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B. Evidence for a Ligand Docking Site in the Heme Pockets of Mb and Hb

B. Evidence for a Ligand Docking Site in the Heme Pockets of Mb and Hb

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Infrared Studies in Heme Proteins



211



Figure 6 Time-resolved mid-IR spectra of photolyzed h-Mb13 CO:D2 O (gray

line), sw -Mb13 CO:D2 O (filled circles), and Hb13 CO:D2 O (black line). The spectra

were recorded at 100 ps and 283 K with the A-state region (A) and B -state region

(B) collected independently. The A- and B -state designations follow the convention

of Alben et al. (33). To facilitate comparison among the spectra, the constant and

linear contributions to the polynomial fit of the background have been subtracted

from the measured spectra.



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appears to be conserved across the proteins h-Mb, sw -Mb, and the ˛ and ˇ

chains of Hb, suggesting that this environment is functionally relevant. The

relatively narrow B-state linewidths suggest that these proteins fashion a

docking site that imposes orientational constraints on “docked” CO. Moreover, this docking site may mediate the binding and escape of ligands.

To justify these conclusions, we discuss the temperature dependence of

B -state spectra and the theoretical basis for interpreting the near-ambient

temperature spectra.

1. Temperature Dependence of B-State Spectra

The B states have also been cryogenically trapped and characterized with

static IR spectroscopy (33). The IR spectrum of MbŁ CO, obtained by photolysis of sw -MbCO:G/W at 5.5 K, reveals three features denoted B0 , B1 , and

B2 , whose integrated absorbance is 21.7 š 1.6 times smaller than that for

the A states. The B0 state contains approximately 17% of the integrated Bstate absorbance and has a vibrational frequency that is the same, within

experimental error, as gas phase CO (2143.3 cm 1 for 12 CO; 2096 cm 1

for 13 CO). It was concluded that B0 corresponds to CO that is “free” within

the heme pocket. Because the center frequencies of B1 and B2 are modestly

red-shifted from the gas phase CO frequency (12.8 and 24.3 cm 1 shifts for

B1 and B2 , respectively), the CO is not covalently bound but does interact

weakly with residues in the heme pocket. The integrated B1 absorbance

was found to be about 1.96 š 0.05 times that of B2 , but upon warming the

photolyzed sample above ¾13 K, B1 grew at the expense of B2 while the

total B-state integrated absorbance remained constant. The relative populations of B1 and B2 do not revert upon cooling the thermally annealed sample

back down to 5.5 K. These results suggest that (1) B2 is higher in energy

than B1 , (2) B1 and B2 share the same oscillator strength, and (3) B1 and B2

do not correspond to different protein conformations, but rather to CO in a

similar proximity but in a different orientation.

The B states generated by photolyzing various heme proteins at 283 K

(11) are similar but not identical to those measured when photolyzing

sw -MbCO:G/W at 5.5 K (33). The B0 state is not seen in any of the

three proteins shown in Fig. 6B. The integrated absorbance of B1 and

B2 at 283 K is 32.8 š 1 times smaller than that for the A states. In sw MbCO:D2 O, the integrated absorbance of B1 is approximately 1.94 times

that for B2 , but in h-MbCO and HbCO that ratio is closer to 1.5-1.6. The

linewidths of B1 and B2 at 283 K are approximately twice as broad as

those at 5.5 K. The center frequencies of B1 and B2 in sw -MbCO:G/W

at 283 K were estimated to be 1-2 cm 1 blue shifted compared to those



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Infrared Studies in Heme Proteins



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at 5.5 K. [Although the B states for sw -MbCO:G/W at 283 K were not

measured directly, we could predict their center frequencies by comparing

the frequencies of sw -MbCO:D2 O vs. h-MbCO:D2 O (species-dependent

shift) and those for h-MbCO:D2 O vs. h-MbCO:G/W (solvent-dependent

shift) (11).]

The 1-2 cm 1 blue shift in the near-ambient spectra can be readily

explained. Both B1 and B2 were found to blue shift nonexponentially with

time after photolysis, with B1 experiencing a larger shift. This time-dependent

blue shift is a consequence of the conformational relaxation that can occur

under ambient conditions (39,46–48) but is inhibited at temperatures below

the ¾185 K glass transition of the protein (24). Due to the similarity between

the cryogenic and near-ambient temperature B-state center frequencies, the

cryogenic B states appear to be the same as those measured near ambient

temperature, but trapped within a conformationally unrelaxed protein.

The major differences between the cryogenic and near ambient

temperature B1 and B2 states are their linewidths and integrated

absorbances. In discussing integrated absorbance, a distinction must be

made between the integrated absorbance and the integrated oscillator

strength for the transition. The integrated absorbance is a measured

quantity that relates to experimentally resolved features, while the integrated

oscillator strength refers to a theoretical quantity that is intrinsic to the

vibrational transition. For example, the measured integrated absorbance will

be deficient when a portion of the oscillator strength resides in a feature

broad enough to be subsumed by the background.

Because absolute absorbances are difficult to determine experimentally, we confine ourselves to relative absorbances and use the A states as

an internal standard. The ratio of integrated A- to B-state absorbance at

5.5 K was reported to be 21.7 š 1.6 (33). What should the measured ratio

be at 283 K? In general, the integrated oscillator strength is intrinsic to a

vibrational transition, and if it is only weakly coupled to other oscillators,

it can be assumed to be temperature independent (provided the population resides primarily in its ground vibrational state). Therefore, we assume

the integrated B-state absorbance at 283 K should be the same as that

at 5.5 K. In contrast, the A-state integrated absorbance exhibits a weak

temperature dependence with the integrated absorbance at ³283 K being

0.8 of that measured at 30 K (49). It has been suggested that the loss of

integrated oscillator strength at elevated temperature is due to a temperaturedependent change in electron-nuclear coupling (23). If one assumes the

B-state integrated absorbance measured at low temperature to be equivalent to its integrated oscillator strength (a reasonable assumption given



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Lim et al.



the spectral broadening expected due to thermal motion at 5.5 K is less

than the measured linewidth), then the integrated A- to B-state ratio at

283 K should be approximately 17. Instead, the average of the integrated

A- to B -state ratios measured for Mb and Hb at 283 K is 32.8 š 1. Consequently, the B-state features of Fig. 6B contain only about 53% of the

integrated absorbance expected. Where is the “missing” half of the integrated B-state absorbance at 283 K? The answer becomes clear in the next

section, where the relationship between motional dynamics and spectra is

considered.

2. Relationship Between B-State Spectra and CO Motional Dynamics

Because the rapid initial decay of the orientational dipole correlation function is independent of environment, the spectrum of CO trapped in a protein

at 283 K should exhibit a feature approximately as broad as the envelope

containing its gas phase P and R branches. One would also expect to see a

narrower feature or features on top of the broad pedestal with the integrated

oscillator strength of CO partitioned between the broad and narrow features

in proportion to the relative amplitudes of the fast and slow decay of the

orientational correlation function. Finding approximately half of the integrated CO oscillator strength under the narrow features suggests that the

relative amplitudes of the fast and slow decay of the orientational dipole

correlation are comparable. This conclusion is in near-quantitative agreement with the dipole correlation function of CO in Mb obtained from

molecular dynamics simulations using CHARMM (50). The “missing” half

of the integrated oscillator strength is not missing at all, but is contained

within a broad pedestal under the narrow features. Because the width of

the pedestal is more than an order of magnitude broader than the narrow

features, its amplitude is approximately an order of magnitude smaller and

is, therefore, difficult to extract from the solvent background in the experimental time-resolved IR absorbance spectra.

The relatively large amplitude of the slow-decaying contribution to

the orientational correlation implies that the protein environment imposes

relatively severe constraints on the orientational freedom of CO. Indeed,

the mean angular displacement from equilibrium has been estimated to

be ³38 degrees (11). Consequently, we have concluded that the highly

conserved protein residues on the distal side of the heme fashion a

“docking” site where ligands can become trapped (11). This docking site

mediates the transport of ligands to and from the active binding site and

may function to discriminate between ligands such as O2 , CO, and NO.



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Infrared Studies in Heme Proteins



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C. Orientation of Bound and ‘‘Docked’’ CO



Time-resolved polarized IR absorbance spectra of photolyzed MbCO are

shown in Fig. 7. The A-state spectra reveal two overlapping features,

denoted A1 and A3 after Ormos et al. (17), with A1 blue-shifted relative

to A3 . The ratio of the polarized absorbance, A? /Ajj , is nearly constant



Figure 7 Polarized IR absorption spectra and their ratio measured 100 ps after

photolysis of Mb13 CO in D2 O. The left axis corresponds to the photolysis-induced

absorbance changes, A? (thick lines) and Ajj (thin lines); the right axis

corresponds to their ratio A? /Ajj (open circles). The ratio is plotted where the

absorbance exceeds ¾25% of its maximum and has been corrected for fractional

photolysis. The dashed lines correspond to the average A- and B -state ratios. The

A- and B -state spectra were collected at 10.8% and 20% photolysis of Mb13 CO,

respectively. The background and hot band contributions to the B -state spectra have

been removed. (Adapted from Ref. 51.)



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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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