From the Duke University Marine/Freshwater Biomedical
Center, School of the Environment Marine Laboratory,
Beaufort, North Carolina 28516, the ¶ Institute of Cell and
Molecular Biology, TMC, 250 Wu-Hsing Street, Taipei 110, Taiwan, Republic of China,
IBM,
San Jose, California 95193, and the ** Department of Physiology and
Biophysics, Albert Einstein College of Medicine,
Bronx, New York 10461
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ABSTRACT |
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Hb Chico is an unusual human hemoglobin variant
that has lowered oxygen affinity, but unaltered cooperativity and anion
sensitivity. Previous studies showed these features to be associated
with distal-side heme pocket alterations that confer increased
structural rigidity on the molecule and that increase water content in
the Exquisite molecular adaptations match the hemoglobins of widely
diverse organisms to their respective physiological needs and
environments. As a model protein and paradigm of allosteric control
mechanisms, Hb continues to provide investigators with information on
how proteins control and modify the properties of active-site metals.
Studies of normal and variant human hemoglobins and model heme
compounds have shown that various combinations of electronic and steric
factors can alter the ligand-binding affinity of the heme iron (1-5).
Our recent studies demonstrated that it was possible to differentiate
electronic from steric effects by comparing oxidation curves of various
types and states of Hb with oxygenation curves under the same
conditions (6). Such studies led us to conclude that the extent and
frequency of conformational fluctuations play a significant role in the
anionic modulation of oxygen affinity of T-state Hb, with increased
steric hindrance and lower affinity associated with greater structural
rigidity (7).
This study further characterizes the functional consequences of changes
in the heme pocket of Hb Chico (Lys Increased structural rigidity of residues in the heme pocket was
invoked as part of the explanation for the fact that the entire oxygen-binding curve of Hb Chico is shifted toward
lower affinity. The structure-function changes in this protein lower the ligand affinity of both its low-affinity (T-state) and
high-affinity (R-state) conformations without loss of cooperativity or
anion sensitivity. The substitution of Lys with Thr at position The conserved distal His(E7) residue, with which Thr This report describes the results of a further exploration of the
functional properties of Hb Chico and its isolated Whole blood from which Hb Chico was isolated was obtained from
members of the affected family. Hb Chico and Hb A0 were
prepared and stripped of residual anions, particularly phosphates, as
described previously (9). Hemoglobin concentrations were determined
spectrophotometrically (17). Isolation of the An excimer-pumped dye laser (Lambda Physics) was used both in the
photolysis experiments (as a photodissociation light source) and in the
resonance Raman experiments. The laser was operated at 50 Hz and
yielded 30 milliwatts of light at 430 nm with an 8-ns pulse (full-width
at half-maximum). The laser passed through a prism assembly to remove
any amplified stimulated emission or fluorescence. This light beam was
focused onto the sample with a spot size of ~1 × 3 mm. To avoid
heating the sample and to present a fresh spot for each laser pulse,
the samples were placed in a rotating quartz cell. The Raman scattering
experiments were set up with back-scattering geometry. A holographic
grating filter placed between a 2-inch diameter f3 collection lens and
a 2-inch diameter f8 focusing lens removed unwanted laser scattering
from entering the spectrometer. The reflected light was dispersed and detected by a 1.5-m single spectrograph upon whose exit slit was mounted an intensified diode array detector (750 diodes, Princeton Instruments IR4). Wavelength calibration was done with the known lines
of an argon lamp. The spectral response of the system was ~3
cm For the transient absorption experiments, the Hb concentrations were
all between 100 and 250 µM in heme and were loaded into a
0.75-mm thick quartz cell. An excimer-pumped dye laser tuned to 540 nm
was used to photodissociate the samples, and a continuous wave
helium-cadmium ion laser at 441.6 nm was used as a monitor beam. A
green-reflecting, blue-transmitting dichroic window was used to reflect
the excitation pulse onto the sample and to allow the monitor beam to
pass through co-linearly. A low-resolution spectrometer (~5-nm
full-width at half-maximum) was placed in front of the detection
photomultiplier tube to remove room lights and any stray light from the
photodissociation pulse. A Hamamatsu RG928 photomultiplier tube wired
for high current (10-kilowatt dividers) and short pulses
(0.1-microfarad capacitors) was used to detect the light. The data were
collected with a 1-GHz oscilloscope (Tektronics 7104) and a digitizing
camera (Tektronics DCS01). The system resolution was ~8 ns.
Transient Absorption Studies--
Transient absorption
measurements following photolysis initiated with a pulsed laser
revealed functional differences between Hb A0 and Hb Chico
and their isolated
Further information can be ascertained as to the functional differences
between Hb A0 and Hb Chico by modeling ligand migration behavior. In the most simplified model of geminate rebinding, photolysis is immediately followed by an interval when the ligand occupies the distal heme pocket. From there the ligand can either rebind, with rate kb, or migrate out toward the
solvent, with rate km. It is easily shown that
the rate of the geminate rebinding phase (kg) is
determined by kb + km and
that kb = fgkg and
km = fskg. These parameters
are shown in Table I. In almost all cases, the values of km are similar for Hb Chico
and Hb A0 as well as for their subunits. For O2
escape from the heme pocket, km(Hb
A0)/km(Hb Chico) = 0.96 and
km(
In contrast to the unchanged rates of ligand escape from the heme
pocket, the differences in kb for O2
rebinding are appreciable, with kb(Hb
A0)/kb(Hb Chico) = 2.4 and
kb( Resonance Raman Studies--
To better understand the structural
causes of the lowered ligand-binding affinity for Hb Chico
versus Hb A0, resonance Raman spectra were
obtained from the same protein-ligand systems as discussed above. The
probe wavelength of 430 nm couples to the
The difference in fractional intensity of the 1356/1375
cm
The The objective of this study was to determine the nature and origin
of consequences for ligand binding and dissociation within the heme
pocket that result from the point mutation that causes Hb Chico to have
dramatically lowered ligand affinity in both low-affinity (T-state) and
high-affinity (R-state) conformations. The nanosecond geminate
rebinding experiments showed that one or more heme pocket barriers to
rebinding of oxygen are indeed increased in Hb Chico and in its
isolated The process of geminate recombination provides a direct window into the
molecular events that shape ligand dynamics within the local
environment of the heme. Geminate recombination at room temperature was
first reported for nanosecond time-resolved absorption (20, 21) and
Raman spectroscopy (22). These studies revealed that the quantum yield
for photodissociation of the CO forms of Hb and Mb differ due to a
100-ns geminate rebinding process that is more pronounced in Hb.
Many workers, starting with Frauenfelder and co-workers (31), have
contributed to our understanding of the barriers to ligand binding and
dissociation. Recent room-temperature studies provide convincing
evidence for there being spatially distinct regions from which geminate
recombination can occur (32-35). Subsequent to photodissociation,
ligand diffusion occurs in the progressive spatial separation of the
ligand and heme. In Mb, the ligand, still within the heme pocket, can
occupy accessible cavities or docking sites; and geminate rebinding
from these sites gives rise to different geminate rebinding phase(s).
Scott and Gibson (35) have shown that xenon atoms can alter the pattern
but not the yield of geminate rebinding by occupying, and thereby
blocking access to, cavities within the distal pocket of Mb.
A reaction-coordinate diagram for three spatially separated heme pocket
barriers is shown in Fig. 4. The
potential energy of Well A represents the condition for the iron-bound
ligand. Wells B and C correspond to the potential energy wells for the ligand as it moves from a position very close to the heme (the proximate well) to more distant regions of the heme pocket (the distal
well). S represents the ligand in the solvent. The potential energy of Barrier I controls iron-ligand bond formation. Barrier II
controls the ligand motion between the site of iron-ligand bond
formation and transient docking sites on the distal side, spatially
removed from the iron. Barrier III regulates ligand escape from the
heme pocket into the bulk solvent. It is likely that Process 1, B -chain heme pocket. We report here that the extent of nanosecond
geminate rebinding of oxygen to the variant and its isolated
-chains
is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering
rates of migration out of the heme pocket. Data analysis indicates that
one or more barriers that impede rebinding of oxygen from docking sites
in the heme pocket are increased, with less consequence for CO
rebinding. Resonance Raman spectra show no significant alterations in
spectral regions sensitive to interactions between the heme iron and
the proximal histidine residue, confirming that the functional
differences in the variant are due to distal-side heme pocket
alterations. These effects are discussed in the context of a schematic
representation of heme pocket wells and barriers that could aid the
design of novel hemoglobins with altered ligand affinity without loss
of the normal allosteric responses that facilitate unloading of oxygen
to respiring tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
66(E10)
Thr), a
naturally occurring, low-affinity Hb variant that has an increased rate
of autoxidation (8). The partial pressure of oxygen required for
half-saturation of Hb Chico is approximately twice that required for Hb
A0. The oxygen affinity of isolated
-chains of Hb Chico
is similarly lowered relative to normal
-chains. These equilibrium
properties are mirrored by changes in the transient kinetics of both
ligand binding and ligand dissociation. The CO binding rates for Hb
Chico in the millisecond time region are about half those for Hb
A0, and oxygen dissociation occurs about twice as fast
(9).
66 was inferred to result in increased structural rigidity on the distal
side of the heme pocket as a result of hydrogen bonding between the
distal histidine of the
-chains and Thr
66(E10)
through a bridging water molecule. This interaction would also be
expected to reduce any ligand-stabilizing effects associated with
hydrogen bonding between the distal histidine and bound oxygen, although the extent to which hydrogen bonding stabilizes bound ligands
in the
-chain of Hb A0 is still debated. Lowered oxygen affinity is observed for both isolated
-chains of Hb Chico and Hb
Chico tetramers, indicating that this functional change has a tertiary
basis apart from any alteration of the normal quaternary equilibrium
between the R- and T-states (9).
66
in Hb Chico interacts, has long been considered a candidate for the
modulation of oxygen affinity. Its functional role has been
experimentally tested (for recent reviews, see Refs. 10 and 11). In
myoglobin (Mb)1 and in the
-chains of Hb A0, this histidine contributes to
increased oxygen affinity by stabilizing the heme-bound oxygen through
either direct hydrogen bonding or polar interactions with the oxygen. The distal histidine can also play a role in controlling ligand access
to the heme by participating in the control of fluctuations that result
in an open and accessible pocket (10, 12-14). It can also modulate the
occupancy of the distal pocket with respect to water molecules (15). It
has been shown that the presence or absence of water can influence the
polarity within the heme pocket, which also influences the stability of
the bound oxygen (15, 16).
-chains by the
techniques of resonance Raman spectroscopy and nanosecond laser
photolysis followed by ligand recombination. These techniques allowed
us to show that one or more heme pocket barriers in Hb Chico are
increased. As a consequence of this change, in both intact Hb Chico and
its isolated
-chains, the fraction of oxygen that rebinds from
within the heme pocket after a nanosecond flash (geminate
recombination) is significantly diminished relative to Hb
A0 and normal human
-chains. Analysis of the geminate
rebinding kinetics following laser flash photolysis allowed us to
quantify the magnitude of differences in O2 and CO
rebinding shown by the Hb variant and to suggest where the mutation
exerts its influence along the reaction coordinate for ligand rebinding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
-chains of Hb
A0 and Hb Chico was accomplished with the method described
by Geraci et al. (18). Regeneration of the
SH-chains was
assured by carrying out spectrophotometric titrations of the
regenerated sulfhydryl groups as described by Boyer et al.
(19). Samples were stored in the CO form and packed in ice (0 °C)
prior to the analyses reported here. Sodium dithionite (Merck) at a
concentration of ~0.5% was used to deoxygenate samples prior to
kinetic and resonance Raman experiments with the CO derivative. The
oxygenated forms were prepared at room temperature by bubbling the HbCO
samples with air while illuminating them with light whose IR spectrum
was filtered out.
1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
-subunits in their O2 and CO ligand
rebinding kinetics. Fig. 1 is a plot of
the fraction of surviving unbound ligands versus time,
n(t), following photolysis by a laser with a pulse width of
~8 ns for the oxy forms of Hb A0 and Hb Chico. The
signals were normalized to their respective values at 8 ns by dividing
the change in optical absorption from the pre-photolysis value by the
change measured at 8 ns. The fast process that occurs in ~100 ns is
attributed to geminate rebinding of O2 molecules that have
not escaped from the heme pocket (20-22). This geminate phase is
characterized by a fraction of geminately rebinding ligands
(fg) and a rate (kg). The
fraction remaining unbound beyond ~0.5 µs
(fs) is attributed to ligands that have escaped
into the solvent. Fig. 2 summarizes the
differences in O2 rebinding kinetics for Hb A0
and Hb Chico as well as for their isolated
-subunits. A consistent
result is that fg and kg
for O2 rebinding are appreciably smaller for the Hb Chico
samples. For CO rebinding, a similar but much reduced protein-specific difference is observed.
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Fig. 1.
Time courses of recombination after
photolysis of oxy forms of Hb A and Hb Chico. The fraction of
surviving unbound ligands versus time, n(t), is
shown following photolysis by a laser with a pulse width of ~8 ns for
the oxy forms of Hb A and Hb Chico. An excimer-pumped dye laser tuned
to 540 nm was used to photodissociate the samples, and a continuous
wave helium-cadmium ion laser at 441.6 nm was used as a monitor beam.
The signals were normalized to their respective values at 8 ns by
dividing the change in optical absorption from the pre-photolysis value
by the change measured at 8 ns. See "Experimental Procedures" for
further details.
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Fig. 2.
Graphical representation of differences in
O2 rebinding kinetics for Hb A0 and Hb Chico
and their isolated -subunits using data of
Table I.
of Hb
A0)/km(
of Hb Chico) = 0.97. CO
migration out of the heme pocket is also largely unaffected by the
substitution.
Fraction of ligands rebinding geminately (fg) or escaping to
the solvent (fs), the rate of binding (kb) or migrating
out of the heme pocket (km), and the observed germinate rate
(kg)
of Hb
A0)/kb(
of Hb Chico) = 1.9. Similar but much smaller differences in kb for
CO rebinding are also apparent. Thus, the structural differences between Hb Chico and Hb A0 result in barrier increases that
affect the rate of oxygen rebinding to the active site, rather than
migration out of the heme pocket. The small difference between samples
of Hb A0 and Hb Chico for CO rebinding from the heme pocket
must be reconciled to the fact that the slower solution-phase CO
binding to R-state Hb Chico and its isolated
-chains is half as fast as for Hb A0 and normal
-chains (see
"Discussion").
* Soret absorption band
of the heme (23-27), resulting in resonance enhancement of several
protein conformation-sensitive Raman vibrational bands associated with
the heme and the linkage between the heme and the proximal histidine.
We focused on two bands: 1) the porphyrin band labeled
4 (1350-1385
cm
1) (24) and 2) the iron-proximal histidine band,
(Fe-His) (200-240 cm
1) (26, 27). The
4 band
involves a symmetric vibrational breathing mode of the porphyrin made
up largely of C-N stretches. It is very sensitive to changes in heme
ligation. For liganded ferrous Hb,
4 is ~1374 cm
1,
whereas for deoxy-Hb and photolyzed Hb,
4 is ~1352
cm
1. The frequency of
4 for the five-coordinate
species is sensitive to both quaternary and tertiary structure of
hemoglobins (27). Fig. 3 shows
4 for
HbO2 A0 and HbO2 Chico photolyzed
and probed with a 10-ns laser pulse at 430 nm (50 Hz, 30 milliwatts).
The band at ~1356 cm
1 is characteristic of photolyzed
Hb, whereas the band at ~1376 cm
1 is characteristic of
either liganded or methemoglobin. The absorption spectrum indicates
that there is no measurable met formation in these samples. The
fractional integrated area of the 1376 cm
1 band was found
to be 0.8 for HbO2 A0 and 0.7 for
HbO2 Chico. For deoxy forms, no band at ~1375
cm
1 was detected, whereas for the easily photodissociated
carboxy forms, the 1375 cm
1 band was significantly
decreased, indicative of nearly complete photodissociation.
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Fig. 3.
Comparison of the 4
Raman bands for partially photodissociated oxy derivatives of Hb
A0 and Hb Chico. The lower and higher frequency bands
correspond to the five-coordinate oxy photoproduct and the
six-coordinate unphotolyzed oxy species, respectively. The spectra were
generated from identical concentrations of protein and identical
excitation conditions. They are normalized with respect to the
photoproduct peaks at ~1312 cm
1.
1 band between HbO2 Chico and
HbO2 A0 (for samples having the same optical
density) means that it is harder to photodissociate the latter. This
difference in the ease of photodissociation with an 8-ns pulse suggests
that there is either a subnanosecond geminate phase for which Hb
A0 has a higher geminate yield or that the actual intrinsic
quantum yield for photodissociation is lower for Hb A0.
Future studies are planned to examine this effect.
(Fe-His) bands for the deoxy forms as well as the photoproducts
of the carboxy and oxy forms of Hb A0 and Hb Chico were measured. The frequency of the
band is very sensitive to structural changes on the proximal side of the heme that have been shown to
correlate with ligand affinities (26-29). No discernible differences in
(Fe-His) could be detected between the Hb Chico structures and
those measured or reported for Hb A0 (28, 30).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
-chains. Knowledge of the site at which the mutation has
occurred and the existing x-ray crystallographic data implicate
tertiary-level changes on the distal side of the heme as the source of
the modified ligand reactivity. This view is further strengthened by
the Raman data that indicate that the structural parameters associated
with the proximal side of the heme are the same in Hb A0
and Hb Chico. The question remains as to where along the reaction
coordinate for ligand binding the distal pocket perturbation in Hb
Chico exerts its influence. To address that question, we must consider the protein- and ligand-specific pattern of changes observed in the
geminate recombination process.
A, can be further subdivided since rebinding processes occurring from
about one to several hundred picoseconds have been observed. However
complex the underlying reality, many ligand- and protein-specific
processes within the heme pocket can be compared within the context of
this reaction-coordinate diagram and the associated kinetic scheme
given below.
Considerable information has been obtained about the relative
potential energy barriers in the heme pocket of Hb A0.
Ligand specificity in the geminate rebinding process has shown that
Barrier I is much higher for CO compared with O2 and NO
(36-39). In Hb A0, oxygen exhibits geminate rebinding on
the time scales of several picoseconds, hundreds of picoseconds, and
hundreds of nanoseconds, whereas CO displays negligible rebinding on
subnanosecond time scales. This ligand specificity is reflected in the
differences shown for O2 and CO in Fig. 4. As a result of
this ligand dependence of the inner barrier, on-rates for oxygen and
especially NO binding can approach the diffusion limit, whereas CO
binding is slower (40). Similarly, the lower yield of geminate
recombination for CO relative to O2 is understandable in
terms of a higher Barrier I for CO.
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Fig. 4.
Reaction-coordinate diagram for ligand
dissociation and recombination in Hb. This qualitative energy
coordinate diagram illustrates barrier differences for O2
and CO ligands as described under "Discussion." Proximate and
distal geminate wells refer to the heme pocket sites occupied by the
dissociated ligand that are kinetically (with respect to
rebinding) close to or far from the heme iron, respectively.
Friedman and co-workers (28, 41, 42) presented a mechanistic explanation for the origin of this ligand-specific barrier-height difference based upon differences in the nature of the transition state for CO and O2. The claim is that CO requires a nearly planar heme transition state, whereas oxygen can form a bond with the heme when the iron is still partially out of the heme plane. Thus, much larger fluctuations in structure are required for the formation of the CO-heme transition states. This assessment of the transition state is similar to the kinetically and thermodynamically derived conclusion of Szabo and Karplus (43) that asserts that CO has a more product-like transition state, like that when ligand is bound, whereas O2 has a more reactant-like transition state. There is also evidence that the orientation of the CO contributes to the energy of the transition state. In particular, the picosecond IR absorption studies of Anfinrud and co-workers (44, 45) show that bond re-formation proceeds in myoglobin for an upright transition-state conformation of the photodissociated CO.
For a given ligand, the height of Barrier I is both protein- and conformation-dependent. It has been shown that in Hb A0, the height of Barrier I is highly sensitive to both tertiary and quaternary structure (46). The geminate yield decreases as the heme pocket becomes more T-like, consistent with increases in Barrier I. The decrease in the geminate yield in these instances correlates with spectral changes that are attributed to proximal strain as reflected in Raman measurements of the iron-proximal histidine linkage (28-30, 38, 47). These proximal effects appear decoupled from distal-side effects, as seen in a study on Hb Zurich where the inositol hexaphosphate effect on the geminate rebinding of CO was found to be similar to that in Hb A0, despite the large distal-pocket alteration created by the replacement of the distal histidine with an arginine in this Hb variant (48). A large part of the inositol hexaphosphate effect on geminate rebinding has been inferred to be due to a proximal-side effect, as indicated by the reduction in frequency of the iron-proximal histidine stretching mode in the photoproduct spectra of both HbCO A0 and HbCO Zurich upon adding inositol hexaphosphate.
The absence of detectable shifts in (Fe-His) between Hb
A0 and Hb Chico for both the deoxy-T and photoproduct R
forms of the protein indicates that the geminate rebinding differences do not have a proximal-strain origin. It has been argued (49) that
factors that increase the rate of tertiary relaxation on the time scale
of geminate rebinding can contribute to a progressive increase in
Barrier I and thus decrease the geminate yield. It is therefore
possible that enhanced relaxation of the initial R-state photoproduct
structure in Hb Chico is responsible for the observed reactivity
differences. The arguments given below that are based on differences in
the distal heme pocket are, however, more plausible in this instance.
Relative heights of the other heme pocket barriers in Hb A0
can be estimated. Picosecond studies of the geminate process with a
30-ps excitation pulse reveal that the quantum yields for the subnanosecond geminate phase for oxygen and CO are ~50 and 2%, respectively, for Hb A0 (38, 48). This result indicates
that from Well B, Barriers I and II are comparable for oxygen
(kba/kbc ~ 1). For CO,
however, Barrier I is clearly much greater than Barrier II
(kba/kbc 1). Since
kg, the geminate rate from Well C (but not the
quantum yield), is essentially ligand-independent, the barrier
controlling escape into the solvent (Barrier III) and the barrier
controlling the movement of the ligand back into Well B are, in first
approximation, ligand-independent for the Hb A0
tetramer. Rebinding of oxygen from Well B is much faster than the decay
of the ligand in Well C, with the consequence that the crossing of
Barrier II and ligand escape are rate-determining for the rebinding of
oxygen from Well C. The geminate yield of ~50% for oxygen from Well
C indicates that the heights of Barriers II and III are comparable.
Thus, for O2, kba
kcb
kcs, and the
ratio kcb/kcs determines
the nanosecond geminate yield. For CO, where the height of Barrier I is
much greater than for oxygen and where the rates but not the yields for
the nanosecond process are essentially independent of protein-induced
changes in Barrier I, kba
kcb
kcs, and
kba/kcs determines the
geminate yield.
The role of specific distal heme pocket residues in the partitioning of
the geminate rebinding between the fast and slow phases has been
documented by site-directed mutagenesis studies on Mb (10). Rebinding
studies of genetically engineered mutants in conjunction with dynamic
simulations for MbNO indicate that certain substitutions on the distal
side can appreciably alter the heme pocket barriers. For example, the
substitution Leu(B10) Phe inhibits the motion of dissociated
ligands away from the iron and simultaneously reduces access to the
iron for those ligands that have successfully diffused to the most
distant regions of the heme pocket (32, 34). Many aspects of the
partitioning between fast and slow phases of geminate rebinding are
still unclear. The possibility remains that this partitioning is
controlled by tertiary conformational fluctuations and relaxations that
modulate both distal- and proximal-side effects on heme ligation.
The above considerations, relative to the qualitative pictorial
representation in Fig. 4 of the heme pocket barriers in Hb A0, establish a context for discussion of the functional
modifications of Hb Chico and its isolated -chains. The previous
studies of Hb Chico established that its oxygen affinity is lowered to
about half that characteristic of Hb A0. The oxygen
affinity of its isolated
-chains is similarly lowered relative to
normal
-chains. These equilibrium properties indicate that the well
depth for bound ligand (Well A) in Hb Chico as well as its isolated
-chains is significantly decreased relative to that of Hb
A0 and normal
-chains. The equilibrium situation is
mirrored by changes in the transient kinetics of both ligand binding
and ligand dissociation on the millisecond time scale. The CO binding
rates for Hb Chico in the millisecond time region are about half those
for Hb A0, and oxygen dissociation occurs about twice as
fast (9). The decreased well depth for bound ligands contributes to the
observed 2-fold increased rate of non-photoinduced dissociation of
O2 and the moderately increased rate of CO dissociation
from fully liganded Hb Chico.
The structural differences between Hb Chico and Hb A0
clearly result in increases in one or more barriers that affect the geminate rate of oxygen rebinding to the active site. This is revealed
by the nanosecond geminate rebinding kinetics, where dioxygen rebinding
shows a dramatic decrease. Much smaller decreases are seen in rates of
CO rebinding in going from Hb A0 to Hb Chico. This
contrasts with the large decreases observed in previous millisecond time scale studies where it was shown that the binding of CO from solution shows substantial decreases for both Hb Chico and its isolated
-chains relative to Hb A0 and its
-chains (9). To reconcile these results, we must invoke a structural change that occurs
after photodissociation on a time scale longer than that of the
geminate process, i.e. ~100 ns. We suggest that the
observed reduction in CO "on"-rates in the millisecond time range
can be explained based on the x-ray crystallographic structure of
deoxy-Hb Chico (9). The crystal structure reveals that water is held in
the distal pocket of Hb Chico and acts as a bridge between the distal
histidine and Thr(E10) in the deoxy state. The binding of a ligand at
the iron requires the displacement of the water to a sterically neutral
site. Studies on mutant myoglobins strongly support the claim that
site-stabilized waters in the distal pocket act as steric effectors
that slow down the diffusion time of ligands from the solvent to the
iron (15, 16, 50). It appears that water has not as yet re-entered the
appropriate steric hindering site in the distal pocket of the
photolyzed HbCO Chico on the time scale of the geminate process. This
result suggests that CO must first move from the heme pocket before
water can occupy the site between His(E7) and Thr(E10).
It can be readily seen from Table I that the isolated -chains (as
tetramers) of both Hb A0 and Hb Chico exhibit less geminate rebinding than the corresponding
2
2-tetramers. The effect is especially
noticeable for the CO derivatives, where the
4-tetramers show much slower rates of CO rebinding than the
2
2-tetramers. The simplest models of Hb
reactivity predict that the reactivity of the R-state tetramer should
resemble that of either the isolated chains or the
-dimers. The
limitations of this simple view were indicated by studies showing that
the last available subunit in R-state
2
2-tetramers binds ligands with higher
affinity than
-dimers (51). This phenomenon, termed quaternary
enhancement, was shown in recent studies to be evident in an increased
geminate yield for fully liganded
2
2-tetramers relative to the
corresponding dimers (52). Based on geminate rebinding data and Raman
spectra for the photoproducts of tetrameric and dimeric forms of Hb
A0, it was claimed that the quaternary enhancement effect
arises from a proximal-heme environment in the R-state photodissociated
2
2-tetramer that favors rebinding
relative to that for
-dimers (29). This more favorable
environment is reflected in the higher frequency of
(Fe-His) for the
photoproduct of
2
2-tetramers compared
with either
-dimers or isolated
- and
-chains of Hb
A0 (29, 53). A more favorable proximal environment, with
lower proximal strain, translates into a lower barrier for geminate
rebinding to the extent that the transition state has product-like
character. Since the rebinding of CO relative to O2 is more
likely to require a transition state with an in-plane iron (28, 41,
42), it is probable that the geminate rebinding of CO is more
responsive to proximal perturbations compared with O2,
which would account for the ligand-specific (CO/O2)
differences between
4-tetramers and
2
2-tetramers shown in Table I.
The geminate recombination experiments under consideration were initiated by excitation with a 10-ns pulse. This creates a photoproduct population that has the ligand predominantly in Well C. For a ligand to geminately recombine, it must first overcome Barrier II, followed by bond formation controlled by Barrier I. In competition with the geminate process is the escape of the ligand into the solvent as controlled by Barrier III. One explanation that would account for the pattern of change in the geminate recombination in going from Hb A0 to Hb Chico is that the amino acid substitution in Hb Chico causes a distal-side heme pocket perturbation that increases Barrier II relative to its height in Hb A0 for both O2 and CO. As can be seen from Fig. 4, an increase in Barrier II would have less effect on the geminate rebinding of CO as long as kba/kbc remains small, whereas the nanosecond geminate yield and the nanosecond rebinding of dioxygen should decrease and slow down, as is observed.
Although ligand-independent increases in Barrier II are sufficient to explain most of the data, some evidence points to a ligand-specific increase in Barrier I for O2 rebinding to Hb Chico. Notably, the yield of photodissociation at 10 ns is higher for HbO2 Chico than for HbO2 A0. An increase in Barrier I in Hb Chico could have this result; the apparent increase in degree of photodissociation would result from a decrease in the number of ligands quickly rebound. A degree of uncertainty in using the 10-ns Raman measurement as an indication of small changes in the subnanosecond kinetics yields a less-than-strong argument for a Barrier I increase. Verification of a ligand-specific increase in Barrier I in Hb Chico would require a picosecond rebinding study. If Barrier I has increased, then the yield for both fast and slow geminate phases should decrease. If Barrier II has increased without a change in Barrier I, then the yield for the fast-phase rebinding process should increase at the expense of slower phase rebinding, i.e. the ratio of the quantum yield for the picosecond and nanosecond rebinding should increase.
Recent studies on the effect of distal heme pocket polarity on the bond energies of the iron-ligand bond indicate that the stability of bound oxygen is much more sensitive to polarity effects than that of bound CO (11). If the transition state for the formation of the iron-ligand bond shows a parallel sensitivity, then it might be anticipated that Barrier I would also be more polarity-dependent for O2 than for CO. If this mechanism is operative, then the significant decrease in geminate rebinding for O2 in going from Hb A0 to Hb Chico could arise from a more polar distal heme pocket environment in the latter, providing a more negatively charged environment for the transition state. A more negatively charged distal pocket would also have the expected consequence of increasing the non-photoinduced rates of ligand dissociation for both CO and O2 in going from Hb A0 to Hb Chico, as is observed (9). For oxygen, an increase in the negative charge in the vicinity of the bound oxygen would be expected to weaken hydrogen bonding to the oxygen, which, at least in Mb, contributes strongly to the stability of the iron-oxygen bond. Similarly, an increase in negative charge near the bound CO is known to decrease the stability of bound CO, as reflected in both the Fe-C and inversely correlated CO stretching frequencies (54-56).
A combination of loss of hydrogen-bonding stabilization of bound
ligands and the increased polarity in the heme pocket could bring about
a decrease in well depth for ligands bound to both high- and
low-affinity conformations of Hb Chico. Another mechanism that could
contribute to this distinct functional alteration is an increased
structural rigidity on the distal side of the heme pocket. As noted,
the substitution of Thr for Lys at position 66, on the distal side
of the heme, was inferred to result in increased structural rigidity as
a result of hydrogen bonding between the distal histidine of the
-chains and Thr
66(E10) through a bridging water
molecule. Localized rigidity of the Hb structure in the heme pocket
region, brought about by this hydrogen bonding, could directly affect
the barriers to oxygen rebinding. It has been shown that the distal
histidine can act in conjunction with other heme pocket residues to
control ligand access to the heme by participating in the control of
fluctuations that result in an open and accessible heme pocket (10,
12-14). Accordingly, modifications that restrict motion of the distal histidine could result in altered barriers to ligand rebinding and dissociation.
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CONCLUSIONS |
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This study indicates that the molecular mechanism behind the
altered reactivity of Hb Chico and other mutant hemoglobins can be
dissected by use of a combination of kinetic and spectroscopic probes
that can be viewed as a general scheme for analyzing ligand reactivity
changes in variant hemoglobins. In particular, the changes in the
picosecond and nanosecond geminate phases in conjunction with
ligand-specific behavior provide a means of determining where functional alterations occur along the reaction coordinates for ligand
rebinding. The reaction-coordinate data in conjunction with the CO or
Fe-C stretching frequency for HbCO derivative, which indicates the
effective polarity of the distal pocket, can be used to implicate
charge-stabilization effects. The resonance Raman spectra expose
changes on the proximal side of the heme pocket. A comparison between
solvent-derived ligand binding and geminate rebinding can, in addition,
reflect ligand displacement of localized water that blocks access to
the iron. Such comprehensive studies are of timely importance in that
they can help in developing synthetic strategies based on molecular
biophysics for selectively altering ligand-binding properties of
hemoglobins for pharmaceutical uses. The specific functional
alterations in Hb Chico demonstrate that structural alterations in the
distal heme pocket can alter ligand affinity at the tertiary level,
without loss of the normal allosteric responses that facilitate oxygen
unloading to respiring tissues.
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ACKNOWLEDGEMENT |
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We thank Dr. John Howard (Chico Medical Group of California) for the generous contribution of blood samples.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants ESO1908 and ESO4287 (to C. B.), a grant from the Tobacco Institute (to J. B.), and National Institutes of Health Grants HL5108 and HL58247 (to J. F.).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.: 252-504-7591; Fax: 252-504-7648; E-mail: bona{at}mail.duke.edu.
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ABBREVIATIONS |
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The abbreviation used is: Mb, myoglobin.
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REFERENCES |
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