From the Department of Physiology and Biophysics,
Albert Einstein College of Medicine, Bronx, New York 10461 and
§ Department of Plant Science, University of Manitoba,
Winnipeg, Manitoba R3T 2N2, Canada
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ABSTRACT |
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To elucidate the environment and ligand structure
of the heme in barley hemoglobin (Hb), resonance Raman and electron
paramagnetic resonance spectroscopic studies have been carried out. The
heme is shown to have bis-imidazole coordination, and
neither of the histidines has imidazolate character. Barley Hb has a
unique heme environment as judged from the Fe-CO and C-O stretching
frequencies in the CO complex. Two Fe-CO stretching modes are observed
with frequencies at 534 and 493 cm Barley (Hordeum sp.) hemoglobin
(Hb),1 the first nonsymbiotic
plant Hb to be isolated and characterized from a monocotyledonous plant
(1), is expressed in seed and root tissues under anaerobic conditions.
Because the level of Hb in barley aleurone tissue is of the order of
only 20 µM, an expression system in Escherichia coli was developed to produce a barley Hb fusion protein that is
indistinguishable from the native protein in most properties but
differs in having five extra amino acids at the N terminus (1).
Nonsymbiotic plant Hbs constitute a new class of protoheme proteins
that are expressed at low concentrations (on the order of 1-20
µmol/kg wet tissue weight) in roots, stems, or germinating seeds of
monocotyledonous (2, 3) and dicotyledenous plants (3-6). Nonsymbiotic
Hbs of both monocots and dicots fall into a single coherent gene
family, distinct from the family of genes that encode the symbiotic Hbs
(6). They are functionally distinct from the familiar symbiotic plant
leghemoglobins that are expressed by some plants in nitrogen-fixing
symbiotic associations with the bacterium Rhizobium or the
actinomycete Frankia. Distinguishing characteristics of
nonsymbiotic Hbs (1, 7, 8) are extremely slow dissociation of bound
oxygen and 6-coordinate, low spin ferrous (deoxy) species.
The barley nonsymbiotic Hb gene is induced under conditions of low
oxygen pressure (2). Low oxygen tension, per se, is not
responsible for the induction, because the gene can be induced in the
presence of oxygen by respiratory inhibitors that interfere with
mitochondrial ATP synthesis (9). Induction appears to be initiated by a
decline in ATP within the cell, leading to a Ca2+-mediated
signal transduction process (10). Studies using cultured maize cells
transformed to express the sense and antisense gene (11) have
demonstrated that cells containing the sense construct possessed higher
levels of ATP and adenylates than wild type or antisense-transformed
cells when grown under limiting oxygen, suggesting that Hb acts to
maintain energy status under low oxygen tension (11). It has been
postulated (12) that barley oxyHb, in conjunction with another protein,
could function as an oxygenase, oxidizing NADH to maintain glycolytic
ATP synthesis as an alternative pathway to ethanol or lactate formation.
The recombinant barley Hb studied here and native barley Hb are
homodimers with a subunit molecular mass of 18.5 kDa, having one
protoheme IX per subunit. Extraordinarily slow dissociation of bound
oxygen (koff = 0.027 s The 6-coordinate nature of ferrous barley Hb is reminiscent of the
structure of cytochromes. For this reason, it is of interest to
identify the ligands to the heme iron. Here we present evidence that
the histidine, placed by sequence alignment in the position proximal to
the heme iron, ligates to the iron and has the character of an
uncharged imidazole. We further show that a second histidine residue,
placed by sequence alignment in the position distal to the heme iron,
ligates to the heme. Furthermore, we show that the distal histidine
interacts with exogenous iron-bound CO in a novel manner and may
account for a remarkably high ligand stability associated with the slow
oxygen dissociation rate of barley oxyHb.
Recombinant Barley Hb--
Barley Hb was prepared as described
previously (1). Briefly, the barley root Hb cDNA was cloned into
pUC 19 plasmid (2). E. coli strain DH5- Resonance Raman Spectroscopy--
The Raman instrumentation has
been described in detail elsewhere (13). Briefly, the resonance Raman
measurements were carried out with an excitation wavelength of 413.1 nm
from a Kr-ion laser (Spectra Physics, Mountain View, CA). The sample
cell was spun at 6000 rpm to avoid local heating. The Raman scattered
light was dispersed through a polychromator (Spex, Metuchen, NJ)
equipped with a 1200 grooves/mm grating and detected by a liquid
nitrogen-cooled charge-coupled device camera (Princeton Instruments,
Princeton, NJ). A holographic notch filter (Kaiser, Ann Arbor, MI) was
used to remove the laser scattering. Typically, six 30-s spectra were recorded and averaged after the removal of cosmic ray spikes by a
standard software routine (CSMA; Princeton Instruments, NJ). Frequency
shifts in the Raman spectra were calibrated using
acetone-CCl4 or indene as a reference. The laser power was
maintained at ~1 mW to minimize CO dissociation from HbCO samples. In
photolysis experiments with HbCO samples, partial CO photodissociation
was achieved with 400 mW of laser power.
The concentration of the protein samples used for the Raman
measurements was typically 30-100 µM in 100 mM buffer (sodium acetate, pH 5; sodium phosphate, pH 7.4;
CAPS, pH 10.5). For the ferrous samples, ferric barley Hb was reduced
by the addition of a freshly prepared anaerobic solution of dithionite
to the degassed protein solution. HbCO was prepared by exposing
dithionite-reduced samples to either 12C16O or
13C18O in tightly sealed Raman cells.
13C18O gas was a product of ICON (Mount Marion,
NY). Absorption spectra were recorded before and after the Raman
measurements to verify the stability of the species studied.
Electron Paramagnetic Resonance--
EPR spectra were obtained
at 6 K using a Varian E112 spectrometer equipped with a Systron-Donner
frequency counter and a PC-based data acquisition program. The samples
of ferric barley Hb were dissolved in 20 mM Hepes, pH 7.5, for the EPR measurements. The spectrum was recorded at a microwave
frequency of 9.29 GHz, a microwave power of 10 mW, a modulation
frequency of 100 kHz, and a modulation amplitude of 5 G.
High Frequency Resonance Raman Spectra of Fe(III) and Fe(II) Barley
Hb--
The high frequency region (1300-1700 cm
The ferric form (Fig. 1a) displays a frequency of the
electron density marker,
The spectrum of ferrous barley Hb at pH 7.4 (Fig. 1b) is
dominated by a 6-coordinate low spin heme ( Fe-His Stretching Frequency--
The low frequency region of
resonance Raman spectra of hemeproteins is comprised of several
in-plane and out-of-plane vibrational modes of the heme, including heme
propionate modes and ligand vibrational modes (13, 14). Enhancement of
the axial ligand (bound to the central metal atom) vibrational modes
arises from electronic coupling of the orbitals of the ligand to the
metalloporphyrin electronic orbitals. Assignment of a ligand
vibrational mode is extremely useful because it directly identifies a
particular ligand and the nature of its interactions with amino acid
residues in the heme pocket. As noted above, the ferrous barley Hb
contains both a low spin 6-coordinate form and a smaller population of a deoxy-type 5-coordinate high spin species. The low frequency region
of the resonance Raman spectra of the ferrous species is shown in Fig.
2 (spectrum a). The line at
219 cm
It is to be noted that in peroxidases, the EPR Spectrum of Ferric Barley Hb--
The EPR spectrum of ferric
barley Hb at pH 7.5 shows the presence of both high and low spin
signals (Fig. 3). The high spin signal is
slightly rhombic, with g values of 6.04, 5.63, and 1.99. The g values
for the low spin signal are 3.02, 2.22, and 1.48, values that are
virtually identical to those of bovine liver cytochrome b5 (3.03, 2.23, 1.43; Ref. 20) for which the
x-ray crystal data (21) and solution NMR studies (22) have shown a
bis-histidyl imidazole-ligated heme structure. The EPR
spectrum also resembles that of the bis-imidazole model heme
complexes (23). Furthermore, the g tensor anisotropy of low spin barley
Hb does not resemble that of the alkaline form of cytochrome
b5 and the high pH complex of
bis-imidazole heme that contain axial imidazolate ligands
(20). Thus, the possibility that one or both of the axial ligands of barley Hb is imidazolate is excluded. In addition, a signal at g = 3.32 was resolved for the ferric cyanide complex of barley Hb. This
value is similar to gmax for cyanide complexes of globins (24) but larger than that for peroxidases (25) that have an imidazolate
axial ligand (26). This further supports the assignment of a neutral
histidyl imidazole as the proximal ligand and is consistent with the
resonance Raman observation of a Mb-like (proximal imidazole and not
imidazolate) Fe-His stretching frequency.
Fe-CO Stretching Frequency--
The Fe-CO stretching mode
(
It is to be noted that at pH 7.4 (Fig. 4, spectrum a), the
intensity of the
Fe-CO stretching frequencies at ~495 cm
The origin of such high frequencies for
The H/D isotope effect observed here is not a simple mass effect
because in that case, a D2O-induced shift to lower
frequency would be observed. Whereas the specific mechanism of the
increase in the Fe-CO stretching frequency in D2O remains
to be solved, it has been postulated that the hydrogen bond strength
between the FeCO and the histidine N-H is changed upon the H/D exchange due to a decrease in the zero point energy of the N-D bond (41, 44, 45)
relative to the N-H bond in an anharmonic potential well. The N-D bond
is more stabilized due to a retardation of the vibrational amplitude of
one of its bending modes that primarily represents the wagging motion
of the bridging deuterium. As a result, the CO···D-N assembly
becomes more rigid than the CO···H-N moiety; thus, the
stretching vibrational frequency of the Fe-CO mode is increased.
The above results, which suggest that the conformer associated with the
high frequency of
As discussed above, the location of the distal histidine in close
proximity to the diatomic ligand-binding site of the heme iron should
play a significant role in controlling the ligand stability. This, in
fact, is manifested in the observation of a dramatic decrease
(>440-fold) in the oxygen dissociation rate (koff) relative to sperm whale Mb (1, 48). Such
a low dissociation rate can be explained by the extra stability of the
bound ligand due to a strong hydrogen bonding interaction with the
distal histidine.
Correlation between Fe-CO and C-O Stretching Frequencies--
It
is well established that the Fe-CO and C-O stretching modes follow an
inverse correlation due to Dihistidyl Heme in Nonsymbiotic Plant Hbs--
There are three
histidine residues in the amino acid sequence deduced from the
nucleotide sequence of barley Hb (2). Sequence alignment (2) suggests
that His-105 and His-70 are the proximal and distal ligands,
respectively, to the heme iron atom of barley Hb. Evidence from several
spectroscopies, as discussed above, indicates that the distal histidine
ligates to the heme iron in both the ferrous and ferric forms of the
protein. Growing evidence suggests that all nonsymbiotic plant Hbs may
have dihistidyl ligation of the heme iron. Sequence alignment places
histidine in the distal heme pocket of soybean (6), rice Hb1 and Hb2
(7), Trema (4, 51), Arabidopsis AHB1 (8), and
barley (1, 6) Hbs. Optical spectra of ferrous deoxy rice Hb1 (7),
Arabidopsis AHB1 (8), and barley (1) Hbs indicate that these
proteins are in 6-coordinate low spin form, as expected for ferrous
dihistidyl structures. This was confirmed for rice Hb1 by mutation of
the distal histidine (7).
Conclusions--
Barley Hb has a bis-histidine
coordinated heme in which both the imidazoles are uncharged. Among the
distal histidine-containing globins, barley appears to have a unique
heme environment judged from Fe-CO stretching vibrational frequencies
in the CO complex. Unusual Fe-CO and C-O stretching frequencies (at 534 and 1924 cm1, with relative
intensities that are pH sensitive. The 534 cm
1 conformer
shows a deuterium shift, indicating that the iron-bound CO is
hydrogen-bonded, presumably to the distal histidine. A C-O stretching
mode at 1924 cm
1 is assigned as being associated with the
534 cm
1 conformer. Evidence is presented that the high
Fe-CO and low C-O stretching frequencies (534 and 1924 cm
1, respectively) arise from a short hydrogen bond
between the distal histidine and the CO. The 493 cm
1
conformer arises from an open conformation of the heme pocket and
becomes the dominant population under acidic conditions when the distal
histidine moves away from the CO. Strong hydrogen bonding between the
bound ligand and the distal histidine in the CO complex of barley Hb
implies that a similar structure may occur in the oxy derivative,
imparting a high stability to the bound oxygen. This stabilization is
confirmed by the dramatic decrease in the oxygen dissociation rate
compared with sperm whale myoglobin.
INTRODUCTION
Top
Abstract
Introduction
References
1),
corresponding to a t1/2 of ~25 s,
taken together with an unremarkable combination rate constant, results
in a very high affinity for oxygen (KD = 3.8 nM (1)). The net rate of oxygen
dissociation2 (~30
µM min
1) is far too slow to support a
metabolic function. The concentration of barley Hb within the aleurone
or root cell is too small to store a metabolically significant amount
of oxygen or facilitate oxygen movement, except perhaps within very
limited structural domains. Ferrous deoxy barley Hb, in common with
rice Hb (7), has an optical spectrum characteristic of a low spin
6-coordinate heme, indicating the presence of a ligand in the distal
position (1, 2). It has been suggested that an exogenous ligand, such
as oxygen or carbon monoxide, can react with the heme iron only
following prior dissociation of the bound endogenous ligand (1). In any
hemeprotein, the heme pocket structure plays a crucial role in
controlling protein function, such as heme ligand stability, and thus
determines the class of a particular hemeprotein. Since barley Hb is a
newly discovered hemeprotein, it is important to elucidate the
structure of the heme pocket as a whole, particularly the nature of the
heme axial ligands and the electronic environment of the heme crevice.
Upon unveiling of the specific nonbonding interactions that prevail in
the heme pocket, the origin of the remarkably slow oxygen dissociation
rate may be elucidated.
EXPERIMENTAL PROCEDURES
was used as the
host for the recombinant plasmid. The Hb was extracted and purified
from the cells, and the most pure fractions of Hb were pooled and
concentrated to a final volume of ~200 µl in phosphate buffer, pH
7. The purified protein was stored at
80 °C until used for
spectroscopic measurements.
RESULTS AND DISCUSSION
1) of
the resonance Raman spectra of hemeproteins is comprised of porphyrin
in-plane vibrational modes that are sensitive to the electron density
in the porphyrin macrocycle and also to the oxidation, coordination, and spin state of the central iron atom (13, 14). The resonance Raman
spectra of the ferric and ferrous protein (at pH 7.4) in the high
frequency region are shown in Fig. 1.
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Fig. 1.
Resonance Raman spectra of (a)
ferric and (b) ferrous barley Hb at pH 7.4 in the high
frequency region. The laser power at the sample was ~5 mW.
4, at 1374 cm
1, a
frequency characteristic of the Fe(III) state. The location of
3 (at 1505 cm
1) and
10 (at
1635 cm
1) in the spectrum indicates that the protein
contains a 6-coordinate low spin ferric heme. However, the observation
of a weak
3 line at 1474 cm
1 suggests that
a minor population of a 6-coordinate high spin complex is also present.
The spectrum of the ferric complex did not show any pH dependence in
the pH range 5.0-10.5.
4 = 1361 cm
1;
3 = 1493 cm
1) in
addition to a population of a 5-coordinate high spin heme (
3 = 1470 cm
1). The population of the
5-coordinate heme is much smaller than that of the 6-coordinate heme,
although the intensity of the 1470 cm
1 line is higher
than the 1493 cm
1 line. This results from the fact that
the intrinsic intensity of
3 (1470 cm
1) is
very high for the 5-coordinate species compared with that of the
6-coordinate species and thus makes it difficult to assess the relative
populations by inspection of the resonance Raman spectrum. The optical
absorption spectrum (1) confirms that the 6-coordinate form dominates
at a neutral pH. The population of the ferrous high spin heme observed
at both neutral and acidic pH values becomes immeasurably small at an
alkaline pH value (pH 10.5). Ferrous mammalian Hbs, on the other hand,
remain in a 5-coordinate high spin form over a wide pH range.
1 is assigned to the Fe-His (proximal) stretching
mode (
Fe-His). This assignment is supported by the
appearance of this line at 219 cm
1, generated by
photodissociation of the CO derivative (Fig. 2, spectrum c)
that was completely absent from the spectrum of the CO derivative (Fig.
2, spectrum b). The high frequency region of the photolyzed
species (data not shown) has the characteristics of a typical
5-coordinate high spin heme (
4 = 1356 cm
1;
3 = 1470 cm
1). Observation of a line
attributed to the Fe-His stretching frequency suggests that histidine
is the proximal ligand to the heme in barley Hb. As expected, this line
is not observed in the resonance Raman spectrum of the 6-coordinate
species of ferrous barley Hb at high pH values (data not shown).
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Fig. 2.
Resonance Raman spectra of ferrous barley Hb
in the low frequency region at pH 7.5. a, ferrous (deoxy);
b, CO derivative; c, photolyzed CO derivative.
The three spectra are normalized with respect to the porphyrin mode
7 (at 676 cm
1). The intensity in spectrum
a is amplified by a factor of 2 for the sake of clarity. The
laser powers used were (a) 5 mW, (b) 1 mW, and
(c) 400 mW.
Fe-His mode
is detected at a significantly higher frequency (>240
cm
1) (15-18) compared to that in globins (200-230
cm
1) (13, 18, 19). Whereas the origin of the anomalous
frequency of
Fe-His in peroxidases is not well
understood, it is likely that the high frequency of the
Fe-His mode in peroxidases is due in part to the
imidazolate character of the proximal histidine (13, 18). Consideration
of the above facts suggests that barley Hb in which the frequency of
the
Fe-His mode is similar to that of mammalian Hbs and
Mbs has an uncharged proximal imidazole (histidine) and not an imidazolate.
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Fig. 3.
EPR spectrum of ferric barley Hb in 20 mM Hepes, pH 7.5. The spectrum was recorded at a
microwave frequency of 9.29 GHz, a microwave power of 10 mW, a
modulation frequency of 100 kHz, a modulation amplitude of 5 G, and a
temperature of 6 K.
Fe-CO) has been identified in CO complexes of
hemeproteins. Its frequency is sensitive to interactions of bound CO
with neighboring residues. Fig. 4 shows
the spectra of the CO derivative of barley Hb as a function of pH and
isotopic composition of the bound CO. Interestingly, two Fe-CO
stretching frequencies at 534 and 493 cm
1, respectively,
are observed at pH 7.4 (Fig. 4, spectrum a). Assignment of
these two frequencies is confirmed by isotope replacement with 13C18O, in which the corresponding frequencies
appear at 518 and 486 cm
1, respectively (Fig. 4, spectrum
b). The isotope shift of the 493 cm
1 line is
seen more clearly in the comparison of spectra c and d obtained at pH 5.0. However, determination of the exact
magnitude of the frequency shift of the 493 cm
1 band on
isotope replacement
(12C16O/13C18O) is
difficult because of the overlap of
Fe-CO with a
porphyrin peak at ~490 cm
1. The Fe-C-O bending mode
(
Fe-C-O) associated with the 534 cm
1
species is assigned to the band at 586 cm
1 that shifts to
568 cm
1 in 13C18O.
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Fig. 4.
Resonance Raman spectra of barley HbCO in the
low frequency region as a function of the pH and the isotopic
composition of CO. a, pH 7.4, 12C16O; b, pH 7.4, 13C18O; c, pH 5, 12C16O; d, pH 5.0, 13C18O; e, pH 10.5, 12C16O. The laser power at the sample was ~1
mW.
Fe-CO band at 534 cm
1 is
much stronger than that at 493 cm
1. However, upon
lowering the pH to 5.0, the intensities of the bands are reversed (Fig.
4, spectrum c); the
Fe-CO line at 493 cm
1 becomes dominant. Frequency shifts of both of these
lines by isotope replacement of CO at the lower pH value confirm their assignment as
Fe-CO modes (Fig. 4, spectrum
d) that arise from two different conformations of the
protein that have different electronic environments around the Fe-CO
moiety. More importantly, the transition between these two conformers
is linked to a proton-induced phenomenon in the heme pocket. At a very
alkaline pH value, pH 10.5, only the
Fe-CO line at 493 cm
1 is observed (Fig. 4, spectrum e).
1 have been
observed in the A0 state of Hbs and Mbs at acidic pH values
and in many other hemeproteins as well and are believed to arise from
an open heme pocket in which CO has very little interaction with the
surrounding amino acid residues (27-29). However, a value as high as
534 cm
1 for
Fe-CO is unprecedented in
globins and in other hemeproteins, with the exception of some
peroxidases (13, 15, 16, 30, 31). The terminal oxidases also exhibit a
high frequency of
Fe-CO at ~520 cm
1
(32-34). However, the high frequency of the Fe-CO modes in oxidases is
believed to result from an interaction with the nearby copper atom.
Mammalian Hbs and Mbs containing a distal histidine, on the other hand,
show
Fe-CO modes in the 505-510 cm
1 range
(28, 35-37) at neutral pH value (A1 state), whereas
horseradish peroxidase and cytochrome c peroxidase have
Fe-CO in the range of 530-540 cm
1 (13,
15, 16, 30, 31).
Fe-CO has been
debated extensively. In peroxidases, it is attributed to an increase in
the Fe-CO bond order caused by the imidazolate character of the
proximal histidine ligand and hydrogen bonding interactions of a distal
residue with CO. Because the proximal histidine of barley Hb does not
have imidazolate character, the high frequency for
Fe-CO
must arise from distal interactions. We propose that the origin of the
high frequency of
Fe-CO in barley Hb is due to a strong
interaction of CO with a positively polarized (
+)
residue on the distal side of the heme. It has been suggested that
electrostatic interactions with CO can modulate
Fe-CO
significantly. Specifically, a positively charged environment tends to
increase the frequency of
Fe-CO (37-40). The amino acid
sequence homology (with mammalian globins) of barley Hb suggests that
His-61 is located distal (E7) to the heme. It also suggests that, there are no other positively charged residues present in the distal heme
pocket (Phe at B10, Phe at CD1, Val at E11). It is likely that the
imidazole of this histidine residue interacts with CO. Evidence of a
hydrogen bonding interaction comes from the observation of an isotope
effect on the
Fe-CO line shown in Fig.
5. The
Fe-CO line at 534 cm
1 in H2O (spectrum b) shifts to
535 cm
1 in D2O (spectrum a) with a
concomitant decrease in the intensity of the
Fe-CO
located at 586 cm
1. The difference spectrum
(H2O-D2O) shows the effect of deuterium substitution more clearly (spectrum c), indicating the
presence of a proton in close interaction with CO. A similar isotope
effect has been reported to occur in the CO complex of sperm whale Mb, in which it was suggested that a hydrogen bond exists between the
distal histidine and the iron-bound CO (41). It is well known that such
hydrogen bonding occurs between the distal histidine and the bound
oxygen in mammalian oxyMb (42, 43). In barley Hb, however, we propose
that the hydrogen bonding distance of the histidine N
H
to CO is shorter than that in mammalian Mb. Close proximity of the
polar N
H group to CO should increase the
Fe-CO frequency because it was shown that such an effect is distance dependent; the shorter the distance, the higher the frequency of
Fe-CO (38). The fact that the distal
histidine of barley Hb can bind to the heme iron in both the ferric and ferrous states (in the absence of exogenous ligands) is consistent with
the distal histidine residing closer to the heme than seen in mammalian
Mbs and Hbs.
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Fig. 5.
Resonance Raman spectra of barley HbCO in the
low frequency region at pH 7.4 as a function of isotopic composition of
water. a, D2O; b, H2O;
c, difference spectrum (H2O-D2O).
The laser power at the sample was ~1 mW.
Fe-CO involves an interaction with the
distal histidine, are further supported by observations at acidic pH
values. At pH 5.0, the presumptive open conformer (
Fe-CO at 493 cm
1) becomes the major population at the expense
of the second conformer (534 cm
1) (Fig. 4). This
transition is consistent with protonation-induced changes in the distal
histidine that result in its moving out of the heme pocket, leaving it
open. Similar transitions in
Fe-CO have been seen in
sperm whale Mb at acidic pH values and have been ascribed to an
open-closed transition of the heme pocket supported by the crystal
structure of the acid form of Mb (37, 46, 47). At an alkaline pH value
(pH 10.5), the
Fe-CO line at 534 cm
1 and
the
Fe-C-O at 586 cm
1 are completely lost
from the spectrum of barley HbCO, resulting in the sole appearance of
Fe-CO at 493 cm
1 (Fig. 4). This indicates
an open conformation of the heme pocket in which the hydrogen bonding
network becomes very weak or is completely abolished at strongly
alkaline pH values.
-electron back-donation from the d
(dxz, dyz) of Fe to the empty
*
orbitals of carbon monoxide, which results in an increase of the Fe-CO
bond order and a concomitant decrease in the C-O bond order (13, 16,
36, 49). The correlation between these two frequencies depends on the
nature of the proximal ligand, because the electron density in the
Fe-proximal ligand bond affects the Fe-CO bond order. Back-bonding to
CO is controlled by many factors such as steric crowding of the bound
CO, polarity of the neighboring environment, and hydrogen bonding of
the CO oxygen to an adjacent proton of a distal residue. At pH 7.4, two
C-O stretching frequencies (
C-O) were detected in barley
Hb: 1) a strong line at 1924 cm
1, and 2) a weak line at
~1960 cm
1 (Fig. 6,
spectrum a). With 13C18O, a line at
1836 cm
1 presumably corresponding to the mode at 1924 cm
1 was detected. Although the isotopically shifted line
corresponding to the line at ~1960 cm
1 could not be
detected (Fig. 6, spectra b and c), probably due to the higher noise level in the 13C18O
spectrum, we assign the ~1960 cm
1 line to the
C-O of a minor population of the CO conformer in which
the Fe-CO stretching mode is located at 493 cm
1. Whereas the
C-O line at ~1960 cm
1 is similar to the
C-O line in mammalian Mbs and Hbs with an open
conformation (1965 cm
1), the frequency of the
C-O line at 1924 cm
1 in barley Hb is
significantly lower than that in any state (A1,2, ~1946
cm
1; A3, ~1932 cm
1) of the
closed conformation in mammalian Mbs (37). Some vertebrate Hbs (lamprey
Hb, rabbit Hb) however, have a CO conformer with low frequency of the
C-O line (1927 cm
1 for lamprey Hb; 1928 cm
1 for rabbit Hb) (50). It was proposed that the low
frequency of the
C-O line in rabbit Hb is caused by the
proximity of the CD6 leucine to the distal histidine (50). To determine
whether the electronic interactions in barley Hb correspond to those in other Hbs and Mbs, we plotted
C-O against
Fe-CO (Fig. 7). Both frequencies fall on a correlation line that is characteristic for
hemeproteins that contain histidine as the proximal ligand, showing
that the bond order of Fe-CO is inversely related to that of C-O. The
Fe-CO mode at 534 cm
1 (
C-O
at 1924 cm
1) falls toward the left end of the correlation
line, close to the points for the peroxidases. However, as already
discussed, the frequencies of the Fe-CO and C-O modes in barley Hb
result from distal interactions, not the imidazolate character of the proximal ligand. Thus, for barley Hb, the low frequency of
C-O and the high frequency of
Fe-CO
result from a direct interaction of distal residues with the bound
CO.
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Fig. 6.
Resonance Raman spectra of barley HbCO in the
CO frequency region at pH 7.4 as a function of isotopic composition of
CO. a, 12C16O; b,
13C18O; c, difference spectrum
(12C16O-13C18O). The
laser power at the sample was ~1 mW.
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Fig. 7.
Correlation between Fe-CO
( Fe-CO) and C-O (
C-O) stretching
frequencies for various hemeproteins that have histidine as the
proximal ligand.
, stretching frequencies of globins;
,
those of peroxidases.
(indicated by vertical arrows),
the frequencies of CO barley Hb.
1, respectively) are proposed to arise from an
electrostatic effect of the strong positive polarity of a short
hydrogen bond between N
H of the distal histidine and CO
and are confirmed by isotopic substitution studies. At low pH values,
the distal histidine is believed to move out of the heme pocket due to
protonation, leaving an open heme pocket, just as in mammalian Mbs.
Existence of strong hydrogen bonding between the bound CO and the
distal histidine implies that a similar situation may exist in the oxy
complex of barley Hb. Such strong hydrogen bonding is expected to
impart a great stability to the bound ligand that is in fact manifested by the observation of a very low oxygen dissociation rate.
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ACKNOWLEDGEMENT |
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We thank Doug Durnin for skilled technical assistance.
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FOOTNOTES |
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* This work was supported by Natural Sciences and Engineering Research Council of Canada Grants OGP4689 and STR0149182 (to R. D. H.) and by National Institutes of Health Grants GM54806 and GM54812 (to D. L. R.) and GM40168 and RR02538 (to J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 718-430-4264; Fax: 718-430-4230; E-mail: rousseau{at}aecom.yu.edu.
The abbreviations used are: Hb, hemoglobin; HbCO, carbonmonoxy hemoglobin; Mb, myoglobin; EPR, electron paramagnetic resonance; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
2 The net rate of oxygen dissociation is calculated by multiplying koff by the concentration of Hb (~20 µM) in barley aleurone tissue.
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REFERENCES |
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