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J. Biol. Chem., Vol. 275, Issue 22, 16738-16745, June 2, 2000
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,
, and§**
From the § Howard Hughes Medical Institute, the
Department of Medicine, and the ¶ Nicholas School
for the Environment, Duke University Medical Center, Durham, North
Carolina 27710 and the Department of Chemistry, Montana State
University, Bozeman, Montana 59717
Received for publication, January 20, 2000, and in revised form, March 20, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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S-Nitrosohemoglobin (SNO-Hb) is a
vasodilator whose activity is allosterically modulated by oxygen
("thermodyamic linkage"). Blood vessel contractions are favored in
the oxygenated structure, and vasorelaxant activity is "linked" to
deoxygenation, as illustrated herein. We further show that
transnitrosation reactions between SNO-Hb and ambient thiols transduce
the NO-related bioactivity, whereas NO itself is inactive. One
remaining problem is that the amounts of SNO-Hb present in
vivo are so large as to be incompatible with life were all the
S-nitrosothiols transformed into bioactive equivalents
during each arterial-venous cycle. Experiments were therefore
undertaken to address how SNO-Hb conserves its NO-related activity. Our
studies show that 1) increased O2 affinity of SNO-Hb (which
otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with
ambient thiols for vasorelaxation; 2) some NO groups released from
Cys The NO group can bind to the hemes and cysteine residues of
hemoglobins of microbial, invertebrate, and mammalian origin, forming
alternatively Hb(Fe(II))NO1
and SNO-Hb, respectively (1-4). Iron nitrosylhemoglobin and SNO-Hb are
in a dynamic, redox-dependent equilibrium (1-4) (Equation 1).
93 upon transition to T structure are autocaptured by
the hemes, even in the presence of glutathione; and 3) an
O2-dependent equilibrium between SNO-Hb and
iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a
significant fraction of NO liberated upon transition to T structure, Hb
can conserve NO groups that would otherwise be released in an untimely
or deleterious manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
In mammalian hemoglobin, the position of this equilibrium is
linked to the protein conformation: SNO-Hb formation is favored in the
R (oxy, low spin) structure, and iron nitrosylhemoglobin is formed
preferentially in the T (deoxy, high spin) structure (1, 2, 5-9).
Conversely, NO group release from thiols of Hb is promoted by
deoxygenation and by heme oxidation (T structure, high spin) (1, 5,
10), in keeping with thermodynamic linkage relationships (11-14).
SNO-Hb is also in equilibrium with low mass SNOs (1) such as GSNO
(Equation 2). High concentrations of GSH thus shift the equilibrium in
favor of the deoxy structure (1, 7); where Equation 4 is the effective
sum of Equations 2 and 3.
(Eq. 1)
(Eq. 2)
These conclusions as well as their physiological relevance are
strongly supported by a series of recent findings. 1) SNO-Hb and
Hb(Fe(II))NO have been detected in arterial and venous blood of 41 rats
(1, 5), 19 human fetuses in utero (15), and 7 patients with
sickle cell disease (16). The measurements show that the position of NO
binding in hemoglobin is dependent on both pO2
and location within the circulation. In particular, NO is redistributed
among the hemes and thiols of hemoglobin as it transits the lung,
placenta, and peripheral vascular bed. 2) An umbilical arterial-venous
difference in the amount of low mass SNOs was detected in term infants,
in keeping with an equilibrium between GSNO and SNO-Hb (17). 3)
Kinetic, mass spectroscopic, and crystallographic data obtained in
multiple laboratories (1, 16, 18-23) have unequivocally identified
Cys
(Eq. 3)
93 as a site of NO binding to hemoglobin.
We have further suggested that the equilibria between SNO-Hb and iron nitrosylhemoglobin, on the one hand (Equation 1), and GSNO, on the other hand (Equation 4), have important functional implications for the regulation of blood flow and platelet activity (5, 10). In support of this contention, we have shown both in vitro and in vivo that SNO-Hb dilates blood vessels (1, 5) and inhibits platelets (10) under conditions that promote its T structure, but not under conditions that promote its R structure. Moreover, we have shown that SNO-Hb bioactivity is promoted by low pO2, thiols, and heme oxidation (1, 5, 10). The vasodilator and antiplatelet activities of SNO-Hb and promoting role of exogenous thiols have recently been confirmed by others (23, 24). We have also demonstrated that SNO-Hb can sense the physiological O2 gradient in resistance vessels (small arterioles that control blood flow), thereby coupling the release of vasodilator SNO with regional metabolic needs of the tissue (5). Additional work that supports the importance of SNO-Hb includes the myriad studies of the systemic effects of inhaled NO (25-29), which raises the levels of endogenous SNO-Hb (16). It is tempting to suggest, moreover, that the hypoxia-increased tissue levels of NO in kidneys of animals that had been treated with NO synthase inhibitors (30) implicate SNO-Hb as the source of the elevated NO.
The levels of SNO-Hb in arterial blood are ~1 µM. Release of such an amount of SNO (from Hb) during each arterial-venous cycle would not, however, be compatible with life or NO biology for two reasons. First, it would cause life-threatening hypotension and shunting of blood. This effect can be understood by appreciating that regulation of blood flow, which is a function of arteriolar diameter to the fourth power (31), requires only low nanomolar SNO (1, 5, 32-34). Second, it would impose an insurmountable metabolic burden on an organism that produces less NO, in toto (35), than the amount that would be turned over by Hb alone (6). Only a small fraction (0.1-1%) of SNO bound to hemoglobin is involved in signaling during arterial-venous transit to regulate blood flow (1, 5). The NO unit is thus quantitatively and functionally dissimilar from O2. Whereas millimolar O2 is delivered by Hb to meet basal metabolic requirements, NO/SNO functions as a signal that is dispensed at nanomolar concentrations. The fundamental challenge to our understanding of SNO-Hb function is therefore not whether enough SNO can be released to dilate blood vessels, as recently questioned by some (23, 36, 37), but rather how red blood cells are able to conserve the vast majority of SNO when their Hb is routinely exposed to hypoxic tissue and to ambient intracellular glutathione. We have previously suggested that part of the solution lies in the autocapture by hemes of the NO released from the thiols in Hb (1, 2, 6), i.e. Hb conserves its NO.
In this study, we report on the functional behavior of
(SNO)2-Hb((Fe(II))O2)4 (here called
SNO-Hb) and on the allosteric mechanisms that regulate NO group release
from this molecule. We show that 1) NO group release from SNO-Hb is
favored in the T structure, thus conforming to principles of
thermodynamic linkage; 2) as with other S-nitrosothiols,
vasorelaxation by SNO-Hb is not mediated by free NO (as recently
misapprehended (23)); and 3) by stabilizing thiol-bound NO in the R
structure and autocapturing a significant fraction of NO released upon
transition to T structure, Hb can conserve NO that would otherwise be
released in an untimely, nonspecific, deleterious, or prodigal manner.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Reagents and Hemoglobin Solutions-- Chemical reagents were purchased from Sigma, except where otherwise stated. Human hemoglobin (HbA0; Apex Bioscience, NC) was purified (>99%) from outdated human blood as described previously (1, 7). The buffer used in the final chromatographic purification process was lactated Ringer's solution, pH 7.40. Unless otherwise stated, working solutions of HbA0 were prepared by rapid centrifugation over a Sephadex G-25 chromatography column (20-fold volume excess) equilibrated with PBS, pH 7.40, containing 0.5 mM EDTA.
Synthesis and Measurement of S-Nitrosohemoglobin--
Hb was
dialyzed against 2% aerated borate, pH 9.20, containing 0.5 mM EDTA. S-Nitrosocysteine (0.5 M)
was prepared immediately before use by reacting 1 M
NaNO2 in H2O with 1 M
L-Cys hydrochloride dissolved in 0.5 N HCl and
0.5 mM EDTA. S-Nitrosocysteine was partially
neutralized prior to addition to the hemoglobin solution by dilution in
PBS, pH 8.0, containing 0.5 mM EDTA (7). Hb was
S-nitrosylated by incubation at room temperature with
10-fold molar excess S-nitrosocysteine; the extent of
S-nitrosylation was determined by the duration of
incubation. The reaction was stopped by rapid transfer of the mixture
to a Sephadex G-25 column equilibrated with PBS, pH 7.40, containing
0.5 mM EDTA. Human HbA0 contains three pairs of
cysteine residues: Cys93, Cys112, and
Cys
104. Mass spectroscopic analysis of the product
formed under study conditions revealed that only Cys93
is nitrosylated (data not shown) (16, 19). SNO-Hb samples were made
fresh on the day of an experiment, kept on ice, and protected from
light. The SNO yield was determined by the Saville method in aliquots
from SNO-Hb solutions used for functional studies (1). SNO content is
expressed as a ratio normalized to the spectrophotometrically
determined tetrameric Hb concentration. Tetrameric Hb or SNO-Hb
concentration was adjusted to 15 µM by dilution in PBS,
pH 7.40, except where otherwise stated. Some binding study results were
confirmed at 50 µM in order to check for effects of
dimer/tetramer equilibrium (38, 39).
Equilibrium O2-binding Studies-- SNO-Hb or Hb(Fe(II))O2 was diluted as described above to 15 or 50 µM final tetrameric concentration and added to a sealed glass tonometer fitted with a cuvette. A base-line visible (500-700 nm) spectrophotometric scan in the oxygenated state was obtained (Perkin-Elmer, Beaconsfield, United Kingdom). Rigorous deoxygenation was accomplished through several cycles of alternately flushing with ultra-high purity argon gas and applying a measured vacuum. The tonometer was mechanically rotated for 7 min in a 20 °C water bath, and a spectrum was obtained. A second round of deoxygenation was performed to verify the completeness of conversion to deoxy-Hb and its stability. An airtight syringe (Hamilton, Ontario, Canada) was then used to incrementally deliver measured volumes of room air into the tonometer through a rubber septum. Following each injection, the tonometer was rotated in a temperature-controlled (20 °C) water bath for 7 min to allow equilibration between the head space (~300 ml) and the Hb solution (3 ml), and then a visible spectrum was obtained. Injections of air were repeated until a fully oxygenated spectrum was observed.
The tonometer pO2 resulting with each injection
was calculated, taking into account the cumulative volume injected, the
tonometer volume, the measured barometric pressure, the ambient room
temperature, the water bath (tonometer) temperature, and the relative
humidity. The fractional O2 saturation at each step was
calculated based on a mean of the change in absorbance at each of three
characteristic wavelengths (542, 555, and 577 nm) relative to the
respective absorbances of the fully oxygenated and fully deoxygenated
species studied in the first two and the last steps of the experiment. Fractional O2 saturation (Y) or
log[Y/(1 
Y)] (Hill plot) was plotted as
a function of the logarithm of pO2, except where
otherwise stated.
Analysis of MetHb and Hb(Fe(II))NO Content-- Spectral decomposition procedures using extinction coefficient spectra obtained from standard synthetic solutions of pure Hb(Fe(II))NO, MetHb, Hb(Fe(II))O2, and deoxy-Hb were used to determine the fractional content of MetHb and Hb(Fe(II))NO (6). In addition, Hb(Fe(II))NO and SNO-Hb were measured by the photolysis-chemiluminescence method (7).
Bohr Effect in SNO-Hb-- SNO-Hb and Hb solutions at varying pH were prepared by dilution in PBS solutions pre-titrated to the specified pH with either NaOH or HCl. In the case of the acidification of PBS to achieve pH 6.80, the added concentration of chloride ion (an allosteric effector) was <1.0% of the total anion concentration of PBS maintained at pH 7.40.
Allosteric Effects of Organic and Inorganic Phosphates--
Hb
or SNO-Hb prepared as described above was diluted in PBS solutions (pH
7.40) of monobasic and dibasic sodium phosphate calculated to yield a
final PO43
concentration of either 10 or 100 mM. The change in anion concentration (specifically
[Cl]) with pH adjustment of these solutions was
estimated to be negligible. Inositol hexaphosphate (150 µM), 2,3-diphosphoglycerate (150 µM), or
-NADPH (Calbiochem) was dissolved in Hepes and adjusted to the
specified pH before the addition of Hb/SNO-Hb.
Transnitrosation and O2 Affinity: Influence of GSH on O2 Equilibria of SNO-Hb-- The O2 equilibria of SNO-Hb (1.8 SNO/tetramer) were compared in the presence and absence of varying concentrations of GSH to test the ability of a thiol acceptor to facilitate denitrosylation of SNO-Hb and, consequently, the offloading of O2 as a function of the O2-linked allosteric transition in Hb. In experiments in which GSH was present, the oxidation of hemes routinely observed with the oxygenation-deoxygenation process was accelerated. The formation of MetHb was minimized by using purified stroma-free Hb (PSF-Hb), a partially purified hemolysate containing physiological levels of catalase, superoxide dismutase, methemoglobin reductase, and HbA0. The hemolysates were stripped of phosphates and studied in Hepes containing 5 mM NADPH (naturally present in the red blood cell), included to provide reducing equivalents, but also functioning as an allosteric effector of the organic phosphate class (40, 41).
Bioactivity Assays--
Thoracic aortic rings were harvested
from New Zealand White rabbits as described previously (5). The rings
were mounted on stirrups in tissue baths filled with Krebs-Henseleit
buffer, pH 7.40 (no chelating agent), bubbled with either 21%
O2, 5% CO2, and balance N2
("normoxia") or 5% CO2 and balance N2
("hypoxia"; <1% O2), and maintained at 37 °C. This
buffer contains ~125 mM Cl and ~1
mM PO43. Changes in
isometric tension were measured and recorded with Statham pressure
transducers and a Grass polygraph. Phenylephrine was added to the
tissue baths in concentrations sufficient to elicit an active basal
tension of ~2 g. Tension was equally maintained in normoxia and
hypoxia by adjusting the dose of phenylephrine. Under typical bioassay
conditions (<50 µM Hb, 95% O2), tetrameric hemoglobin will dissociate into dimers, a fundamental problem that NO
pharmacologists have not previously considered. Because dimers do not
transition to T structure, the allosteric effect of hypoxia cannot be
tested. To adequately limit dimerization with dilute solutions of
cell-free Hb in bioassays (38), we have found it necessary to work at
1% O2, a level much lower than that used by Wolzt
et al. (23) to assess the allosteric transition. The
functional integrity of the endothelium was confirmed in each vessel
ring studied by demonstrating vasodilator responses to acetylcholine.
In some experiments, reduced GSH dissolved in PBS, pH 7.40, was added
to tissue baths 15 min prior to the administration of SNO-Hb. Responses
to SNO-Hb are expressed as the percent change from the initial tension
achieved with phenylephrine. Statistical testing for differences was by
analysis of variance for repeated measures or by the paired
t test, with p < 0.05 accepted as the criterion for statistical significance.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Oxygen Affinity of SNO-Hb: Influence of SNO Content--
The
influence of the extent of S-nitrosylation of Hb on
O2 affinity in SNO-Hb was studied in O2-binding
experiments using solutions of Hb S-nitrosylated to varying
degrees. As outlined above, the extent of Hb S-nitrosylation
was varied by arresting the incubation of Hb with
S-nitrosocysteine at predetermined points in time. The
O2-binding curve for SNO-Hb was shifted leftward relative
to that of HbA0 (Fig.
1A). When expressed in the
form of the Hill plot (log pO2 versus
log[Y/(1 Y)], where Y is the fractional O2 saturation), it becomes clear that the
leftward shift for SNO-Hb is asymmetric in that differences in affinity are most pronounced at low pO2 values (Fig.
1B). Accordingly, the n50 (the slope
of the Hill plot at half-maximal O2 saturation, a measure
of cooperativity) is lower in SNO-Hb than in HbA0 (Table I). Oxygen affinity rises (and
P50 decreases) as a function of SNO content
across the full range from 0 to (the maximal) 2 SNO groups/Hb tetramer
(Fig. 1C), in contrast to the findings of Patel et
al. (36) and in agreement with those of Bonaventura et
al. (42). The relationship between O2 affinity
(defined as P50) and SNO content assumes a
weakly parabolic, nearly linear shape (Fig. 1C).
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Bohr Effect in SNO-Hb--
The effect of varying pH on
O2-binding equilibria was compared in Hb and SNO-Hb (SNO/Hb
ratio = 1.72 ± 0.05) in PBS. Progressive increases in pH in
the range from 6.80 to 8.00 shifted the O2-binding curves
for both HbA0 and SNO-Hb leftward to similar degrees (Fig. 2). At each pH studied, the
O2 affinity of S-nitrosylated Hb was greater
than that of unmodified HbA0, although at pH 8.0, the difference between the two molecules was diminished (Fig.
2C). The magnitude of the pH dependence of O2
affinity, expressed as the slope of log P50
versus pH, was similar for the two molecules (Fig.
2C).
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Comparative Allosteric Effects of Organic and Inorganic Phosphates
on Hb and SNO-Hb--
Phosphate anion concentration has been recently
shown to dramatically affect the propensity for NO addition
versus oxidation in reactions with hemoglobin, and an
allosteric mechanism was proposed (6). Increasing the concentration of
phosphate from 10 to 100 mM (in sodium phosphate buffers)
indeed shifted the O2-binding curves to the right in Hb and
to a similar extent in SNO-Hb (Fig.
3A), i.e. both are
similarly responsive to allosteric effectors. The
n50 was increased slightly in SNO-Hb by organic and inorganic phosphate buffers, whereas it was decreased slightly in
some instances in native Hb (Table I). In the presence of the
organic phosphates 2,3-diphosphoglycerate and inositol hexaphosphate, the P50 values for Hb and SNO-Hb (in 0.1 M Hepes) were raised to similar extents as a function of pH
(Fig. 3B).
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Transnitrosation, Iron Nitrosyl Formation, and O2
Affinity: Influence of GSH on O2 Equilibria of
SNO-Hb--
The O2-binding curve for
S-nitrosylated PSF-Hb was similar in character to that of
S-nitrosylated HbA0 (Fig.
4A) and was shifted to the
left relative to that of unmodified PSF-Hb (data not shown). O2-binding curves for S-nitrosylated PSF-Hb and
PSF-Hb were both shifted rightward in the presence of NADPH (data not
shown). The 
log P50 between
S-nitrosylated and unmodified PSF-Hbs was similar to that
between S-nitrosylated HbA0 and unmodified
HbA0. When 1.5 mM GSH was also present during
the deoxygenation-reoxygenation cycle, O2 equilibria were
further displaced to the right relative to those obtained in the
absence of GSH (Fig. 4A). Yields of Hb(Fe(II))NO upon
deoxygenation in these experiments were ~30% of total [Hb] when
measured by spectral deconvolution (6) and ~27% when measured by
photolysis-chemiluminescence (Fig. 4B). Moreover, the amount of NO remaining bound to Hb in the deoxy state was only ~40% of that
in the oxy state (Fig. 4B), showing that
transnitrosation/denitrosylation reactions are favored in low
pO2. 15 mM GSH caused a leftward shift in
O2-binding compared with that obtained in the presence of
1.5 mM (Fig. 4A). This seemingly paradoxical
normalization of the O2-binding curve at very high
GSH/SNO-Hb ratios was associated with and caused in significant part by
GSH-induced heme oxidation of SNO-Hb during the
deoxygenation-reoxygenation cycle (see "Discussion").
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Influence of GSH Concentration and O2 Tension on the Vasoactivity of SNO-Hb-- Vascular responses to SNO-Hb were studied in aortic rings exposed to varying final concentrations of reduced GSH under both 21% O2 and low pO2 conditions that more closely simulate the tissues. The addition of GSH alone produced no change in tension in the majority of experiments; in ~10% of vessel rings, a small but transient vasorelaxant response was seen.
Under normoxic conditions, SNO-Hb (1.7 SNO groups/tetramer;
108 to 105.5 M Hb) elicited
concentration-dependent, sustained increases in vessel
tension as described previously (Fig.
5A) (1, 5). These
vasoconstrictor responses were preserved in the presence of
107 to 105 M GSH (Fig.
5A). At 104 M GSH, vasoactive
responses to SNO-Hb were variable, with relaxation observed in some
preparations and blunted vasoconstriction in others (Fig.
5A). Vasorelaxation to SNO-Hb was, however, observed in the
presence of millimolar (103 M) GSH (Fig.
5A). Relaxation began 3-10 s after the addition of SNO-Hb
to the tissue bath, consistent with response times to other agents in
this system (data not shown).
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In vessel rings made hypoxic (<1% O2), SNO-Hb similarly
produced dose-dependent contractions. However, the addition
of glutathione even at low micromolar concentrations elicited
relaxations (Fig. 5B). Specifically, vasoconstriction to
SNO-Hb (107 to 103 M) was
observed in the presence of up to 106 M GSH,
whereas in vessels exposed to 105 to 103
M GSH, SNO-Hb elicited potent vasorelaxation (Fig.
5B). At millimolar glutathione, SNO-Hb relaxation in hypoxia
was comparable to that seen in normoxia (Fig. 5A), an
indication that sensitivity of vessels to SNO-induced relaxation is not
greater under hypoxic conditions, as we have previously reported (5).
The dose-response curves for SNO-Hb in the presence of millimolar GSH
were very similar (almost superimposable) to the curves generated by
estimated amounts of GSNO formed in the reactions, whereas NO solutions were inactive at these concentrations (data not shown), i.e.
GSNO, not NO as recently suggested (23), is responsible for relaxation in this system; NO is inactive because it is autocaptured at the hemes
of Hb as illustrated in Fig. 4. In summary, SNO-Hb potency was
potentiated 100-fold under hypoxic conditions as compared with normoxic
conditions, but only in the presence of glutathione. This result is
entirely in keeping with thermodynamic linkage relationships, which
predict from the O2-binding isotherms (Fig. 1) that the
equilibrium in Equation 2 will be shifted to the right under hypoxia,
thereby producing vasorelaxant GSNO. For obvious reasons, bioassays can
only reveal shifts in the position of this equilibrium by working under
conditions (e.g. glutathione concentrations) that produce
far less than maximal relaxations (see "Discussion"). This
condition was not met in a recent study (23).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The biological function of Hb Cys93, which is
highly conserved evolutionarily among higher animals, had remained
obscure until recently. Several studies (1, 2, 5, 7) have now made the
case that it "acts as an allosterically controlled NO buffer" (8),
exchanging the NO group with ambient thiols, including glutathione,
thereby regulating blood flow, the critical determinant of
O2 delivery. Thus, the respiratory cycle can be viewed as a "three-gas (NO/O2/CO2)" system (43). One
remaining conundrum is that the amounts of SNO-Hb present in
vivo are seemingly enormous. When viewed from the standpoint of
NO-related bioactivity, red blood cells carry ~1000 times more SNO
than is needed to regulate blood flow (1, 5, 32-34) or to dilate blood
vessels in vitro (1, 5, 44). If this SNO were transformed
into bioactive equivalents during arterial-venous transit, hypotension
would result on the one hand, and NO synthase (NO production rates) would be unable to sustain endogenous SNO-Hb levels on the other hand.
How, then, does SNO-Hb conserve its NO-related activity?
Here we have shown that the O2 affinity of S-nitrosohemoglobin is greater than that of native Hb. SNO-containing Hbs are therefore more resistant to the O2-dependent transition to T structure. Thus, S-nitrosylation mitigates excessive release of the NO group by promoting the R structure.
Molecular modeling suggests that in SNO-Hb,
S-nitrosocysteine 93 is located in a protected pocket
within the R conformation, but is exposed in the T conformation (5).
Transnitrosation to glutathione is therefore disfavored in the R state
relative to the T state. Allosteric modulators that shift the position of the R/T equilibrium toward R would therefore lower the yield of
SNO-Hb transnitrosation reactions and decrease the bioactivity (Equation 4 and Fig. 5). At sufficiently high glutathione
concentrations, however, Equation 2 indicates that GSNO formation will
ultimately prevail. Indeed, our bioassay results confirm the formation
of GSNO in the presence of millimolar glutathione. In the red blood cell, which is rich in glutathione, ascorbate, and other naturally occurring reductants (45), GSNO is highly reactive, liberating NO
(46-50). This momentarily freed NO would be quickly captured at vacant
hemes at a nearly diffusion-limited rate (2, 6). Hb thus limits the net
release of NO and thereby regulates related bioactivity.
The interaction of SNO-Hb with GSH entails not only a transnitrosation
reaction, but an additional process in which the NO group is
reductively transferred to the heme presumably via the transient
release of NO (Fig. 4B). Under experimental conditions in
which all hemoglobin molecules carry ~2 SNO groups and approach full
oxygenation, an inevitable consequence is MetHb formation. With NO/Hb
ratios in the physiological range, however, the NO addition reaction,
which yields Hb(Fe(II))NO, outcompetes the MetHb-forming oxidation
reaction (6) because the concentration of NO is significantly lower
than the concentration of vacant (deoxygenated) hemes in
HbO2, and the binding of NO to HbO2 is cooperative (6). Thus, a major product of GSH interaction with SNO-Hb
in vivo is likely to be iron nitrosylhemoglobin (Equation 5).
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(Eq. 4) |
SNO-Hb exists in equilibrium with Hb(Fe(II))NO (Equations 1 and 5).2 The position of this redox-coupled equilibrium is linked to the allosteric state of Hb as evidenced here by SNO-to-heme iron transfer of NO upon deoxygenation. It has been previously inferred from O2-binding studies that this amount of Hb(Fe(II))NO is very small and inconsequential (36). In these earlier studies, however, SNO-Hb was subjected to deoxygenation in the absence of a reductant. Under such artificial conditions, SNO-Hb would be restricted to an unfavorable homolytic decomposition reaction (that yields NO· and Hb thiyl radical, ·S-Hb(Fe(II))). Moreover, even if a reductant were present, the supraphysiological ratios of NO to heme in this system could retard the transition to T structure upon deoxygenation as NO replaces the departed O2 ligands. In this study, we have shown that the addition of physiological reducing agents (NADPH, GSH) facilitates NO migration from thiols to heme and does so to a greater extent at low pO2 (i.e. in T structure). Specifically, a major fraction of SNO-Hb(Fe(II))O2 is converted into Hb(Fe(II))NO upon deoxygenation. Thus, both the activity-generating transnitrosation reaction that yields vasorelaxant S-nitrosothiol (Equation 4) and the reductive NO-storing reaction yielding Hb(Fe(II))NO (Equations 1 and 5) are facilitated by deoxygenation.
SNO-Hb oxygen-binding curves exhibit diametrically opposite effects at different concentrations of GSH (15 versus 1.5 mM) (Fig. 4). This behavior may be related to differing amounts of mixed disulfide, MetHb, and iron nitrosylhemoglobin, the formation of which is glutathione-dependent. To control these effects, we used a purified stroma-free red blood cell hemolysate containing physiological levels of superoxide dismutase, catalase, and methemoglobin reductase, with NADPH added to provide reducing equivalents. Even under these conditions, oxidation at the heme centers, although slowed, was significant. Nevertheless, concentrations of GSH mimicking those found in the red blood cell under normal conditions clearly shifted the O2 dissociation curve to the right, consistent with denitrosylation of Hb and the transfer of SNO to GSH, forming GSNO (Figs. 4B and 5 and Equation 4). Thus, GSH facilitates the offloading of (S)NO from Hb, lowering its overall affinity for O2.
Our O2-binding data for SNO-Hb are consistent with previous
studies demonstrating that covalent modification of
Cys93 by N-ethylmaleimide, mercurials,
glutathione, or S-nitroso compounds augments heme ligand
affinity (23, 36, 42, 51-53). We observed increases in O2
affinity that were proportional to the extent of
S-nitrosylation, up to 2 SNO groups/tetramer. Patel et
al. (36) have suggested that a plateau in O2 affinity
is reached at 1 SNO group/tetramer, which would have had implications
for the distribution of mono- and di-S-nitrosylated Hbs
among partially S-nitrosylated molecules. The reason for
this discrepancy is unclear. Wolzt et al. (23) have reported
that additional thiols can be modified in Hb and that molecules with
>2 SNO groups might have different functional behavior; however, the
existence of >2 SNO groups/tetramer probably reflects disruption of Hb
tertiary/tetrameric structure by high concentrations of thiol reactants
(23, 54). There may indeed be a diversity of SNO-Hb forms and
functions, but this diversity will be primarily linked to homotropic interactions.
Measurements of SNO-Hb and iron nitrosylhemoglobin from blood reveal ~1 of each per 1000 tetramers (1, 5). The increases in O2 affinity produced by S-nitrosylation are thus unlikely to affect the O2-binding properties of blood. More generally, the same holds true for iron nitrosylhemoglobin, which has been reported to decrease O2 affinity (39), but again, only at supraphysiological NO concentrations and then only under nonphysiological conditions (low pH, added inositol hexaphosphate, low temperature). The importance of NO group binding to hemes and thiols of Hb lies not in an effect on the population of Hb at large, but on the functional behavior of molecules carrying the NO, i.e. S-nitrosylation may serve to conserve SNO by favoring the R structure (as discussed above), whereas migration to hemes limits the escape of NO upon deoxygenation of SNO-carrying molecules. In this way, Hb tightly regulates the dispensing of NO groups. Since blood flow is regulated by nanomolar SNO targeted to smooth muscle (1, 5, 32-34, 44), this dispensing of NO groups (for the purpose of dilating blood vessels) is far more important for O2 delivery than any NO-related change in O2 saturation of Hb, which could only be effected by supraphysiological NO levels.
The O2-binding properties of SNO-Hb, like those of
unmodified Hb, are strongly pH-dependent. The magnitude of
the Bohr effect in SNO-Hb is similar to that seen with unmodified Hb.
Specifically, the sensitivity of SNO-Hb to changes in pH is similar to
that of native Hb. The ability of His146 to bind protons is
reported to account for nearly one-half of the alkaline Bohr effect in
Hb (55-57). Our findings therefore suggest that the behavior of the
salt bridge between His146 and Asp94 is not
altered by S-nitrosylation of the protein. This suggestion is consistent with the modeling work of Stamler et al. (5). Modeling work by Chan et al. (18), on the other hand,
implied that these terminal residues would be disordered in the T as
well as R states of SNO-Hb, thus ruling out a significant contribution to the Bohr effect by these residues. We have reasoned that the formation of this salt bridge will increase the pK of
Cys93 in Hb (5), i.e. protonation of
His146 and Cys93 is linked. It remains to be
seen, therefore, if Cys93 contributes to the Bohr
effect, in which case, protonation of nitrosocysteine 93 may
facilitate NO group release (58). The O2 affinity of SNO-Hb
is also regulated by phosphate anion concentration (Fig. 3), in keeping
with the recent observation that NO chemistry with Hb can be greatly
influenced by the buffers commonly used for studies of Hb function
(6).
The thermodynamic implications of Monod's concept of allostery in the
regulation of protein function were developed by Wyman (13, 14), with
hemoglobin as the model. Numerous reciprocal relationships between
O2 equilibria and the binding of heterotropic allosteric
effectors, required by thermodynamics, have been demonstrated experimentally. For example, the covalent binding of CO2 to
the amino terminus of Hb 
-globin chains lowers O2
affinity, and accordingly, O2 binding at the hemes triggers
the dissociation of CO2 (the Haldane effect) (58). These
linkage principles similarly illuminate the interaction of Hb with the
NO group. Five groups have shown S-nitrosylation to be
favored in the R structure (11, 19, 22, 36, 42). Subsequently, we and
two other groups (36, 42) have demonstrated the reciprocal effect of
S-nitrosylation increasing the O2 affinity, and
we have also reported that heme deoxygenation promotes NO group release
from SNO-Hb (1).
This linkage has implications for the recent report of Patel et al. (36), who determined that transnitrosation of GSH by SNO-Hb is more favorable in the R than T structure. Specifically, they indicated that the equilibrium constant for GSNO formation from SNO-Hb (Equations 2 and 4) is greater in the R structure and concluded that the O2-dependence of the vasorelaxation could not involve a mechanism that is "intrinsic to hemoglobin." They then inferred that NO group release from SNO-Hb would be insufficient to dilate the flow-regulating arterioles in vivo. Wolzt et al. (23) reached much the same conclusion, although it is not entirely clear why from the data that they reported. They found that SNO-Hb potently relaxed blood vessels and verified that vasorelaxation was potentiated by glutathione, but challenged the notion that the bioactivity of SNO-Hb is linked to the allosteric transition. The conclusions of both groups are difficult to reconcile with the apparent thermodynamic linkage of O2 ligation and S-nitrosylation.
These studies have a number of shortcomings. Patel et al. (36) did not actually evaluate either the aerobic or anaerobic equilibrium constants for Equation 2, nor did they actually measure the bioassay responses on which their biological conclusion rests. In fact, their conclusions rest upon a series of suppositions, estimates, and extrapolations that are unjustified. As a most notable and crucial example, they substantially overestimated, with no supporting argument or cited reference, the amounts of NO/SNO that would be needed to dilate blood vessels and substantially underestimated the circulation time of red blood cells.
Wolzt et al. (23) did conduct bioassays, but they worked under conditions in which Hb is significantly dissociated into dimers (see "Experimental Procedures"), which do not transition to T structure (38, 59). Their methods therefore prevented them from rendering any meaningful conclusion as to the allostery-related effects of oxygen tension. Moreover, since Wolzt et al. used only high concentrations of glutathione, they would have masked (as we show in Fig. 4) the potentiating effect of hypoxia on vasodilation. High enough concentrations of glutathione strongly shift the equilibrium toward GSNO regardless of the Hb quaternary structure (Equation 2). For obvious technical reasons, the organ chamber bioassay can only illuminate these differences in equilibrium position at lower concentrations of glutathione (see Fig. 5). Wolzt et al. also concluded that the GSH-dependent relaxations of SNO-Hb are mediated by NO, not by GSNO. The NO signal that they detected (~250 pM), however, was probably an artifact, being well below the sensitivity of the Clark-type NO electrode (manufacturer's specification, 1 nM; typically, >10 nM; World Precision Instruments, Inc., Sarasota, FL) and ~100-fold too low to account for the ~40% relaxation seen under these conditions. The idea that SNO-dependent relaxations are, in general, mediated by release of free NO has been refuted previously (5, 60-62).
In conclusion, we have shown that 1) the binding and release of NO
groups by Hb are governed by allosteric state and the presence of
thiols, which transduce the SNO bioactivity in transnitrosation reactions, in keeping with thermodynamic linkage relationships in Hb;
2) the mechanism of SNO-Hb-induced vasorelaxation in the presence of
GSH is independent of the free NO liberated, in keeping with known
kinetic considerations for NO reactions with Hb; and 3) NO is
autocaptured by hemes of hemoglobin in transition to T state, which we
propose acts to conserve the NO-related signal. Additional mechanisms
contributing to the conservation, packaging, and transport of
erythrocytic NO-related activity are not excluded by these results.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Veronica Lance for performing the O2 equilibria in the presence of organic phosphates and Andrew Gow for deconvolution of spectra.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants R01 HL52529-05 and HL59130-02 (to J. S. S.) and Grant K08 HL04014-01 (to T. J. M.).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: P. O. Box 2612 (MSRB Rm. 321), Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6933; Fax: 919-684-6998; E-mail: STAML001@mc.duke.edu.
Published, JBC Papers in Press, March 23, 2000, DOI 10.1074/jbc.M000532200
2 B. P. Luchsinger, A. J. Gow, T. J. McMahon, J. S. Stamler, and D. J. Singel, manuscript in preparation.
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ABBREVIATIONS |
|---|
The abbreviations used are: Hb(Fe(II))NO, iron nitrosylhemoglobin; SNO-Hb, S-nitrosohemoglobin; SNO, S-nitrosothiol; GSNO, S-nitrosoglutathione; PBS, phosphate-buffered saline; MetHb, methemoglobin; PSF-Hb, purified stroma-free Hb.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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S. C. Rogers, A. Khalatbari, B. N. Datta, S. E |
) and SNO-Hb
(
). B, Hill plots for solutions of HbA0 (
)
and SNO-Hb with 0.7 (
), or 1.8 (
) SNO groups/tetramer
in PBS, pH 7.4, and 0.5 mM EDTA. The results are
representative of five independent experiments. C,
P50 (the pO2 at 50%
O2 saturation) plotted as a function of the number of SNO
groups bound per Hb tetramer (the maximum is 2).
)
mM GSH in 0.1 M Hepes containing 5 mM NADPH, pH 7.4. The data shown are representative of four
similar experiments. Asterisks in A identify
parallel samples analyzed in B for Hb(Fe(II))NO and SNO-Hb
content. B, fractional amounts (expressed as percent initial
SNO) of Hb(Fe(II))NO (
), 10
) M) in either 21%
O2 (A) or low pO2 (<1%
O2; B). Base-line tension among the groups was
not significantly different.