Human Hemoglobin: Identication of a Key Intermediate

Gary K. Ackers and Jo M. Holt,

doi: 10.1002/9780470048672.wecb222

Advanced Article
Article Contents
Introduction Background Chemistry Key Experiments and Observations

Department of Biochemistry & Molecular

Biophysics, Washington University School of Medicine, St. Louis, Missouri

Human hemoglobin binds four oxygen molecules with positive cooperativity in a binding cascade of eight partially ligated intermediates whose oxygen ligands are distributed in different combinations between the hemoglobins two dimers. Traditionally, it had been assumed that, given their close association in forming the hemoglobin tetramer, the two dimers would always respond to oxygen binding in a synchronous and symmetric manner. Using linkage thermodynamics, the intermediate binding constants are evaluated via dimertetramer assembly with ligand congurations within the tetramer xed through the use of hemesite analogs. It is observed that the free energy contribution of the asymmetrically ligated intermediate composed of one fully oxygenated dimer plus one unoxygenated dimer is not the same as other doubly ligated intermediates, which contain at least one bound oxygen on both dimers. Therefore, the dimers within the tetramer respond to oxygenation differently, and cooperativity is dependent on the distribution of ligands between the two dimers.

Introduction
The structural and functional properties of human hemoglobin (Hb) have been the subject of study for decades, stimulated by the intriguing characteristic of positive cooperativity. How do the four subunits that compose the Hb tetramer communicate with one another? The answer to this question has been sought primarily through the comparison of deoxy with oxy Hb. However, to understand the molecular mechanism of a chemical reaction, it is necessary to characterize the intermediate(s) of the process, and the reaction of Hb with O2 is no exception. The binding of four O2 ligands by human Hb occurs through a series of 14 partially ligated intermediates, of which eight are unique in their combinatorial arrangement of bound O2 among the two -subunits and two -subunits. The well-known sigmoidal binding curve that results (see Fig. 2 for an example) is indicative of a strong positive cooperativity of oxygenation, thus, the binding constants for each Hb intermediate are changing as the O2 binding process continues. The individual microscopic binding constants cannot, however, be measured from the binding curve: Only four average, macroscopic binding constants can be directly observed. This result is a result of several factors, foremost of which is the high cooperativity of O2 binding itself, which suppresses the

concentrations of the intermediates. Thus, the binding curve is dominated by the properties of the two end-states, i.e., the fully deoxygenated tetramer and its fully oxygenated counterpart. Other factors that contribute to the low resolution of the binding curve are the lability of the bound O2 and the continuous dissociation of the tetramer to its constituent dimers. Therefore, in a system that binds O2 close to equilibrium to begin with, the rearrangement of bound O2 among the heme binding sites acts only to mask the individual properties additionally (such as a microscopic binding constant) of a given intermediate. This classic problem of disproportionation can be solved experimentally through the use of hemesite analogs that either block O2 binding or O2 dissociation in specic subunits (1 , 1 , 2 , 2 ) within the tetramer (1). The dissociation of tetramer to free dimer, and the resulting dimer rearrangement among tetramers, cannot be blocked but can be measured. The dimertetramer assembly free energy, G asm , can then be applied as a constraint that permits the determination of the Gibbs free energy, G ij , of each intermediate binding reaction by employing thermodynamic linkage analysis. The microscopic O2 binding constants thus determined reveal a particularly strong energetic coupling between the subunits 1

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

within each dimer of the tetramer (2). This intradimer cooperativity is evident particularly in the intermediate composed of one oxy dimer and one deoxy dimer, or the asymmetric doubly ligated species. Identication of this intermediate provided the rst direct experimental evidence of intradimer cooperativity, which challenged the commonly held two-state model of cooperativity, in which the two dimers within the tetramer are assumed to maintain the same structural and energetic properties throughout the binding process. Rather than maintaining dimerdimer symmetry, the dimers each exhibit a unique O2 afnity and continue to modulate the O2 afnity of each other.
O2

dimer-dimer interface

deoxyHb ? species 01

K0

Background
Since the determination of its crystal structure almost ve decades ago, the study of cooperativity and allostery in human Hb was focused primarily on the properties of the two end-states, the deoxy and oxy tetramers. The approach to mechanistic questions of subunitsubunit coupling within the tetramer is now shifting to the characterization of the partially ligated intermediates. Although crystal structures are not yet available for the intermediates, their individual O2 binding constants are now determined for one set of solution conditions.

O2 O2 O2
12a 12b 11b

11a

K1
O2 O2
21a

O2 O2
21b

O2 O2 O2 O2
22a 22b

O2 O2
23

O2 O2
24

K2
O2 O2 O2
31a

O2 O2 O2
31b

O2 O2 O2
32a

O2 O2 O2
32b

Structural elements of the hemoglobin tetramer

The human hemoglobin tetramer is composed of two types of polypeptide chains, designated (with 141 amino acid residues) and (with 146 residues). Both subunit types exhibit a high degree of helical content with no structure, and each contains a noncovalently associated b-type Fe heme to which O2 binds. As a tetramer, the four subunits are organized structurally as two dimers held together by a polar, water-lled dimerdimer interface (Fig. 1) (3, 4). Although the dimerdimer interface dissociates readily under physiologic conditions to produce free dimers, the intradimer interface is hydrophobic and only dissociates appreciably in the presence of certain metal ions or under denaturing conditions. Therefore, the dimers are shared constantly and redistributed among the tetramers. When the Hb tetramer binds O2 , a large change in quaternary structure is observed in which the two dimers reorient relative to one another. From the deoxy or T structure, this reorientation can occur in either one of two major forms, which yields the R or the R2 structure. The R structure is observed by crystallization of oxyHb under high salt conditions, whereas the R2 structure is observed in low salt crystals. Nuclear magnetic resonance analysis has demonstrated that the R and R2 structures can coexist in solution (5). Additional crystal structure conditions have revealed that multiple oxy or R as well as deoxy or T forms are possible (68). All structural forms of the tetramer are ligated symmetrically (or unligated); i.e., the two dimers within the tetramer are always observed as structurally equivalent in available crystal structures. Structural changes that take place in the dimers themselves are referred to in the Hb literature as tertiary and include the movement of the heme Fe into the plane of the 2

K3
oxyHb ? species 41

Figure 1 The cascade of O2 binding to the four subunits of the human Hb tetramer. The polar dimerdimer interface is composed of 1 2 plus 2 1 contacts. Two intradimer interfaces exist, the 1 1 and the 2 2 ; both are nonpolar. Each tetramer is assigned a species designation, which begins with deoxy Hb (species 01) and ending with oxy Hb (species 41). The rst O2 can bind to any one of four subunits; however, because oxygenation of the 1 subunit is indistinguishable from oxygenation of the 2 subunit, the two isomeric tetramer species that result are designated 11a and 11b. This labeling is likewise the case for the subunits. Similar isomeric oxygenation microstate tetramers are also generated in the second and third binding steps. Crystal structures are from the Arnone laboratory 14, 15.

heme when oxygenation begins; subsequent movement of helices are close to the heme and to the dimerdimer interface. A signicant structural change in the intradimer interface is not observed in crystal structures, which has led to the conclusion that oxygenation-induced tertiary structural changes are not communicated between the subunits within a dimer, i.e., between 1 and 1 or between 2 and 2 . Although this belief has spanned the course of several decades, more subtle structural changes in the intradimer interface have not been ruled out. Recently, Arnone et al. have pointed out that the intradimer structure has not been analyzed thoroughly in modern crystal structures of Hb

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

(6). Very few attempts to crystallize the partially ligated intermediates of Hb have been reported, and structural information is still not available. When O2 binding occurs, the quaternary reorientation of the two dimers forms the primary basis for the popular two-state model of Hb cooperativity. In this model, O2 binding to a heme Fe causes signicant structural change only in the dimerdimer interface. The bonds of the deoxy or T interface, held by the model to be signicantly stronger than those of the oxy or R interface, maintain the tetramer in the low-afnity conformation. Oxygenation of deoxy Hb causes bonds in the T dimerdimer interface to break, which weakens the low-afnity T state relative to the high-afnity R state. Therefore, the O2 afnity of the tetramer is controlled by the strength of the dimerdimer interface, as modulated by the number of O2 ligands bound to the subunits. The particular conguration of the bound O2 among the four hemesites is not signicant in this model. For example, in a tetramer that bears two bound O2 , six possible congurations of the bound ligands exist among the four hemesites (see Fig. 1). In the two-state model, all six of the doubly ligated tetramers have the same O2 afnity because the presence of two ligands results in the same number of bonds broken in the dimerdimer interface, regardless of the exact distribution of the ligands among the four hemesites.

process (such as the historic two-state model or models with multiple T and R forms) requires that the microscopic stepwise binding constants for each binding step be equal to one another:

K0111 = K0112 K1121 = K1221 = K1122 = K1222 = K1123 = K1224 K2131 = K2231 = K2431 = K2132 = K2232 = K2332 K3141 = K3241 (2)
An exception to these equalities can be made to accommodate inherently different O2 afnities of - and -subunits; however, because differences between - and -subunit binding constants are not signicant in normal human Hb, this variation is not additionally considered here.

Chemistry
The dimertetramer assembly constant K asm is very sensitive to O2 binding by human Hb, which ranges over 130,000-fold among the intermediates, in comparison with the 400-fold change in O2 binding constant under conditions of this study (pH 7.4, 21.5 C) (9). The equilibrium between free dimer and assembled tetramer is an integral property of Hb in solution, and has a marked impact on the O2 binding curve observed experimentally.

Relationship of macroscopic to microscopic binding constants

Oxygen-binding curves can be analyzed directly to yield four macroscopic binding constants: K 1 , K 2 , K 3 , and K 4 . Usually, the macroscopic constants are dened as product constants, i.e., products of the stepwise macroscopic constant K ii+1 (where i is the number of bound O2 ):

The concentration-dependent oxygen isotherm

The sigmoidal shape of the O2 binding isotherm, i.e., the cooperativity of O2 binding, is dependent on the concentration of the Hb solution (Fig. 2). As the solution is diluted, the relative concentration of free dimers increases, and unlike the tetramer, the free dimer binds O2 noncooperatively with high afnity. Thus, the true tetramer-binding curve is observed only at the highest Hb concentrations: At lower concentrations, the experimental isotherm reports a mixture of tetramer and free dimer (1). The thermodynamic scheme that links the ligation of the free dimer to the ligation of the assembled tetramer (Fig. 2) shows all reaction equilibriums that contribute to the concentrationdependent isotherms. Binding to the free dimer is designated by G int , which denotes the change in intrinsic (noncooperative) free energy. To solve the linkage scheme experimentally, the assembly free energy change for the deoxy tetramer, 0 G asm , is determined in an independent kinetic measurement using haptoglobin trapping of the free dimer. In addition, the corresponding 4 G asm (for the oxy tetramer) is measured independently by large-zone size-exclusion chromatography (1).

K1 K2 K3 K4

K01 K02 K03 K04

= K01 = (K01 )(K12 ) = (K01 )(K12 )(K23 ) = (K01 )(K12 )(K23 )(K34 )

(1)

The constant K ii+1 is composed of microscopic constants, as each O2 binding step is composed of multiple microscopic reactions, which is illustrated by the reaction arrows in Fig. 1. Thus, 4 ways exist to bind the rst O2 , 12 ways to bind the second O2 , 12 ways to bind the third O2 , and 4 ways to bind the fourth O2 . Each microscopic constant is designated by the notation ij of the species formed in the binding process (Fig. 1, Table 1). For each binding step i = 1,2,3, and 4, the macroscopic constant K ii+1 represents the average of the microstate constants k ij(i+1)j , with accompanying statistical factors that account for the different isomeric forms of the microstate tetramers, as shown in Table 1. Any scenario in which the tetramer is assumed to maintain symmetry between the two dimers throughout the O2 binding

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

Table 1 Relationship between macroscopic and microscopic O2 binding constants Average microstate binding constants k0111 = k0112 = k1121 = k1221 = k1122 k1222 k1123 k1224 k2131 k2431 k2132 k2232 k2332 = = = = = = = = =
k0111a + k0111b 2 k0112a + k0112b 2 k11a 21a + k11b 21b 2 k12a 21a + k12b 21b 2 k11a 22a + k11b 22b 2 k12a 22a + k12b 22b 2 k11a 23 + k11b 23 2 k12a 24 + k12b 24 2 k21a 31a + k21b 31b 2 k22a 31a + k22b 31b 2 k2431a + k2431b 2 k21a 32a + k21b 32b 2 k22a 32a + k22b 32b 2 k2332a + k2332b 2 k31a 41 + k31b 41 2 k32a 41 + k32b 41 2

Overall binding step 01

Microstate binding step 01 11 01 12 11 21

Macro Ki i +1 k01 =
k0111 + k0112 2

Micro kij (i +1)j

12

12 21 11 22 12 22 11 23 12 24 21 31

K12 =

k1121 + k1221 + k1122 + k1222 + k1123 + k1224 6

23

22 31 24 31 21 32 22 32 23 32

k2231 =

k23 =

k2131 + k2231 + k2431 + k2132 + k2232 + k2332 6

34

31 41 32 41

k3141 = k3241 =

k34 =

k3141 + k3241 2

Each binding step is composed of multiple microstate binding steps, and each microstate binding step represents an average of isomeric forms of the microstates, yielding the average microstate binding constants. The macrostate binding constant is then the average of the binding constant for each microstate.

The macroscopic thermodynamic linkage scheme

The concentration dependence of the O2 binding curve is a result of thermodynamic linkage between O2 binding and dimertetramer assembly. Consider the rst binding step as illustrated in the linkage scheme in Fig. 2. Conservation of free energy dictates that the change in free energy during assembly followed by ligation must equal the change in free energy during ligation followed by assembly:
0

necessary to x the hemesite ligand to prevent disproportionation caused by ligand rearrangement among the hemesites. Hemesite analogs employed for either the deoxy heme (which replaces Fe2+ ) or the oxy heme (which replaces Fe2+ O2 ) are:

Native Fe2+ /Fe2+ O2

Analog Zn2+ /Fe2+ O2 Fe2+ /Fe3+ CN Co2+ /Fe2+ CO Co2+ /Fe3+ CN Fe2+ /Mn3+

Gasm + G1 = Gint + 1 Gasm

(3)

Therefore, the change in the tetramer assembly free energy during O2 binding is equal to the change in the O2 binding constant during tetramer assembly:
1

Gasm 0 Gasm = G1 Gint

(4)

Each stepwise microscopic binding reaction follows the same formula, as thermodynamic linkage holds for all binding steps.

Forming hybrid tetramers from parent tetramers

The equilibrium between free dimer and tetramer can be exploited to provide a means of forming partially ligated hybrid tetramers by mixing any two-parent tetramers. However, it is 4

Each hemesite analog perturbs the Hb tetramer in some manner: The Fe3+ CN and Mn3+ analogs are susceptible to electron exchange over very long incubation periods (10). The Co2+ analog exerts a specic effect on -subunit binding constants (11), and the use of Zn2+ imparts a light sensitivity to the solution (12). However, the relative relationship between each measured microstate-binding constant is found to be invariant among the analog species (9). Using the Zn2+ /Fe2+ O2 analog as an example, when deoxy ZnHb (species 01 ) is mixed with an equimolar amount of native FeHb (species 41 ), a mixture is formed that contains the asymmetrically doubly ligated species 21 (Fig. 3a). Likewise, species 11 or 12 are formed by mixing species 01 with 23 or 24 , respectively (see Fig. 1 for illustrations of each species). Species 22 is formed by mixing species 23 with 24 . And

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

+ 8.3 1.0
fraction hemesites bound
O2 dilute Hb O2

14.3
0G asm

Gint + 11.4
1G asm

G1

5.4 O2

O2 8.3 2 O2 8.3

concentrated Hb

Gint +
2

G2 8.8 Gasm G3 7.2

3G asm

5.7 2 O2

0.5

O2 O2 O2 O2

Gint + O 2

6.7 3 O2

0.0 0 4 8 12 O2 8.3
O2 O2

ln pO2 (mm Hg)

Gint + O2
O2

G4 8.0
4G asm

9.1

O2 O2 O2 O2

Figure 2 The dependence of O2 binding on Hb concentration. Binding curves are shown (solid black lines) for Hb concentrations of 0.005, 0.04, 0.10, 0.27, 1.0, 5.4, and 38 M (from left to right). Theoretical binding curves (broken red lines) are shown for a pure tetramer solution and a pure dimer solution. The macroscopic, thermodynamic linkage scheme relates the dimer tetramer assembly constants to the O2 binding constants for free dimer and assembled tetramer. The brackets around gurines indicate that the O2 ligand may be bound at any one of the available deoxy hemesites. Thus, the macroscopic constants are average values for multiple microscopic processes.

species 31 or 32 are formed by mixing species 41 with 24 or 23 , respectively. In this way, all possible combinatorial forms of the partially ligated intermediates can be formed. Only the parent tetramers 01 , 23 , 24 , and 41 are present in pure form in solution: All other species are present in equilibrium with their respective parent tetramers. Assembly of tetramers from free dimers occurs very rapidly with a rate constant k on of 1.1 0.1 106 M1 s1 . This assembly is referred to as the consensus on constant, which is not dependent on the number of bound ligands or their conguration, the presence of hemesite analogs, or the presence of mutations. Therefore, it is in the tetramerfree dimer dissociation constant, k off , that the sensitivity of the assembly constant is manifest, because
ij

measured ij G asm of the parents. In the kinetic approach, the tetramerdimer dissociation constant is measured by trapping free dimers kinetically with the plasma protein haptoglobin.

Assembly free energy of hybrid tetramers

Low-temperature isoelectric focusing
Species 21 represents a unique halfway point in oxygenation of Hb in that one of its dimers is fully ligated and the other is fully deoxygenated. The 21 hybrid is formed in vitro by mixing species 01 and 41 , as in the example in Fig. 3. One of the two parent Hbs carries an electrophoretic tag to enhance separation based on charge, typically the HbS variant (6 GluVal). At equilibrium, the assembly free energy of species 21 is related to the assembly free energies of each parent by
21

Gasm = RT ln

kon koff

(5) Gasm =

01 G

asm

+ 41 Gasm 2

+ 21

(6)

Key Experiments and Observations

Two experimental approaches are taken to measure the assembly free energy of partially ligated Hb intermediates: an equilibrium method and a kinetic protocol. In the equilibrium method, symmetrically ligated tetramers are mixed to generate asymmetrically ligated hybrid tetramers. Then, the relative stability of the hybrid to its parents is measured, which permits the hybrid assembly free energy to be calculated from the independently

where 21 is the free energy deviation from the average of the parent tetramer assembly free energies. The deviation free energy is measured directly from the fraction of each tetramer at equilibrium:

21 = RT ln

f21 2 f01 f41

(7)

The relative fractions of hybrid and parent tetramers are measured by quenching the dissociation of tetramer to free 5

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

(a)
O2 O2+ O O2 + 2

(b) additive

01 (01G
asm)

41 (41Gasm) 2 2 O + 2
21G asm

O2

observed

2 O 2
+

O2

21 01

O2 O + 2

O2 O2+ O O2 + 2

21

41

Figure 3 Hybridization of deoxy and oxy Hb to form the asymmetrically ligated Hb species 21. (a) Each parent tetramer is in equilibrium with two free dimers. The free dimers may reassemble to the original parent tetramer or may assemble with one another to form the hybrid. The oxy Hb tetramer carries the sickle Hb mutation, on the surface of each -subunit (yellow). (b) The populations of the three-tetramer species are at equilibrium. Their electrophoretic separation is made possible by their charge difference. If the assembly free energy of the hybrid was the average of that of the two parents, the hybrid population would be 50% of the hybrid mixture, or additive, rather than the 3% observed experimentally.

dimers with low temperature (25 to 35 C). Electrophoretic focusing is then conducted at the same low temperature to maintain a quenching environment. The equilibrium population of species 21 lies far from the average of the two parents, which would be observed as a 1:2:1 binomial distribution and 48% of the mixture as hybrid. Instead, species 21 is observed at only 3% of the hybrid mixture (Fig. 3), which translates to a deviation free energy of 2.0 kcal/mol and an assembly free energy 21 G asm of 9.2 0.2 kcal/mol (10). In contrast, the assembly free energy of species 22 , which is also determined by low-temperature isoelectric focusing, was measured at 7.7 0.3 kcal/mol, a value similar to the other doubly ligated species 23 and 24 . The results from low-temperature isoelectric focusing for the remaining partially ligated intermediates yielded the following pattern of microscopic binding constants for each binding step: For the rst binding step, k 0111 = k 0112 ; for the second binding step, (k 1121 = k 1221 ) > (k 1122 = k 1222 = k 1123 = k 1224 ); for the third step, (k 2131 = k 2231 ) < (k 2431 = k 2132 = k 2232 = k 2332 ); and for the nal step, k 3141 = k 3241 . These results show that binding two ligands to the same dimer within a tetramer occurs with a greater positive cooperativity than binding one ligand to each dimer. This distinction between the distribution of O2 ligands within the tetramer does not agree with Equation 2, which demonstrates that one of the basic tenets of all symmetric (multistate or two-state) models of cooperativity is not veried by experiment.

change in absorbance, can be monitored by uorescence spectroscopy. Thus, mixing Hb with a slight excess of Hp results in complete conversion to the Hp(dimer)2 complex by pulling the tetramer dissociation reaction to the right:

Hb 2 dimers + Hp Hp( dimer)2 k

on

koff

kHp

(8)

Haptoglobin trapping
The tetramerfree dimer dissociation constant, k off , is measured in the presence of haptoglobin (Hp), which is a plasma protein that binds two free dimers rapidly and essentially irreversibly (13). The reaction of Hp with Hb that contains deoxy subunits can be followed by UV/visible spectroscopy, whereas the Hp reaction with fully ligated Hb, which has no appreciable 6

Because both k on and k Hp are very rapid (essentially diffusion-controlled) processes, the overall rate-limiting step in Equation 8 is k off . The k off for the asymmetrically ligated species 21 was measured rst by forming the unligated version of the hybrid by mixing native Fe-heme deoxy Hb with Zn Hb (Fig. 4). Because both parents and hybrid (the unligated species 21 or 21u ) have the same assembly free energy, 01 G asm , mixing the parent tetramers in a 1:1 ratio generates an equilibrium hybrid mixture that contains approximately 50% hybrid 21u . Because of the slow k off for both parents and hybrid (7.5 hours), equilibrium is attained after 3 days of incubation. In practice, the amount of hybrid present after 24 hours is sufcient for detection in the reaction with Hp. The anaerobic hybrid mixture is mixed with an oxygenated solution of Hp in a stopped-ow instrument. The absorbance is monitored for 20 seconds, and the resulting observed rate constant is measured at 0.20 0.02 s1 . This results in an assembly free energy, 21 G asm , of 9.1 0.1 kcal/mol when combined with the consensus on constant in Equation 6 (9). This value is in excellent agreement with the results of the equilibrium low-temperature isoelectric focusing experiment.

Model-independent distribution of Gc among the hemoglobin intermediates

The measurements described here show that binding O2 ligands to only one dimer within the Hb tetramer occurs with a

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

+ native Fe

Zn Zn Zn Zn

Zn analog dimer exchange

Zn Zn Zn Zn Zn Zn

+ Fe

FeZn hybrid + O2 + Hp

Zn

An extensive kinetic analysis of ligand binding in normal Hb carried out by Goldbeck et al. has demonstrated agreement with the unique binding constant for the asymmetric doubly ligated Hb (14). Thermodynamic experiments from the Ackers laboratory that employs asymmetrically modied human Hbs have conrmed the asymmetric character of Hb cooperativity (15). This discovery generates critical energetic and structural questions, particularly with respect to the relationship of intradimer to cross-dimer cooperativity, in a classic system that was once thought to be well understood.

O2 O2 O2 O2

O2 Zn O2 Zn

Zn Zn Zn Zn

References
1. Ackers GK, Holt JM, Burgie ES, Yarian CS. Analyzing intermediate state cooperativity in hemoglobin. In: Methods in Enzymology. Volume 379. Energetics of Biological Macromolecules Part D. 2004. Holt Jo M., Johnson, MJ, and Ackers GK, eds. Elsevier, San Diego, CA. pp. 328. 2. Ackers GK, Doyle ML, Myers D, Daugherty MA. Molecular code for cooperativity in hemoglobin. Science 1992;255:5463. 3. Kavanaugh JS, Rogers PH, Case DA, Arnone A. High resolution x-ray study of deoxyhemoglobin Rothschild 37b Trp to Arg: a mutation that creates an intersubunit chloride-binding site. Biochemistry 1992;31:41114121. 4. Silva MM, Rogers PH, Arnone A. A third quaternary structure of human hemoglobin A at 1.7 A resolution. J. Biol. Chem. 1992;267:1724817256. 5. Lukin J, Kontaxis G, Simplaceanu V, Yuan Y, Bax A, Ho C. Quaternary structure of hemoglobin in solution. Proc. Nat. Acad. Sci. USA 2003;100:517520. 6. Kavanaugh JS, Rogers PH, Arnone A. Crystallographic evidence for a new ensemble of ligand-induced allosteric transitions in hemoglobin: the T-to-T(high) quaternary transitions. Biochemistry 2005;44:61016121. 7. Mueser T, Rogers P, Arnone A. Interface sliding as illustrated by the multiple quaternary structures of liganded hemoglobin. Biochemistry 2000;39:1535315364. 8. Samuni U, Juszczak L, Dantsker D, Khan I, Friedman AJ, P erez-Gonz alez-de-Apodaca J, Bruno S, Hui HL, Colby JE, Karasik E, Kwiatkowski LD, Mozzarelli A, Noble R, Friedman JM. Functional and spectroscopic characterization of half-liganded iron-zinc hybrid hemoglobin: evidence for conformational plasticity within the T state. Biochemistry 2003;42:82728288. 9. Holt JM, Klinger AL, Yarian CS, Keelara V, Ackers GK. Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 1. Cooperative and noncooperative oxygen binding in Zn-substituted hemoglobin. Biochemistry 2005;44:1192511938. 10. Ackers GK, Holt JM, Huang Y, Grinkova Y, Klinger AL, Denisov I. Conrmation of a unique intra-dimer cooperativity in the human hemoglobin a1 b1 half-oxygenated intermediate supports the symmetry rule model of allosteric regulation. PROTEINS: Struct. Func. Gen. 2000;(Suppl. 4):2343. 11. Huang Y, Ackers GK. Transformation of cooperative free energies between ligation systems of hemoglobin: resolution of the carbon monoxide binding intermediates. Biochemistry 1996;35:704718. 12. Scholler DM, Wang M-YR, Hoffman BM. Metal-substituted hemoglobin and other hemoproteins. Meth. Enzymol. 1972;76: 487493. 13. Nagel RL, Gibson QH. The hemoglobin-haptoglobin reaction as a probe of hemoglobin conformation. Biochem. Biophys. Res. Commun. 1972;48:959966.

FeO2 t1/2 = 1 s
O2 O2
1.00

FeO2/Zn hybrid

Zn t1/2 = 7.5 h Hp
Zn Zn

Hp

Abs424

0.99

Hp

Zn t =4s Zn 1/2

0.98

0.97

10

15

20

time (sec)
Figure 4 Chemical strategy for the measurement of the dissociation rate constant for the hybrid tetramer species 21. The unligated version of the hybrid is formed from a mixture of Fe Hb and Zn Hb. When exposed to an oxygenated solution of Hp, the Fe hemes bind O2 , which destabilizes the tetramer immediately, and it begins to dissociate into free dimers. Hp traps all free dimers, but the absorbance change is caused by the production of free dimer by dissociation of the hybrid.

unique binding constant that differs signicantly from that for the other doubly ligated intermediates (Table 2). This nding does not agree with the historically dominant presumption of a symmetric T/R-based model for cooperativity, which requires that the hemesites in both dimers maintain equal O2 afnity at each binding step. This asymmetric doubly ligated Hb intermediate, species 21 , is considered a key intermediate in that its unique conguration of ligands reveals the presence of functional differences between the two dimers. Symmetric models for cooperativity have been supported by the observation that only two functional states of the Hb tetramer, the low-afnity T and high-afnity R state, are required to describe O2 binding curves obtained over a range of solution conditions. However, the O2 binding curves are dominated by the properties of the two end-states, largely because of the presence of strong cooperativity, and thus cannot provide a clear distinction between most allosteric models. To begin to understand the rules for coupling between the subunits in Hb, it is necessary to measure the microscopic binding constants experimentally, as they cannot be determined from the binding curves.

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Human Hemoglobin: Identication of a Key Intermediate

Table 2 The distribution of binding free energy among the individual binding steps in Hb Stepwise binding free energy, G(ii+1)j kcal/mol 0.3 0.4 0.4 0.3 0.3 0.5 0.5 0.3 Stepwise microscopic binding constant, k(ii+1)j M1 1 3 8 400 1 1 70 400 e e e e e e e e + + + + + + + + 4 4 4 4 4 4 4 4

Binding steps

Oxygenation through the asymmetric doubly ligated tetramer 11 or 12 5.5 1 01 + O2 21 6.1 2 (11 or 12 ) + O2 31 or 32 6.6 3 21 + O2 41 8.9 4 (31 or 32 ) + O2 Oxygenation through the symmetric doubly ligated tetramer 1 01 + O2 11 or 12 22,23 or 24 2 (11 or 12 ) + O2 31 or 32 3 (22,23 or 24 ) + O2 41 4 (31 or 32 ) + O2 5.5 4.8 7.9 8.9

The binding cascade shown in Fig. (1) is essentially composed of two different pathways, passing through either an asymmetrically ligated species at the second binding step or a symmetrically ligated species. 14. Goldbeck RA, Esquerra RM, Holt JM, Ackers GK, Kliger DS. The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 1. Microstate linear free energy relations. Biochemistry 2004;43:1204812064. Ackers GK, Dalessio PM, Lew GH, Daugherty MA, Holt JM. Single residue modication of only one dimer within the hemoglobin tetramer reveals autonomous dimer function. Proc. Natl. Acad. Sci. USA 2002;99:97779782. Royer WEJ, Zhu H, Gorr T, Flores J, Knapp J. Allosteric hemoglobin assembly: diversity and similarity. J. Biol. Chem. 2005;280: 2747727480. Wyman J Jr. Linked functions and reciprocal effects in hemoglobin: a second look. Adv. Protein Chem. 1964;19:223286. Wyman J, Gill SJ. Binding and Linkage. Functional Chemistry of Biological Macromolecules. 1990. University Science Books, Mill Valley, CA.

15.

Further Reading
Ackers GK. Deciphering the molecular code of hemoglobin cooperativity. Adv. Prot. Chem. 1998;51:185253. Ackers GK, Holt JM. Asymmetric cooperativity in a symmetric tetramer: human hemoglobin. J. Biol. Chem. 2006;281:1144111443. Jayaraman V, Spiro TG. Structure of a third cooperativity state of hemoglobin: ultraviolet resonance Raman spectroscopy of cyanomethemoglobin ligation microstates. Biochemistry 1995;34:45114515.

See Also
Bioenergetics of Self-Assembly Energetics of Protein Folding Ligand-Operated Membrane Channels Protein-Protein Interactions Thermodynamics in Living Systems

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.