From the
The iron complex of
The heme in hemoproteins is a tetrapyrrole compound cyclized
through the four meso carbons. Replacement of one or more of the
bridging carbon(s) with nitrogen(s) leads to the special porphyrinoids
known as azaporphyrins (Jackson, 1978). The most familiar azaporphyrin
is the tetraaza compound, phthalocyanine (Hoffman and Ibers, 1983;
Lever et al., 1993).
Monoazaporphyrin (Jackson, 1978), a
formal 1:3 hybrid of phthalocyanine and porphyrin, has received very
limited attention as compared with the parent macrocycles. Fischer and
Friedrich(1936) first synthesized monoazaporphyrin in extremely low
yield. The synthesis has been much improved by Harris et
al.(1966) and Engel et al. (1978). The efficient and
general synthesis is essential because it first allows us to prepare
sufficient quantity of the monoazaporphyrin bearing a molecular
architecture like native porphyrins. Attachment of the two propionyl
residues at the 6th and 7th sites of heme periphery, although not
always essential (Neya et al., 1988, 1993), suggests steady
incorporation of the synthetic azaporphyrin into the protein matrix.
Nitrogen atom has a smaller van der Waals radius and is more
electronegative than carbon atom. Both conformational distortion at
meso nitrogen and electron withdrawing capacity of the nitrogen should
coincidentally affect the reactivity of the chelating metal ions, as
has been extensively examined in phthalocyanines (Jackson, 1978;
Thompson et al., 1993). Thus, it is likely that the
reactivities of monoazahemin-bound protein toward the ligands are
affected to a considerable extent. We employ in the present work
The iron complex was prepared
by refluxing the azaporphyrin with
FeCl
We recorded the pH-dependent optical
spectra to deduce the coordination environment. The 385-nm absorption
decrease in Fig. 2can be fitted with a single-proton equilibrium
of pK = 8.5. The result suggests the presence of an
iron-bound water molecule, which dissociates into hydroxide with
increasing pH, as in many other aquomet Mbs (Tamura et al.,
1973a; Sono and Asakura, 1976). We examined the proton NMR spectrum to
further deduce the coordination state in the deoxy Mb. The NMR spectrum
in Fig. 3agrees with those of the native (La Mar et
al., 1977) and etioheme-substituted Mbs (Neya et al.,
1989). The exchangeable 85.2 ppm signal is unambiguously assigned to
the iron-bound proximal histidine because such a large shift can be
induced solely by the direct iron-spin transfer through the
iron-histidine bond (La Mar et al., 1977). Appearance of the
single peak provides evidence for the selective interaction between the
azahemin and apoMb, the formation of the iron-histidine bond, and
5-coordinate high spin state of the ferrous heme iron.
The ferric protein is easily reduced with sodium
dithionite or enzymic reducing system to form stable ferrous Mb that
reversibly binds O
The C-N(meso) bond in chloro Fe(III)
phthalocyanines, 1.317-1.320 Å (Palmer et al.,
1985; Fitzgerald et al., 1992) is shorter than the C-C(meso)
bond in corresponding porphyrins, 1.378-1.401 Å (Koenig,
1965; Scheidt and Finnegan, 1989) due to a smaller atomic radius of
nitrogen atom. The bond angle at the meso nitrogen, 121° in
tetraazaporphyrin (Palmer et al., 1985; Fitzgerald et
al., 1992), is slightly smaller than 124-126° at meso
carbon in porphyrin (Koenig, 1965; Scheidt and Finnegan, 1989).
Combination of these two factors results in a smaller central cavity
and stronger in-plane ligand field of the phthalocyanine iron atom.
According to the recent x-ray result, the structural features found in
the tetraazaporphyrins are moderately conserved in Fe(III)
monoazaporphyrin (Balch et al., 1993b). The average
Fe(III)-N(pyrrole) distance, 2.044 Å in monoazahemin (Balch
et al., 1993b), is smaller than 2.055-2.062 Å in
ordinary hemins (Koenig, 1965; Scheidt and Finnegan, 1989), so that the
hole size of monoazahemin is somewhat more constricted. Consequently,
an intermediate (S = 3/2) spin character found in the
aquomet derivative of azaheme Mb directly reflects a narrower
coordination hole, as found in some other hemoproteins (Maltempo et
al., 1979; Weber, 1982).
Another support for a narrower metallo
cavity in azaheme Mb comes from the acid-alkaline transition
(Fig. 2). The pK
Additional
evidence arises from greater reluctance of the oxy Mb to autoxidation.
Consistent with the high equilibrium affinity and much slower
dissociation rate for O
The altered
ligand-binding behaviors in ferrous azaheme Mb (Tables I and II) may be
similarly explained. Narrower cavity causes greater
Fe(II)-to-macrocycle back bonding and increases the effective positive
charge on the Fe(II). The x-ray results of tetraazaporphyrin supports
this interpretation. The Fe(II)-N(imidazole) bond length in
phthalocyanine (1.946 Å; Ercolani et al., 1991) is
appreciably shorter than in porphyrin (2.014 Å; Scheidt and
Gouterman, 1983). Consistently, ferrous tetraazaheme (Fitzgerald et
al., 1992) has much larger affinities for pyridine and imidazole
than ferrous porphyrin (Brault and Rougee, 1974). It is also notable
that CN dissociates from Fe(II) with 0.002 s
As shows,
O
We are much indebted to Drs. Tetsuhiko Yoshimura and
Hiroshi Fujii (Yamagata Research Institute of Technology) for helpful
discussions. We also thank anonymous reviewers for valuable comments on
the original manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-azamesoporphyrin XIII was combined
with apomyoglobin to investigate influence of the meso nitrogen on
ligand binding properties in the reconstituted protein. Stoichiometric
complex formation between the two components was confirmed, and
conservation of the native coordination structures in the resultant
myoglobin was established with spectroscopic criteria and apparently
normal ligand binding. The visible absorption spectra of various ferric
and ferrous derivatives are characteristic with less intense Soret
peaks and enhanced visible bands. The electron paramagnetic resonance
spectrum with g = 5.2 suggests an anomalous
intermediate spin (S = 3/2) character for the aquomet
protein. The oxygen affinity of reduced azaheme myoglobin, 0.010 mm Hg,
is 50 times larger than that of the native myoglobin. In addition,
azaheme myoglobin forms stable complexes with imidazole, pyridine, or
cyanide in ferrous state. All of these new properties were consistently
explained in terms of stronger equatorial ligand field of the heme iron
in a narrower coordination cavity. Similarities of azaheme to verdoheme
were also pointed out.
-azamesoporphyrin XIII (see inset of Fig. 1), a new
compound derived through the established synthetic route (Engel et
al., 1978), as the prosthetic group of Mb.
(
)
The iron complex, as we demonstrate below, forms stable
complex with apoMb. In consequence, one may inquire about the
biological activity and spectral properties of the monoazahemin in Mb
under various ligation and oxidation states. The Mb could contribute to
the new biological chemistry of Mb as well as the development of the
coordination chemistry of azahemins.
Figure 1:
Electronic spectrum of purified
ferric azaheme Mb in 0.1 M Tris buffer at pH 7.0 and 20
°C. The inset denotes the structure of the prosthetic
group.
1,19-Dideoxy-3,17-diethyl-8,12-di(methoxycarbonylethyl)-2,7,13,18-tetramethyl-1,19-dibromobiladiene-ac dihydrobromide (100 mg), prepared from
5-bromo-4-ethyl-3-methyl-2-formylpyrrole (Siedel and Fischer, 1933) and
3,3`-di(methoxycarbonylethyl)-4,4`-dimethylpyrromethane-5,5`-dicarboxylic
acid (Fuhrhop and Smith, 1975), was refluxed in 20 ml of methanol for
16 h in the presence of 500 mg of sodium azide (Engel et al.,
1978). The resultant crude -Azamesoporphyrin XIII and the Iron
Complex
-azamesoporphyrin XIII, after
evaporation of the solvent, was purified with silica gel column using
2% methanol in dichloromethane as eluent. Slow evaporation of the
concentrated solution from methanol-dichloromethane mixture (1:1, v/v)
under low heating afforded the title azaporphyrin as deep violet
micro-needles (20 mg, 39%).
C
H
N
O
requires C,
70.57; H, 6.94; N, 11.74: Found C 70.57; H, 7.04; N, 11.45. m/e 595 (M
) for 595.7.
-2.73
(2H, br s, NH), 1.81 (6H, t, CH
CH
), 3.22
(4H, t, CH
CH
CO), 3.53 and 3.54 (each 6H, s,
ring CH
), 3.64 (6H, s, OCH
), 3.96 (4H, q,
CH
CH
), 4.32 (4H, t,
CH
CH
CO), 9.85 (2H, s, meso H) and
10.04 (1H, s, meso H).
(
) in chloroform; 376 nm
(95 mM
cm
), 503 (6.5),
534 (18.8), 559 (6.7), and 610 (20.0).
nH
O in dimethylformamide
under nitrogen atmosphere (Adler et al., 1970), oxidized with
air, and purified on a silica gel column with chloroform-methanol
mixture (9:1, v/v).
(
) in chloroform: 363 nm
(85 mM
cm
), 457 (8.7),
486 (8.8), 552 (11.9), 630 (3.6), and 660 (3.5). The monoazahemin ester
was hydrolyzed in a refluxing methanol-water mixture containing 1% KOH
as described by Fuhrhop and Smith(1975) for ordinary porphyrin
diesters.
Reconstituted Mb
Sperm whale Mb was available from
Sigma (type II). ApoMb (Teale, 1959), mixed with a 1.2-equivalent
amount of the azahemin, was dialyzed against cold 10 mM
bis-Tris buffer, pH 6.0 (Asakura, 1978). The solution was applied on a
short CM-cellulose (Whatman, CM-52) column previously equilibrated with
10 mM Tris buffer, pH 7.0. The purified Mb was eluted from the
column with a linear gradient of Tris buffer, 10-100 mM,
at pH 7.0 and 4 °C. Fractions with an absorbance ratio
A/A
> 3.3 were
collected and combined. All of the above manipulations smoothly
proceeded to afford a reconstitution yield of 80-90%. The protein
solution was concentrated for proton NMR measurements with an Amicon
ultrafiltration apparatus equipped with PM-10 membrane. Concentration
of the eluted Mb was determined with 105 mM
cm
at 385 nm on the basis of the pyridine
hemochromogen spectrum with 73.2 mM
cm
at 394 nm and 48.0 mM
cm
at 553 nm in 0.1 M NaOH-pyridine
mixture (1:1, v/v).
Spectroscopy
Optical absorption measurements were
carried out on a Shimadzu MPS-2000 instrument equipped with
thermostatted sample holder. Proton NMR spectra were recorded at 300
MHz with a Varian XL-300 spectrometer. EPR spectra were obtained at
X-band (9.35 GHz) microwave frequency by using a Varian cavity with a
home-built EPR spectrometer with 100 kHz field modulation (0.5 mT). An
immersion double Dewar flask was used for liquid-helium temperature EPR
measurements.
Ligand Binding
Ligand binding experiments were
achieved with the visible absorption spectrometer. An oxygen
equilibrium curve was recorded on an automatic recording apparatus
(Imai, 1981) in the presence of an enzymic reducing system (Hayashi
et al., 1973). The CO association constant k was measured by a UNISOK flash-photolysis apparatus as described
previously (Neya et al., 1994). The dissociation constant
k
for CO was determined by the CO-NO replacement
method. The association constant of O
was obtained by the
stopped-flow measurements. The k
for O
was estimated by dividing k
with the
equilibrium constant. Attempted flash photolysis for the NO Mb was
unsuccessful because the derivative was not photodissociable. Rapid NO
binding could not be monitored with the stopped-flow apparatus. The
slow cyanide binding to deoxy Mb was monitored with a Shimadzu MPS-2000
spectrophotometer.
Autoxidation Rates
The ferric azaheme Mb was
reduced with 3-fold molar excess of sodium dithionite powder, loaded
onto and eluted from a Sephadex G-25 (Pharmacia Biotechnology Inc.)
column with air-saturated 0.1 M Tris, pH 7.0, in a cold room.
The oxy Mb, diluted with the same buffer, was used immediately for the
autoxidation measurements at 30 °C on the spectrophotometer.
Absorbance of the Soret peak was monitored. The end point was
determined after complete oxidation of the oxy Mb by adding excess
potassium ferricyanide. The oxidation of mesoheme-substituted oxy Mb
was similarly monitored. The results were analyzed with an equation
d(MbO)/dt =
-k(MbO
), where k is the apparent
autoxidation rate.
Prosthetic Group
We first determined the
pK (Hambright, 1975) of
-azamesoporphyrin
XIII to quantitatively evaluate the influence of electronegative meso
nitrogen. With lowering pH of the porphyrin solution from 5 to 1 in
2.5% aqueous sodium laurylsulfate at 20 °C, the Soret absorption in
a 340-460 nm region reversibly changed with isosbestic points at
377, 388, and 426 nm (results not shown). The optical shifts were
ascribed to the third protonation at the pyrrole nitrogen, rather than
at meso nitrogen, because the meso nitrogen is protonated only at high
sulfuric acid concentrations (Grigg et al., 1973; Jackson,
1978). The analysis of the 398-nm transition with a single-proton
equilibrium afforded a pK
of 3.0 ± 0.15.
Thus the introduction of a single nitrogen atom at the meso bridge
sufficiently decreases pK
= 5.8 of
mesoporphyrin (Hambright, 1975) like the strongly electron-withdrawing
formyl groups in the diformyl porphyrin (Sono et al., 1976).
Reconstituted Mb
The spectrophotometric titration
of the ferric azahemin to apoMb confirmed a 1:1 binding between the two
components. In the optical spectrum of the purified Mb in Fig. 1,
the absorbance ratio A/A
= 3.5 was reproducibly constant for several preparations.
The entire spectral profile with main bands at 455, 495, 540, and 637
nm looks like that of catalase (Schonbaum and Chance, 1976). The
decreased absorbance ratio
A
/A
= 3.5, as
compared with 5.0 of native Mb (Rothgeb and Gurd, 1978), arises from
inherently lower Soret absorption of the prosthetic group containing a
meso nitrogen (Jackson, 1978).
Figure 2:
Acid-alkaline transition of ferric azaheme
Mb (9 µM) in 5 mM Tris at 20 °C. A,
the 385-nm peak decreases with increasing the pH from 7.03 to 7.19,
7.34, 7.54, 7.73, 7.93, 8.13, 8.28, 8.43, 8.56, 8.83, 9.08, 9.47, 9.81,
and 10.91. B, analysis of the 385-nm transition with a pK = 8.51 ± 0.32 and a slope = 0.98 ±
0.03.
Figure 3:
Proton
NMR spectrum of deoxy azaheme Mb. The spectrum was recorded in
HO-D
O (9:1, v/v) mixture containing 3
mM Mb and 0.1 M Tris at pH 7.0 and 20 °C. The
85.2 ppm resonance is an exchangeable NH proton from the proximal
histidine. DSS, sodium
2,2-dimethyl-2-silapentane-5-sulfonate.
EPR is
particularly sensitive to the spin and coordination states of Mb. Our
initial measurement for metMb at 80 K was unsuccessful to afford no
signals. The signal with g = 5.3 (part A of
Fig. 4
), appeared only at liquid helium temperature, is similar
to that of an intermediate spin protein, Aplysia metMb (Hori
et al., 1990), and quite distinct from the typical ferric high
spin spectrum of native aquomet Mb. Addition of sodium fluoride or
sodium azide to azaheme Mb produced the typical high and low spin
spectra as illustrated. The EPR spectrum of the ferrous NO
complex was anomalous (part B of Fig. 4). Contrary to
native Mb, the fleshly prepared azaheme Mb exhibits a typical
5-coordinate type spectrum with g
= 2.098
and g
= 2.010 and a hyperfine coupling
constant A
= 2.3 mT, similar to that found
in the hemoglobin bound with inositol hexaphosphate (Maxwell and
Caughey, 1976). The spectrum gradually replaced with that of
6-coordinate nitrosyl heme. On standing the sample in ice bath for
>10 h, only the signal from 6-coordinate nitrosyl Mb with
A
= 3.1 mT remained.
Figure 4:
EPR spectra of azaheme Mb derivatives.
A, ferric complexes at 5 K. The topspectrum is for native aquomet Mb. B, ferrous NO
complexes at 80 K. A 0.8 mM Mb in 0.1 M Tris buffer
at pH 7.0 was used for both observations.
Ligand Binding and Absorption Spectra
Ferric
azaheme Mb binds a variety of exogenous ligands. With addition of
sodium azide, a new Soret peak appeared at 398 nm with well-defined
isosbestic points at 394 and 448 nm. Analysis of the intensity decrease
at 385 nm resulted in an affinity of 1440
M. Optical titration confirmed the cyanide
and fluoride binding to the Mb as well with 140,000
M
and 3.3 M
,
respectively.
, CO, and NO. The analysis using the Hill
plot in Fig. 5revealed an unprecedentedly high O
affinity of P
= 0.01 mm Hg. This is
indeed 40-100-fold higher than those reported for
mesoheme-substituted or native Mbs (Sono and Asakura, 1975). The oxy
azaheme Mb was fairly stable; the autoxidation rate was 0.34
10
min
at 30 °C while we obtained
5.2
10
min
for oxy mesoheme Mb
under the same conditions (results not shown). The CO binding profiles
of azaheme Mb were comparable with those of native Mb. Tables I and II
summarize the ligand-binding results.
Figure 5:
A Hill plot of oxygen equilibrium for
ferrous azaheme Mb. P, partial oxygen pressure in mm Hg;
Y, fractional saturation with oxygen. The measurement were
made in 0.1 M phosphate buffer at pH 7.0 and 20 °C in the
presence of enzymic reducing system. P at half saturation is
0.01 mm Hg and the Hill coefficient n is
0.97.
The electronic spectra of the
ferric and ferrous derivatives are presented in Fig. 6and
I. The visible bands are unusually prominent relative to
the Soret region, being about 10-60% as intense. The Soret peaks
are broader and move toward blue by 10-30 nm than those of
mesoheme Mb (Tamura et al., 1973a, 1973b). The CO complex
exhibits two split Soret bands, in contrast to a single prominent peak
of native Mb (Rothgeb and Gurd, 1978).
Figure 6:
Visible
spectra of representative azaheme Mb derivatives. A, ferric
complexes (CN, - - - -; F
, -;
OH
,
). B, ferrous
complexes (deoxy, - - - -; O
, -; CO,
). The spectra were recorded in 0.1 M
Tris buffer at pH 7.0 and 20 °C in the presence of saturating
amounts of the ligands.
Azaheme Mb exhibits novel and
unique ligand-binding behaviors. We found that cyanide, pyridine, and
imidazole bind to the ferrous Mb. Although Keilin and
Hartree(1955) reported weak cyanide binding to native deoxy Mb at pH
9.3, the affinity at neutral pH is extremely low so that the cyano
complex is formed only transiently upon reduction of cyanmet Mb (Olivas
et al., 1977; Bellelli et al., 1990). Pyridine and
imidazole have never been appreciated as the ligands for native ferrous
Mb. On the other hand, the cyanide, pyridine, and imidazole compounds
of azaheme Mb were fairly stable. The imidazole and pyridine
derivatives were reddish-violet and the cyano Mb was bright blue-green
in solution. Fig. 7shows the imidazole titration to the deoxy Mb
examined under nitrogen atmosphere. The affinities for pyridine,
imidazole, and cyanide were determined to be 350
M, 7.1 M
, and
440 M
, respectively. We obtained a
k
= 0.70 M
s
for cyanide at pH 7 and 20 °C. No EPR
signals were detectable for these three derivatives, suggesting their
ferrous low spin configuration. The visible spectra and binding
constants of the new complexes are summarized in Fig. 8and
. Azide, thiocyanate, and fluoride did not bind to the
deoxy Mb.
Figure 7:
Imidazole binding to deoxy azaheme Mb.
A, imidazole concentration increases from 0 to 9.30, 19.4,
36.2, 54.4, 75.4, 97.9, and 124 mM in 0.1 M Tris at
pH 7.0 and 20 °C. B, analysis of the ligand binding
equilibria monitored at the wavelengths where the largest changes were
observed.
Figure 8:
Visible spectra of the ferrous complexes
of azaheme Mb. These were recorded in 0.1 M Tris buffer at pH
7.0 and 20 °C in the presence of high concentrations (0.5
M KCN, 0.3 M pyridine, and 3 M imidazole) of
the ligands.
Mb with Azahemin
The 1:1 binding stoichiometry
of the azahemin with apoMb and the formation of the iron-histidine bond
provide solid experimental support for the normal Mb reconstitution.
The apparently normal binding of oxygen and other ligands is consistent
with regular azaheme insertion. It is very likely that the entire
globin fold and the side chain conformations around the prosthetic
group are least perturbed after insertion of azamesohemin, as in the
hemoproteins reconstituted with mesohemin (Padlan, 1975; Seybert and
Moffat, 1977) or even with iron porphine (Neya et al., 1993).
Smooth preparation of the reconstituted Mb may be understood from the
structural similarity of the azaheme bearing two propionyl side chains
with natural hemes.
Contracted Iron Cavity
The EPR signal at g = 5.2 (Fig. 4) indicates dominant intermediate spin
(S = 3/2) character (Weber, 1982; Maltempo et
al., 1979) for azaheme-substituted aquomet Mb, in contrast with
native aquomet Mb with high spin (S = 5/2) heme iron.
The anomalous spin state of azaheme Mb primarily comes from insertion
of a meso nitrogen.
= 8.5 is
about one pK unit larger than 7.3 for 2,4-diformylhemin Mb
(Sono and Asakura, 1976), despite an identical pK
of 3.0 for the two free-base porphyrins. The larger
pK
of azaheme Mb suggests stronger H-OH
bond of the coordinating water molecule and hence weaker
Fe(III)-H
O interactions. The weaker axial ligand field in
tazaheme Mb is consistent with the stronger Fe(III)-N(pyrrole)
interactions in the contracted central cavity of azaheme.
(Springer et al., 1989;
Brantley et al., 1993), azaheme Mb is 15-fold more stable than
mesoheme Mb. The stability of oxy azaheme Mb may due to enhanced back
bonding from Fe(II) into
orbitals of azaporphyrin, as has been
pronounced in the tetraazaporphyrin case (Ercolani et al.,
1983; Fitzgerald et al., 1992). The enhanced back bonding from
Fe(II) reflects contraction of the iron hole.
Meso Nitrogen and Ligand Binding
It is well known
that phthalocyanine with a smaller cavity is a better -donor than
porphyrin (Kennedy et al., 1986; Fitzgerald et al.,
1993). Inherent phthalocyanine character found in the monoazaheme in
Mb, as pointed out in the preceeding discussion, rationally suggests
increased ability of the pyrrole as electron donor. This could induce
repulsive interactions between iron
d
for
azaheme Mb while 0.14 s
is reported for native Mb
(Bellelli et al., 1990) under comparable conditions. A larger
iron-to-azaheme back bonding accounts for increased stability of the
Fe(II)-ligand complexes of azaheme Mb.
is much more susceptible to the narrower iron cavity of
azaheme than CO. The results may be rationalized by the difference in
the
and
bonding properties of the two ligands. It is
genarally accepted that CO is a better
acceptor and poorer
donor than O
(Shriver et al., 1994). The altered
discrimination between CO and O
in azaheme Mb suggests
weakened
bonding and/or enhanced
interaction in the
Fe(II)-ligand bonds. It is also notable that the NO azaheme Mb
initially forms a 5-coordinate complex (Fig. 4). We propose that
the Fe(II)-NO bond in azaheme Mb is much stronger than that in native
6-coordinate Mb. The NO azaheme Mb correspondingly is not
photodissociable. Since NO is a better
-acceptor than CO, the
cleavage of the Fe(II)-histidine bond in azaheme Mb indicates enhanced
-bonding character of the Fe(II)-NO interactions.
Similarity with Verdoheme
In the liver and spleen
of many animals, protoheme IX is degraded into biliverdin by heme
oxygenase. In the degradation process, an oxygen atom is inserted to
the meso bridge to produce an intermediate compound called
-oxoprotoheme or verdoheme (Saito and Itano, 1986; Balch et
al., 1993a). Replacement of the porphyrin meso bridge by an oxygen
atom infers close similarity of oxoheme with azaheme because both
elements are more electronegative and smaller in atomic radii than
carbon atom. Indeed, azaheme (Balch et al., 1993b) has a
narrower metallo cavity and forms the bis-halogen adduct in the ferric
state as oxohemin does (Balch et al., 1993a). It is notable
that ferrous azaheme Mb binds cyanide like the ferrous Mb containing
verdoheme (Fujii, 1990). The visible spectrum of ferrous cyano azaheme
Mb is a typical verdoheme-type spectrum (Saito and Itano, 1986; Fujii
1990) to support spectroscopic similarity between azaheme and
verdoheme. In ferrous azaheme Mb, a 60 nm blue-shift of the visible
band is observed upon the ligand replacement from cyanide to CO, in
good agreement with the results that the CO and cyanide adducts of
verdohemochrome in Mb show visible bands at 655 and 707 nm,
respectively (Fujii, 1990). The verdoheme in heme oxygenase is not easy
to characterize due to the spontaneous degradation into biliverdin.
Oxoheme and azaheme are chemically similar to each other except for the
enhanced stability of the latter against reducing agents and molecular
oxygen. The possibility exists that azaheme is a stable substitute for
labile verdoheme in heme oxygenase. A neutral imidazole has been
proposed as the iron-bound ligand in heme oxygenase (Takahashi et
al., 1994a, 1994b; Sun et al., 1993) as in Mb. This
observation suggests that azaheme Mb itself serves as a close model to
investigate unexplored physicochemical properties of the heme oxygenase
substituted with verdoheme.
Table:
Comparison of the ligand affinities in
M between azaheme and native Mbs
in 0.1 M Tris at pH 7.0 and 20 °C
Table:
Rate and
equilibrium (accurate to ±10%, at pH 7.0 and 20 °C) for
O and CO binding to the ferrous Mbs containing the
porphyrins with pK
= 3.0
Table:
Visible absorption parameters of the Mb
containing -azamesohemin XIII
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.