C. Orientation of Bound and ‘‘Docked’’ CO
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1.931 š 0.02 across these features, demonstrating that the transition
moments for the two A states are oriented at a similar angle. From the
measured polarization ratio, the equilibrium angle Âeq for the transition
moment was estimated to be Ä7° (51). The upper limit of 7° obtains in
the limit where the heme is a perfectly flat circular absorber, and this
value is in excellent agreement with static measurements of the polarization
anisotropy in ambient temperature MbCO crystals (52–54). The two spec˚ crystal structure of
troscopic results are significantly different from a 1.5 A
P21 MbCO, where the highly conserved distal histidine purportedly caused
CO to bind in a nonoptimal bent geometry of 39° and thereby inhibited the
˚ crystal structure of P6
binding of CO (55). In contrast to that result, a 2 A
MbCO reported an angle of 19° (56). Spiro and Kozlowski (18) suggested
that the discordant results between IR spectroscopy and x-ray crystallography might be rationalized by density functional theory, which was used to
explore the relationship between the direction of the CO transition moment
and the C–O bond axis when bound to Mb. They found that a 7° angle
between the heme plane normal and the transition moment of bound CO
could correspond to a C–O angle as large as 15° , but certainly not 39° .
The discrepancy between IR spectroscopy and x-ray crystallography
˚
became negligible when Bartunik and coworkers reported a 1.15 A
resolution structure of MbCO at ambient temperature and found the C–O
angle to be approximately 12° with respect to the heme plane normal (57).
The near-quantitative agreement obtained between IR spectroscopy and
atomic resolution x-ray crystallography leaves little doubt that the primary
source of ligand discrimination between CO and O2 is something other than
steric hindrance. Rather than suppression of the binding affinity of CO, it
has been suggested that ligand discrimination arises from enhancement of
the binding affinity of O2 by formation of a hydrogen bond with the distal
histidine (58).
The B -state spectra reveal two features, denoted B1 and B2 after
Ormos et al. (17), with B1 blue-shifted relative to B2 . According to Fig. 7,
the polarized absorbance ratio A? /Ajj for “docked” CO is much closer
to 0.75 than it is to 2, demonstrating that CO rotates substantially upon
dissociation from the heme iron. According to Fig. 7, the two B states
reveal a similar ratio, A? /Ajj D 0.856 š 0.03, which was found to be
consistent with Âeq ¾ 90° (11,51). X-ray structures of MbŁ CO at cryogenic temperatures reveal electron density assigned to unbound CO that
˚ from the binding site (59,60). The CO orientation from
is displaced Ä2 A
those structures is not inconsistent with the polarized IR results.
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Infrared Studies in Heme Proteins
217
Because the electrostatic field in the vicinity of the docking site is
anisotropic, the vibrational frequency of “docked” CO should be Stark
shifted and the direction of that shift should depend on the CO orientation.
Consequently, the two B-state features are interpreted as Stark-shifted
spectra with B1 and B2 corresponding to CO pointing in opposite directions.
Unlike the polarized absorbance ratio measured for the A states, the ratio
for the B states is vibration-frequency dependent with the polarization ratio
a maximum at the midpoint between the two features. Because the center
frequency of CO depends on its orientation, its vibrational frequency should
shift smoothly from one B state frequency to the other as it undergoes endto-end rotation. Consequently, one might expect the vibrational frequency
of CO near the transition state for end-to-end rotation to be centered
between the peaks of the two B states. At the midpoint, the ratio A? /Ajj
exhibits a maximum of ¾1, demonstrating that the trajectory for end-to-end
rotation passes through a transition state that has a component out of the
plane of the heme. If this trajectory were to lie in a plane, the transition
state would be oriented at least 55° from the heme plane normal (51). Such
a trajectory would maintain a ligand orientation far from that for bound
CO, even at the transition state for end-to-end rotation, thereby inhibiting
CO binding while permitting end-to-end rotation.
D. Ligand Translocation Trajectories
To experimentally probe the CO trajectory after dissociation, ultrafast
time-resolved polarized mid-IR spectra of photolyzed h-MbCO in G/W
were recorded (34), the results of which are plotted in Fig. 8A. This
study was performed in G/W primarily because the flatness of the solvent
absorbance spectrum near 2100 cm 1 minimizes temporal distortion of the
transmitted femtosecond IR probe pulse, thereby maximizing the effective
time resolution of the measurement. Two features are already apparent at
0.2 ps, the earliest time shown, and these features rapidly develop into
the “docked” states denoted B1 and B2 . The development of the “docked”
CO spectrum is further quantified by the time dependence of the polarization anisotropy, as defined in Equation (2). The B1 and B2 polarization
anisotropies, plotted in Fig. 8B, evolve exponentially with time constants
of 0.20 š 0.05 ps and 0.52 š 0.10 ps, respectively, and converge to the
same anisotropy of approximately 0.2. According to Fig. 8C, ligand translocation is accompanied by a 1.6 š 0.3 ps growth of the integrated isotropic B-state absorbance.
Copyright © 2001 by Taylor & Francis Group, LLC
218
Lim et al.
Figure 8 (A) Femtosecond time-resolved IR absorbance spectra of CO measured
after photodissociation from the heme of h-Mb in G/W. Spectra were recorded
with the photolysis and probe pulses polarized parallel Ajj and perpendicular
A? to one another. The polarized absorbance spectra reveal the time dependence
of the ligand orientation as well as the protein surroundings (see text). The
features labeled B1 and B2 (bottom) correspond to the least-squares fit of the
Ajj spectrum measured at 10 ps. For clarity, the background and hot band
contributions to the time-resolved spectra have been removed and the spectra
have been offset from one another. (B) Time dependence of the polarization
jj
?
B?
anisotropy, r t D [Bjji t
i t ]/[Bi t C 2Bi t ], after photodissociation from
the heme of Mb. Bi t represents the integrated absorbance under state i at time t
with the polarization denoted by a superscript. The polarization anisotropies of
B1 and B2 appear to evolve exponentially (solid lines) with time constants of
0.2 ps and 0.52 ps, respectively. (C) Time dependence of the isotropic B -state
absorbance (filled squares). The relative contribution of B1 to the total absorbance
is time independent out to 10 ps (open squares) and averages 56% (dashed
line). To generate these data, the isotropic absorbance was synthesized from
the polarized absorbance spectra according to the “magic” angle prescription:
AMA t D [Ajj t C 2A? t ]/3. (Adapted from Ref. 34.)
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Infrared Studies in Heme Proteins
219
How do we rationalize these observations? Photoexcitation of MbCO
renders the Fe–CO coordinate repulsive, causing CO to acquire translational
kinetic energy as it moves up and away from the heme iron. Before the
CO rotates, its polarization anisotropy should be approximately 0.19, and
its mid-IR absorbance should be confined to a single, broad feature. Upon
colliding with the surrounding protein, the ligand rebounds back toward the
heme and proceeds along one of two different trajectories. Because ligand
translation and rotation are not slow compared to 0.2 ps, the polarization
anisotropy measured at 0.2 ps is not the theoretical minimum of 0.19, nor
is the spectrum manifested as a single broad feature. As the CO translates
and rotates, the two trajectories become spectroscopically distinguishable,
owing to the vibrational Stark shift that arises from the electrostatic field
surrounding the ligand. The fact that B1 and B2 evolve at different rates
demonstrates that the trajectories leading to B1 and B2 are distinguishable
kinetically as well as spectroscopically. The polarization anisotropy for both
B1 and B2 converges within a few ps to ¾0.2. Had the polarization anisotropy
been measured using a wavelength where the heme is a perfectly flat circular
absorber, the polarization anisotropy would rise to only about 0.05. By
photolyzing at a wavelength where the heme is an elliptical absorber, the
range of the polarization anisotropy was enlarged, thereby improving the
signal-to-noise ratio of the measurement. Moreover, the fact that the polarization anisotropy exceeds 0.05 suggests that the major axis of the elliptical
heme absorber must be at least partially aligned with the C–O axis. If the
orientation of the major axis of the heme transition moment were known
as a function of wavelength, polarization anisotropy measurements at more
than one wavelength would permit a determination of both the azimuthal
CO orientation and its angle with respect to the heme plane normal.
The prompt appearance and independent development of the two B
states suggest that the two trajectories are deterministic in nature, with
the outcome (B1 or B2 ) established promptly after photodetachment. What
do the two limiting states correspond to structurally? From a kinematic
argument, it was rationalized that the faster B1 trajectory has CO sliding
into the docking site with the O end of C–O pointing toward the heme
iron (34). This structural assignment is supported by geminate rebinding
studies of photolyzed MbCO at 20 K, where B1 predominates: the geminate
rebinding of 13 C16 O was found to be slower than 12 C18 O, in spite of the
latter being heavier (61). Because geminate rebinding at 20 K is dominated
by tunneling (61) and the tunneling rate depends on distance as well as
mass, this surprising isotope effect can be rationalized by orienting C–O
such that the O end is pointing toward the heme iron. The orientation of the
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Lim et al.
CO in the docking site is too fine a detail to be extracted from the electron
density maps of photolyzed MbCO (59,60).
Interestingly, the proportion of the integrated absorbance ascribed
to B1 (plotted in Fig. 8B) remains largely unchanged during the protein
conformational reorganization. Assuming the integrated area under each
B state is proportional to its population, an assumption for which there
is experimental support (33), the partitioning between B1 and B2 is 1.3:1.
This ratio is not far from the statistical (1:1) distribution expected with
two possible CO orientations. The time independence of this ratio (out to
10 ps) provides added support for the suggestion that the ligand dissociation trajectories are deterministic. The ratio does not remain constant out to
longer times, however, but increases to 1.7:1 by 100 ps (51), demonstrating
that B1 is lower in energy than B2 and that end-to-end rotation between 10
and 100 ps renders the distribution thermodynamic rather than statistical.
If the integrated areas under B1 and B2 are indeed proportional to population, then the ratio of 1.7:1 corresponds to a free energy difference of
about 1.2 kJ/mol with B1 lower in free energy. Evidently, there is a modest
preference for the trajectory leading to B1 , which also turns out to be the
more stable state thermodynamically.
Because the interior of Mb is densely packed, ligand translocation
from the active binding site to the docking site requires some degree of
protein rearrangement, a process that should affect the vibrational spectrum
of “docked” CO. Moreover, the conformational response of the protein
should be more sluggish than the motion of the ligand. Might the 1.6 ps
growth of the integrated isotropic B -state absorbance be assigned to protein
rearrangement, or might it arise from other causes? One often equates
changes in integrated absorbance with changes in population, however, that
is not the case here: all CO produced photolytically is generated in less than
0.2 ps, the time resolution of the measurement. Might the growth be due
to thermal cooling of the CO and its environment? Because “docked” CO
is in contact with the heme, its kinetic temperature would be expected to
cool at a rate similar to the heme, which was found to thermally relax
with a time constant of 6.2 š 0.5 ps (22). Because the 1.6 ps growth of
the integrated B-state absorbance is longer than the 0.2 and 0.5 ps rotation times and shorter than the 6.2 ps cooling rate, it cannot be ascribed
to population or cooling dynamics. Rather, it most likely arises from reorganization of the neighboring protein residues about the nascent “docked”
CO. Recall that the integrated absorbance of CO is partitioned between
the narrow B states and a broad unresolved pedestal, the partitioning of
which is determined by the orientational constraints imposed on CO by the
Copyright © 2001 by Taylor & Francis Group, LLC
Infrared Studies in Heme Proteins
221
surrounding protein. The more constrained the CO, the smaller the amplitude of its librational motion, and the greater the integrated absorbance
measured under the narrow B-state features. A 1.6 ps time constant for
protein rearrangement that serves to constrain “docked” CO appears to be
quite reasonable. Evidently, this process helps to establish a steric barrier to
the reverse rebinding process. That this steric barrier arises before the electronic state of the heme relaxes to its ligand-receptive ground state (3.4 ps)
may explain the absence of ultrafast geminate ligand rebinding.
E. Origin of the Barrier to CO Rebinding
Photolysis of MbNO and MbO2 is followed by substantial geminate recombination (28,62–64) with NO rebinding on the sub-ns time scale. On the
other hand, geminate recombination of CO in Mb is minimal and occurs on
the few hundred ns time scale (65). The lack of significant CO rebinding is
˚ away from the
remarkable considering the ligand remains “docked” Ä2 A
binding site for several hundred ns. It had been suggested that the kinetic
differences among these ligands arise from differences in the electronic
barrier to binding, with CO having the highest electronic barrier and NO
having the lowest (66). The discovery of a ligand docking site that can
constrain the orientation of “docked” ligands as small as CO (11) lead us
to consider another possibility. The docking site might slow the rebinding
rate of CO by strongly hindering access to the transition state for CO
rebinding. Because O2 and NO both bind in a bent configuration, access to
their transition state for rebinding is far less hindered.
To explore the possibility that slow CO rebinding is a consequence
of a steric, not an electronic barrier, the geminate rebinding dynamics of
CO to Mb and microperoxidase were compared (67). Microperoxidase is
an enzymatically digested cytochrome c oxidase that consists of a heme
with a “proximal” histidine that is part of an 11-peptide fragment. This
peptide renders the heme soluble under the neutral conditions used in the
Mb studies. When reduced to Fe(II), 13 CO binds to microperoxidase and
a vibrational stretch near 1908 cm 1 appears. This frequency is virtually
identical to that found in Hb13 CO and is similar to the 1900 cm 1 transition
found in Mb13 CO. Consequently, extracting the heme out of the protein
appears to have only a minor effect on the heme-CO interaction and, one
might assume, the electronic barrier to ligand binding. Because the peptide
is not long enough to wrap around and fashion a docking site on the distal
side of the heme, photodissociated CO will be surrounded by disordered
solvent, not a highly organized docking site. Any differences in the rates
of geminate rebinding to Mb and microperoxidase might, therefore, be
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222
Lim et al.
ascribed primarily to the steric constraints imposed by the docking site
in Mb.
The geminate-rebinding dynamics measured after photolysis of
MbCO and microperoxidase-CO are shown in Fig. 9. The survival fraction
denotes the fraction of photolyzed hemes that remain in the deoxy form
after CO dissociation. The population was determined by measuring the
time dependence of the vibrational absorbance of bound CO. According to
Fig. 9, CO rebinds to microperoxidase much more rapidly than to Mb.
Figure 9 Geminate recombination after photolysis of MbCO (ž) and microperoxidase-CO ( ). The survival fraction refers to the population that remains unbound
after photolysis. The population was determined by measuring the IR absorbance
at frequencies corresponding to the peak of the bound CO stretch. (Adapted from
Ref. 67.)
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