From the Department of Biology, Northeastern University, Boston, Massachusetts 02115
The characteristic properties of hemoglobin are
due to the manner in which its individual subunits bond to one another
first as an
INTRODUCTION
Top
Introduction
References
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 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),1 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
- 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), (
(Eq. 1)
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.
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Subunit Interfaces of Human Hemoglobin |
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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).
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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 (
)
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
O2 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 O2 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 O2
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 O2 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 O2 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 HbF1 and unacetylated HbFo (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). HbF1 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,
HbA2, or HbF1 in the equilibrium concentration
range (Fig. 2), consistent with a tighter
structure (23-25). Except for HbA2
(2
2), a minor Hb in normal individuals,
Kd 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 HbA2, 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
HbA2 and the displacement of the maximum from its
Kd may be because of the simultaneous presence of
all three species, monomers, dimers, and tetramers.
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Sickle Hemoglobin Polymerization |
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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 non-sickling 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).
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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. Recombinant 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.
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Fish Hemoglobins |
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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 O2 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 O2 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 O2; 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 filtration method at this pH together with analysis of the elution profiles (25, 32). To prevent the spontaneous loss of the O2 ligand and simultaneous formation of deoxyhemoglobin tetramers, CO was 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.
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Could different dimer stabilities of human and fish dimers at pH 6.3 account for their physiological differences in O2 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 (1-3). 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.
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Conclusions |
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This minireview illustrates the advantages in studying different types of hemoglobin to determine the various mechanisms by which heme proteins carry O2, 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 O2 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.
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ACKNOWLEDGEMENTS |
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We acknowledge the contributions of those whose work could not be cited because of space limitations but who nevertheless have contributed significantly to our knowledge of hemoglobin.
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FOOTNOTES |
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* This minireview will be reprinted in the 1998 Minireview Compendium, which will be available in December, 1998. This work was supported in part by National Institutes of Health Grants HL-18819 and HL-58512.
To whom correspondence should be addressed: Dept. of Biology,
Mugar Life Sciences Bldg., Rm. 414, Northeastern University, 360 Huntington Ave., Boston, MA 02115. Tel.: 617-373-5267; Fax: 617-373-4496; E-mail: jmanning{at}lynx.neu.edu.
1 The abbreviations used are: 2,3-DPG, 2,3-diphosphoglycerate; HbS, sickle hemoglobin.
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REFERENCES |
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