From the Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19194-6089
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ABSTRACT |
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-Nitrosyl hemoglobin,
(Fe-NO)2
(Fe)2, which is frequently
observed upon reaction of deoxy hemoglobin with limited quantities of
NO in vitro as well as in vivo, has been
synthetically prepared, and its reaction with O2 has been
investigation by EPR and thermodynamic equilibrium measurements.
-Nitrosyl hemoglobin is relatively stable under aerobic conditions
and undergoes reversible O2 binding at the heme sites of
its
-subunits. Its O2 binding is coupled to the
structural/functional transition between T- (low affinity extreme)
and R- (high affinity) states. This transition
is linked to the reversible cleavage of the heme Fe-proximal His bonds
in the
(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol
hexaphosphate. In fact,
(Fe-NO)2
(Fe)2
is exceptionally sensitive to protons, as it exhibits a highly enhanced
Bohr effect. The total Bohr effect of
-nitrosyl hemoglobin is
comparable to that of normal hemoglobin, despite the fact that the
oxygenation process involves only two ligation steps. All of these
structural and functional evidences have been further confirmed by
examining the reactivity of the sulfhydryl group of the
Cys
93 toward 4,4'-dipyridyl disulfide of several
-nitrosyl hemoglobin derivatives over a wide pH range, as a probe
for quaternary structure. Despite the halved O2-carrying
capacity,
-nitrosyl hemoglobin is fully functional (cooperative and
allosterically sensitive) and could represent a versatile low affinity
O2 carrier with improved features that could deliver
O2 to tissues effectively even after NO is
sequestered at the heme sites of the
-subunits. It is concluded that
the NO bound to the heme sites of the
-subunits of hemoglobin acts
as a negative allosteric effector of Hb and thus might play a role in
O2/CO2 transport in the blood under
physiological conditions.
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INTRODUCTION |
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When deoxy hemoglobin
(Hb)1 is exposed to
less-than-stoichiometric amounts of NO ([NO]/[heme] 0.5) in
solution (1-3) and in the erythrocytes (4-6), the predominant species
formed upon equilibrium are
-nitrosyl Hbs, i.e.
(Fe-NO)
(Fe)
(Fe)2 or
(Fe-NO)2
(Fe)2. Such compounds were
readily identified by their EPR spectra with a set of sharp triplet
14N hyperfine structures (Az = 17 Gauss) around gz = 2.009, which is derived from the
5-coordinate nitrosyl hemes in the
-subunits. When rats or mice had
been exposed to doses of lipopolyscaccaride, tumor necrosis factor,
nitroglycerin, nitrite, or NO (7-12), their plasma concentration of NO
was known to increase. Venous bloods from the treated animals
invariably exhibited EPR spectra with distinct triplet hyperfine
signals. Such EPR spectra cannot be expected from tetranitrosyl Hb,
(Fe-NO)2
(Fe-NO)2, in the absence of IHP
at a physiological pH of 7.4 (13). Therefore, it is obvious that the
primary nitrosyl products formed upon reaction of deoxy Hb with NO
under physiological conditions, where [NO]
[heme], are
-nitrosyl Hbs (6, 12, 14, 15). In order to assess physiological
roles of such compounds, we have investigated its O2
binding properties of
(Fe-NO)2
(Fe)2 by
EPR and O2 binding measurements as well as the reactivity
of Cys
93 toward 4-PDS as a probe for the quaternary
structure. We have found that its oxygenation characteristics and
allosteric functions make
(Fe-NO)2
(Fe)2 a
unique cooperative low affinity O2 carrier with full
allosteric sensitivity that could deliver O2 to tissues efficiently under physiological conditions. This study has also provided a new insight into the molecular mechanism of cooperativity and allostery in Hb, particularly the major role of the
-heme Fe-F
helix linkage in the quaternary structural transition, and the mode
interaction of Hb with NO.
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EXPERIMENTAL PROCEDURES |
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Reagents-- 2,3-Biphosphoglycerate, IHP, pMB, 4-PDS, Tris, bis-Tris, bis-Tris propane, dithiothreitol, catalase, superoxide dismutase (Sigma), and argon (grade 5 gas; BOC gases, Murray Hill, NJ) were used without further purification. Nitric oxide (99.00% pure; MG Industries, Malvern, PA) was purified by passing through a series of gas-bubble washing bottles containing 1 M NaOH and deoxygenated distilled water and another bottle containing deoxygenated distilled water. Purified NO gas was used under strict anaerobic conditions. Anaerobic conditions were obtained by removing O2 from the media with repeated evacuation and flushing with water-saturated argon gas or by continuous flushing with water-saturated argon gas over the surface of stirred reaction media. The use of dithionite as reductant was avoided as much as possible to prevent inducing unknown side reactions.
Preparation of
(Fe-NO)2
(Fe-O2)2--
All
preparation procedures were carried out at 4 °C. Freshly outdated
adult human Hb was obtained from a local branch of the American Red
Cross; it was purified according to the method of Drabkin (16) and
stripped from organic phosphates by the method of Berman et
al. (17). The Hb solution was stored in the CO form, and no
further attempt was made to strip Hb from its minor components.
Temperature Dependence of the Aerobic met Hb Formation of
Nitrosyl Hb Derivatives--
To determine the optimal temperature for
quantitative O2 equilibrium and EPR studies, the rates of
aerobic formation of met Hb for -nitrosyl Hb, tetranitrosyl Hb, and
oxy Hb samples were measured. The reaction was followed by the
absorbance increase at 630 nm of met Hb over time with a
Hewlett-Packard 8452A diode array spectrophotometer (Hewlett-Packard,
Palo Alto, CA). To avoid artifacts due to turbidity or water vapor
condensation at low temperatures, readings were corrected with a second
wavelength at 800 nm. Sample concentration was 60 µM heme
in 50 mM bis-Tris-propane containing 0.1 M
Cl
, pH 7.4. Data were collected every second for 20 min
and analyzed according to a first-order kinetic scheme.
Oxygen Equilibrium Measurements--
Oxygen equilibrium curves
were measured by an improved version of Imai's automatic method (19)
with the following modifications. Absorbance was monitored using a
computer-controlled Olis-Cary 118 spectrophotometer (Olis, Bogart, GA).
Oxygen concentrations were monitored with a low noise, high response
electrode (O2 Sensors, Gadwyne, PA), using a custom-made
amplifier (Biomedical Instrumentation Shop, University of Pennsylvania
Medical Center, Philadelphia, PA). The signal was then digitized using
a 12-bit A/D converter. Absorption changes were monitored at 560 nm.
Sample concentration was 120 µM heme in 50 mM
bis-Tris-propane buffer, containing 0.1 M
Cl, and small amounts of catalase and superoxide
dismutase. Measurements were carried out at 15 °C. Analyses of
oxygenation data were performed according to a two-step model,
corresponding to the third and fourth O2 bindings in Hb, as
reported previously (20).
Kinetic Studies of the Sulfhydryl Reactivity of
Cys93 of
-nitrosyl Hb Derivatives toward
4-PDS--
This method (21) was carried out as described previously
(22, 23), with the following modifications for a quantitative measurement. A standard solution of 4-PDS was prepared by dissolving approximately 10 mg in 10 ml of deoxygenated distilled water at 60 °C. The concentration of 4-PDS was calculated using an extinction coefficient of 16.3 mM
1 cm
1 at
247 nm and pH 7.0.
EPR Measurements--
Buffers used for EPR measurements were 0.1 M sodium acetate buffers, pH 4.8-5.8, and 0.1 M bis-Tris propane buffers, pH 6.0-pH 9.0, containing 0.1 M Cl. Oxygen binding equilibria for EPR
samples were obtained at 15 °C in a modified Imai cell (19), in
which O2 concentrations of samples were continuously
monitored by a sensitive O2 electrode (O2
Sensors). Aliquots of sample were taken at determined partial pressures
of O2 and anaerobically transferred into EPR tubes and immediately frozen by immersion into liquid nitrogen. EPR measurements were carried out with a Varian X-band EPR spectrometer, model E109
(Varian Associates, Palo Alto, CA), integrated with the
data-acquisition system (Scientific Software Services, Normal, IL). EPR
samples (300 µl of 500 µM heme) in quartz EPR tubes
(3-mm precision bore) were frozen by immersion into liquid nitrogen and
measured at liquid nitrogen temperature. The spectrometer was operated
at a microwave frequency of 9.11GHz, microwave power of 20mW,
modulation frequency of 100kHz, modulation amplitude of 2.0 Gauss,
magnetic field scan rate of 125 Gauss/min, and time constant of 0.2. Recorded EPR data were manipulated with the EPR software (Scientific
Software Services) for quantitative analyses and plotted using Origin
for Windows, Version 5.0 (Microcal, Northampton, MA).
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RESULTS |
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Temperature Dependence of the Met Hb Formation of Nitrosyl Hb
Derivatives--
Native oxy Hb,
(Fe-NO)2
(Fe-O2)2, and
tetranitrosyl Hb were slowly oxidized to respective met forms under
aerobic conditions. Both nitrosyl Hb derivatives were less stable than
native oxy Hb, and tetranitrosyl Hb was the least stable of the
species. Half-life times at 37 °C were 15 h, 2 h, and 41 min for oxy Hb,
(Fe-NO)2
(Fe-O2)2, and
tetranitrosyl Hb, respectively. However, these values increased to
42 h, 22 h, and 16 h, respectively, at 15 °C.
Accordingly, all experiments were conducted at this temperature.
Coordination States of Nitrosyl Hemes as Measured by
EPR--
Isolated (Fe-NO) and
(Fe-NO) subunits exhibited EPR
spectra around g = 2.0 of the 6-coordinate nitrosyl hemes at pH
7.4, as shown in Fig. 1A, in
agreement with previous reports (14, 15, 24). However, the overall line
shape of their EPR spectra was distinctly different from one to the
other: the isolated
(Fe-NO) subunits (Fig. 1A,
broken line) showed a more rhombically distorted line shape
than the isolated
(Fe-NO) subunits (dotted line). These
spectra were independent of pH in a range from pH 6.0 to pH 9.0. The
EPR spectrum of tetranitrosyl Hb (solid line) was essentially a sum of those of the isolated
(Fe-NO) and
(Fe-NO) subunits, as previously reported (14, 15, 24) and did not change
significantly over a pH range from 7.0 to 9.0. The EPR spectrum of the
-deoxy,
-nitrosyl hybrid,
(Fe)2
(Fe-NO)2 was reported to be
practically identical with that of isolated
(Fe-NO) subunits
(spectrum B in Fig. 1A) (14, 24) and was pH-independent over
a pH range from 6.0 to 9.0.
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Oxygen Binding Characteristics of
(Fe-NO)2
(Fe-O2)2--
Results
obtained from spectrophotometric O2 equilibrium
measurements are shown in Fig. 3. At pH
5.8 (Fig. 3A),
-nitrosyl Hb showed a strikingly
diminished O2 affinity, virtually absent cooperativity, and
decreased effect of BPG and IHP. In the absence of organic phosphates,
the lower asymptote of the Hill plot for
-nitrosyl Hb matched that
for Hb, indicating that despite
-nitrosyl Hb having both the
-subunits ligated with NO, the O2 affinity of the
complementary
-subunits remained as low as at initial ligation
stages of native Hb. In the presence of IHP, the lower asymptote for
the
-nitrosyl Hb was also similar to that of Hb under the same
conditions. On the other hand, at pH 8.2 (Fig. 3C), the
upper asymptote of the curve for
-nitrosyl Hb approached that for
Hb. This indicates that the affinity for O2 of this
derivative increased with pH, while showing a trend of being comparable
but not completely equaling the O2 affinity at the last
oxygenation steps of Hb under similar conditions. In other words,
-nitrosyl Hb at this pH exhibited characteristics of a high affinity
species. Inositol hexaphosphate, as well as BPG, to a lesser degree,
had the effect on this derivative of shifting the curve to the right. Cooperativity was present and comparable to that for a Hb species with
two binding sites. At pH 7.4 (Fig. 3B), the oxygenation
curve for
-nitrosyl Hb shifted toward the upper asymptote of that
for Hb, that is, the high affinity side. However, BPG and IHP decreased its O2 affinity by shifting the curve toward the low
affinity side.
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Reactivity of the Sulfhydryl Group of Cys93 as a
Probe for the Quaternary Structure--
In the presence of excess of
4-PDS, reactions were first-order with respect to concentrations of Hb
derivatives. The apparent rate constants (kapp)
for both oxy and deoxy Hb increased monotonically above pH 7. Moreover,
rate constants for oxy Hb were about 10 times those corresponding to
deoxy Hb at any pH studied (not shown). To ease comparison, the
relative reactivity of a derivative at any pH was normalized with
respect to rate constants for deoxy Hb corresponding to the same pH, as
shown in Fig. 6. Thus, rate constants for
deoxy Hb were equal to 1, and those for oxy Hb were approximately 10. In the same figure, reactivity profiles for various derivatives of
-nitrosyl Hb were included.
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DISCUSSION |
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Stability of -Nitrosyl Hb--
Nitrosyl derivatives of Hb are
less stable than native Hb under aerobic conditions. Their oxidative
decomposition leads to the formation of fully and partially met Hbs and
NO3
.
-Nitrosyl Hb
was converted to met Hb more rapidly than native Hb but more slowly
than tetranitrosyl Hb. Moreover, the rate for all derivatives
diminished as the temperature was reduced. At 15 °C, the half-life
time for
-nitrosyl Hb was 16 h; this time span was reasonably
long enough for all experiments to be conducted on this derivative
without the concern of formation of significant amounts of met Hb. This
rather moderate stability of
-nitrosyl Hb derivative upon exposure
to air might conflict with common expectation. We have observed,
however, that
-nitrosyl Hb formed within intact and fully functional
erythrocytes was much more rapidly decomposed at 37 °C
(t1/2
21 min) than in solution
conditions.2 This could
explain why this derivative has been seldom found in vivo
under normal conditions, but only observed when large amounts of NO
were produced in the blood, such as in shock, in inflammation, or upon
administration of cytokines, nitrite, and organic nitrates (7-12).
Under such conditions, steady-state concentrations of
-nitrosyl
hemes so formed could reach several percent of the total hemes of Hb,
which corresponds to steady-state intra-erythrocyte concentrations of
over 400 µM
-nitrosyl heme. This indicates an active
role of Hb in removing NO from the plasma and
sequestering/concentrating NO as
-nitrosyl hemes, as plasma
concentrations of NO range only from ~10
7 to
~10
5 M.
The Action of NO on the -Hemes of Hb--
The tenet of the
cooperative mechanism of Hb is that the quaternary structural
equilibrium of deoxy Hb in the T (low affinity) state (1<
L <
) reversibly shifts toward the R-
(high affinity) state upon successive binding of four ligands to its
heme groups (30, 31), as schematically illustrated in Fig.
7. (L is an allosteric
parameter defined as [To]/[Ro] or a ratio
of molar concentrations of deoxy Hb in the T- and R-states,
respectively (31).) The stronger the affinity of the ligand, the more
effective is the shift of the equilibrium toward the R state. Such a
homotropic (or positively allosteric) property of ligand has been
unequivocally proven for diatomic ligands like O2 and CO.
Low concentrations of CO, which has more than 10-fold higher affinity
than O2, effectively compete with O2 for the
hemes of deoxy Hb under physiological conditions. In addition, its
binding shifts the quaternary structure of Hb toward the R- (high
affinity) state, so that partial binding of CO renders Hb ineffective
in its delivery of O2 to tissues, which is the primary
cause of CO poisoning. The United States Environmental Protection
Agency sets the Federal occupational limits of CO to be 8 and 35 ppm
for 9-h and 1-h inhalation, respectively (32). Such a homotropic
behavior has been generally assumed for NO, another diatomic ligand,
which has an extremely high affinity of ~10
12
M for deoxy Hb (33). Thus NO has more than
103-fold stronger affinity for deoxy Hb than CO. However,
several doses of 1-h inhalation of ~80 ppm NO have been successfully
prescribed for clinical treatments of persistent pulmonary hypertension
for the newborn and other pulmonary distress syndrome for the adult with no apparent adverse effect (34). Therefore, one must wonder why
there have been neither observation nor report of NO poisoning.
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Effect of Cleavage/Elongation of the -Heme
F Helix Linkage on
the Quaternary Structure of Hb--
Several years ago, we demonstrated
that the weakening/cleavage of the
-heme-His (F8) bonds causes the
permanent shift of the quaternary structure of Hb to a T- (low affinity
extreme) state (43), where L =
(31), as exemplified
by
(porphyrin)2
(Fe)2 (43),
HbMIwate(His
87
Tyr) (44), and
HbMBoston (His
58
Tyr) (45). The recently
reported recombinant Hb (His
87
Gly) (46) probably
belongs to this category. The
(Ni)2
(Fe)2 hybrid, the
(Ni)-subunits of which are predominantly in a
4-coordinate state, having Ni bonding with neither a distal ligand nor
the His
87 (F8) residue (47), shows similar structural
and functional characteristics
(47).3 Therefore, it can also
be considered to belong to this category as well.3 These
artificial and natural hybrid Hbs exhibit the lowest O2 affinity attainable for the
-subunits of Hb with no or substantially diminished cooperativity and allosteric responses toward proton and
organic phosphates, characteristics associated with the T- (low
affinity extreme) state with L =
(Fig. 7). The
common structural denominator among these hybrid Hbs is the cleaved
-heme Fe-proximal bonds or an extended linkage between the
-heme
Fe and the
-carbon of the F8 residue (43). This permanent quaternary
structural shift is surprisingly independent of the nature/oxidation
state of the porphyrin metal ion, the presence and absence of the
porphyrin metal ion, and/or the presence and absence of distal
ligation. It solely depends upon the geometry (distance/orientation) of the proximal linkage between the
-porphyrin metal and F helix. This
might imply that the F helices in the
-subunits of Hb have an
inherent tendency to move away from the
-heme planes. Thus, if the
-heme-F helix linkage is removed, Hb would shift its quaternary structure to the energetically more stable T- (low affinity extreme) state with L =
. The presence of this linkage might
be actually constraining and preventing Hb to settle into such a stable
state with L =
(in nonequilibrium) and keeping
deoxy Hb at the energetically more dynamic T- (low affinity) state with
1 < L <
(in equilibrium), which can readily
and reversibly shift to the R- (high affinity) state in response to
ligand binding and/or to interaction with allosteric effectors.
Effect of Ligation of NO at the -Hemes on the Tertiary and
Quaternary Structural Equilibria and O2 Binding
Characteristics of Hb--
It is, therefore, obvious from the
preceding discussion that the NO-induced cleavage of the
-heme
Fe-His (F8) bonds would transform
(Fe-NO)2
(Fe)2 into a T- (low
affinity extreme) state, despite ligation of two molecules of a high
affinity ligand (NO) per tetramer. In other words, two NO molecules
bound to the
-hemes act as a negative allosteric ligand rather than
as a homotropic (or positive allosteric) ligand by breaking the
-heme Fe-His (F8) bonds and thus, shifting the quaternary structural
equilibrium from the T- (low affinity) state with 1 < L <
(in equilibrium) toward the far end of the
T-state, i.e. the T- (low affinity extreme) state with
L
(in nonequilibrium), so that the
O2 affinity in the
-subunits of
(Fe-NO)2
(Fe)2 is substantially reduced. In the above-mentioned hybrids,
(porphyrin)2
(Fe)2,
HbMIwate(His
87
Tyr),
HbMBoston (His
58
Tyr), and recombinant
Hb (His
87
Gly), the
-heme Fe-F helix linkages
are irreversibly either cleaved or elongated by chemical modification
or mutation. Therefore, their quaternary structure is permanently
locked into the T- (low affinity extreme) state with L =
(in nonequilibrium), and thus their O2 binding
process is not only of low affinity but also noncooperative and
allosterically insensitive (Fig. 7). On the other hand, the cleavage of
the
-heme Fe-His (F8) bonds in
(Fe-NO)2
(Fe)2 is reversible. Varying
degrees of re-formation of the
-heme Fe-His (F8) bonds upon ligation
in the
-subunits, in the absence of IHP, and/or at higher pH (Fig.
2) represent the transition of the quaternary structural equilibrium
toward the R- (high affinity) state (Fig. 7). Thus, it is
evident that the reversible, two-step O2 binding to the
-subunits of
(Fe-NO)2
(Fe)2 is
accompanied by the T- (low affinity extreme)
R (high affinity)
quaternary transition and is a cooperative and allosterically sensitive
process (Fig. 7).
The Enhanced Bohr Effect in -Nitrosyl Hb--
The enhanced Bohr
effect, namely, an increased pH effect on the O2 affinity
of the
-subunits observed in
-nitrosyl Hb (Fig. 4) is striking,
where only the two-step ligation was involved. Its Bohr coefficient,
H+, around physiological pH of 7.4 in the absence of
organic phosphates (Fig. 4) is almost double that for native Hb, which
involves the four-step ligation. This indicates that the
ligation-linked Bohr groups of
-nitrosyl Hb ionize more easily than
in native Hb, or that additional groups such as His
87
are involved in the process. In the presence of IHP, the alkaline Bohr
effect was similar to that in the absence of IHP, except that the curve
was shifted toward the alkaline side about 1.5 pH units. Comparable
features were also detected in EPR experiments. Because the top and
bottom on each bar (Fig. 2, open and closed rectangles, respectively) are roughly related to
K3 and K4, respectively, estimated from oxygenation experiments, a midpoint between these two
extremes (Fig. 2, open and closed circles) could
be a convenient way to indicate qualitatively the O2
affinity. It should be remarked here that there is no attempt to imply
any strict correlation between these parameters. Note that the curve
connecting the midpoints does not follow a linear dependence over pH.
The shape of this curve reflects how the ligation-linked allosteric
transition takes place in the
-nitrosyl Hb by varying pH and
indirectly reflects its Bohr effect. Under acidic conditions, the
enhanced effect of protons on the trans-axial bond cleavage in the
-subunits is such that the molecule shifts to the extreme side of
the T- (low affinity) conformation, i.e. the T- (low
affinity extreme) state with L
(Fig. 7). This makes
its quaternary structure and functions virtually insensitive to any
allosteric effectors (such as homotropic ligation in the
-subunits
and/or negatively allosteric organic phosphates). Under alkaline
conditions, where the
-heme Fe-His (F8) bond is partially restored,
the cooperativity between two
-subunits seems to be re-established.
It becomes then evident that the overall O2 affinity in Hb
is closely related to the coordination equilibrium of the
-heme
Fe-His (F8) bonds in
-nitrosyl Hb and consequently to its
quaternary state. For
-nitrosyl Hb, all allosteric effectors studied
in the present work have proved to trigger this transition. The
breakage of the
-heme Fe-His (F8) bonds implies the ionization of
the His
87 side chains; this could cause a change in pK
values of other Bohr groups, or rather be the consequence of these
alterations. More stretched/tilted or even absent
-heme Fe-His (F8)
bonds promote the transition of the molecule to T-states, either (low affinity) or (low affinity extreme) (43). Earlier resonance Raman
studies (42, 49) suggested that that the protein control of
O2 binding in Hb is regulated by the state of the heme
Fe-proximal His bonds without specifying the identity of the subunits
involved.
The Mode of Reaction of Hb with NO--
The fact that NO reacts
with the -hemes of Hb as a negative allosteric ligand rather than as
a high affinity, homotropic ligand explains the previously unexplained
puzzle that although
(Fe-NO)2
(Fe)2 is in
the T- (low affinity extreme) state, the NO in the
(Fe-NO) subunits
is very tightly bound, because it is in a 5-coordinate state (Table I).
Nitric oxide binds to the
-hemes of Hb as a conventional homotropic
ligand in a 6-coordinate state, so that it is released readily from the
(Fe-NO) subunits of Hb in the T- (low affinity) state, as the
two-state model predicts under acidic conditions. In other words, the
interaction of two molecules of NO with the
-hemes of
(Fe-NO)2
(Fe)2 involves a quaternary
structural transition between T- (low affinity extreme) and R- (high
affinity) states and thus conforms quite well to a two-state model
under acidic conditions. However, a simple two-state model cannot
adequately explain the four-step reaction of deoxy Hb with NO at acidic
and/or neutral pH, because the first two molecules of NO act as a
negative allosteric ligand and the subsequent two molecules of NO
behave as a positive allosteric ligand. Therefore, NO present in low
concentrations in the blood would react with deoxy Hb only as a
negative allosteric effector. Thus, NO is not detrimental to the
physiological functions of Hb as an O2/CO2 transporter in the blood, although the O2-carrying capacity
of Hb would decline as much as 50% by ligation of NO at the
-heme sites.
Physiological Scavenging of NO by Hb--
The NO present in the
blood is synthesized primarily by NO synthases, particularly those in
the endothelial cells of blood vessels throughout the circulatory
system upon local chemical and physical stimulation. It activates
soluble guanylyl cyclases in adjacent cells as a paracrine signal
transducer. Excess NO so produced in the rapidly moving blood must be
scavenged as quickly as possible in order to prevent its action at
unintended locations elsewhere downstream. Low concentrations of NO in
the blood (~107 M < [NO] < ~10
5 M) can be effectively
(KD < 10
12 M) and rapidly
(kon = 107
M
1 s
1) sequestered by Hb
through eventual coordination at its
-hemes. The
(Fe-NO)2
(Fe)2 thus formed becomes a
cooperative, low affinity O2 carrier that can deliver
O2 to tissues as efficiently as native Hb under
physiological conditions, although it can carry only two molecules of
O2 per tetramer. In contrast, the partially CO-bound Hb,
which enters a higher-affinity state, is less effective in delivery of
O2 to tissues. The
-nitrosyl hemes in
(Fe-NO)2
(Fe)2 are eventually oxidized by
O2 to met hemes and
NO3
ions at reasonable rates
(t1/2
21 min in erythrocytes at
37 °C).2 The partially met Hb thus formed will be
effectively reduced to deoxy Hb by Hb reductase in the erythrocytes to
complete the process of NO scavenging. We have shown that during the
scavenging of NO through binding at the
-subunits, Hb transforms
itself into
-nitrosyl Hb, a cooperative, low affinity O2
carrier. Thus, the ability of effective O2 delivery to
tissues of Hb would not be impaired in the presence of low
concentrations of NO. To put simply, Hb will not be poisoned in the
presence of low concentrations of NO. This may explain in part why NO
causes no acute adverse effect on newborn infants during clinical
treatments with inhaled NO (34), even though NO has a substantially
higher (>103-fold) affinity for Hb than CO. Thus, we find
Hb to be much more agile than we have previously assumed. Hemoglobin
can function simultaneously as a NO sequestering agent as well as an
efficient O2 carrier in the hostile environment of the
blood, where NO, a high affinity ligand, is always present in low
concentrations.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL14508 and GM48130.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: B605 Richards Bldg.
6089, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19194-6089. Tel.: 215-898-8787; Fax: 215-898-8559; E-mail: yonetant{at}mail.med.upenn.edu.
§ Present address: Dept. of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China.
The abbreviations used are:
Hb, hemoglobin; IHP, inositol hexaphosphate; BPG, 2,3-biphosphoglycerate; pMB, p-hydroxymercuribenzoate4-PDS, 4,4'-dipyridyl disulfide,
-subunit
,
-subunits(Fe), deoxy heme(porphyrin), protoporphyrin IX(Ni), nickel protoporphyrin IX(Fe-NO), nitrosyl
heme(Fe-O2), oxy heme, and (Fe-CO), carbonmonoxy hemeEPR, electron paramagnetic resonance.
2 T. Yonetani, A. Tsuneshige, Y. Zhou, and X. Chen, unpublished results.
3 A. Tsuneshige and T. Yonetani, unpublished results.
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