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J. Biol. Chem., Vol. 277, Issue 28, 25783-25790, July 12, 2002
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From the Department of Physiology and Biophysics, Albert Einstein
College of Medicine, Bronx, New York 10461
Received for publication, January 10, 2002, and in revised form, April 25, 2002
We have used sol-gel encapsulation protocols to
trap kinetically and spectroscopically distinct conformational
populations of native horse carbonmonoxy myoglobin. The method
allows for direct comparison of functional and spectroscopic properties
of equilibrium and non-equilibrium populations under the same
temperature and viscosity conditions. The results implicate tertiary
structure changes that include the proximal heme environment in the
mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of Myoglobin (Mb)1
continues to be used as a model protein system for investigations into
how structure, dynamics, and reactivity interconnect to give rise to
functionality. Recently there has been a renewed interest in myoglobin
based on several new developments. From a functional point of view
there are indications and suggestions that, apart from its
physiological importance in facilitating oxygen diffusion, myoglobin
has functions arising from its ability to participate in catalytic
multisubstrate reactions involving such molecules as NO,
O2, and H2O2 (1-3). In addition
the presence of apolar intra-protein substrate docking sites (the
so-called Xe cavities) (4, 5) have recently been linked both to these newly proposed functions and to the kinetic patterns associated with
geminate and bimolecular ligand binding (3, 6-13).
These new developments highlight fundamental biophysical issues that
have not been fully addressed despite the extensive research effort
that has been directed toward myoglobin. The issue that we address in
this work relates to ligand reactivity as a function of conformation.
Several studies show that myoglobin can adopt different tertiary
conformations. In particular there is evidence that the equilibrium
distribution of tertiary conformations is different for the deoxy
(ferrous five coordinate) and CO derivatives of Mb. X-ray
crystallographic studies show that there is a small clam shell-like
rotation of the E and F helices in going from deoxy to COMb (14) as
well as adjustments in the positioning of residues in the distal heme
pocket (8-10, 15, 16) that may or may not be related to the clam shell
motion. Recent time-resolved x-ray crystallographic studies of
photodissociated COMb (17) indicate that these motions can occur on the
subnanosecond time scale. Raman (18-23) and absorption studies
(24-30), in which the equilibrium deoxy derivative is compared with
the non-equilibrium deoxy derivative derived from the photodissociation
of COMb under conditions where there is either no or slowed relaxation
of the photoproduct, indicate that the proximal heme environment is
different in the two cases. How and through what mechanism these
different tertiary conformations impact ligand reactivity are uncertain.
A complication in addressing the role of conformational relaxation
arises from the difficulty in trapping COMb derivatives having
different tertiary structure distributions at the same temperature
under ambient conditions. In this study, we describe the use of sol-gel
encapsulation as a method of trapping different tertiary conformations
of COMb that allows for both spectroscopic and functional
characterization at the same ambient conditions.
Relatively inert and transparent amorphous sol-gel matrices, composed
of a porous network of silica-oxygen-silica bonds have been prepared
under conditions that allow for the non-destructive encapsulation of
proteins (31-33). In most instances the trapped protein molecules are
isolated and immobile, preventing any possible complications due to
aggregation. Advantageously, the porous matrix does allow water and
small molecules to diffuse in and out. Thus, when the matrix is placed
in a bathing buffer, the encapsulated proteins are solvated and their
function can be examined and compared with solution phase work. Studies
have revealed that the encapsulated proteins remain intact and
functional. For example encapsulated hemeproteins undergo ligand
binding and dissociation as well as redox reactions (32, 34). Many
encapsulated enzymes have been shown to retain enzymatic activity
within the sol-gel matrix (35). The sol-gel matrix also seems to confer
enhanced structural stability (36-38).
Most significantly, with respect to the present project, sol-gel
encapsulation has been shown to limit conformational change in
hemoglobin. Oxygen binding (39-42), kinetic (43, 44), and spectroscopic (45, 46) studies all indicate that the sol-gel can be
used to stabilize the quaternary state conformation of the initially
encapsulated derivative to the extent that subsequent addition or
removal of ligand does not induce the usual quaternary state transition
that occurs in solution. As a result non-equilibrium species associated
with the liganded T state and the deoxy R state can be generated and
studied. Furthermore, the sol-gel-induced slowing of the conformational
dynamics is dramatically dependent on temperature (40, 45, 46). The
tertiary and quaternary relaxation times can be tuned from essentially
days or weeks at 4 °C to hours and minutes as the temperature is
progressively raised (46).
Potentially, this sol-gel property opens the way to overcome the
"diffusional barrier" that exists in rapid mix experiments, where
the diffusion time of ligands/reactants is often longer than the time
it takes for conformational relaxation. Encapsulation can extend the
relaxation time beyond the diffusion time for small substrate
molecules. Thus, encapsulation of proteins in sol-gel may become a new
method both for trapping non-equilibrium species and for studying the
evolution of short-lived non-equilibrium structures. In this work we
show that the sol-gel-based approach of "locking in" of the
quaternary structure of Hb can be applied to the much smaller amplitude
motions associated with the ligation-dependent tertiary
structure of myoglobin. As a result we are able to expose the
ligation-induced conformational and functional changes in myoglobin at
a temperature well above the glass transition. The Soret band-enhanced
resonance Raman spectrum is used to characterize the proximal
environment of the 8-ns photoproduct as a function of trapped
conformation (47, 48), whereas the geminate and solvent phase rebinding
of CO is used to expose the functional consequences of conformation
(11, 12).
Buffered solutions of filtered horse Mb (Sigma Chemical Co.)
were prepared in either BisTris acetate, pH 6.5, or the same with 25%
or 50% glycerol by volume. All chemicals were from Aldrich. To improve
the degree of locking in, the original sol-gel preparation protocol (32) was modified as previously described (43) by avoiding
sonication and by changing the buffer to 50 mM BisTris acetate, pH 6.5, diluted 3:1 with glycerol to yield 25%
glycerol by volume. The gels were cast as thin films in 10-mm NMR
tubes. Equal volumes of tetramethylorthosilicate, buffer, and Mb were combined and added to the NMR tubes (New Era and Willmad). The tubes
were then spun using a high speed spinner (Princeton Photonics Inc.,
Princeton, NJ) to generate the thin film that eventually gelled after
approximately 1 h of spinning. The samples were flushed and
covered with buffer and then allowed to age a minimum of 1 day at
4 °C. The final concentration of Mb was 0.5 mM. DeoxyMb samples were prepared from nitrogen-purged solutions of metMb to which
a slight excess of sodium dithionite (solution) was added. All
solutions and gels were prepared in and stored under anaerobic conditions. The spinner was contained and operated within a
nitrogen-purged glove box.
The preparations for both visible resonance Raman and transient
absorption were as described earlier (38). In addition to the visible
resonance Raman measurements described below, absorption, front-face
fluorescence, and UV resonance Raman measurements on samples of Mb
encapsulated using this protocol all indicate that the native structure
is maintained.
Scheme 1 shows the two types of encapsulated samples that were
prepared. In one case COMb is directly encapsulated in sol-gel. This
protocol or sample type is denoted [COMb]. For the other protocol,
deoxyMb is encapsulated in the sol-gel, and is denoted as [deoxyMb].
The Mb encapsulated in sol-gels was found to be intact and functional
by exhibiting the unperturbed absorption spectra of the
corresponding solution phase samples (not shown). The samples were
aged at ~4 °C, to allow the evolving gel to "template" around
the equilibrium structure of the encapsulated Mb derivative to maximize
the likelihood of locking in the initial distribution of conformations.
After the encapsulated deoxyMb sample was allowed to age (1-5 days)
and its spectra were recorded it was then exposed to CO. Rapid (within
5 min or less) ligation with CO occurred as determined by the
characteristic changes in the visible absorption spectrum. Such samples
are denoted [deoxyMb]+CO to indicate that the CO is added after
encapsulation. A similar two-protocol approach was used to prepare the
COHbA locked in either the R or the T quaternary state (43).
Visible Resonance Raman Spectra, A Comparison of
Sol-Gel-encapsulated Samples--
Fig. 1
contains segments of Soret-enhanced resonance Raman spectra for both
myoglobin and hemoglobin samples. The resonance Raman spectra from the
sol-gel-encapsulated samples are very similar to those of the
corresponding solution phase samples (as shown in the inset
of Fig. 1) indicating that the protein conformation is not altered by
the sol-gel matrix to any significant degree. Nonetheless, for some of
the sol-gel-encapsulated samples there are differences in the frequency
of the iron-proximal histidine mode,
The frequency of
Fig. 1 also shows the Fe-His bands of sol-gel-encapsulated HbA,
prepared using similar protocols ([deoxyHbA] (H);
[deoxyHbA]+CO (I); and [COHbA] (J)). Based on
the kinetics and UV resonance Raman spectra from such samples (43, 45,
46), we have unambiguously established that, in the first two samples,
HbA is locked in the T quaternary structure and in the last it is
locked in the R quaternary structure. The spectra from the two
equilibrium forms, [deoxyHbA] and the 8-ns photoproduct of [COHbA],
are both identical to the corresponding spectra obtained in solution
(see inset in Fig. 1 and Table I). It can be seen that the
frequency of Geminate and Solvent Phase Rebinding Kinetics--
The question
now arises as to whether the spectroscopically distinct encapsulated
COMb samples are also functionally distinct. For Hb such a correlation
exists. Earlier work on encapsulated HbA clearly shows a progression in
the geminate recombination that parallels the changes in the quaternary
state (43, 44). The geminate yield for T state COHbA is substantially
lower than that of R state COHbA. The kinetics for the solvent phase
rebinding associated with the encapsulated CO derivatives of HbA show a direct correspondence to kinetic phases that are observed in solution (70). Whereas in solution both the slow T state and fast R state solvent phases are observed, for the encapsulated HbA samples only one
solvent phase is seen. The encapsulated T state COHbA derivative,
[deoxyHbA]+CO, and the encapsulated R state COHbA derivative,
[COHbA], exhibit the slow T state and fast R state solvent phase
recombination phases, respectively.
Fig. 2 shows a comparison of the kinetic
traces of CO rebinding to photodissociated COMb under different
conditions. In the top panel, there is a comparison of the
kinetics at 3.5 °C for COMb in solution (trace a),
[deoxyMb]+CO bathed in buffer (trace b), and [COMb]
bathed in buffer (trace c) where the CO saturated buffer in
all three cases contains 25% glycerol. It can be seen that the
geminate yield increases in going from solution, to [deoxyMb]+CO, to
[COMb]. It can also be seen that the solvent phase kinetics for the
encapsulated samples are different both from each other and from the
solution sample. The near vertical decay seen for the solution sample
is characteristic of the expected exponential kinetics. Both
encapsulated samples exhibit solvent phase kinetic traces that show a
sloping decay suggestive of a distribution of rates. The distribution
for [COMb] clearly consists of a faster rebinding kinetic population
compared with that of [deoxyMb]. Again the pattern is similar to what
is seen for the comparison of kinetics from [COHbA] and
[deoxyHbA]+CO, but the magnitude of the protocol-specific differences
is smaller for the Mb samples as might be anticipated from the Raman
results. The Mb kinetic differences are similar in magnitude to the
variation in the kinetic pattern within a given quaternary state of
encapsulated Hb due to such factors such as the presence or absence of
allosteric effectors or chemical modification (e.g.
modification of
The second panel in Fig. 2 depicts the kinetic traces at
3.5 °C for the same three types of samples as above, but in each case the buffer used both for the solution phase sample and for the
bathing solvent for the two sol-gel samples contains 50% glycerol. The
pattern observed in the upper panel is further enhanced in this comparison. The geminate yield increases in going from solution (trace d) to [deoxyMb]+CO (trace e) to [COMb]
(trace f). The geminate yield for both of the encapsulated
samples is enhanced over that of the corresponding samples bathed in
buffer containing 25% glycerol, but the protocol-specific difference
remains. It is also apparent that the solvent phase rebinding is again
faster for the [COMb] sample.
In the bottom panel a kinetic comparison is shown for the
two encapsulated samples, but in this case the bathing solvent is CO-saturated glycerol and the temperature is reduced to
The two kinetic traces shown in the bottom panel of Fig. 2
are from samples of [deoxyMb]+CO (trace g) and [COMb]
(trace h), which had been stored at 4 °C for a period of
several months. The kinetic traces of [deoxyMb]+CO and [COMb]
exhibit a clear difference. The kinetic traces show that the amplitudes
for the faster geminate phases are larger for the [COMb] sample. This observation indicates that the dissociated ligand has a greater probability of rebinding from the more distant cavities for the conformation(s) associated with the [deoxyMb]+CO sample.
The sample-specific difference seen in the bottom panel is
observed to very slowly decrease with time. A comparison with the kinetic traces obtained shortly after the samples were bathed with
glycerol reveals a larger difference due to the kinetics from the
[deoxyMb]+CO sample having an even lower amplitude for the faster
geminate phases. The change in kinetics over time indicates that there
is a small amount of relaxation associated with the non-equilibrium
[deoxyMb]+CO sample over a period of many months. This relaxation is
in the direction of the [deoxyMb]+CO sample evolving toward a
population similar to that of the [COMb] sample. In the absence of
added glycerol, the kinetic difference between the two sol-gel samples
exhibits substantial decay over a period of days to weeks.
The Fe-His Stretching Frequency--
To assess the significance of
the Determinants of the Equilibrium Frequency of
The orientation of the histidine determines the magnitude of the
repulsive interaction between the imidazole and the heme. Orientations
of the imidazole that have increased repulsive interactions (e.g. the tilted histidine for the
The positioning of the F helix can be expected to modulate the
orientation effects of the proximal histidine with respect to the
frequency of
In addition to the above two conformational factors, there are an
additional two physical properties that can modify the frequency of
Determinants of the Frequency of
Under the experimental conditions employed in the present study, the
photoproduct frequency of Encapsulated Mb: Conformational Consequences--
The spectra of
deoxyMb in solution and in the sol-gel (with buffer as the bathing
medium) are essentially the same. In particular there is no obvious
frequency shift in
The photoproduct spectrum of [COMb] resembles the spectra obtained
from COMb in solutions that contain significant amounts of
viscosity-enhancing reagents such as 75% or higher glycerol (by
volume) (23). It is also similar to the unrelaxed spectrum of the COMb
photoproduct occurring within several picoseconds after
photodissociation (51). Thus the sol-gel clearly creates an environment
that slows or stops the subnanosecond relaxation, which occurs in low
viscosity solutions. The addition of pure glycerol as the bathing
buffer further increases the frequency of
Both static and time-resolved x-ray crystallographic studies of Mb (14,
17) implicate the shifting of the F helix as a major contributor to the
relaxation dependence of
The frequency of Evidence That the Sol-Gel Traps Conformational Populations of
Mb--
If the sol-gel merely slows relaxation, then the end point
spectra for [COMb] and [deoxyMb]+CO should eventually be the same both for the buffer-bathed and glycerol-bathed samples. The results clearly show that the spectra from the two samples are not the same.
Although both samples exhibit a frequency increase for Comparison between [COHbA] and [COMb]--
That the sol-gel
imposes protocol-specific limits on accessible proximal conformations
for the encapsulated Mb populations is very suggestive (but on a
smaller scale) of the behavior observed in hemoglobins. For hemoglobins
there is a bounded range of accessible tertiary proximal hemepocket
conformations that are distinctly different for the T and R quaternary
states. As with Mb, the low frequency limit for the frequency of
Within either the T or the R state quaternary structure, ligand binding
induces tertiary structure changes that result in an increase in the
frequency of
Although the 8-ns photoproduct frequency of
An obvious question arises: Why is the tertiary relaxation in
myoglobin so much faster than in hemoglobin even though both tertiary
relaxations are purported to be a similar clam shell type rotation of
the E-F helices? A likely explanation is that for hemoglobin there is
an extensive series of hydrogen bonds and salt bridges functioning as
scaffolding that maintains the spacing between pairs of
Thus, in R state HbA, we attribute the increase in the frequency of
Functional Consequences--
The sol-gel kinetic results parallel
the Raman results. There is a consistent difference in the kinetics
between the [deoxyMb]+CO and [COMb] samples independent of the
bathing buffer. For both samples, the geminate yield (GY) increases
when the bathing buffer contains increasing amounts of glycerol (25 to
50%); nonetheless, the GY is always higher for the [COMb] sample.
Similarly, the distribution of rates for the solvent phase rebinding is
skewed toward faster values for the [COMb] sample. When the bathing
solvent is 100% glycerol (CO-saturated), the kinetic trace consists of several geminate phases that have been attributed to rebinding arising
from the CO localized in different xenon cavities (65). Under
this high viscosity limit it can be seen that the GY for the faster
phases is greater for the [COMb] sample. These results are consistent
with the sol-gel trapping populations that are not only
spectroscopically distinct but also functionally distinct.
The protocol dependence of the Raman suggests a possible basis for the
difference in the kinetics. The frequency of Conclusions--
A new sol-gel encapsulation protocol is
shown to successfully maintain (lock) both equilibrium and
non-equilibrium conformational distributions of myoglobin. It is not
clear how the sol-gel locks the conformational populations of Mb. A
pure viscosity effect seems unlikely given the ease with which ligands
can enter and escape from the protein. A likely possibility, based on
the templating behavior of the gel around encapsulated proteins (34,
37), is that the cavity surrounding the encapsulated protein
preferentially limits only certain categories of conformational
changes. Transitions between protein conformations that require a
transition state that has an enhanced volume with a change in the
hydration pattern as in hemoglobin (116) may be especially vulnerable
to being damped. This vulnerability could arise from both spatial
constraints of the sol-gel matrix and possible solvent shell
stabilization effects by the Si-O groups lining the templated cavity
surrounding the hydrated protein. The dramatic slowing in the
relaxation properties of encapsulated Hb below 10 °C (46) is
suggestive of a mechanism involving the immobilization of hydration
shell water molecules.
Spectroscopically distinct encapsulated populations are seen to have
different ligand rebinding kinetics. The spectroscopic differences
implicate differences in the proximal heme environment as contributing
to the observed functional differences. The retention of kinetic
differences when the two populations are exposed to the osmotic stress
of 100% glycerol argues against variation in water occupancy within
the distal pocket as a source of the kinetic differences. Recent
findings3 on
sol-gel-encapsulated distal heme pocket mutants of Mb indicate that the
reported effects are not due to the different conformational substates associated with the distal histidine.
A comparison between sol-gel-encapsulated Mb and HbA derivatives
reveals a similar pattern of tertiary structure change upon ligand
binding. For sol-gel-trapped forms of both the R and T quaternary
states of HbA, ligand binding induces tertiary structure changes that
increase the frequency of
This general approach for locking in of equilibrium and non-equilibrium
conformational populations allows for spectroscopic and kinetic
comparisons under identical environmental conditions. The results pave
the way for the isolation and the eventual full characterization of the
pure conformational contribution to the inverse temperature effect in
which the kinetics slow instead of accelerate with increasing temperature.
The approaches described herein for Mb are anticipated to be of general
applicability for comparing the conformational and functional
properties of substrate-binding proteins by maintaining the
substrate-bound and substrate-free equilibrium conformations when
substrate is subsequently removed or added, respectively. The general
usefulness of this approach is further enhanced by the observations
that the combination of the new encapsulation protocol with the
addition of glycerol as the bathing material for the sol-gel greatly
extends the lifetime of non-equilibrium populations.
*
This work was supported by National Institutes of Health
Grants RO1 HL65188, PO1 GM58890, and RO1 HL58247 and by the W. M. Keck
Foundation.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: Dept. of Physiology and
Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave.,
Bronx, NY 10461. Tel.: 718-430-3591; Fax: 718-430-8819; E-mail:
jfriedma@aecom.yu.edu.
Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M200301200
2
T. Das and J. M. Friedman, unpublished result.
3
U. Samuni, D. Dantsker, and J. M. Friedman,
unpublished results.
The abbreviations used are:
Mb, myoglobin;
COMb, carbonmonoxy myoglobin;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol;
GY, geminate yield.
Spectroscopically and Kinetically Distinct
Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy
Myoglobin
A COMPARISON WITH HEMOGLOBIN*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the
degree of separation between the F helix and the heme is assigned as
the conformational coordinate that modulates both this frequency and
the innermost barrier controlling CO rebinding. A comparison with the
behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are
qualitatively similar to the tertiary changes that occur within both
the R and T quaternary states. Protein-specific differences in the time
scale for the proposed F helix relaxation are attributed to variations
in the intra-helical hydrogen bonding patterns that help stabilize the
position of the F helix.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Scheme 1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Fe-His). Table
I contains a summary of the relevant peak frequencies for (Fe-His). Fig. 1 displays a section of the
visible resonance Raman spectrum, comparing the Raman band arising from (Fe-His) for [deoxyMb] (A), the 8-ns photoproduct
of [deoxyMb]+CO (B), and the 8-ns photoproduct of [COMb]
(C). Also shown are the solution phase spectra for deoxyMb
(D) and the 8-ns photoproduct of COMb (E). The
absolute peak positions are accurate to ~0.5 cm
1 based
on repeated spectral acquisitions. Peak position differences for
(Fe-His) obtained when comparing spectra from different samples are accurate to within 0.2 cm1 on average when the
spectra are generated under similar conditions on the same day.
View larger version (30K):
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Fig. 1.
Visible (Soret-enhanced) resonance
Raman spectra of deoxy derivatives and of the 8-ns photoproduct of
CO-liganded derivatives of solution and sol-gel-encapsulated Mb and
HbA. The Raman spectra of the region showing the
(Fe-His) band for: A, [deoxyMb] (blue
trace); B, [deoxyMb]+CO (red trace);
C, [COMb] (green trace); D, deoxyMb
(blue trace); E, COMb (green trace);
F, [deoxyMb]+CO+100% glycerol stored for 1 year
(red trace); G, [COMb]+100% glycerol stored
for 1 year (green trace); H, [deoxyHbA]
(blue trace); I, [deoxyHbA]+CO (red
trace); J, [COHbA] (green trace). The
inset shows a larger Raman shift range for: a,
[COMb] (green trace); b, [deoxyMb]+CO
(blue trace); c, [COHbA] (green
trace); d, [deoxyHbA]+CO (blue trace). The
traces were baseline-corrected and normalized to the
(Fe-His) band (to 7 for the traces
in the inset). All solutions were 50 mM BisTris
acetate, pH 6.5; all sol-gels were bathed in a 50 mM
BisTris acetate, pH 6.5, buffer with 25% glycerol (except for
traces F and G, which are bathed in 100%
CO-saturated glycerol). Throughout this report, bracketed specie names
signify the state of the protein at the time of encapsulation;
whereas, the plus sign is followed by the chemicals added to the sample
after the sol-gel has undergone aging for at least 24 h.
(Fe-His) frequency for Mb and Hb generated from either
equilibrium deoxy derivatives or the 8-ns photoproduct of
the CO-saturated derivative
(Fe-His) for [deoxyMb] is very close to that
of the corresponding solution phase sample. Whereas in solution, the
frequency of (Fe-His) for the 8-ns photoproduct of COMb is only
a fraction of a cm1 higher than for the equilibrium
deoxyMb sample as previously reported (23, 49, 50), in the sol-gel it
is higher by ~2 cm1. This increase is also seen for
COMb samples in highly viscous media (23) and for the unrelaxed
picosecond photoproduct of COMb in solution (51). The frequency of
(Fe-His) for the 8-ns photoproduct of [deoxyMb]+CO is slightly
higher than for the [deoxyMb] sample but is still clearly lower than
for [COMb]. Traces F and G illustrate the
consequence of replacing the bathing buffer with 100% CO-saturated
glycerol for the 8-ns photoproduct Raman spectrum of [deoxyMb]+CO and
[COMb], respectively. The added glycerol has the effect of increasing
the frequency of (Fe-His) for both samples (see Table I);
nevertheless, the frequency difference between the two photoproduct
populations is still maintained. It is noted that traces F
and G are for samples that have been kept for about a year
in a cold room, after CO exposure, demonstrating that trapping of
non-equilibrium structures for extended periods of time can be achieved.
(Fe-His) for the photoproduct of the liganded T
state derivative, [deoxyHbA]+CO, is clearly higher than that of the
deoxy T state but substantially lower than that of the R state
photoproduct. This frequency is similar to what has been reported for
either mutant Hbs stabilized in the T state even when fully liganded
(52) and for the early time photoproduct of T state NOHbA+IHP
(53). Not shown is the spectrum obtained from a sample of [oxyHbA],
which had been deoxygenated through the addition of dithionite after
gelation and aging. In this case the frequency of (Fe-His) is
~223 cm1, a value much lower than that of the R state
photoproduct but at a value in the frequency range for mutant or
modified ferrous Hbs and transient forms that are in the R state
despite being ligand free (54-58).
93-SH) (44).
View larger version (17K):
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Fig. 2.
The recombination kinetics of CO to
photodissociated COMb in sol-gel and solution environments subsequent
to an 8-ns, 532-nm laser pulse. The kinetics values are displayed
on a log-log plot of normalized absorbance versus time. The
top panel shows the kinetics at 3.5 °C from COMb in
solution (trace a), [deoxyMb]+CO (trace b), and
[COMb] (trace c) with the bathing buffer containing 25%
by volume glycerol. The middle panel shows the kinetics at
3.5 °C from COMb in solution (trace d), [deoxyMb]+CO
(trace e), and [COMb] (trace f) with the
bathing buffer containing 50% by volume glycerol. The bottom
panel shows the kinetics at 
15 °C from [deoxyMb]+CO
(trace g), and [COMb] (trace h) with the
bathing buffer consisting of CO-saturated pure glycerol. Traces
g and h were obtained from glycerol bathed sol-gel
samples stored at 4 °C for a period of several months. In the
top two panels the bathing buffer used for the sol-gel
samples in each panel was also the solvent used to prepare the solution
phase sample whose kinetics are displayed in the corresponding
panel.
15 °C to
further increase the viscosity. The addition of 100% glycerol and the
further cooling is used both to eliminate possible differences due to
conformational relaxation and to further enhance the geminate rebinding
phase (23, 28, 59-64). The results obtained from kinetic studies of
[COHbA] in the presence of glycerol clearly indicate that the
glycerol diffuses into the sol-gel and imparts substantially enhanced
local viscosity to the encapsulated protein (43). An earlier study on a
COMb sample in 90% glycerol at ~270 K showed that relaxation of the
photoproduct does not occur at least out to 10 µs and that the
geminate yield is greatly enhanced (25). In a recent study (65) of the
rebinding kinetics of COMb and COMb mutants in a trehalose glass, the
different kinetic phases seen in the third panel were
assigned to geminate rebinding arising from CO localized in different
cavities within the protein (see below). Rebinding directly from the
distal heme pocket gives rise to the fastest phase, and progressively
slower phases derive from progressively more distant xenon
cavities (e.g. Xe4 and Xe1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Fe-His) frequency differences seen for the Mb samples
requires an understanding of the conformational factors effecting this
frequency. The following description is a framework for evaluating
these shifts. This description is derived from a syntheses of the many
experimental and theoretical contributions that have focused either on
the behavior of (Fe-His) or elements of structure, which are
directly related to the positioning of the iron and the proximal
histidine (47, 48, 50, 51, 53, 54, 66-77).
(Fe-His)--
There are two major conformational factors that
contribute significantly to the frequency differences among equilibrium
deoxy derivatives of myoglobins and hemoglobins. These two factors are the relative orientation of the proximal histidine with respect to the
heme and the packing or positioning of the F helix above the heme.
heme in T state
deoxyHbA) favor a configuration of the heme that has the iron with a
greater displacement out of the heme plane. An increase in the
displacement of the iron is associated with a decrease in the frequency
of (Fe-His) (66, 77).
(Fe-His). It is likely that the strong repulsive interactions between the imidazole and the heme dictate the
displacement of the iron above the heme plane. For a given
displacement, there is an optimal iron-imidazole bond length that would
be achieved for the protein-free imidazole derivative. If, through
intra-protein interactions, the F-helix is highly constrained at a
fixed position above the heme, the iron-imidazole bond length may be
either compressed or expanded relative to the optimal length that would
occur either in a model system or in a protein where the positioning of
the F-helix is fully responsive to the iron displacement. Compression and expansion of the bond will increase and decrease the frequency of
(Fe-His), respectively.
(Fe-His): the hydration status of the heme environment and the
strength of the hydrogen bond associated with the N
of the proximal
histidine. For deoxyMb, the loss of water from the heme environment,
either through osmotic effects (23) or the replacement of distal
residues with more hydrophobic residues (72), results in a maximum
2-cm1 decrease in the frequency of (Fe-His).
Enhancing and weakening of the hydrogen bond associated with the
N of the proximal histidine have been shown to increase and decrease
the frequency of (Fe-His), respectively (69, 78), although these
effects are not likely to be a factor for Mb and Hb.
(Fe-His) for the Early Time
Photoproduct--
The spectra of the photoproduct of COMb and COHb are
determined by the initial events occurring subsequent to
photodissociation. It has been shown that the displacement of the iron
out of the heme plane upon photodissociation occurs within a fraction
of a picosecond (79, 80). Under most circumstances this seemingly ballistic motion will precede any relaxation involving movements in the
helices that have been triggered by the dissociation. Thus, in
general, there is an adiabatic type partitioning of the early events in
that, within the first picosecond, the iron has undergone a
displacement out of the heme plane and the overall tertiary structure
remains that of the initial liganded species. At some later time, the
tertiary structure begins to respond. Whereas the time scale for the
protein relaxation is highly dependent on solution conditions to the
extent that they can be greatly slowed by enhancing the solution
viscosity, the rapid motion of the iron appears insensitive to
environmental factors. There is Raman evidence that the iron
displacement can be inhibited only at liquid helium temperatures (18,
20, 21, 48).
(Fe-His) will reflect a species (or
population) where the iron has undergone displacement and the key
variable in determining frequency differences will be the extent to
which the protein tertiary structure has evolved. Under conditions
where the encapsulated samples are bathed with 100% glycerol, the
expectation is that the photoproduct spectra will reflect a population
that has the iron out of the heme plane but with the tertiary structure
still largely reflective of the distribution of conformations
associated with the initial liganded derivative.
(Fe-His). This result indicates that the
encapsulation protocol does not perturb the equilibrium conformational
distribution of deoxyMb and does not exert any undue osmotic stress on
the heme environment. The addition of high concentrations of
glycerol to the encapsulated deoxyMb sample does induce a reduction in
the frequency2 that is seen
in solution (23).
(Fe-His) over the
increase achieved through encapsulation. This "upper limit"
frequency is the same as that which we obtain from samples of COMb
embedded in an ultra high viscosity glass matrix derived from
trehalose. Earlier studies show that increasing the viscosity slows and
eventually stops the tertiary relaxation of photodissociated COMb (23,
25, 28, 59, 60, 62, 64, 81-86). These findings all indicate that the
distribution of photoproduct conformations being probed for the
[COMb] +100% glycerol sample is reflective of the initial liganded
protein but with the iron displaced out of the heme plane (and the heme geometry appropriately modified).
(Fe-His). It appears that there is a
clam-shell type rotation of the E and F helices upon switching from the
deoxy to CO derivative. For the CO derivative the F helix is more
tightly packed over the heme. Relaxation of the F helix away from the
heme upon photodissociation of COMb in a crystal has been observed to
occur within nanoseconds of photodissociation (17). Picosecond IR
absorption studies on photodissociated COMb also indicate that
substantial relaxation of the F helix occurs on the picosecond time
scale (87). Based on these findings, we attribute the high frequency
end point for the 8-ns photoproduct (Fe-His) ([COMb]+100%
glycerol, COMb in trehalose glass) as arising from a situation where
the F helix has not yet shifted from its COMb position. As a
consequence, the Fe-His bond is compressed yielding an increased
frequency for (Fe-His). In solution the F helix shifts on a
subnanosecond time scale accounting for the rapid picosecond decrease
in the frequency of (Fe-His) subsequent to photodissociation of
COMb (51) and the minimal frequency difference seen between deoxyMb and
the 8-ns photoproduct of COMb (23, 49, 50). This rapid shifting is
likely to account for at least some of the rapid picosecond relaxation
observed (88) for Band III, a near IR charge transfer band
sensitive to both proximal and distal effects (18, 72, 89-91).
(Fe-His) for the 8-ns photoproduct of the
[COMb] sample bathed with buffer containing 25% glycerol is lower than that of the [COMb]+100% glycerol sample yet higher than that of
COMb in just the buffer (also containing 25% glycerol). In the context
of the above discussion, it would appear that the sol-gel allows some
relaxation of the initial conformation within the 8-ns time
period but clearly not to the extent seen in solution. The
question now arises as to whether the sol-gel is merely slowing the
full relaxation process that occurs within solution or is imposing
limitations as to the extent of relaxation, i.e. whether the
sol-gel actually traps a conformational population that has a bounded
range of accessible (Fe-His) frequencies.
(Fe-His) upon addition of the 100% glycerol, the frequency for the [COMb] sample is always higher. This result is consistent with the sol-gel biasing the encapsulated population toward that of the initially encapsulated Mb derivative. Thus the range of accessible frequencies for (Fe-His) for the [deoxyMb]+CO sample is bounded by the
constraints of the overall deoxyMb tertiary structure, whereas for
[COMb] the bounded range is determined by the tertiary conformation
of COMb. The glycerol-bathed samples yield different upper frequency limits for the 8-ns photoproduct, reflecting a different upper limit
for each of the initially trapped populations.
(Fe-His) is achieved for the deoxy derivatives, but the T state
has a lower limit (~214 cm1) than the R state (~ 220 cm1). Similarly the upper limit for the range of
accessible values for this frequency is achieved for the unrelaxed
photoproduct of fully liganded derivatives, with the unrelaxed R state
species having a higher upper limit (230 cm1) than the
unrelaxed T state species (~222 cm1). A comparison of
the behavior of Mb and Hb with respect to ligand binding-induced
tertiary conformational changes as reflected in the proximal heme
pocket indicates a similar trend for both proteins.
(Fe-His). As suggested from x-ray crystallographic
data (92-97), it appears that the quaternary structure sets boundaries
for a range of accessible ligation state-dependent tertiary
conformations. The pattern observed for Mb is very similar to that seen
for Hb within either quaternary state. Ligand binding induces tertiary
structure changes that increase the frequency of (Fe-His). This
similarity is consistent with the proposed E-F helix clam-shell type
rotational motions for both Mb (14) and HbA (58, 98). It is also
interesting that both the high and low frequency end point values for
Mb and Hb are not the same. The higher frequency for the R state
photoproduct (230 vis à vis 223 cm1) has
been attributed to the quaternary enhancement effect due to a
quaternary structure-induced compaction of the Fe-His bond (69, 98);
whereas, the lower deoxy T state value (~205 cm1 for
the T state deoxy subunit vis à vis 220 cm1 for deoxyMb) is due to a quaternary constraint that
induces a proximal strain on the subunit through a tilting of the
proximal imidazole (47, 48, 52, 54, 55, 99).
(Fe-His) for
[COHbA] is identical to the solution phase value, the value is higher for [COMb]. This difference in the behavior of the two proteins is
readily explained based on their respective relaxation properties. Both
proteins undergo a substantial slowing in the tertiary relaxation of
the photoproduct with increasing viscosity (23, 25, 62, 64, 83-85, 88,
100). In the case of aqueous COMb, the relaxation is largely complete
within tens of picoseconds but with some residual components persisting
well beyond 10 ns (23, 28, 48, 50, 51, 62, 64, 82, 88, 101-103). In
dramatic contrast, for COHbA, the tertiary relaxation does not begin
until several nanoseconds subsequent to photodissociation (48, 57, 58,
103-106). Consequently, slowing down the relaxation will, in the case
of Mb, eventually allow the unrelaxed photoproduct to persist to beyond
nanoseconds, whereas for HbA, the photoproduct already persists beyond
nanoseconds. This assessment is consistent with our observation that,
for COHbA, there is no change in the 8-ns photoproduct frequency of
(Fe-His) in going from aqueous buffer to either a solution with
added glycerol (100) or to an ultra high viscosity trehalose glass
matrix (107). In contrast, the addition of glycerol to the solution or
the use of a trehalose glass results in an increase in the frequency
for the 8-ns photoproduct of COMb.
helices
(71, 98, 108, 109). By damping rapid change in inter-helical spacing,
the scaffolding will slow the response time of the E-F helices to the
sudden local changes associated with ligand dissociation/association.
Enhancement of this scaffolding has been invoked to account for
dramatic slowing in tertiary relaxation seen in the mutant hemoglobin,
Hb(Ypsilanti) (98).
(Fe-His) in going from the deoxy R derivative to the 8-ns
photoproduct to a compression of the Fe-His bond for the photoproduct
due to the intra-protein damping of the F helix motion away from the
heme. In contrast, for COMb the increase in the frequency for the 8-ns
photoproduct is apparent only when the external viscosity damps the
rapid motion of the F helix that leads to the subnanosecond
decompression of the Fe-His bond that is seen in solution.
(Fe-His) has been
related to proximal strain that is a measure of the protein-related amount of extra work needed to move the iron into the heme plane upon
ligand binding (47, 48, 52-54, 99, 110-112). The lower the frequency,
the greater is the energetic cost of creating a stable six-coordinate
ferrous heme derivative. To the extent that the kinetic barrier is
determined in part by a transition state that has the iron at least
partially moved toward the heme plane, as is believed to be the case
for the CO ligand (47, 113, 114), the binding rate will be responsive
to proximal strain. This assessment is consistent with pressure studies
on Mb in which increased pressure both increases the frequency of
(Fe-His) and increases the geminate yield (115). In the case of
the Mb samples, the difference in the kinetics arising from proximal
effects would stem from the positioning of the F helix. In the case of
the photodissociated glycerol-bathed [COMb] sample, the F helix is
already at the equilibrium COMb position and consequently there is no
extra "proximal" work associated with moving the iron back into the
heme plane upon CO rebinding. For samples whose photoproduct spectra
exhibit frequencies for (Fe-His) that are less than the upper
limit, the rebinding of CO requires the extra work of moving the F
helix. In the absence of any modification of the heme-histidine
stereochemistry (e.g. tilting or rotation), which alters the
steric interactions between the heme and the proximal imidazole, the
range of accessible values of (Fe-His) is an indication of the
dynamic range for the positioning of the F helix above the heme.
(Fe-His). Although the pattern of
ligand-induced change is similar to that of Mb, both the magnitude of
frequency change and the range of accessible frequencies for HbA are a
function of the quaternary state.
FOOTNOTES
Present address: Dept. of Chemistry, Bowdoin College, Brunswick,
ME 04011.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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