an increase at around 280 nm, which is
3
Figure 1. Structures and CPK models of 1 and protoporphyrin IX.

250
Y. Hitomi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 248–251
that coordinates to the heme iron and the distal histidine
in the vicinity of the heme iron. Taking into account the
structural homology of 1 with protoheme, these histidine
residues might be located close to nitrogen atoms of the
phenanthroline moiety of 1. Thus, the spectral change
of 1 suggests a possible interaction between nitrogen
atoms of the phenanthroline moiety of 1 and the proximal
and/or distal histidine residue(s).
Circular dichroism (CD) spectra measurements provid-
ed another evidence to support the localization of 1 in-
side the heme-pocket. Figure 1 shows the CD spectra
of apomyoglobin in the absence and presence of 1. A
very small amount of alpha-helix induction was ob-
served in the presence of 1 (Fig. 4A). This result shows
that 1 does not work as a denaturing reagent but does
stabilize apomyoglobin to a small extent, which might
correlate to the smaller binding affinity of 1 with apo-
myoglobin compared to that of the native prosthetic
group, hemin. More interestingly, the 1–apomyoglobin
complex exhibited a positive induced CD signal in the
absorption region of 1 (Fig. 4B), which clearly supports
the location of 1 in chiral environments. Thus, the CD
experiments clearly indicate the localization of 1 inside
the heme-pocket of apomyoglobin.
Figure 2. (A) Absorption spectral changes of 1 upon addition of
apomyoglobin. Conditions: 10 mM HEPES buffer, pH 8.1, 25 ꢁC.
In 1987, Zemel reported the hydrolytic activity of
15
apomyoglobin. The reaction proceeds more efficiently
as the substrate becomes more hydrophobic; that is,
[
1] = 10 lM. [apomyoglobin] = 0–20 lM. Inset: absorption as a func-
tion of added apomyoglobin monitored at 317 and 370 nm (molar
ratio plot). (B) Calculated spectrum of 1 inside apomyoglobin (solid
line), together with that of 1 (dotted line).
p-nitrophenyl acetate < p-nitrophenyl hexanoate (PNH)
p-nitrophenyl nonate. Thus, the hydrolytic reaction
<
is promoted by histidine residues in the hydrophobic
heme-pocket, which is also supported by no hydrolytic
activity of holomyoglobin. Therefore, it is expected that
the incorporation of 1 in the heme-pocket would effect
this hydrolytic reaction. In this study, we examined the
catalytic hydrolytic activity of sperm whale apomyoglo-
bin by using PNH as a hydrophobic substrate in the
presence and absence of 1 in 20 mM HEPES buffer
Figure 3. Absorption spectral changes of 1 upon addition of apo-
tetrazole-myoglobin. Conditions: 10 mM HEPES buffer, pH 8.1, 25 ꢁC.
[
1] = 10 lM. [apo-tetrazole-myoglobin] = 0–20 lM. Inset: absorption
as a function of added apomyoglobin monitored at 317 and 370 nm
molar ratio plot).
(
Armaroli and co-workers reported the redshift of the
absorption bands of 1,10-phenanthroline derivatives
upon protonation of the phenanthroline nitrogens due
1
14
to stabilization of the lowest pp level. Thus, it is sug-
*
gested that nitrogen atoms of the phenanthroline moiety
of 1 become protonated upon the incorporation into the
heme-pocket. The heme-pocket environments surround-
ing 1 might affect the equilibrium between 1 and its pro-
tonated form. There are two histidine residues in the
heme-pocket of myoglobin; that is, the proximal histidine
Figure 4. CD spectra of apomyoglobin in the absence and presence of
1
. Conditions: 20 mM phosphate buffer, pH 6.9, 10 ꢁC. [apomyoglo-
bin] = 10 lM, [1] = 11 lM (solid line); [apomyoglobin] = 10 lM (dot-
ted line); [native myoglobin] = 10 lM (dashed line).

Y. Hitomi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 248–251
251
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Figure 5. Initial rate of the PNH hydrolysis as the PNH concentration.
Conditions: 10 mM HEPES buffer, pH 8.1, 10 ꢁC. [apomyoglo-
bin] = 10 lM without (filled circle) 1 and with 1 (11 lM) (open circle).
2
Chem. Lett. 2003, 32, 496; (f) Matsuo, T.; Hayashi, T.;
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(
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(
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3
Figure 5 shows a plot of the initial rate constant as a
function of the PNH concentration, which shows satu-
ration behavior in the absence of 1, where the intrinsic
hydrolysis rate of PNH (k = 1.0 · 10
from the observed reaction rate constants following the
reported procedure. The Michaelis–Menten parame-
ters were estimated as follows: K = 0.086 ± 0.017 mM
and kcat = (3.9 ± 0.2) · 10
in agreement with those reported by Zemel;
K = 0.074 mM and k = 48 · 10
2
tial reaction rate does not show saturation behaviors but
increases almost linearly with substrate concentrations,
which leads to the second-order rate constant of
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s ) is subtracted
1
6
m
À3 À1
s . These parameters are
À3 À1
s
(pH 8.0,
m
cat
1
5
5 ꢁC). In the presence of 1, on the other hand, the ini-
7
À1 À1
k2 = 1.5 ± 0.1 M
side the heme cavity inhibits the hydrolysis of PNH by
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4
The result presented here shows that the water-soluble
highly conjugated phenanthroline ligand 1 is readily
incorporated into the hydrophobic heme-pocket of
sperm whale apomyoglobin probably due to the struc-
tural similarity to protoheme. The incorporation of 1
into the heme-pocket inhibits the hydrolytic activity of
apomyoglobin toward activated esters. Thus, the present
study demonstrates that even non-porphyrinic mole-
cules can be smoothly inserted into the heme cavity of
apomyoglobin through proper molecular design. The
resulting 1–apomyoglobin complex could provide a nov-
el metal-binding site in the heme cavity. The construc-
tion and characterization of artificial metalloproteins
based on the 1–apomyoglobin complex are currently
undergoing in our laboratory.
1
9
. Characterization of 1: H NMR (DMSO, 400 MHz):
d 8.61 (d, J = 8.4 Hz, 2H), 8.52 (t, J = 1.8 Hz, 2H), 8.45 (d,
J = 1.2 Hz, 4H), 8.14 (d, J = 7.6 Hz, 2H), 8.08 (s, 2H),
NMR (DMSO, 100 MHz): d 165.6, 144.9, 141.8, 137.0,
1
FAB MS (DEA/DMSO): m/z 556 [M ]; HRMS (FAB,
1
3
C
36.0, 132.1, 130.5, 128.2, 127.3, 127.1, 122.4, 90.9, 87.6;
À
À
DEA/DMSO, [M ÀH]) Calcd for C H O N 555.0828.
3
2
15
8
2
Found 555.0840.
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