Altered Ligand Rebinding Kinetics Due to Distal-side Effects in Hemoglobin Chico (Lysbeta 66(E10) right-arrow  Thr)*

Celia BonaventuraDagger §, Joseph BonaventuraDagger , Daniel Tzu-bi Shih, E. Timothy Ibenparallel , and Joel Friedman**

From the Dagger  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, parallel  IBM, San Jose, California 95193, and the ** Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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 beta -chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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 (Lysbeta 66(E10) right-arrow 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 beta -chains of Hb Chico is similarly lowered relative to normal beta -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).

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 beta 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 beta -chains and Thrbeta 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 beta -chain of Hb A0 is still debated. Lowered oxygen affinity is observed for both isolated beta -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).

The conserved distal His(E7) residue, with which Thrbeta 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 alpha -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).

This report describes the results of a further exploration of the functional properties of Hb Chico and its isolated beta -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 beta -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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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 beta -chains of Hb A0 and Hb Chico was accomplished with the method described by Geraci et al. (18). Regeneration of the beta  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.

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-1.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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 beta -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 beta -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 beta -subunits using data of Table I.

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(beta of Hb A0)/km(beta of Hb Chico) = 0.97. CO migration out of the heme pocket is also largely unaffected by the substitution.

                              
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Table I
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)

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(beta of Hb A0)/kb(beta 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 beta -chains is half as fast as for Hb A0 and normal beta -chains (see "Discussion").

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 pi pi * 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 nu 4 (1350-1385 cm-1) (24) and 2) the iron-proximal histidine band, nu (Fe-His) (200-240 cm-1) (26, 27). The nu 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, nu 4 is ~1374 cm-1, whereas for deoxy-Hb and photolyzed Hb, nu 4 is ~1352 cm-1. The frequency of nu 4 for the five-coordinate species is sensitive to both quaternary and tertiary structure of hemoglobins (27). Fig. 3 shows nu 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 nu 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.

The difference in fractional intensity of the 1356/1375 cm-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.

The nu (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 nu  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 nu (Fe-His) could be detected between the Hb Chico structures and those measured or reported for Hb A0 (28, 30).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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 beta -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.

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 right-arrow 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.
<UP>A</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>ba</UP></SUB></LL><UL>k<SUB><UP>ab</UP></SUB></UL></LIM> <UP>B</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>cb</UP></SUB></LL><UL>k<SUB><UP>bc</UP></SUB></UL></LIM> <UP>C</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>sc</UP></SUB></LL><UL>k<SUB><UP>cs</UP></SUB></UL></LIM> <UP>S</UP>
<UP><SC>Scheme</SC> I</UP>
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 nu (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 congruent  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 congruent  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) right-arrow 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 beta -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 beta -chains is similarly lowered relative to normal beta -chains. These equilibrium properties indicate that the well depth for bound ligand (Well A) in Hb Chico as well as its isolated beta -chains is significantly decreased relative to that of Hb A0 and normal beta -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 beta -chains relative to Hb A0 and its beta -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 beta -chains (as tetramers) of both Hb A0 and Hb Chico exhibit less geminate rebinding than the corresponding alpha 2beta 2-tetramers. The effect is especially noticeable for the CO derivatives, where the beta 4-tetramers show much slower rates of CO rebinding than the alpha 2beta 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 alpha beta -dimers. The limitations of this simple view were indicated by studies showing that the last available subunit in R-state alpha 2beta 2-tetramers binds ligands with higher affinity than alpha beta -dimers (51). This phenomenon, termed quaternary enhancement, was shown in recent studies to be evident in an increased geminate yield for fully liganded alpha 2beta 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 alpha 2beta 2-tetramer that favors rebinding relative to that for alpha beta -dimers (29). This more favorable environment is reflected in the higher frequency of nu (Fe-His) for the photoproduct of alpha 2beta 2-tetramers compared with either alpha beta -dimers or isolated alpha - and beta -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 beta 4-tetramers and alpha 2beta 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 beta 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 beta -chains and Thrbeta 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.

    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES

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.

    ACKNOWLEDGEMENT

We thank Dr. John Howard (Chico Medical Group of California) for the generous contribution of blood samples.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviation used is: Mb, myoglobin.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSIONS
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