Blue myoglobin reconstituted with an iron porphycene shows extremely high oxygen affinity

Article abstract of DOI:10.1021/ja0265052

Myoglobin will be a good scaffold for engineering a function into proteins. To modulate the physiological function of myoglobin, almost all approaches have been demonstrated by site-directed mutagenesis, however, there are few studies which show a signifi

Full text of DOI:10.1021/ja0265052

Published on Web 08/28/2002
Blue Myoglobin Reconstituted with an Iron Porphycene Shows Extremely
High Oxygen Affinity
Takashi Hayashi,*,†,‡ Hirohisa Dejima,† Takashi Matsuo,‡ Hideaki Sato,‡ Dai Murata,† and
Yoshio Hisaeda*,†
Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu UniVersity, and PRESTO in
Japan Science and Technology Corporation, Fukuoka 812-8581, Japan
Received April 11, 2002
Scheme 1. Reconstitution of Myoglobina
Myoglobin, one of the well-known hemoproteins, will be an
attractive biomolecule for engineering a function into the protein,
since a prosthetic group, heme, bound in the protein matrix shows
a variety of reactivities and unique physicochemical properties. The
strategy of the myoglobin modification can be mainly divided into
two approaches: (i) amino acid mutation by site-directed mutagen-
esis and (ii) replacement of the native heme with artificially created
metalloporphyrins. The former method enables us to modulate the
physiological function of the myoglobin1,2 or convert the myoglobin
into peroxidase or peroxygenase.3-5 In contrast, the latter method
has the advantage of introducing a new function on the myoglobin
surface by modification of heme-propionate side chains as shown
in Scheme 1[A].6-12 Furthermore, as can be seen in Scheme 1[B],
the modification of the heme framework will be another way to
improve the physiological function of myoglobin,13-16 although
there have been few examples that demonstrate drastic changes in
the inherent myoglobin function by the chemical modification of
the heme. Toward this end, we focused on a study involving the
use of an iron porphycene, a structural isomer of iron porphyrin,
a [A] Modification of heme-propionate and [B] modification of heme
as an artificial prosthetic group.17,18 Here, we demonstrate that the
framework.
reconstituted myoglobin exhibits an extremely high O2 affinity and
low autoxidation rate without any changes in the amino acid
sequence.
To establish the affinity of an artificially created prosthetic group
into the heme pocket, we designed and prepared an iron porphycene,
2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis-carboxyethylporphycena-
toiron (1), which has peripheral alkyl groups and two propionate
side chains at the porphycene pyrrole rings. The reconstituted
metmyoglobin with 1, met-rMb(1), was obtained by a standard
reconstitution protocol from horse heart apomyoglobin and purified
by gel and ion-exchange chromatographies.19,20 As can be seen in
Figure 1. Visual color image of metmyoglobins: (a) nMb(2) and (b) rMb-
(1).
Figure 1, the solution of met-rMb(1) is light blue due to the iron
porphycene color, and the electronic absorption spectrum of met-
rMb(1) shows an axially coordinate iron(III) character with wave-
lengths of 387, 563, and 624 nm.21,22 The ESI-TOF mass spectrum
of rMb(1) gave a mass number of 17 572 which is identical to that
expected for the holoprotein. The pH corresponding to 50% un-
folding, obtained by monitoring the Soret band at 387 nm of met-
rMb(1), is 3.1. The value is 1.4 pH units lower than that of the
+52 mV determined for nMb(2). These results support the fact
that the coordination of proximal His93 to 1 is stronger than to 2.
The UV-vis spectrum of the reconstituted deoxymyoglobin,
deoxy-rMb(1), was observed upon the addition of dithionite. After
the removal of excess dithionite under aerobic conditions by Seph-
adex G25, a greenish-blue solution was obtained, indicating the
formation of oxymyoglobin, oxy-rMb(1). The reduction of met-
rMb(1) under a CO atmosphere exhibits a spectrum different from
native metmyogobin, met-nMb(2), at 25 °C, suggesting that ferric
oxy-rMb(1), showing the formation of CO-myoglobin. The revers-
1 is more stable against dissociation from the protein matrix than
ibility between the two species was detected by blowing the
is the ferric heme 2. The UV-vis spectroelectrochemical experi-
corresponding gas stream.
ments demonstrate that rMb(1) was electrochemically reversible
The O2 binding parameters for ferrous rMb(1) are compared in
and the redox potential (Fe3+/Fe2+) was found to be -193 mV vs
Table 1 with those for nMb(2). The O2 association for rMb(1) is
NHE which is significantly lower than the corresponding value of
5-fold faster than that observed for nMb(2). Furthermore, it is of
particular interest that oxy-rMb(1) shows a 250-fold reduction in
the O2 dissociation rate constant compared with oxy-nMb(2). Thus,
the binding kinetics demonstrate the extremely high O2 affinity of
* To whom correspondence should be addressed. E-mail: thayatcm@
mbox.nc.kyushu-u.ac.jp
† Kyushu University.
‡ Member of PRESTO in JST.
9
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J. AM. CHEM. SOC. 2002, 124, 11226-11227
10.1021/ja0265052 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Table 1. O2 Binding Parameters and Autoxidation Rate Constants
for Native and Reconstituted Myoglobins
propose that the replacement of the native heme 2 with iron
porphycene 1 dramatically improves the function as detailed above
without any changes in the amino acid sequence of the horse heart
myoglobin. Thus, the modification of the heme framework in the
present study will serve as an effective method for the creation of
a unique functionalized hemoprotein.
myoglobin
k
on (µM-1 s-1 a,b
)
k
off (s-1 a,c
)
KO2 (M-1 d
)
k )
auto (h-1 e
nMb(2)
rMb(1)
22( 1
120 ( 10
27 ( 2
0.11 ( 0.01
8.1 × 105
0.18 ( 0.01
0.026 ( 0.001
1.1 × 109
a Reaction conditions: 100 mM phosphate buffer (pH 7.0) at 25 °C.
b Association process of O2 ligand was measured by a laser flash photolysis
system. c Dissociation rates were measured by a stopped-flow spectropho-
tometry upon the addition of excess amount of ferricyanide. d O2 binding
constants were calculated from the measured kon and koff values. e The rate
of autoxidation was determined by the spectral changes to metmyoglobin
at 37 °C in 100 mM phosphate buffer (pH 7.0).
Acknowledgment. We thank Prof. E. Vogel for his warm
encouragement. This work was supported by the Japan Science and
Technology Corporation (JST), the Ministry of Education, Sports,
Culture, Science and Technology, Japan, and the Mazda Foundation.
Supporting Information Available: Experimental details, mass
spectrum of rMb(1), and kinetic data (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
rMb(1), 1.1 × 109 M-1, indicating a significant 1400-fold increase
in O2 affinity for rMb(1) relative to that for the native horse heart
myoglobin. The determined binding constant is as high as that of
several oxygen-avid hemoglobins such as Ascaris hemoglobin,23,24
which has special distal Tyr and Gln as strong hydrogen-bonding
donors to stabilize the bound O2, whereas rMb(1) has no trick in
the protein matrix. However, it is noteworthy that the enhancement
of the O2 affinity is mainly derived from a very slow O2 dissociation
both in Ascaris hemoglobin and rMb(1). The reason for the strong
O2 binding to rMb(1) is suggested as follows. According to several
model studies of iron porphyrins without a protein matrix, the O2
off-rate decreases with increasing electron donation of the axial
base ligand, because of the increasing favorable character of the
Fe(III)-O2- charge separation species.25,26 In our case, the observed
lower redox potential of the iron in rMb(1) relative to that in nMb-
(2) supports the fact that the strength of the proximal His93-iron
heme coordination increases and then the charge separation species
is predominant over Fe(II)-O2 complex due to the increasing
π-back-donation to the bound O2.27 Indeed, it is known that an iron
porphycene binds imidazoles more strongly than an iron porphyrin.22
In addition, it is likely that the remarkable charge separation gives
rise to the tight hydrogen-bonding between the negatively charged
oxygen and imidazolyl NH of the distal His64 in rMb(1). Therefore,
the very small rate constant of O2 dissociation for rMb(1) is clearly
explained by the stabilization of the bound O2 via a strong
hydrogen-bonding interaction with His64.28
Next, we investigated the stability of oxy-rMb(1) by monitoring
the autoxidation from oxy-rMb(1) to met-rMb(1) in 100 mM
phosphate buffer, pH 7.0, at 37 °C. The spectral changes in the
Q-band region exhibited clear isosbestic points, and the decay at
621 nm provides the exact first-order kinetics. The rate constants
of two proteins are summarized in Table 1. Interestingly, the
autoxidation of oxy-rMb(1) is 7-fold slower than that of nMb(2).
The significantly slow oxidation will come from the slow dissocia-
tion of the O2 ligand, although the autoxidation mechanism is
complicated. This explanation is supported by previous reports
where the O2 affinity is inversely related to the autoxidation rate
constant.29
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