Distal Histidine Stabilizes Bound O₂ and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

Abstract

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O₂, CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20–500-fold increases in the rates of O₂ dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O₂ in both the α and β chains (ΔG_{His(E7)H-bond} ≈ −8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k_O2′, k_NO′, and calculated bimolecular rates of CO entry (k_entry′) are observed. Replacing His(E7) with Trp causes further decreases in k_O2′, k_NO′, and k_entry′ to 1–2 μm⁻¹ s⁻¹ in β subunits, whereas ligand rebinding to αTrp(E7) subunits after photolysis is markedly biphasic, with fast k_O2′, k_CO′, and k_NO′ values ≈150 μm⁻¹ s⁻¹ and slow rate constants ≈0.1 to 1 μm⁻¹ s⁻¹. Rapid bimolecular rebinding to an open α subunit conformation occurs immediately after photolysis of the αTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed αTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.

Introduction

In 1970, Perutz (1) proposed that the distal histidines located at the E7 helical positions,³ αHis-58 and βHis-63, play crucial structural roles for regulating both the affinities and rates of O₂ binding to adult human hemoglobin (HbA).⁴ These ideas were based on the suggestion by Pauling (2) that His(E7) could stabilize bound O₂ by donating a hydrogen bond to the partial negative charge on the superoxide-Fe(III)-like FeO₂ complex and on the idea by Perutz and Mathews (3) that the distal histidine could also be acting as gate for ligand entry and exit. Studies of model heme compounds and naturally occurring globins with His(E7) replacements suggested strongly that the distal histidine also plays a key role in discrimination between O₂ and CO binding (4–9).

The first mutagenesis studies on sperm whale Mb and human HbA reported that His(E7) to Gly mutations in α subunits and Mb cause marked increases in the rates of O₂ dissociation and significant decreases in affinity, both of which indicate strong stabilization of the FeO₂ complex by proton donation from the wild-type His(E7) side chain (7, 10, 11). In addition, the association rate constants for O₂ and CO binding increased 5–10-fold. The latter results were interpreted in terms of the distal histidine gate mechanism, with the His(E7) to Gly mutation resulting in an open E7 channel. In contrast, neither the dissociation nor association rate constants for O₂ binding to the R state βGly(E7) mutant of HbA appeared to increase significantly, implying no electrostatic stabilization of bound ligands by the native His(E7) in β subunits and an already open gate or alternative pathway. These surprising kinetic results were explained by the first high resolution structure of human oxyhemoglobin published by Shaanan (12), in which the NϵH atoms of distal histidine in β subunits seemed to be further away from the bound O₂ atoms and pointing toward the heme plane.

Between 1989 and 1999, our group and others constructed large libraries of mammalian Mb mutants as a model system for understanding the structural mechanisms of ligand binding to vertebrate globins involved in O₂ transport and storage. A detailed molecular mechanism for O₂ binding has emerged. Ligand entry into myoglobin occurs through the distal histidine gate; hydrogen bond donation by the neutral NϵH tautomer of His(E7) regulates oxygen affinity, and electrostatic stabilization of bound oxygen represents the main determinant of discrimination between O₂ and CO binding (13–18). During the same 10-year period, Champion and co-workers (19–21) came to similar conclusions when examining opening of the E7 channel by protonation of His(E7) in Mb. Their conclusions were verified by the low pH crystallographic studies of Yang and Phillips (22).

Similar mechanisms have been proposed to occur in the α and β subunits of HbA. However, the experimental evidence has been weak, due in part to the technical difficulties of generating large mutant Hb libraries, assigning rate and spectral parameters to the individual subunits within mutant/wild-type hybrid tetramers, and working with distal histidine mutants, which are highly unstable with respect to autoxidation, hemin loss, and denaturation (see Refs. 23, 24).

In 2000, Ho and co-workers (25) used heteronuclear NMR spectra of chain-selectively labeled samples to show that the Nϵ and not the Nδ atoms of the His(E7) imidazole rings are protonated and capable of donating hydrogen bonds to bound O₂ in both the α and β subunits of HbO₂. In 2006, Tame and co-workers (26) reported a new high resolution crystal structure (1.25 Å) of HbAO₂. The distances between Nϵ of His(E7) and bound O₂ in this structure are 2.6 and 3.0 Å in the α and β subunits, respectively. These structural observations indicate significant hydrogen bonding with bound ligands in both subunits, a result that is at odds with the lack of effect of the βHis(E7) to Gly mutation reported 20 years ago (11, 27).

To resolve the question of hydrogen bonding in β subunits and test the E7 gate hypothesis in HbA systematically, we constructed His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutations in both genes of recombinant human hemoglobin, expressed wild-type/mutant hybrids, and isolated individual mutant subunits to verify assignments of kinetic and spectral properties within the initial tetramers. New methods were designed to measure rate constants for O₂, CO, and NO binding under conditions that avoid autoxidation of these unstable mutants. The new kinetic results show unambiguously that bound O₂ is stabilized by hydrogen bonding in both subunits of human hemoglobin and that ligands appear to enter each Hb subunit through the distal histidine gate.

EXPERIMENTAL PROCEDURES

Preparation of Recombinant Hemoglobin Mutants

All of the wild-type and mutant recombinant human hemoglobins were expressed and isolated using the plasmid, fermentation, and purification systems developed at Somatogen, Inc. (28), as described in supplemental Section A. In these recombinant Hbs, the N-terminal Val codons in both α- and β-globins are replaced by the initiator Met codon to yield the α(V1M)/β(V1M) double mutant. This wild-type recombinant Hb control has R state rate parameters that are virtually identical to those of native R state HbA (28). The Leu(E7) mutants were also expressed from the pHE2 plasmid designed by Ho and co-workers (29, 30) to examine the effects of the V1M mutation (see supplemental Table S1). Individual α and β mutants were separated and isolated using procedures initially developed by Parkhurst and Parkhurst (31) (see supplemental Section A).

Kinetic Measurements of O₂, CO, and NO Binding

Parameters for O₂ binding to the Hb E7 mutant tetramers and monomers were measured using laser flash photolysis of the stable HbCO complex in O₂/CO mixtures to avoid prolonged exposure to oxygen and autoxidation (32). Descriptions of the 0.5-μs dye laser system and sample preparations are given in supplemental Section B.

In these experiments, the reaction is started by photolyzing HbCO to generate a deoxyHb species that can then react with the ligands present in solution (Scheme 1 and Fig. 1A). Two distinct phases are observed after photolysis. The initial fast phase represents bimolecular ligand binding with the transient deoxyHb species. The observed rate (k_fast, Equation 1) is dominated by O₂ binding because in most cases k_O2′ > k_CO′ (where k_O2′ and k_CO′ are CO and O₂ association rate constants) (33). The slow replacement phase represents the displacement of transiently bound O₂ by CO, and r_obs (Equation 2) is determined by the rate of O₂ dissociation attenuated by the relative rates of O₂ versus CO association. The bimolecular binding reaction is best observed at 436 nm, which is near the peak of deoxyHb, and the replacement reaction is best observed at 425 nm, near the peak for HbCO.

SCHEME 1.

FIGURE 1.

Time courses for O₂ binding to and displacement from isolated human Ala(E7) αCO subunits at pH 7. 4, 20 °C. A, reactions were carried out in buffers equilibrated with 1 atm of O₂ and air, and no free CO, until after photolysis of the αCO sample. The time courses were fitted to single exponential expressions with an offset representing the 1,000-fold slower CO replacement reaction. B, time courses for the slower replacement reaction measured at 425 nm at 4 different [O₂]/[CO] ratios. The amplitude of the slow phase decreases as [CO]/[O₂] increases because CO begins to compete with O₂ in the initial association reaction. C, dependence of the observed replacement rate on [CO]/[O₂] and fits to Equation 2.

Partial photolysis (≤10% of the total absorbance change obtained for complete photolysis) was used for mutant/wild-type hybrid HbCOs to ensure that only rebinding to the R state tetramers was being examined. Full photolysis was used for isolated subunits, which are intrinsically in an R state-like conformation (see Fig. 1). The value of k_CO′ was determined in a separate experiment by photolyzing HbCO in the presence of 1 mm CO with a small amount of dithionite added to eliminate residual oxygen and when necessary at various [CO] values. The slow rate for O₂ displacement by CO was measured at 425 nm after 100% photolysis for both tetramers and monomers because, on these time scales, all Hb tetramers have either 3 or 4 ligands bound and remain in the R quaternary state (24, 33, 34).

The rate constants for CO dissociation from mutant Hb tetramers and monomers were determined by rapidly mixing the HbCO complexes with excess NO. Under these conditions, the observed rate for displacement of CO by NO equals k_CO (where k_CO is the carbon monoxide dissociation rate constant) (supplemental Section B) (33).

NO binding to the wild-type and mutant HbA samples was measured using a flow-flash system to allow initiation of the reaction by photolysis of FeCO complexes, which have quantum yields for photodissociation of CO into solvent of 0.4 to 1.0. In these experiments, a sample of HbCO is rapidly mixed with concentrated NO and then photodissociated 50 ms after flow stopped with a 0.5-μs excitation pulse (supplemental Section B). Bimolecular NO binding to the newly generated deoxyHb is followed at 436 nm. As shown in Scheme 2, the observed rate, k_obs, is given by k_NO′[NO] + k_CO′[CO] ≈ k_NO′[NO] (where k_NO′ is the nitric oxide association rate constant), because the concentration of CO was kept low (∼50 μm) and k_NO′ > k_CO′ for all the samples examined. The rates of NO binding to HbA are roughly independent of quaternary state (35), and the observed rates of NO binding are linearly dependent on [NO] with an intercept effectively equal to 0 and a slope equal to k_NO′ (see Fig. 5).

SCHEME 2.

FIGURE 5.

NO binding to E7 mutants of HbA subunits. A, time courses for NO binding to isolated native α and β chains. HbCO samples (∼25 μm, after mixing) were rapidly mixed with NO (1000 μm in final reaction mixture) and then photolyzed ∼50 ms after flow stopped with a 500-ns dye laser pulse. Bimolecular NO binding was observed at 436 nm, and the time courses were normalized by the absorbance change immediately before and after photolysis. The offset after the reaction is complete represents the difference in absorbance between the final product HbNO and the starting HbCO sample. The open circles and open squares represent native α and β subunits, respectively. The black lines represent fits to single exponential expressions for the isolated α (k_obs = 28,000 s⁻¹) and β (k_obs = 68,000 s⁻¹) subunits. The inset shows the dependence of the observed pseudo first order rate constants of native α and β subunits and HbA tetramers on [NO]. The observed rates show a straight line dependence with a y-intercept equal to 0 because the NO dissociation rate constant is on the order of 10⁻⁴ s⁻¹ (13). The open symbols in the inset correspond to the rates for isolated subunits, and the closed symbols represent the rates for the fast (β) and slow (α) HbA phases. The slopes represent the k_NO′ values, which are given in Table 3. B, comparison of calculated rates of CO entry with the bimolecular rate constants measured for NO binding to recombinant Hb. Data were taken from Table 3. The closed and open circles represent isolated α and β subunit rate constants, respectively, and the open and closed triangles represent values for the closed α and slowly reacting βTrp(E7) conformers.

FTIR Measurements and Geminate Recombination for HbCO Complexes

Vibrational spectra for the bound C-O stretch were measured as described in supplemental Section C. Time courses for geminate CO recombination within isolated native, wild-type, and mutant Hb subunits were measured after photolysis with a 7-ns YAG laser. Complete descriptions of the instrument and experiments are given in supplemental Section D.

Geminate CO recombination in the individual α and β Hb subunits appears to be a simple first order process. A two-step binding scheme was assumed for analysis and involves internal bond formation between the ligand and the iron atom (k_bond) and ligand escape (k_escape) from or bimolecular return (k_entry′) to the geminate state (Hb···CO) in Scheme 3. The rate parameters for these processes define the observed rates (k_gem, Equation 4) and fractions (F_gem, Equation 3) of geminate recombination and the overall bimolecular association rate constants (k_X′, Equation 5) (18, 36):

SCHEME 3.

RESULTS

Effects of E7 Mutations on O₂ Association

In the past, O₂ binding to hemoglobin was measured after flash photolysis of HbO₂ complexes. This approach is difficult when examining distal histidine mutants because of the following: 1) the quantum yield for complete photodissociation of O₂ into solvent is inherently small, ≤0.1, and decreases even further when His(E7) is replaced with an apolar amino acid; 2) the E7 apolar mutants autoxidize rapidly with half-lives ranging from a few minutes to less than 1 h; and 3) complete removal of CO from His(E7) mutants is very difficult because these variants have K_CO/K_O2 ratios in the range 4000–40,000 (where K_CO is the carbon monoxide affinity constant and K_O2 is the oxygen affinity constant). For example, CO can only be removed from Leu(E7) mutants by oxidation with ferricyanide under an intense light, and then the oxygenated complex has to be generated by reduction with dithionite in air followed by gel filtration to rapidly remove excess reducing agent.

We have addressed the photodissociation yield, autoxidation, and CO removal problems by examining O₂ binding and release starting with HbCO complexes in mixtures of O₂ and CO. Direct comparisons between our new data and older rate constant determinations are given in supplemental Section E and supplemental Table 1S.

Time courses for bimolecular O₂ binding to isolated subunits in photolysis experiments with HbCO are shown in Fig. 2 for the complete set of E7 mutations. Similar experiments were carried out with hybrid tetramers at 10% photolysis. In the case of hybrid tetramers, the observed time courses were fitted to two exponential expressions, one for the wild-type subunit and one for the mutant subunit (34, 37). Although physiologically more relevant, the assignment of the phases to α and β subunits within Hb tetramers can be ambiguous, whereas the assignment of rate parameters is straightforward for isolated α and β chains. In some cases, the monomeric subunits do show slightly different ligand binding properties from those of the corresponding subunits in heterotetramers, but these differences are always small (≤2-fold, Table 1) (23).

FIGURE 2.

Bimolecular binding of O₂ to native and E7 mutants of isolated α and β subunits of recombinant human HbA. In these experiments, ∼50 μm HbCO was photolyzed with a 0.5-μs dye laser excitation pulse in a mixture of 625 μm O₂ and 500 μm CO in 0.1 m phosphate buffer, pH 7.0, or 50 mm HEPES, 0.1 m NaCl, 0.1 mm EDTA, pH 7.4, 20 °C (no differences were observed between these conditions for isolated subunits and R state tetramers in partial photolysis or replacement experiments). Under these conditions, the observed first order rate constant, k_obs, equals k_O2′[O₂] + k_O2 + k_CO′ [CO] and, in most cases, is dominated by O₂ binding. Bimolecular binding was monitored at 436 nm, and the time courses were normalized from the absorbance changes for the fast bimolecular phases for ease of comparison between the mutants. A, time courses for bimolecular O₂ binding to mutant α subunits, where E7 refers to position 58; B, time courses for O₂ binding to mutant β subunits, where E7 refers to position 63.

TABLE 1.

Rate and equilibrium constants for O₂ binding to R state native HbA, mutant hybrid tetramers, and isolated mutant subunits with E7 substitutions

Conditions were 0.1 m phosphate buffer, pH 7.0, or 50 mm HEPES, 0.1 m NaCl, 0.1 mm EDTA, pH 7.4, 20 °C. Rates of bimolecular O₂ binding and first order displacement were measured after laser flash photolysis in O₂/CO mixtures. Oxygen association/dissociation rate constants were obtained by fitting sets of observed k_fast and k_slow as a function of [O₂] and [CO] using Equations 1 and 2. Equilibrium association constants were calculated as K_O2 = k_O2′/k_O2. The superscripts M and T refer to isolated monomers or WT/mutant hybrid tetramers, respectively.

Subunit	kO2′	kO2	KO2	Subunit	kO2′	kO2	KO2
	μm−1s−1	s−1	μm−1		μm−1s−1	s−1	μm−1
αGly(E7)M	250	540	0.45	βGly(E7)M	160	680	0.24
αGly(E7)T	190	500	0.38	βGly(E7)T	95	310	0.31
αAl▵E7)M	160	880	0.18	βAl▵E7)M	120	860	0.14
αAl▵E7)T	170	320	0.53	βAl▵E7)T	120	520	0.23
αLeu(E7)M	91	450	0.2	βLeu(E7)Ma	110	4600 (40%) 630 (60%)	0.02 0.18
αLeu(E7)T	100 ± 60	680 ± 180	0.14 ± 0.10	βLeu(E7)T	110 ± 23	600 ± 170	0.18 ± 0.1
αGln(E7)M	43	42	1.0	βGln(E7)M	70	26	2.7
αGln(E7)T	35	86	0.41	βGln(E7)T	72	42	1.7
α NativeM	40 ± 2	22 ± 6	2.5 ± 1.2	β NativeM	63 ± 12	16 ± 1.6	2.3 ± 1.0
α NativeT	32 ± 4.1	13 ± 2.9	2.6 ± 0.4	β NativeT	82 ± 15	28 ± 6.1	3.1 ± 1.0
α WTM	40	16	2.5	β WTM	52	14	3.7
α WTT	29 ± 11	14 ± 8	1.9 ± 0.5	β WTT	60 ± 12	31 ± 13	3.9 ± 0.6
αPhe(E7)Mb	51 (63, 28)	9800 (13,500, 6600)	0.0052	βPhe(E7)Mb	25 (13, 36)	2400 (3500, 1900)	0.01
αPhe(E7)T	47	3500	0.013	βPhe(E7)T	33	1700	0.02
αTrp(E7)M	210	450	0.47	βTrp(E7)M	1.7	190	0.009
	∼0.5c		0.001c
αTrp(E7)T	260	940	0.27	βTrp(E7)T	4.1	200	0.02

^a The replacement reactions for isolated Leu(E7) β subunits showed two measurably different phases, whereas the βLeu(E7)/WT mutant tetramers showed only one phase. The relative amplitudes of the two phases for the βLeu(E7) monomer O₂ dissociation traces are given in parentheses.

^b Although fits to a single exponential function are satisfactory approximations, the observed time courses for O₂ binding to and dissociation from isolated α- and βPhe(E7) chains are slightly biphasic, and calculated k_O2′ and k_O2 values for each phase are presented in parentheses. We could only resolve one phase for the same mutants in hybrid tetramers. Because the heterogeneity was not dramatic (≤2–3-fold), we used the single values for the Phe(E7) monomers in all remaining analyses.

^c The fast phases for bimolecular O₂ binding to the αTrp(E7) monomer in the O₂/CO mixtures appeared monophasic. Slow O₂ binding to a closed conformer, similar to that seen for CO binding to this mutant (Fig. 3), is obscured in the oxygen binding experiments because the slow displacement phase (HbO₂ + CO → HbCO + O₂) occurs on roughly the same time scale. A slow bimolecular O₂ binding phase was observed when we prepared oxygenated αTrp(E7) subunits in 1 atm of O₂ and examined rebinding using the 0.5-μs dye laser system without any displacement phase. In this case, we observed a very small slow phase that suggested a bimolecular rate constant of ∼0.5 μm⁻¹ s⁻¹, which was similar in magnitude to that observed in the CO binding experiments. This rate constant was assigned to O₂ binding to a closed αTrp(E7) conformer. The oxygen affinity of the closed conformer (K_O2) was estimated as the ratio of the closed k_O2′ value and k_O2 from the replacement analysis.

A summary of the data for both isolated subunits and R state hybrid tetramers is given in Table 1. In β subunits, the rate constant for bimolecular O₂ binding decreases significantly with increasing size of the E7 amino acid, from 160 μm⁻¹ s⁻¹ for βGly(E7) to 1.7 μm⁻¹ s⁻¹ for βTrp(E7) (Fig. 2B and Table 1). This dramatic ∼100-fold decrease strongly supports the idea that His(E7) is acting as a gate for ligand entry into β subunits. As expected, the His(E7) to Gln mutation has little effect on the association rate constants for O₂ binding because it is similar in size and polarity to the wild-type histidine.

The results for α subunits are more complex. The value of k_O2′ for αGly(E7) subunits in either R state tetramers or isolated subunits is also very large, 200–250 μm⁻¹ s⁻¹, and decreases as the amino acid size is increased from Ala to Leu to Phe and His(E7). The value of k_O2′ for native αHis(E7) subunits is 35 μm⁻¹ s⁻¹ (Fig. 2A and Table 1).

The αPhe(E7) mutant binds O₂ slightly more rapidly than the wild-type α subunit (∼50 versus ∼35 μm⁻¹ s⁻¹, respectively), even though the phenyl ring is bigger than the imidazole ring. This difference can be explained by the presence of a water molecule hydrogen-bonded to His(E7) in wild-type deoxy α subunits (26). Distal pocket water must be displaced from the active site of wild-type α chains before ligands can enter the heme pocket and coordinate to the iron atom. The distal pockets of Phe(E7) mutants are presumed to be anhydrous, and the only hindrance to binding is the large aromatic side chain of the E7 residue. The smaller k_O2′ value for wild-type α subunits indicates that water displacement offers a larger barrier to ligand binding in this subunit than the benzyl side chain. In the case of the βPhe(E7) mutant, the value of k_O2′ is 2-fold smaller than that for wild-type β subunits (∼30 μm⁻¹ s⁻¹ versus ∼ 60 μm⁻¹ s⁻¹, respectively). In this case, the lower rate of the mutant subunit is explained by the larger size of the Phe(E7) side chain because no water molecule is present in the distal pocket of native deoxy β subunits or, if present, is highly disordered and easily displaced (26).

The most remarkable result in Fig. 2A and Table 1 is the rapid rate of bimolecular O₂ binding to the photolyzed αTrp(E7) mutant in either monomers or tetramers. The observed rate constant is 200–250 μm⁻¹ s⁻¹ for αTrp(E7), which is ∼5 times larger than k_O2′ for wild-type α subunits and identical to that for the αGly(E7) and αAla(E7) mutants, in which the E7 gate is completely open. This result suggests that the indole side chain of αTrp(E7) adopts an outward pointing conformation when a ligand is bound and, after photolysis, cannot rotate back into the E7 channel on the time scales for bimolecular O₂ binding. In contrast, O₂ binding to photolyzed βTrp(E7) mutants is slow, indicating rapid relaxation of the indole ring into the E7 channel after photolysis of bound CO.

Bimolecular CO Binding and Movement of Trp(E7)

Time courses for bimolecular CO binding to mutant and native isolated α and β subunits are shown in Fig. 3, and fitted values of k_CO′ are given in Table 2. The bimolecular rate constants for CO binding to isolated α and β subunits also decrease with increasing size for the Gly, Ala, Leu, and His series, from 50–100 to ∼5–10 μm⁻¹ s⁻¹. Again, CO binds slightly faster to the αPhe(E7) mutant than to the WT subunit (∼5.5 versus 4.5 μm⁻¹ s⁻¹), whereas the opposite order is observed for βPhe(E7) and WT subunits, presumably because of water in the distal pocket of unliganded wild-type α but not β subunits.

FIGURE 3.

Time courses for bimolecular CO binding to wild-type and E7 mutants of isolated human HbA subunits after laser photolysis. A, CO binding to mutant α subunits, and B, CO binding to mutant β subunits, both at [CO] = 1000 μm (1 atm). C, CO binding to isolated αTrp(E7) subunits at four different [CO] labeled beside each time course. The observed bimolecular rates for the fast phases of the reactions were as follows: 16,500 s⁻¹, 50 μm CO; 46,000 s⁻¹, 350 μm CO; 90,000 s⁻¹, 750 μm CO; and 125,000 s⁻¹, 1000 μm CO. The subunit concentration was 50 μm. D, more detailed comparison between the time courses for bimolecular CO binding to monomeric α- and βTrp(E7) mutants on both long and short time scales at [CO] = 1000 μm. Buffer conditions were the same as in Fig. 2. Several crystals of sodium dithionite were added into each cuvette to scavenge any residual O₂.

TABLE 2.

Rate and equilibrium constants for bimolecular CO binding to native HbA, mutant hybrid tetramers, and isolated mutant subunits with E7 substitutions

Conditions were the same as in Table 1. The pseudo first order rate of CO association was measured after laser flash photolysis of 50 μm Hb at 1 atm CO (∼1000 μm), and this datum along with the fast and slow rates observed in the O₂/CO mixture experiments were fitted simultaneously to obtain k_CO′ (k_O2′ and k_O2) values. CO dissociation rate constants were measured independently by mixing HbCO in anaerobic buffer with 1 atm NO (∼2000 μm) in a stopped-flow spectrophotometer. CO equilibrium association constants for the R state, K_CO, were calculated from k_CO′/k_CO. The superscripts M and T refer to isolated monomers or WT/mutant hybrid tetramers, respectively.

Subunit	kCO′	kCO	KCO	Subunit	kCO′	kCO	KCO
	μm−1 s−1	s−1	μm−1		μm−1 s−1	s−1	μm−1
αGly(E7)M	100	0.013	7700	βGly(E7)M	40	0.016	2500
αGly(E7)T	77	0.0078	9900	βGly(E7)T	53	0.0084	6300
αAl▵E7)M	93	0.014	6600	βAl▵E7)M	64	0.012	5300
αAl▵E7)T	90	0.0096	9400	βAl▵E7)T	48	0.01	4800
αLeu(E7)M	53	0.0027	20,000	βLeu(E7)Ma	24	0.0056	4300
αLeu(E7)T	22	0.0019	12,000	βLeu(E7)T	29	0.0014	21,000
αGln(E7)M	6.3	0.0071	890	βGln(E7)M	10	0.0082	1200
αGln(E7)T	4.8	0.011	440	βGln(E7)T	13	0.0056	2300
α NativeM	5.2 ± 0.5	0.016 ± 0.003	330 ± 100	β NativeM	11 ± 3.3	0.010 ± 0.001	1100 ± 240
α NativeTa	2.9 ± 0.5	0.005 ± 0.002	600 ± 260	β NativeTa	7.1 ± 2.4	0.007 ± 0.003	1000 ± 550
α WTM	5.5	0.014	390	β WTM	7.2	0.0094	740
α WTT	4.0 ± 1.1	0.011 ± 0.004	360 ± 200	β WTT	7.1 ± 2.0	0.008 ± 0.001	890 ± 440
αPhe(E7)Mb	5.7 (3.5, 7.5)	0.041	140	βPhe(E7)Mb	3.9 (1.8, 6)	0.012	330
αPhe(E7)T	5.1	0.028	180	βPhe(E7)T	3.9	0.012	330
αTrp(E7)Mc	125 (77%)	0.008	16,000	βTrp(E7)Mc	150 (≤15%)
	0.11 (23%)		14		0.7 (70%)	0.0037	190
					0.08 (15%)		22
αTrp(E7)T	53	0.0048	11,000	βTrp(E7)T	1.3	0.0058	220

^a Rates for native tetrameric subunits were taken from Mathews et al. (11). A summary of these values is given in supplemental Table 1S.

^b The observed time courses for CO binding to isolated α- and βPhe(E7) chains were slightly biphasic and analyzed by one and two exponential expressions. We used the single exponential fits in our analyses, and the results of the two exponential analyses are given in parentheses. Note that the differences between fast and slow rate parameters were not dramatic (≤2–3).

^c The traces for CO rebinding to the monomeric Trp(E7) mutants contain at least two phases. For α subunits, the fraction of the fast phase is large in 1000 μm CO (77%), but the slow phase becomes dominant at low [CO] (Fig. 3). For β subunits, there is predominantly one phase (∼70%), which is relatively slow (k_CO′ = 0.7 μm⁻¹ s⁻¹), and the parameter for this phase was used in further analyses. However, both very small, ultrafast, and ultraslow phases were detected. The ultrafast phase occurs on time scales similar to the major phase for bimolecular CO binding to αTrp(E7) subunits but can only be detected at the highest CO concentrations and was not analyzed further. The slow phase could be analyzed and gave the apparent bimolecular rate constant and amplitude shown.

The time courses for CO binding to the α- and βTrp(E7) mutants demonstrate the complexity implied in the results for O₂ binding. Binding to αTrp(E7) subunits at a high ligand concentration (i.e. 1000 μm, see Fig. 3A) is dominated by a rapid bimolecular phase, which defines a k_CO′ value equal to ∼120 μm⁻¹ s⁻¹ based on the dependence of k_obs,fast on [CO]. However, a slow phase is clearly present, even at 1000 μm CO. When [CO] is decreased from 1000 to 50 μm in laser photolysis experiments, the fraction of the fast phase decreases from 77 to ∼20% (Fig. 3C). In the case of partial photolysis experiments with mutant hybrid tetramers, multiple phases are also seen but are almost impossible to resolve because of the presence of CO binding to wild-type subunits.

The slow phases in Fig. 3C for isolated αTrp(E7) subunits indicate a bimolecular rate constant of 0.11 μm⁻¹ s⁻¹ based on the dependence of the observed pseudo first order rate on [CO]. The presence of such a phase in the O₂ binding experiments in O₂/CO mixtures is obscured by the O₂ replacement phase, which occurs on similar time scales. To look for this phase, we prepared a fully oxygenated sample of isolated αTrp(E7) subunits in the absence of CO and measured bimolecular O₂ binding after laser photolysis of HbO₂ samples. Two phases for bimolecular O₂ rebinding were observed, and k_O2′ values for the fast and slow phases were calculated to be 230 and 0.5 μm⁻¹ s⁻¹ (see Table 1). The latter rate constant is similar to the k_CO′ value obtained from the slow phases shown in Fig. 3C.

All of these results suggest that the indole side chain of αTrp(E7) adopts an outward “open” conformation when ligands are bound. This conformation persists on microsecond time scales after photodissociation. The fast bimolecular phases after photodissociation represent O₂ and CO binding before the indole side chain has moved back into the E7 channel. The slow phases represent ligand binding to the equilibrium conformational state of the α-deoxyHb mutant, in which the indole side chain fills the E7 channel and blocks ligand entry. Rotation of the indole side chain into the entry channel occurs at a rate that competes with bimolecular CO binding to the open conformation (∼10⁴–10⁵ s⁻¹). When the external [CO] is high, ligand binding to the open conformer dominates, whereas at low [CO] the opposite is true.

In β subunits, Trp(E7) relaxation after complete photodissociation must be greater than ∼10⁵ s⁻¹ because only a small rapid bimolecular phase (∼150 μm⁻¹ s⁻¹, <15%) is observed at the highest CO concentration and is difficult to define (Fig. 3, B and D, inset). The majority of the bimolecular βTrp(E7) CO rebinding phase is slow with k_CO′ equal to ∼0.7 μm⁻¹ s⁻¹ (70%). A small third phase is seen on longer time scales with a rate similar to that for αTrp(E7) subunits, ∼0.08 μm⁻¹ s⁻¹ (15%), indicating that slow relaxation to an even more blocked and hindered conformation occurs in β subunits.

This competition between Trp(E7) relaxation and ligand rebinding after photolysis in α subunits is similar to the competition between bimolecular ligand rebinding and internal His(E7) coordination that is seen after photolysis of the CO and O₂ complexes of nonsymbiotic plant Hbs and neuroglobins. These globins are hexacoordinate in their equilibrium deoxyHb states (38–41). Tian et al. (20) observed a similar competition between both geminate and bimolecular CO rebinding and inward relaxation of His(E7) at low pH values where the imidazole side chain is in the protonated open conformation when ligands are bound and in the deprotonated closed state when the heme is unliganded.

Determination of Rate Constants for Ligand Entry into α- and β-E7 Mutants

Two other sets of experiments were carried out to verify that the E7 mutations are directly affecting the rate of ligand entry into hemoglobin. Internal CO geminate recombination was examined on nanosecond time scales to determine the fraction of geminate recombination and to estimate rates of internal ligand bond formation and escape using Equations 3–5. CO is the ligand of choice for hemoglobin subunits, because the quantum yield for formation of the initial CO geminate state is close to unity, and the observed rate of geminate CO recombination, 2–70 μs⁻¹, is readily measured on nanosecond time scales. Geminate O₂ rebinding to α and β chains in the R state is too rapid to measure readily with a 7-ns YAG laser pulse and exhibits a quantum yield of ≤0.2 for formation of the first geminate state (27). The rates of CO entry and exit appear to be the same as those for O₂ and NO because all three diatomic gases have almost the same size and polarity (17, 18, 36, 42). The major differences are the iron coordination rates, k_bond, with NO ≫ O₂ > CO (36, 43, 44).

Time courses for geminate CO rebinding to isolated α- and β-E7 mutants are shown in Fig. 4. As in the case of Mb, the dependence of geminate recombination on the size of the E7 residue is complex (17, 45–47). Replacement of His(E7) with Gly, Ala, and Leu increases the fraction of internal recombination (F_gem) by removal of steric hindrance for ligand return to the iron atom, which results in 5–10-fold increases in k_bond with little change in the computed rate constant for escape (Table 3) (45, 48). In contrast, the extents of geminate recombination in the wild-type, Gln(E7), and Phe(E7) subunits are all small values. As in Mb, the His(E7) and Gln(E7) side chains are close enough (≤3 Å) to donate hydrogen bonds to bound ligands, but this proximity results in steric hindrance of the initial photodissociated ligand, pushing it back into the distal pocket, inhibiting its return to the iron atom, and causing both k_bond and F_gem to be small (45, 49). The same steric effect appears to occur for the Phe(E7) mutants.

FIGURE 4.

Geminate CO recombination in wild-type and E7 mutants of isolated α and β subunits of recombinant human HbA. In these experiments, ∼50 μm HbCO was photolyzed with a 7-ns Nd:YAG laser excitation pulse (at time 0.0) in 1000 μm CO. Buffer conditions were the same as in Fig. 2. Geminate recombination was monitored at 436 nm, and the absorbance changes were normalized for comparison between the mutants. Time courses were fitted to single exponential expressions; F_gem is calculated as (total ΔA₄₃₆ − offset)/(total ΔA₄₃₆); k_gem is equal to the observed rate of the first order internal rebinding phase. A, time courses for geminate CO recombination to mutant α subunits, where E7 refers to position 58; B, time courses for geminate CO recombination to mutant β subunits, where E7 refers to position 63.

TABLE 3.

Rate constants for bimolecular CO and NO binding and parameters for CO geminate recombination to isolated α- and βE7 mutants

k_NO′^tetr represents NO binding to mutant subunits within Hb tetramers. All other parameters presented are for ligand binding to isolated subunits, and the various geminate parameters were computed as described under “Experimental Procedures” using Scheme 3 and Equations 3–5. Native α and β subunits have His(E7). Conditions were the same as in Table 1. ND means not determined.

Subunit	kCO′	Fgem	kgem	kbond	kescape	kentry′	kNO′	kNO′tetr
	μm−1s−1		μs−1	μs−1	μs−1	μm−1s−1	μm−1s−1	μm−1s−1
αGly(E7)	100	0.63	25	17	8.6	150	170	140
αAl▵E7)	93	0.57	37	21	16	160	150	160
αLeu(E7)	53	0.66	45	30	16	80	82	64
αGln(E7)	6.3	0.23	22	5.1	17	27	45	42
α Native	5.2	0.14	24	3.4	21	36	31	25
αPhe(E7)	5.7	0.13	69	8.9	60	44	94	71
αTrp(E7)a	125 (77%)	0.77	67	52	15	160	150	ND
	0.11 (23%)					0.14	∼0.4
βGly(E7)	40	0.45	20	9	11	89	140	120
βAl▵E7)	64	0.39	16	6.4	9.6	160	170	130
βLeu(E7)	24	0.55	14	7.7	6.3	44	91	68
βGln(E7)	10	0.22	6	1.3	4.7	45	69	63
β Native	9.2	0.22	7.6	1.7	6	42	68	66
βPhe(E7)	3.9	0.14	37	5.2	32	28	53	44
βTrp(E7)a	150 (<15%)					250
	0.7 (70%)	0.61	70	43	27	1.2	5.4	6.4
	0.08 (15%)					0.13

^a The values of k_entry′ for the Trp(E7) mutants were calculated using the single F_gem value listed in column 3 and the k_CO′ values for all the observed phases.

The rate constant for ligand entry, k_entry′, into the individual subunits of Hb, k_entry′, is calculated empirically as the observed bimolecular rate constant divided by the experimentally determined fraction of internal recombination (k_entry′ = k_CO′/F_gem, Equation 5), regardless of the number of steps in the mechanism. Unlike the values of k_bond and k_escape, which assume only a two-step mechanism, k_entry′ is relatively model independent (17). Our calculations assume that there are no CO geminate phases that cannot be observed on nanosecond time scales. Ultrafast picosecond processes do not occur for native HbCO and its subunits (27), and although they cannot be ruled out, we did not observe any significant decreases in nanosecond yields of photodissociation for the His(E7) variants.

As shown in Table 3, there is a general decrease in k_entry′ with increasing size of the E7 amino acid. Again, the results for the Trp(E7) mutants warrant more careful consideration. In the case of α subunits, the large fractions of geminate rebinding and values for k_bond almost certainly represent geminate CO recombination in an open conformation with the indole side chain pointing outward and with little or no steric hindrance near the iron atom. These parameters in combination with the rate for the fast bimolecular CO binding phase predict that k_entry′ for the open conformation is ∼160 μm⁻¹ s⁻¹, which is similar to that for αGly(E7) and close to the diffusion limit, ∼200–500 μm⁻¹ s⁻¹, which is observed for pentacoordinate model hemes ((50, 51) and hemoglobins with unhindered active sites (i.e. leghemoglobins (52) and Cerebratulus lacteus mini-globin (32)). However, when αTrp(E7) relaxes to its equilibrium deoxyHb conformation, the rate of entry is markedly reduced, with a computed value of k_entry′ ≈ 0.14 μm⁻¹ s⁻¹. In the case of isolated βTrp(E7) subunits, both slow phases for bimolecular CO binding after laser photolysis predict small values for k_entry′ (Table 3).

To verify the k_entry′ calculations based on CO kinetics, we measured the bimolecular association rate constants for NO binding to both mutant monomers and tetramers. Many workers have shown that the fraction of geminate recombination of NO in mammalian Hbs and Mbs is ≥0.99, with bond re-formation occurring on picosecond time scales (45, 47, 53) due to the high reactivity of the NO radical with high spin ferrous iron. In terms of Equation 5, F_gem ≈ 1.0 and k_entry′ = k_NO′. In the case of Mb, Scott et al. (17) showed that the rate constants for ligand entry computed from analysis of geminate and bimolecular O₂ binding correlate linearly with the corresponding association rate constants for NO binding to ∼30 different mutants.

Time courses for NO binding to native α and β subunits are shown in Fig. 5A; summaries of all the bimolecular NO rate constants are listed in Table 3; and a strong linear correlation between k_NO′ and the k_entry′ values calculated from CO binding data is observed (Fig. 5B). These results confirm the following: 1) the ∼2-fold greater rate of ligand association with native β subunits than with α subunits; 2) the ∼200-fold decrease in the bimolecular rate constant for ligand entry in HbA with increasing size of the E7 side chain; and 3) the competition between αTrp(E7) relaxation to the closed conformation and ligand binding from solvent. In the latter case, two phases are observed for bimolecular NO binding to isolated αTrp(E7) subunits, with the dependence on [NO] suggesting fast and slow k_NO′ values ≈150 and 0.4 μm⁻¹ s⁻¹, respectively. The slow phase k_NO′ value is similar to that obtained from the slow phases for O₂ and CO bimolecular binding to αTrp(E7) mutants (Tables 1 and 2).

Rate Constants for O₂ and CO Dissociation from the α- and β-E7 Mutants

Rate constants for O₂ dissociation were obtained by analyzing the slow phase time courses for the displacement of transiently bound O₂ by CO after laser photolysis of the CO complexes of both isolated chains and mutant/wild-type recombinant HbA tetramers in O₂/CO mixtures (Equations 1 and 2 and see Fig. 1). Sample time courses are shown in Fig. 6, and fitted k_O2 (where k_O2 is the oxygen dissociation rate constant) values for R state HbA are listed in Table 1.

FIGURE 6.

Time courses for O₂ replacement by CO in native and E7 mutant α and β subunits. The solutions contained a mixture of 625 μm O₂ and 500 μm CO. Buffer conditions were the same as in Fig. 2. Absorbance changes were detected at 425 nm and normalized for comparison between the mutants. In these experiments, HbO₂ is transiently formed after photolysis of HbCO with a 0.5-μs dye laser pulse, and then O₂ is slowly displaced by CO present in the solution. Only the slow O₂ replacement phase of the reaction is shown, and for these time courses, the observed first order rate constant, r_obs, equals k_O2/(1 + k_O2′[O₂]/k_CO′ [CO]). A, O₂ dissociation from α subunits, where E7 refers to position 58; B, O₂ dissociation from α subunits, where E7 refers to position 63.

Replacement of His(E7) with apolar amino acids causes marked 20–500-fold increases in the rate of O₂ dissociation from both subunits. As expected, the conservative His(E7) to Gln mutations cause the least effect, 2–4-fold increases in k_O2, whereas the Phe(E7) mutations cause the largest increases, from ∼20 s⁻¹ in WT HbO₂ to ∼2,400 s⁻¹ in βPhe(E7) chains and 9,800 s⁻¹ in αPhe(E7) chains (Table 1). Similar trends were observed for the corresponding mutant subunits in hybrid tetramers, although the numerical values are almost always smaller.

The results for the Gly, Ala, and Leu(E7) mutations show that hydrogen bonding to the native His(E7) side chain stabilizes bound O₂ roughly 20–30-fold in both subunits. In the case of βLeu(E7), heterogeneous O₂ displacement time courses are observed for isolated mutant subunits but not in hybrid tetramers (Fig. 6 and Table 1). This heterogeneity for isolated Leu(E7) β subunits was verified in two independent preparations and does not depend on subunit concentration, ruling out differences between β monomers and tetramers (see supplemental material).

The much greater increase in k_O2 observed for the Phe(E7) mutants suggests direct hindrance by the benzyl side chain, causing a greater rate of thermal bond disruption (k_−bond in Scheme 3) and restriction of ligand return to the iron atom after dissociation. This interpretation is supported by the low fractions of geminate ligand rebinding to the Phe(E7) mutants (Fig. 4 and Table 3).

The rate constants for O₂ dissociation from the Trp(E7) mutants are smaller than those for the Phe(E7) mutants and more similar to those of the other apolar mutants. Presumably, the indole ring cannot be accommodated in the active site when a ligand is bound and, in the “out” conformation, cannot directly clash with the bound ligand. In addition, the large indole ring may also block ligand exit from the distal pocket, even in the out conformation. Both of these effects increase geminate recombination and lower overall rates of ligand dissociation (see Fig. 4).

Rate constants for CO dissociation from isolated chains and intact tetramers are listed in Table 2. Unlike O₂ dissociation, there is only a small variation in k_CO, from ∼0.04 to 0.002 s⁻¹ for the His(E7) mutants, with the native value of k_CO being ∼0.01 s⁻¹ for both subunits. There is little change in the rate of CO dissociation for the Gly and Ala(E7) mutations, but a 5-fold decrease in k_CO is observed for both Leu(E7) mutations, giving these mutants unusually high affinities as has been observed for Leu(E7) mutants in several different mammalian Mbs (Table 3) (13, 14).

FTIR Spectroscopy

To confirm electrostatic stabilization of bound O₂ by His(E7), we measured the C-O stretching frequencies of bound CO, ν_C-O, for the complete series of mutant subunits (Fig. 7 and Table 4). Many authors have shown that the positive electrostatic field from the NϵH atoms of His(E7) causes lower ν_C-O values for MbCO and HbCO complexes and that this parameter increases upon replacing the E7 residue with nonpolar amino acids (14, 21, 44, 54–57).

FIGURE 7.

FTIR spectra of wild-type and E7 mutants of human HbA. Buffer conditions were the same as in Fig. 2. A, spectra for wild-type HbA (solid black) and mutant hybrid Hb tetramers (solid spectra represent Hb tetramers containing mutant α chains; dashed spectra represent Hb tetramers containing mutant β chains). B, spectra of isolated native and mutant α (solid) and β (dashed) subunits.

TABLE 4.

Bound C-O stretching frequency peaks, ν_C-O, for WT and mutant α and β subunits of native and recombinant human hemoglobin

Conditions were the same as in Table 1. The superscripts M and T refer to isolated monomers or WT/mutant hybrid tetramers, respectively.

Subunit	νC–O	Subunit	νC–O
	cm−1		cm−1
α NativeM	1950	β NativeM	1950
α NativeT	1950	β NativeT	1950
α WTM	1950	β WTM	1951
α WTT	1950	β WTT	1951
αGln(E7)M	1945,1950	βGln(E7)M	1950
αGln(E7)T	1945,1951	βGln(E7)T	1950
αGly(E7)T	1965,1973	βGly(E7)T	1965,1971
αAl▵E7)M	1972	βAl▵E7)M	1971
αAl▵E7)T	1971	βAl▵E7)T	1969
αLeu(E7)M	1969	βLeu(E7)M	1970
αLeu(E7)T	1969	βLeu(E7)T	1970
αPhe(E7)M	1967	βPhe(E7)M	1967
αPhe(E7)T	1967	βPhe(E7)T	1967
αTrp(E7)M	1970	βTrp(E7)M	1969
αTrp(E7)T	1970	βTrp(E7)T	1969

Representative FTIR spectra for wild-type and mutant hybrid tetramers in the region of the C-O stretching frequency (1900–2000 cm⁻¹) are shown in Fig. 7A, and spectra for isolated Hb subunits with His (native), Phe, and Trp residues at the E7 position are shown in Fig. 7B. The values of the corresponding peak maxima for all the distal His mutants are summarized in Table 4. Mutation of His(E7) to apolar amino acids in HbA causes shifts of the ν_C-O peak, from 1950 cm⁻¹ for wild-type and native subunits to ∼1970 cm⁻¹ for the mutant subunits, both as monomers and in tetramers.

The FTIR spectra of the Gly(E7) mutants show a much broader high frequency peak, which in α subunits is clearly split into two peaks. The crystal structure of Gly(E7) metMb shows discrete distal pocket water molecules in positions similar to those of the Nδ and Nϵ atoms of His(E7) in the wild-type protein (46). Thus, multiple electrostatic fields exerted by different water conformers give rise to broadening and splitting of all the Gly(E7) mutant ν_C-O peaks. As expected, replacing His(E7) with Gln does not significantly change the position of the IR band. However, in both subunits, the peak is broader than that of wild-type protein, indicating greater conformational heterogeneity of the amide side chain. In the case of the αGln(E7) mutant subunits, a low frequency shoulder is observed at 1945 cm⁻¹, implying a small fraction of a conformer with a more positive field near bound CO than that in native α subunits containing His(E7) (Fig. 7 and Table 4).

Twenty years ago, Yu and co-workers (58) obtained very similar ν_C-O values for CO bound to αGln and Gly(E7) and to βGln, Gly, Val, and Phe (E7) mutant/wild-type hybrid tetramers using resonance Raman techniques. Our new results and their previous data suggest strongly that positive electrostatic fields occur near bound ligands in both HbA subunits and are lost when His(E7) is mutated to an apolar amino acid.

DISCUSSION

O₂ Affinity and Hydrogen Bonding

The 1.25 Å resolution crystal structures of the α and β subunits in oxygenated human HbA are shown in Fig. 8 (26). Even a cursory examination shows that the imidazole side chains in both subunits have roughly the same conformation and are equidistant from the bound O₂ atoms. The reported distances between the His(E7) NϵH atoms and the ligand atoms in the α and β subunits are 2.7 and 3.0 Å, respectively (26). These distances are similar to the values reported for MbO₂, 2.6–2.8 Å (46, 59). The effects on oxygen affinity of the apolar E7 amino acid substitutions allow an assessment of the relative strengths of the apparent hydrogen bonds within HbA subunits and myoglobin.

FIGURE 8.

Structures of the α and β active sites in HbO₂. The figures were constructed from the high resolution structure of human oxyhemoglobin A (2DN1) determined by Park et al. (26). The key residues of the heme pocket are labeled and shown in sticks. The atoms of the key amino acids are colored as following: white, carbon; blue, nitrogen; red, oxygen; yellow, the atoms of other amino acids lining the binding site; red, O atoms of bound dioxygen; and orange, heme iron. The blue dotted lines in the α subunits represent H-bonds between His (CE3) and the carboxylate oxygen atoms of the heme 6-propionate, and Lys (E10) and the carboxylates of the heme 7-propionate.

The free energy released when O₂ binds to a globin can be divided into four components: 1) the intrinsic free energy change for binding to an unhindered pentacoordinate heme in an apolar environment, ΔG_heme⁰; 2) the free energy changes associated with movement of the iron into the plane of the heme, which is governed by constraints on the geometry of the Fe-His(F8) complex and back-bonding by the proximal imidazole, ΔG_prox⁰; 3) hindrance with surrounding distal pocket amino acids, ΔG_distal⁰, which excludes possible steric clashes with E7 residue; and 4) both favorable and unfavorable interactions with the E7 side chain, ΔG_E7⁰ (Equation 6).

Interactions with the E7 amino acid side chain can be divided into three categories (Equation 7): (a) stabilization of the bound ligand by hydrogen bond donation, ΔG_(E7)H-bond⁰; (b) inhibition of ligand binding by distal pocket water, which in the native deoxyHb subunits can be hydrogen-bonded to the imidazole side chain, ΔG_(E7)water⁰; and (c) direct or indirect steric hindrance with the bound ligand, ΔG_(E7)steric⁰.

When His(E7) is mutated to smaller apolar amino acids, i.e. Gly, Ala, and Leu, the values of ΔG_heme⁰, ΔG_prox⁰, and ΔG_distal⁰ should be unaltered. The effects of small apolar E7 substitutions on the overall free energy of O₂ binding, δΔG_mutant⁰, can be calculated from the ratio of the mutant to wild-type equilibrium constants (Equation 8) and should equal δΔG_E7⁰.

Because these amino acid side chains are small and apolar, they do not interact with bound ligands nor do they stabilize internal water molecules, and all three terms in Equation 7 should be zero for O₂ binding to these variants. As a result, δΔG_E7⁰ in Equation 8 should be approximately equal to −(ΔG_His(E7)water⁰ + ΔG_{His(E7)H-bond}⁰), which represents the free energy difference between the loss of the unfavorable requirement to displace water noncovalently attached to His(E7) and the loss of favorable electrostatic stabilization of the bound ligand by His(E7) (13). In this analysis, it is assumed that there are no unfavorable steric interactions between bound ligands and His(E7) (ΔG_{His(E7)steric}⁰ = 0) and that the close proximity of the imidazole side chain to bound O₂ and CO is due to favorable hydrogen bonding interactions.

Values of −δΔG_E7⁰for α and β subunits of HbA were calculated using the average values for the Gly, Ala, and Leu(E7) mutants for K_O2(mutant) in Equation 7 and are compared in Table 5 with that computed for sperm whale Mb using the average K_O2 value for Ala, Val, and Leu(E7) mutants. Gly(E7) Mb was excluded because there are discrete water molecules in the distal pocket that clearly stabilize bound O₂ (60). The results in Table 5 demonstrate that net stabilization of bound O₂ by His(E7) (ΔG_{His(E7)H-bond}⁰ + ΔG_His(E7)water⁰) is roughly the same, −5.6 kJ/mol, in both subunits of HbA and ∼2-fold weaker than the net stabilization observed in mammalian Mb, ∼−10 kJ/mol at 20 °C, pH 7.0.

TABLE 5.

Effects of small apolar E7 mutations on ΔG⁰ for O₂ binding to HbA and sperm whale Mb

δΔG_mutant = δΔG_E7 = −RTln(K_O2(mutant)_avg/K_O2(native)), where K_O2(mutant)_avg represents the average O₂ affinity for unhindered E7 mutants containing Gly, Ala, and Leu at the E7 position (Equations 6 and 7). Oxygen affinities for these calculations were taken from Table 1. The K_O2 values for sperm whale Mb (Mb SW) were taken from Ref. 17 as described in the text. Superscript M and T stand for monomer and tetramer, respectively. As described in the text, −δΔG_E7 for small apolar mutations defines the net electrostatic stabilization of bound O₂ His(E7) defined as the sum, Δ G_His(E7)water⁰ + Δ G_{His(E7)H-bond}⁰. Estimates of free energy for water displacement from native deoxyHb or deoxyMb were computed from −(−RTln(k_O2′(mutant)_{T,M, avg}/k_O2′(native,WT)_avg)), where k_O2′(mutant)_{T,M, avg} is the average O₂ association rate constant for Gly, Ala, and Leu(E7) mutants in monomers and tetramers of Hb subunits and Ala, Val, and Leu(E7) mutants of Mb. Estimates of the free energy for H-bond donation to bound O₂ were computed from −RTln(k_O2(mutant)_{T,M, avg}/k_O2(native,WT)_avg), where k_O2(mutant)_{T,M, avg} is the average O₂ dissociation rate constant for Gly, Ala, and Leu(E7) mutants in monomers and tetramers of Hb subunits and Ala, Val, and Leu(E7) mutants of Mb. All rate parameters for HbA subunits were taken from Table 1, and those for Mb were taken from Scott et al. (17).

Globin	E7 substitutions	KO2,avg	KO2(mutant)avg/KO2(native, WT)avg	δΔGE7	− δΔGE7(avg)	ΔGHis(E7)water0	ΔGHis(E7)H-bond0
		μm−1		kJ/mol	kJ/mol	kJ/mol	kJ/mol
αHbA	His	2.5 ± 1.2
	Gly, Ala, LeuM	0.28 ± 0.15	0.11 ± 0.08	5.4 ± 1.8
	HisT	1.9 ± 0.5			− 4.7 ± 1.2a	+3.7 ± 1.0	− 8.6 ± 1.0
	Gly, Ala, LeuT	0.35 ± 0.20	0.18 ± 0.11	4.1 ± 1.5
βHbA	HisM	2.3 ± 1.0
	G,A,LM	0.18 ± 0.05	0.08 ± 0.04	6.2 ± 1.3
	HT	3.9 ± 0.6			− 6.5 ± 0.7a	+1.5 ± 0.7	− 8.0 ± 1.2
	Gly, Ala, LeuT	0.24 ± 0.07	0.06 ± 0.02	6.8 ± 0.8
Mb SW	His	1.1 ± 0.2
	Ala, Val, Leu	0.020 ± 0.008	0.018 ± 0.008	9.8 ± 1.0	− 9.8 ± 1.0a	+4.1 ± 1.0	− 14.5 ± 2.1

^a The value of −δΔG_E7(avg) can also be estimated from the sum of the ΔG_His(E7)water⁰ and ΔG_{His(E7)H-bond}⁰ values calculated from an analysis of the effects of mutagenesis on the O₂ association and dissociation rate constants, respectively. The correspondence between values of −δΔG_E7(avg) calculated from the sum of the entries in last two columns and that calculated from the ratio of observed equilibrium constants is quite good indicating that the analyses are self-consistent.

Olson and Phillips (13) have suggested that increases in O₂ association rate constants due to apolar amino acid substitutions provide a measure of the loss of distal pocket water. They estimated the value of ΔG_His(E7)water⁰ from −RTln(k_O2′ (small apolar E7 mutant)_avg/k_O2′(WT, native)), arguing that, in Mb, a significant barrier to ligand entry is displacement of distal pocket water. ΔG_His(E7)water⁰ was estimated using average values of k_O2′ for the Gly(E7), Ala(E7), and Leu(E7) mutants of Hb subunits and Ala(E7), Val(E7), and Leu(E7) mutants of Mb (Table 5).

Olson and Phillips (13) also argued that the strength of His(E7) hydrogen bonding to bound O₂ could be estimated from a comparison of the apolar mutant dissociation rate constants with those for wild-type and native Mb (i.e. ΔG_{His(E7)H-bond}⁰ ≈ −RTln(k_O2(small apolar E7 mutants)/k_O2 (WT, native)); last column in Table 5). This calculation assumes that k_O2 reflects primarily the thermal rate of Fe-O₂ bond breakage, which will be reduced markedly by the favorable free energy of hydrogen bonding. The overall value of k_O2 is reduced by geminate recombination and increased by direct steric hindrance, but these effects are normally small compared with the 10–100-fold reduction in rate due to electrostatic interaction with His(E7) (Tables 1 and 3) (13).

The results in Table 5 are clear and supported by the published structures of Mb and HbA. Both α subunits and Mb contain distal pocket water (26, 46), which reduces the free energy liberated during O₂ binding by ∼ +4 kJ/mol. In contrast, no observable water is present in the distal pocket of deoxygenated β subunits (26), and ΔG_His(E7)water⁰ is only +1.5 kJ/mol, which is roughly zero considering the errors in the measurements. The extents of electrostatic stabilization of bound O₂ in both subunits of HbA are roughly the same, ∼−8 kJ/mol, and almost half that observed in Mb, where G_{His(E7)H-bond}⁰ is ∼−14.5 kJ/mol.

The enhanced H-bonding in myoglobin is supported by the lower ν_C-O value of native MbCO (∼1941 cm⁻¹) compared with those of tetrameric and isolated αCO and βCO subunits (∼1950 cm⁻¹, see Table 4). In contrast, the ν_C-O values for the apolar mutants of all three globins are in the 1965–1968 cm⁻¹ range. Clearly, there is a stronger positive electrostatic field adjacent to bound ligands in Mb than in the subunits of human HbA, accounting for the greater net stabilization of bound O₂.

Correlations between K_CO and K_O2

Fig. 9A shows a plot of logK_CO versus logK_O2 for all the mutants listed in Tables 1 and 2. The apolar mutant equilibrium constants fall on a straight line with a slope of 1. The smaller amino acid substitutions show K_CO and K_O2 values that cluster together in the upper right of Fig. 9A. Increasing the size of the E7 side chain from Leu to Phe and Trp causes, with one exception, uniform decreases in both affinity constants. In the case of αTrp(E7), two sets of constants were estimated, one for the transient open conformer observed directly after laser photolysis and one for the equilibrium closed conformation. The transient open conformer shows affinities and rate constants for CO and O₂ binding that are similar to those of the Gly(E7) and Ala(E7) mutants (Fig. 9A, upper open circles), whereas the closed conformer shows the lowest affinity constants (Fig. 9A, lower open circles). The polar variants (His and Gln(E7)) have much higher oxygen affinities due to preferential electrostatic stabilization of bound O₂ (Fig. 9A, solid circles), and the K_O2 and K_CO values cluster significantly to the right of the line for the apolar E7 variants.

FIGURE 9.

Effects of E7 mutants on K_CO,K_O2, and their ratio M. A, correlation between logK_CO and logK_O2 of wild-type and E7 mutant HbA subunits and tetramers. Closed circles represent data for subunits containing polar His and Gln(E7) side chains; open circles represent data for apolar E7 mutants. There is strong linear correlation between logK_CO and logK_O2 values for the apolar mutants, whereas the polar E7 variants are clearly outliers and show little correlation as a group. B, effects of mutagenesis on ligand discrimination expressed as log(K_CO/K_O2) or log(M) and plotted as function of the size of the E7 amino acid. Black bars represent α-E7 mutants, and the gray bars represent β-E7 mutants. The upper dashed line represents the logarithm of the average M value for all the apolar mutants, and the lower dashed line represents the logarithm of the average M for HbA subunits. (M) and (T) in the name of the mutant specifies whether log(K_CO/K_O2) was calculated for isolated subunit monomers or the mutant subunit in hybrid tetramers, respectively. NatHisE7 and WTHisE7 stand for native HbA (derived from red blood cells) and wild-type recombinant HbA (expressed in E. coli). Data for the diagrams were taken from Tables 1 and 2.

Ligand Discrimination and Polarity

The traditional way of looking at discrimination in favor of O₂ versus CO binding is to examine the dependence of the ratio K_CO/K_O2 (M value) on ligand size and polarity as shown in Fig. 9B (K_CO and K_O2 values from Tables 1 and 2). The results are very clear. The average M value for all the apolar E7 mutants is 34,000, and although the variation in the K_CO/K_O2 ratio appears large, ±85%, this σ value is small compared with 2–3 orders of magnitude variation in the absolute values of K_CO and K_O2 (Fig. 9A). When Gln is present at the E7 position, the average M value for the α and β subunits is ∼900 ± 400, and the average M value for the wild-type His(E7) subunits is 260 ± 150. The latter value is 10 times greater than that for mammalian Mb, for which M ≈ 30. The increase in M value for the α and β subunits correlates well with the less negative values of ΔG_(E7)H-bond and the higher ν_C-O frequencies of the HbCO complexes compared with the corresponding parameters for sperm whale Mb. The strength of the positive electrostatic field near bound ligands, which preferentially enhances O₂ binding, is clearly larger in Mb than in the subunits of HbA.

Steric Hindrance by Phe(E7) and Trp(E7)

The marked decreases in K_O2 for βPhe(E7), βTrp(E7), αPhe(E7), and the closed αTrp(E7) mutants shown in Fig. 9A and Table 1 imply a large ΔG_(E7)steric⁰ term in Equation 7 for these mutants. This increase is due to either direct hindrance of the bound ligand by the aromatic E7 side chains or indirect inhibition by the need to displace the side chain from the active site before incoming ligands can be captured. The extent of this “extra” hindrance by the benzyl and indole side chains can be estimated by computing δΔG_Phe(E7) or δΔG_Trp(E7) from −RTln(K_O2,mutant/K_O2,native) and then subtracting the δΔG_{small,apolar E7} term determined from effects of the Gly, Ala, and Leu(E7) mutations, which take into account the loss of hindrance by distal pocket water and the loss of stabilization of bound O₂ by the native His(E7) side chain (i.e. the ΔG_{His(E7)H-bond}⁰ + ΔG_His(E7)water⁰ terms in Equation 7). The results of these calculations for the Phe and Trp(E7) mutants are summarized in Table 6.

TABLE 6.

Effects of Phe(E7) and Trp(E7) mutants on O₂ binding to HbA and sperm whale Mb

δΔG_Phe(E7) or δΔG_Trp(E7) values were calculated as −RTln(K_O2,mutant/K_O2,native) and found to be significantly larger than the δΔG_E7 values computed for the smaller Gly, Ala, and Leu mutants, implying steric hindrance by the larger Phe and Trp side chains. The extent of this effect was estimated as ΔG_(E7)steric = δΔG_Phe(E7) or δΔG_Trp(E7) − δΔG_{small, apolar E7}, where δΔG_{small, apolar E7} was taken from monomer and tetramer values for δΔG_E7(avg) in Table 5 for each subunit. O₂ affinities for the Phe(E7) and Trp(E7) were taken from Table 1. K_O2 values for sperm whale Mb (Mb SW) were taken from Ref. 17. Superscripts M and T stand for monomer and tetramer, respectively. The estimated errors for δΔG_(E7)steric are assumed to similar to those for the δΔG_E7 values in Table 5, i.e. ≤ ±2 kJ/mol. Thus, the values of ΔG_(E7)steric for the open conformation of αTrp(E7) and the Phe(E7) and Trp(E7) mutants of Mb are very close to zero, and the decreases in K_O2 for these mutants are similar to those for the corresponding small apolar E7 mutants.

Globin	E7 mutants	KO2(mutant)/KO2(native)	δΔGPhe or δΔGTrp(E7)	δΔGsmall, apolar E7	ΔG(E7)steric
			kJ/mol	kJ/mol	kJ/mol
αHbA	Phe(E7)M	0.0021	15.0	5.4	9.6
	Phe(E7)T	0.0068	12.1	4.1	8.0
	Trp(E7)M closed	0.0004	19.1	5.4	13.7
	Trp(E7)M open	0.24	3.4	5.4	−2.0
	Trp(E7)T open	0.14	4.7	4.1	0.6
βHbA	Phe(E7)M	0.0043	13.2	6.2	7.0
	Phe(E7)T	0.0051	12.8	6.8	6.0
	Trp(E7)M	0.0039	13.5	6.2	7.3
	Trp(E7)T	0.0051	12.8	6.8	6.0
Mb SW	Phe(E7)	0.0067	12.1	9.8	2.4
	Trp(E7)	0.064	6.7	9.8	−3.1

For α and β subunits, the introduction of the benzyl side chain causes both the loss of net electrostatic stabilization and significant hindrance by the aromatic ring, with ΔG_{Phe(E7)steric}⁰ ≈ +7 to +9 kJ/mol. As a result, the O₂ affinities of these Phe(E7) mutants are very small, i.e. K_O2 ≈0.01 μm⁻¹ or P₅₀ ≈ 100 μm in the R state. The βTrp(E7) mutation causes a similar amount of added hindrance, ΔG_{Trp(E7)steric}⁰ ≈ +7 kJ/mol.

The closed unliganded conformer of αTrp(E7) shows a dramatic amount of steric inhibition, leading to an estimated O₂ association equilibrium constant equal to ∼0.001 μm⁻¹ (P₅₀ ≈ 1000 μm) and ΔG_{Trp(E7)steric}⁰ ≈ +14 kJ/mol. In contrast, the transient open conformation of αTrp(E7) in both monomers and tetramers has an estimated K_O2 value of ∼0.4 μm⁻¹, which is similar to the K_O2 values for the smaller Gly, Ala, and Leu(E7) mutants, and it exerts no steric resistance to ligand binding (ΔG_{Trp(E7)steric}⁰ ≈ 0 kJ/mol, Table 6).

The inhibitory effect of the Phe(E7) and the closed Trp(E7) conformers occurs roughly to the same extent for both O₂ and CO binding (Tables 1 and 2 and Fig. 9A). Decreases in K_O2 and K_CO for Phe(E7) mutants are caused by increased dissociation rate constants (k_O2 and k_CO, see Tables 1 and 2), suggesting that the negative steric effect is due to the direct hindrance of the bound ligand by the benzyl side chain. The low ligand affinities for equilibrium deoxyHb Trp(E7) conformers are due to very low k_O2′ and k_CO′ values. The latter case is an example of indirect steric inhibition of ligand binding due to the requirement for displacement of the bulky E7 side chain from the binding site.

In contrast to the HbA replacements, the Phe(E7) and Trp(E7) mutations in Mb cause little or no hindrance (i.e. small or negative ΔG_(E7)steric values for the Mb mutants in Table 6). These results suggest that the Mb active site can either accommodate both the aromatic side chains and a bound ligand or, more likely, that these large side chains are prevented from entering the active site and directly hindering bound ligands.

E7 Gate Appears to be the Pathway for Ligand Entry into HbA

With one exception (the transient, open αTrp(E7) conformer), there are dramatic decreases in the bimolecular rates of O₂, CO, and NO binding to the subunits of Hb with increasing size of the E7 side chain (Tables 1–3). These results suggest that ligands enter and exit through a channel gated by the distal histidine. We used overall CO association rate constants and the fraction of geminate CO recombination to estimate bimolecular rate constants for ligand entry into isolated HbA subunits and then measured association rate constants for NO binding to both monomers and tetramers to verify these estimates of k_entry′ experimentally (Table 3). The dependence of k_NO′ on the size of the E7 side chain in monomeric α and β subunits and sperm whale Mb is shown in Fig. 10.

FIGURE 10.

Dependence of rate constant for NO association on E7 mutations in monomeric α subunits, β subunits, and Mb. Native HbA contains His at the E7 position. Only the slow phase values for k_NO′ for αTrp(E7) was used because it represents NO binding to the closed, physiologically relevant equilibrium state of the mutant. Data for sperm whale Mb mutants were taken from Ref. 17, and k_NO′ for HbA mutants were taken from Table 3.

The Gly and Ala(E7) mutations open up both HbA subunits and Mb, causing bimolecular rate constants on the order of 200 μm⁻¹ s⁻¹, which is close to the diffusion limit (32, 38, 51, 61). The Trp(E7) mutations markedly decrease k_NO′ to values ≤6 μm⁻¹ s⁻¹, effectively blocking the E7 channel, and there is a roughly monotonic decrease in the rate of NO binding with increasing size of the E7 side chain for other mutants. Scott et al. (17) carried out a much more extensive mutagenesis mapping study of Mb using 90 mutants at 27 different positions, and their results confirmed quantitatively the E7 gate mechanism for ligand entry into Mb, which was originally developed using just E7 mutations (60). By analogy with the more extensive results for Mb, it seems clear that the E7 channel is also the major route for ligand entry into both subunits of HbA.

The most striking difference between the α and β subunits is the appearance of a rapidly reacting, transient intermediate after photolysis of Trp(E7) αCO subunits. Movement of the indole side chain to its equilibrium unliganded conformation in α subunits appears to be relatively slow (∼10⁴ s⁻¹) compared with bimolecular ligand binding at high CO or O₂ concentrations. The small amplitude of the fast phase after complete photolysis of Trp(E7) βCO subunits suggests more rapid side chain relaxation. The cause of the slow relaxation in α subunits appears to be a narrower channel opening, due in part to the large His(CE3) side chain in α subunits (Fig. 8). In β subunits, the channel is larger due to a smaller Ser side chain at the CD3 position, which presumably facilitates inward rotation of the E7 side chain after ligands leave the active site.

As is the case for the Mb mutant (17), the low rates of ligand binding to the equilibrium forms of the Trp(E7) deoxyHb subunits do not rule out alternative trajectories for ligand entry that have been identified by molecular dynamics simulations (62, 63). However, the 10–30-fold decreases in k_entry′ and k_NO′ due to replacement of His(E7) with Trp (Table 3) imply that the alternative pathways may account for only 3–10% of the binding events in the native Hb subunits.

The larger opening in β subunits may also contribute to the 2-fold higher bimolecular rate constants for O₂ and NO binding to native or wild-type β subunits, particularly in tetrameric hemoglobin where protein fluctuations are dampened by the quaternary structure (see supplemental Table 1S). The CD3 amino acid in Mb is Arg and the rate of entry into Mb does increase up ∼2-fold when this residue is decreased in size (17). However, the differences in rate constants between the native α and β subunits are small compared with the effects of the Gly and Trp(E7) mutations, which cause ∼3–5-fold increases and 15–80-fold decreases, respectively, in the rates of ligand entry into the equilibrium R state forms of deoxygenated HbA (Fig. 10).

Conclusions

Our new results for E7 mutants of HbA demonstrate unambiguously that the distal histidine stabilizes bound O₂ to roughly the same extent (∼−5.6 kJ/mol) in both the α and β subunits of R state HbA, in agreement with recent high resolution NMR and crystal structures of oxyHbA (25, 26), as well as theoretical calculations of the energy of interaction between His(E7) and O₂ (64). The strength of this net, favorable electrostatic interaction in HbA, appears to be roughly half that observed for the interaction of His(E7) with O₂ in mammalian myoglobin, where the net electrostatic stabilization is ∼−10 kJ/mol. In both HbA subunits and Mb, ligand discrimination occurs by preferential electrostatic stabilization of bound O₂ rather than steric hindrance of CO. When steric hindrance occurs due to the large aromatic rings of Phe(E7) and Trp(E7), the affinities of both ligands decrease to roughly the same extent in both HbA subunits, and the M value remains constant (Fig. 9).

The bimolecular rate constants for ligand binding to α and β subunits of HbA decrease markedly with increasing size of the E7 side chain. By analogy to Mb, these results suggest that diatomic ligands enter and exit native HbA through a channel created by outward motion of the distal histidine. Thus, the basic stereochemical mechanisms governing the rates and affinities of ligand binding to R state HbA are identical to those that occur in Mb. There are subtle differences between the ligand binding properties of the α and β subunits that appear to be due to the size of the E7 channel opening, which is smaller in α subunits because of His at the CE3 position compared with Ser in β subunits, and the lack of ordered water in the distal pocket of deoxygenated native β subunit.