220

THOUGHTS AND PROGRESS

Built-in heme
oxygen carrier.

aor_1238

220..233

Inherently Distorted Heme as a Novel

Tool for Myoglobin-Based Oxygen Carrier
*Saburo Neya and Akira T. Kawaguchi
*Department of Physical Chemistry, Graduate
School of Pharmaceutical Sciences, Chiba
University, Inage-Yayoi, Chiba; and School of
Medicine, Tokai University, Isehara, Kanagawa,
Japan
Abstract: The two types of artificial myoglobins (Mbs)
containing inherently distorted a-ethyl-2,4-dimethyldeuteroheme and undistorted 2,4-dimethyldeuteroheme
were prepared to examine the influence of nonplanar heme
deformation on the reactivity of Mb. In ferrous deoxy
proteins, the paramagnetic proton nuclear magnetic resonance spectra showed that the deformed heme caused a
3.2-ppm lower-field shift of the proximal histidine signal,
indicating a larger iron displacement from the heme plane
upon the nonplanar deformation. The Mb with the nonplanar heme exhibited a markedly lower oxygen affinity as
compared with the Mb containing the planar heme. The
result suggests the utility of heme distortion to design
the Mb-based oxygen carrier. Key Words: Myoglobin

doi:10.1111/j.1525-1594.2011.01238.x
Received July 2010; revised December 2010.
Address correspondence and reprint requests to Professor
Saburo Neya, Department of Physical Chemistry, Graduate School
of Pharmaceutical Sciences, Chiba University, Inage-Yayoi, Chiba
263-8522, Japan. E-mail: sneya@p.chiba-u.ac.jp
Artif Organs, Vol. 36, No. 2, 2012

distortionIron

movementArtificial

In the development of artificial oxygen carriers,

various functional materials have been developed.
The typical examples are modified hemoglobins,
fluorocarbon-based oxygen carriers, lipidheme
vesicles, and synthetic metal chelates (1). However,
myoglobin (Mb), the oxygen reservoir in muscle, has
been scarcely employed as an oxygen carrier despite
its structural similarity to hemoglobin. This is because
Mbs with high oxygen affinity do not allow efficient
oxygen transport to peripheral tissues. It may be
expected, however, that Mbs could serve as oxygen
carriers if the oxygen affinity were properly reduced.
We have recently demonstrated that the oxygen
affinity of native Mb could be effectively lowered
when the heme was replaced with iron corrphycene, a synthetic heme analog (2). The heme substitution of Mb with unnatural iron porphyrin is a
simple and inexpensive procedure. The corrphycenesubstituted Mb is indeed a candidate for a synthetic
oxygen carrier because it could unload 20% of bound
oxygen during blood circulation. The successful
results with corrphycene-Mb inspired us to further
modify the heme.
It has been established that the out-of-plane distortion of planar porphyrin macrocycle modulates
the axial ligand affinity, oxidation potential, and
electron transfer rate of hemoproteins (3). We
adopted this methodology to design a new type of
Mb-based oxygen carrier with an intrinsically distorted heme.

MATERIALS AND METHODS

The nonplanar (1) and planar (2) porphyrins in
Fig. 1 were synthesized according to the literature
methods (4,5). The iron complexes were coupled
with apoMb, prepared from sperm whale Mb
(Sigma-Aldrich, St. Louis, MO, USA), and purified
with chromatography on cellulose column (6). The
energy-minimized structure of heme was calculated
with Gaussian-03 program (Gaussian, Inc., Wallingford, CT, USA) at B3LYP/6-311++G level.
The oxygen equilibrium curve was determined
with the automatic recording apparatus of Imai (7).
The carbon monoxide (CO) affinity for Mb was
measured by spectrophotometric titration on a Shimadzu MPS-2000 (Shimadzu, Kyoto, Japan). The
kinetic measurements for CO binding were carried
out on a Unisok TPS-1000WK time-resolved spectrometer (Unisok, Inc., Osaka, Japan). Laser pulses

THOUGHTS AND PROGRESS

221

heme 1
4

2
1

0.2

5
6

Fe

CO2H

N
N

N1

CO2H

0.1

-0.1

N2

N3

N4

-0.2

heme 2
0.1

N
N

Fe

CO2H

N1

N2

N3

N4

-0.1

CO2H

-0.2

FIG. 1. Prosthetic groups for myoglobin. Molecular structure of heme 1 (a-ethyl-2,4-dimethyldeuteroheme) and 2 (2,4dimethyldeuteroheme), and side view of the energy-minimized structures. The values are in angstrom units. The chloro ligand is omitted
for clarity.

with 6-ns duration were irradiated to the MbCO

sample, and the monophasic absorption increase at
418 nm was analyzed according to the textbook (8).
X-band electron paramagnetic resonance (EPR) for
ferrous Mb nitrogen monoxide (NO; 1 mM, 0.1 mL)
was recorded on a Bruker EMX X-band spectrometer (Bruker Daltonik, Bremen, Germany) at 35 K.
Proton NMR at 400 MHz for ferrous deoxyMb
(3 mM, 0.5 mL) was recorded on JEOL a-400 spectrometers (JEOL Co., Tokyo, Japan).
RESULTS
Heme distortion and visible absorption spectra
Figure 1 compares the molecular structure of the
hemes 1 and 2. They are closely similar to each
other except that an ethyl group is attached to the
a-meso-carbon of heme 1. Although an ethyl group is
a small substituent with two carbons, it potentially
influences the planarity of the heme ring. The methylene (-CH2-) group in the ethyl substituent has a van
der Waals radius of 2.0 (9) making severe atomic
contacts with the adjacent heme methyl groups. To
relieve the steric constraint, the heme plane is forced
to deviate from planarity. In Part B of Fig. 1, the
side view of the energy-minimized structures
of iron(III) complexes of the hemes is shown. Heme
1 is expectedly nonplanar with a mixed domed/
ruffled conformation. In contrast, heme 2 retains a
nearly flat structure with an insignificantly domed

character. In the ruffled conformation of heme 1, the

four meso-carbon atoms are alternatively placed
above and below the plane. In contrast, all of the four
meso-carbons in heme 2 are slightly above the heme
plane. The nonplanarity of heme 1 is approximately
threefold larger in magnitude as compared with
heme 2, demonstrating that the ethyl group significantly deforms the heme.
The visible absorption maxima of the deoxy
(428 nm), O2 (427 nm), and CO (420 nm) derivatives
of Mb1, the Mb containing heme 1, appeared at
longer wavelength side by about 1015 nm as compared with those of Mb2 (10). The visible absorption
of porphyrin generally indicates that the energy
gap between the highest-occupied and lowestunoccupied molecular orbitals decreases upon the
nonplanar deformation (3). The red-shifted Soret
peaks of Mb1 derivatives accordingly indicate that
heme 1 retains a nonplanar conformation in the
protein pocket of Mb.
Proton NMR and EPR
We examined the proton NMR of ferrous deoxy
derivatives of Mb1 and Mb2 to analyze the iron coordination structure. Figure 2A compares the NMR
spectra of ferrous deoxyMb derivatives in the H2O
solvent. Mb1 and Mb2 exhibited signals at 82.0 and
78.8 ppm, respectively. Based on the NMR result of
native Mb (11), these exchangeable signals were
assigned to the imidazole NH protons of the ironArtif Organs, Vol. 36, No. 2, 2012

222

THOUGHTS AND PROGRESS

B

90

80

ppm

70

FIG. 2. (A) Proton NMR spectra of

ferrous deoxy derivatives of Mb1 (top)
and Mb2 (bottom). Peaks at 82.0 and
78.8 ppm are from the imidazole NH of
the proximal histidine. Measured in 0.1 M
Tris in H2O : D2O = 10:1 v/v at pH 7.0 and
20C. (B) EPR spectra of Mb1NO (top)
and Mb2NO (bottom) in 0.1 M Tris at pH
7.0 and 35 K.

60

330

320

340

350

Magnetic field (mTesla)

bound proximal histidine. The larger chemical shift in

the Mb1, as compared with Mb2, indicates that the
influence of paramagnetic iron is more directly transmitted to the proximal histidine. In other words, a
larger electron density is placed in the proximal histidine of Mb1. Thus, it is likely that the ironhistidine
bond is stronger in Mb1 than in Mb2.
Ferrous NO Mb
NO is a radical molecule with a single unpaired
electron. Owing to the paramagnetic nature, NO has
been employed in EPR spectroscopy to probe the
iron center in hemoprotein (12). Figure 2B compares
the EPR spectra of the ferrous NO derivatives of
Mb1 and Mb2. Mb1 exhibited a three-line pattern
while Mb2 did not. According to the empirical relationship, the split of the EPR g-components is small
and large in the six- and five-coordinate NO hemes,
respectively (12). The increased g-anisotropy in fivecoordinate NO heme causes the appearance of a
triplet signal (12). Accordingly, the EPR of Mb2
suggests the presence of a six-coordinate NO-Fehistidine structure. In contrast, Mb1 exhibited a characteristic three-line pattern from the five-coordinate
NO heme on the background signal from the sixcoordinate NO heme. The coexisting two types of
EPR signals in Mb1 indicate that the histidineFe
linkage is broken in some fraction. The ratio of the
five- and six-coordinate NO hemes for Mb1 was estimated to be approximately 50:50 from the spectrum.
Functional aspects of intrinsic heme deformation
The nonplanarity in heme 1, as inferred from the
above light-absorption, NMR, and EPR observations, was found to induce a notable functional
anomaly. The oxygenation for Mb1 was characterized
by a Hill coefficient n = 0.97 and the partial O2 presArtif Organs, Vol. 36, No. 2, 2012

sure at half-saturation P50 = 1.79 mm Hg. The P50

value of Mb1 is indeed 25-fold larger than P50 =
0.07 mm Hg of the Mb2 (10), demonstrating that the
nonplanar heme 1 significantly lowers the O2 affinity
of Mb. The functional anomaly of Mb1 was also
found in CO binding. Mb1 exhibited a P50,CO =
12.4 mm Hg for CO. The CO affinity in Mb1 is only
1/470 as compared with P50,CO = 0.026 mm Hg for
Mb2 (10). We examined the kinetic CO binding of
Mb1, and we obtained the association and dissociation constants of kon = 2.40 106/M/s and koff =
4.0 10/s, respectively, for CO. For Mb2 containing
the flat heme 2, Chang and coworkers reported the
CO binding results of kon = 0.67 106/M/s and koff =
0.022 10/s (10). The comparison of these kinetic
parameters between Mb1 and Mb2 indicates that the
decreased CO affinity of Mb1 solely comes from
marked increase in the koff rate of Mb1.

DISCUSSION
Role of the ethyl substituent
The calculated structures in Fig. 1 indicate that
heme 1 is significantly deformed. The heme deformation in Mb is experimentally supported by the redshift of the Soret absorption band. Nonplanar heme
deformation affects the ironporphyrin interactions.
It is very likely that the proximal histidine pulls the
iron atom more from the heme plane in the intrinsically distorted heme 1. The spectroscopic observations support the above idea. The proton NMR in
Fig. 2A shows that the chemical shift of the proximal
histidine signal is larger in Mb1 due to an increased
iron displacement and a stronger ironhistidine
bond. The EPR in Fig. 2B further shows that the
heme iron is largely displaced toward the NO in the

THOUGHTS AND PROGRESS

223

Mb1NO complex to break the opposite iron

histidine linkage.

the reconstituted Mb. Our research is in progress to

develop this idea, and the results will be reported in
due course.

Functional outcome
It is now apparent that the inherently deformed
heme 1 enhances the iron displacement, and that the
proximal histidine pulls the iron more from the heme
plane in ferrous deoxyMb. The larger iron displacement by the proximal histidine, in turn, decreases the
oxygen binding at the opposite coordination site. The
selective increase in the kinetic koff rate of Mb1 for
the CO binding supports the weaker CO ligation due
to a large iron displacement. It is worth pointing out
that the O2 and CO affinities of Mb1 are largely
decreased down to 1/25 and 1/470, respectively, as
compared with those of Mb2.

Acknowledgments: This work was supported by

the research grant-in-aid (A)-20249072 for A. T. K.
from the Promotion of Scientific Research, Japan. We
also thank the support by the SPECT probe program
from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.

CONCLUSIONS
It is apparent that Mb is locked into the tense
(T)-state by the nonplanar heme 1 just like
deoxyHb. The T-structure in Hb is primarily governed by the globin quaternary structure. In contrast, the T-state Mb is produced by the heme
equipping built-in nonplanarity. Heme 1 is easily
prepared by organic synthesis. It smoothly couples
with apoMb to afford the stable protein in good
yield because heme 1 has two propionate side
chains like natural protoheme. These are the
advantages in the preparation of the Mb-based
oxygen carrier. However, we must admit that the
ability of Mb1, which could release 2% of the bound
oxygen under physiological conditions, is not necessarily sufficient. The attachment of more mesosubstituents or the bulkier meso-group to heme
periphery will be desirous to deform heme further
and to improve the oxygen-transporting ability of

REFERENCES
1. Kim HW, Greenburg AG. Artificial oxygen carriers as red
blood cell substitutes: a selected review and current status.
Artif Organs 2004;58:81328.
2. Neya S, Suzuki M, Hoshino T. Novel controlling mechanism of
oxygen affinity in myoglobin with isomeric porphyrins. Artif
Organs 2009;33:18993.
3. Shelnutt JA, Song XZ, Ma JG, Jiu SL, Jentzen W, Medforth CJ.
Nonplanar porphyrins and their significance in proteins. Chem
Soc Rev 1998;27:3141.
4. Maruyama K, Nagata T, Osuka A. Study on 5,15dialkylporphyrins interconversion between two conformers in
solution. J Phys Org Chem 1998;1:6373.
5. Smith KM, Minnetian OM. Cyclizations of 1,8-dimethyl-a,cbiladiene salts to give porphyrins. J Chem Soc Perkin Trans 1
1986;27780.
6. Asakura T. Hemoglobin porphyrin modification. Methods
Enzymol 1978;52:44755.
7. Imai K. Measurement of accurate oxygen equilibrium curves
by automatic oxygenation apparatus. Methods Enzymol
1981;76:43849.
8. Antonini E, Brunori M. Hemoglobin and Myoglobin in Their
Reactions with Ligands. Amsterdam: North Holland Publishing, 1971;188218.
9. Gordon AJ, Ford RA. The Chemists Companion. New York:
John Wiley & Sons, 1972;109.
10. Chang CK, Ward B, Ebina S. Kinetic study of CO and O2
binding to horse heart myoglobin reconstituted with symmetric hemes lacking methyl and vinyl side chains. Archiv
Biochem Biophys 1984;231:36671.
11. La Mar GN, Budd DL, Goff H. Assignment of proximal histidine NMR peaks in myoglobin and hemoglobin. Biochem
Biophy Res Commun 1977;77:10410.
12. Palmer G. In: Lever ABP, Gray HB, eds. Iron Porphyrins, Part
II. London: Addison-Wesley, 1983;4388.

Artif Organs, Vol. 36, No. 2, 2012