Normal and Abnormal Protein Subunit Interactions in Hemoglobins*

The characteristic properties of hemoglobin are due to the manner in which its individual subunits bond to one another first as an ab dimer and then as an a2b2 tetramer. These subunit interactions also control the binding of allosteric regulatory molecules because of sites they create as they interact with one another. Some of these interactions in hemoglobin change in the transition between its tetrameric oxy (R, for “relaxed”) or deoxy (T, for “tense”) conformational states; adult human hemoglobin A (a2b2) functions as the physiological carrier of O2 between the arterial and the venous circulation in these two conformations, respectively. The transition between these quaternary states is accompanied by concerted changes in the tertiary structure of the individual subunits upon O2 binding known as cooperativity, which is responsible for the sigmoidal shape of the O2 equilibrium curve (1–3). Myoglobin delivers O2 during muscle contraction, as described in a recent minireview (4), and it has a hyperbolic O2 equilibrium profile, i.e. no cooperative interactions because it is a single subunit protein. In tetrameric hemoglobin certain sites between the subunits at the quaternary level have the precise geometry or chemical reactivity to bind 2,3-diphosphoglycerate (2,3-DPG), protons, and chloride preferentially to the deoxy conformational state and hence shift the equilibrium away from the oxy conformation, thereby favoring O2 release. In each quaternary tetramer the oxy and deoxy dimer pairs interact differently to form the two types of tetramer-dimer interfaces in the R and T states. The strength of these interactions influences O2 binding or release in these respective states and determines how easily the tetramer dissociates to dimers. In human Hb, dimers themselves are held together by strong interactions between their aand b-subunits that do not differ significantly for the two R and T conformations.

The characteristic properties of hemoglobin are due to the manner in which its individual subunits bond to one another first as an ␣␤ dimer and then as an ␣ 2 ␤ 2 tetramer. These subunit interactions also control the binding of allosteric regulatory molecules because of sites they create as they interact with one another. Some of these interactions in hemoglobin change in the transition between its tetrameric oxy (R, for "relaxed") or deoxy (T, for "tense") conformational states; adult human hemoglobin A (␣ 2 ␤ 2 ) functions as the physiological carrier of O 2 between the arterial and the venous circulation in these two conformations, respectively. The transition between these quaternary states is accompanied by concerted changes in the tertiary structure of the individual subunits upon O 2 binding known as cooperativity, which is responsible for the sigmoidal shape of the O 2 equilibrium curve (1)(2)(3). Myoglobin delivers O 2 during muscle contraction, as described in a recent minireview (4), and it has a hyperbolic O 2 equilibrium profile, i.e. no cooperative interactions because it is a single subunit protein. In tetrameric hemoglobin certain sites between the subunits at the quaternary level have the precise geometry or chemical reactivity to bind 2,3-diphosphoglycerate (2,3-DPG), 1 protons, and chloride preferentially to the deoxy conformational state and hence shift the equilibrium away from the oxy conformation, thereby favoring O 2 release. In each quaternary tetramer the oxy and deoxy dimer pairs interact differently to form the two types of tetramer-dimer interfaces in the R and T states. The strength of these interactions influences O 2 binding or release in these respective states and determines how easily the tetramer dissociates to dimers. In human Hb, dimers themselves are held together by strong interactions between their ␣and ␤-subunits that do not differ significantly for the two R and T conformations.
All stages of interactions between subunits in the assembly process (known as association) and their reciprocal dissociations are in continuous and rapid equilibrium as shown in Equation 1. In this minireview, we focus first on how subunit interactions between the two dimer pairs result in formation of the central cavity (also referred to as the crystallographic dyad axis), which is involved in the binding of some allosteric regulators to one state but not the other. We then consider the details of aggregation of tetramers into polymers of sickle hemoglobin (HbS), (␣ 2 ␤ 2 ) n also in one state but not the other, and finally we describe an example of the complete dissociation of tetramers to monomers in fish hemoglobin, which is unlike human hemoglobin where dissociation essentially stops at the dimer.

Subunit Interfaces of Human Hemoglobin
Of the two types of interfaces between human hemoglobin subunits, the one between the individual ␣and ␤-subunits (referred to as the ␣ 1 ␤ 1 or the ␣ 2 ␤ 2 interface; equilibrium 1 in Equation 1) does not undergo significant changes during the conformational transition between the oxy and deoxy tetrameric states. In contrast, the interface between the two ␣␤ dimer pairs that form the tetramer (referred to as the ␣ 1 ␤ 2 or the ␣ 2 ␤ 1 interface; equilibrium 2 in Equation 1), which has been the subject of many studies especially regarding cooperative subunit interactions (1-7), undergoes significant structural rearrangements of subunit contacts between amino acid side chains as the tetramer moves between the oxy and deoxy conformations. When the dimer pairs combine to form the tetramer, the central cavity is formed (1) as a region for the binding of allosteric regulatory molecules (Fig. 1).
Most human hemoglobins are tetramers but a few exist as stable dimers, e.g. the natural variant Hb Rothschild (8) and the recombinant hemoglobin D99K (␤) (Asp-99 (␤) 3 Lys) (9), which have disruptive amino acid substitutions preventing formation of the tetramer-dimer interface and thus the central cavity. However, Hb from some clams is a functional dimer (10,11), and it has a cooperative mechanism that is completely different from that found in human hemoglobin because its monomers fit together differently than in vertebrate hemoglobins. Such findings demonstrate the advantages of studying other hemoglobin types to further our understanding of the different mechanisms that have evolved in heme proteins to facilitate O 2 transport.

Anions in Central Cavity of Tetramer
Ever since the structure of hemoglobin was solved it has been known that the central cavity was larger in the deoxy conformation than in the oxy conformation ( Fig. 1), but the reason was not known. At one end of this cavity 2,3-DPG binds with high affinity between the two ␤-chains in the deoxy conformation to shift the equilibrium toward that state to promote O 2 release (12,13). An important binding site for chloride, which is also a physiological regulator (3, 14 -18), is at the other end between the two ␣-chains. A second important chloride site was recently discovered between these two regions in the middle of the central dyad axis (19,20). To identify it, "random" (in contrast to "specific") chemical modification was developed where the objective was to react minimally all sites having a common functional feature (e.g. chloride binding) while preserving the functionality of hemoglobin (21). In random chemical modification the experimental goal is to obtain a mixture of hemoglobin tetramers with both major and minor sites modified (19,21). The strength of chloride-binding sites is also readily measured because the extent of the random modification of the amino groups is related to their binding affinity for chloride anion as long as the modification at any one site has not reached its maximum.
Symmetry in Central Cavity Influences O 2 Affinity-In the deoxyhemoglobin conformation, the symmetrical relationship between the amino groups that bind chloride functionally in the central cavity (identified by random modification described above) is shown in red in Fig. 1; this is likely a major factor in maintaining the central cavity in the more open deoxy conformation because of charge repulsion of bound chloride anions; in the restricted central cavity of oxyhemoglobin, such symmetry does not exist. This mechanism is reminiscent of the tight binding of 2,3-DPG because of symmetrically opposed pairs of positively charged side chains on each ␤-chain in the deoxy conformation (13).
Overlapping Effects of Allosteric Regulators in Central Cavity (Functional Redundancy)-It has long been realized that there was mutual competition between allosteric regulatory anions in their ability to shift the conformational equilibrium toward the deoxy conformation. For example, when O 2 release was maximized by DPG, addition of chloride had little further effect, but less than stoichiometric amounts of each showed additive effects (3,14,22). This behavior can be explained in terms of overlapping regions for anion binding in the central cavity (DPG at one end and chloride at the other end and in the middle) to form the open central cavity so that the redundant effects reflect different regional contributions of these allosteric regulators.

Different Subunit Interactions in Adult and Fetal Hemoglobins
Represent a Probe for Relationships within the Tetramer Hemoglobin F (fetal Hb) composed of ␣ and ␥ subunits is known to have interactions between its dimer pairs different from those between ␣ and ␤ subunits in HbA, as well as a decreased 2,3-DPG response compared with HbA (23,24). It has recently been found that the tetramer-dimer interactions are much stronger in HbF than in HbA (25). From a physiological point of view, these features of HbF are likely involved in the efficient transfer of O 2 from maternal to fetal circulation. Biochemically, these differences in tetramer strength and in DPG response provide a useful experimental tool to study the long range relationship between different regions of the tetramer. Indeed, such a relationship has recently been demonstrated in a recombinant Hb in which the five amino acid differences between the HbA and HbF interfaces (of the total 39 between ␤ and ␥ subunits) were replaced by those in HbF, but the remainder of the HbA sequence was retained (25). This recombinant HbA/F had tetramer dissociation properties and a DPG response that were intermediate between those of hemoglobins A and F, indicating that other amino acids elsewhere in the sequence have a role in controlling the properties of the interfaces themselves.

The Role of N-terminal Acetylation in Subunit Assembly
Nascent eukaryotic proteins universally begin with an N-terminal methionine, which is frequently removed to expose a subsequent amino acid as the new N terminus in a poorly understood process of progressive N-terminal acetylation and deacetylation (26). Therefore, mature proteins have a variety of either free or acetylated N-terminals but whether there is a biological purpose for one or the other is not known. Human fetal hemoglobin occurs in both forms, N-acetylated HbF 1 and unacetylated HbF o (or simply HbF), as minor components in normal adult blood (27). Some embryonic hemoglobins (28) and one adult hemoglobin variant (Hb Raleigh) (29) also contain N-terminal acetyl groups; many fish hemoglobins have this modification (30). HbF 1 was found recently to differ significantly from HbF in its propensity to dissociate to dimers; its dissociation constant is like HbA rather than HbF (25). The presence of N-acetyl groups at one end of the central cavity probably weakens the contacts between the dimer pairs in the tetramer by abolishing positive charges of the N-terminal amino groups. Indeed, acetylation may represent the general mechanism of destabilizing either protein-protein or protein-nucleic acid interactions; acetylation of histone lysine amino groups leading to gene expression is an example of the latter process (31).

Hemoglobin Tetramer Strength
The strength of the hemoglobin tetramer, i.e. the bonding between the dimer pairs, is usually measured by its tetramer-dimer dissociation constant (Refs. 7 and 32, and references therein) or its reciprocal value, the association constant. These values are readily determined for liganded Hb (CO or oxy), but the dissociation of human deoxyhemoglobin tetramers is difficult to estimate because it occurs at concentrations too low for current detectors. Information on subunit interactions can also be obtained from the width of the eluted peak during high resolution gel filtration (25,32), e.g. HbF elutes as a more narrow peak than HbA, HbA 2 , or HbF 1 in the equilibrium concentration range (Fig. 2), consistent with a tighter structure (23)(24)(25). Except for HbA 2 (␣ 2 ␦ 2 ), a minor Hb in normal individuals, K d values are close to the maximum of the bell-shaped curve representing the highest relative concentrations of equilibrating dimers and tetramers. Peak width may reflect the dynamics of the equilibrium process (Equation 1) or a small proportion of trailing dimers. For HbA 2 , the large amino acid substitution (Arg) at its ␣ 1 ␦ 1 subunit interface may prevent efficient dimer and tetramer assembly as a result of the linked equilibria in Equation 1. Thus, the broad peak width of HbA 2 and the displacement of the maximum from its K d may be because of the simultaneous presence of all three species, monomers, dimers, and tetramers.

Sickle Hemoglobin Polymerization
Normal adult hemoglobin A is a very soluble protein at its high concentration in the erythrocyte (5 mM; 320 mg/ml). In contrast, sickle deoxyhemoglobin (HbS) is quite insoluble because of extensive intertetrameric subunit interactions (Fig. 3) leading to polymerization (33-36) (equilibrium 3 in Equation 1); these interactions do not occur in deoxy or oxy HbA or in oxy HbS. Such polymerization of deoxy HbS deforms erythrocytes in the venous circulation of sickle cell anemia patients leading to clinical episodes known as sickle cell crises (see Ref. 36) for a recent review). Early attempts to prevent polymerization of deoxy HbS tetramers were directed at lowering the population of deoxygenated tetramers by increasing the relative amount of oxygenated tetramers, the nonsickling form (37,38). Although this strategy remains feasible in principle, clinically useful pharmacological agents directed solely at sickle hemoglobin have so far proved difficult to achieve in practice perhaps because of a lack of information on the most important sites involved in polymerization. An alternate approach, currently being tested clinically, is to increase the population of HbF tetramers (39,40) because HbF does not have favorable contacts for polymerization (39 -41).
The structure of sickle Hb has been determined both by x-ray (Fig. 3) (42, 43) and by electron microscopic analysis (44,45) so that the interaction sites in the polymer are well established. However, the strength of many of these interactions is not known. Recombi- nant DNA methods are now being used to map their relative strengths to obtain a complete map of those that enhance and those that inhibit polymerization (46,47) to provide a basis for the development of new therapeutic modalities. The initial results with this approach are shown in Fig. 4, and from it several conclusions can be made: 1) some sites contribute more than others to polymer inhibition because of their location in the tetramer, e.g. Lys-95 (␤) compared with Leu-88 (␤) where there is lack of additivity between these sites; 2) polymer enhancement sites are generally on the exterior of the tetramer, and the effects of these substitutions are additive. Sites that contribute most to polymerization or to its inhibition are candidates for the design of future anti-sickling agents using covalent protein modifiers of HbS itself.

Fish Hemoglobins
The blood of most fish contains multiple hemoglobins that are usually classified by their electrophoretic behavior as anodic or cathodic. Many anodic fish hemoglobins and some cathodic types differ from human Hb in their oxygen affinity at low pH, i.e. they release more O 2 as the pH continues to decrease (known as the Root effect or an extended alkaline Bohr effect) (30,48). In contrast, human Hb undergoes a transition to an increased O 2 affinity below about pH 6.5 (known as the acid or reverse Bohr effect). The Root effect enables fish to survive at low pH by facilitating release of O 2 ; its molecular mechanism is not understood. Recently, the structures of two hemoglobins that exhibit the Root effect have been solved (49,50), and both show significant differences in the interactions between their subunits compared with human HbA. At pH 6.3, the spectrum of Root effect hemoglobin is primarily that of deoxyhemoglobin even in oxygenated buffers. We studied subunit interactions in fish hemoglobins using a high resolution gel filtra-tion method at this pH together with analysis of the elution profiles (25,32). To prevent the spontaneous loss of the O 2 ligand and simultaneous formation of deoxyhemoglobin tetramers, CO was FIG. 5. Fish Hb dissociation. The anodal component of Hb from four species of fish Hb (brook trout, brown trout, rainbow trout, and Atlantic salmon) was isolated on an FPLC Mono Q column at pH 8.3. At pH 6.3, the visible spectrum of each had distinct deoxyhemoglobin features as described in Ref. 30 for Root effect hemoglobins. High resolution gel filtration on Superose-12 was performed as described in Ref. 32, but the buffer was pH 6.3, 150 mM Tris-Ac, saturated with CO gas. Samples were also saturated with CO before being applied to the column. The dissociation profile shown is for brook trout Hb. used as the ligand and also maintained in the pH 6.3 elution buffer. Under such conditions, tetramers of the anodic components of brook trout Hb and of rainbow trout Hb dissociate ultimately to monomers as a smooth dissociation profile (Fig. 5). Maximum peak width for this dissociation process (Fig. 5, inset) occurs near the expected position of dimers where tetramer, dimer, and monomer species would be at their highest relative concentrations. Other anodic Root effect fish hemoglobins from Atlantic salmon and from brown trout exhibit distinct tetramer and monomer components but no dimer during dissociation. Indeed, a notable feature of these four types of fish hemoglobins is the absence of dimers in contrast to human hemoglobins where the dimers elute as a discrete component without further dissociation to monomers.
Could different dimer stabilities of human and fish dimers at pH 6.3 account for their physiological differences in O 2 binding at low pH? For human hemoglobins, several studies have shown that dissociation of tetramers to dimers is increased 10-fold for each pH unit decrease (Ref. 25, and references therein), and an increased population of dimers leads to an increased oxygen affinity (1)(2)(3). Further studies are needed to determine whether the weak subunit interactions between monomeric subunits in fish hemoglobins (49,50) are involved in the important physiological process called the Root effect.

Conclusions
This minireview illustrates the advantages in studying different types of hemoglobin to determine the various mechanisms by which heme proteins carry O 2 , as controlled by different subunit interactions. Certain general features such as the central cavity have evolved among human hemoglobin tetramers as an important region that enables control by allosteric regulators. However, other hemoglobins such as clam Hb have evolved a different mechanism to support O 2 requirements. Fish hemoglobins represent a class that have some properties in common with human hemoglobins but possess some fundamental differences as well. Sickle cell anemia is probably the best known example of a disease that can be explained at the molecular level because of anomalous subunit interactions brought about by a mutational event at an important exterior site. It is likely that the methods of recombinant DNA as well as those of classical protein modification that we and others are applying to hemoglobins will continue to contribute to our knowledge on this very important protein.