Oxygen Binding Proteins PDF

CHAPTER 5 PROTEIN FUNCTION 5.1 Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins 5.2 Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins 5.3 Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors Knowing the three-dimensional structure of a protein is an important part of understanding protein function, and structural biology o en offers insights into molecular interactions. However, the protein structures we have examined so far are deceptively static. Proteins function by interacting dynamically with — binding to — other molecules. We divide these interactions into two types. In some interactions, the result is a reaction that alters the chemical conﬁguration or composition of a bound molecule, with the protein acting as a reaction catalyst, or enzyme; we discuss enzymes and their properties in Chapter 6. In other interactions, neither the chemical conﬁguration nor the composition of the bound molecule is changed; such interactions are the subject of this chapter. ^ ligands! 614 Transient int: breaks and forms easily It may seem counterintuitive that a protein’s interaction with another molecule could be important if it does not alter the associated molecule. Yet, transient interactions of this type are at the heart of many complex physiological processes, such as oxygen transport, transmission of nerve impulses, and immune function. Deﬁning which molecules interact and quantifying such interactions are common and illuminating tasks in every biochemical subdiscipline. The study of proteins that function through reversible interactions can be organized around six key principles of protein function, some of which will be familiar from Chapter 4: The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. A ligand binds a protein at a binding site that is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. The interaction is speciﬁc: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few types. A given protein may have separate binding sites for several different ligands. These speciﬁc 615 molecular interactions are crucial in maintaining the high degree of order in a living system. Proteins are ﬂexible. Changes in conformation may be subtle, reﬂecting molecular vibrations and small movements of amino acid residues throughout the protein. Changes in conformation may also be more dramatic, with major segments of the protein structure moving as much as several nanometers. Speciﬁc conformational changes are frequently essential to a protein’s function. The binding of a protein and a ligand is o en coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced ﬁt. In a multisubunit protein, a conformational change in one subunit o en affects the conformation of other subunits. Interactions between ligands and proteins may be regulated. The themes in our discussion of noncatalytic functions of proteins in this chapter — binding, speciﬁcity, and conformational change — are continued in Chapter 6, with the added element of proteins participating in chemical transformations. This discussion excludes the binding of water, 616 which may interact weakly and nonspeciﬁcally with many parts of a protein. 617 5.1 Reversible Binding of a Protein to a Ligand: OxygenBinding Proteins Myoglobin and hemoglobin may be the most-studied and bestunderstood proteins. They were the ﬁrst proteins for which threedimensional structures were determined, and these two molecules illustrate almost every aspect of that critical biochemical process: the reversible binding of a ligand to a protein. This classic model of protein function tells us a great deal about how proteins work. Oxygen Can Bind to a Heme Prosthetic Group Oxygen is poorly soluble in aqueous solutions (see Table 2-2) and cannot be carried to tissues in sufﬁcient quantity if it is simply dissolved in blood serum. Also, diffusion of oxygen through tissues is ineffective over distances greater than a few millimeters. The evolution of larger, multicellular animals depended on the evolution of proteins that could transport and store oxygen. However, none of the amino acid side chains in proteins are suited for the reversible binding of oxygen molecules. This role is ﬁlled by certain transition metals, among them iron and copper, that have a strong tendency to bind oxygen. Multicellular organisms exploit the properties of metals, most commonly iron, for oxygen transport. However, free iron 618 promotes the formation of highly reactive oxygen species such as hydroxyl radicals that can damage DNA and other macromolecules. Iron used in cells is therefore bound in forms that sequester it and/or make it less reactive. In multicellular organisms, iron is o en incorporated into a protein-bound prosthetic group called heme (or haem). (Recall from Chapter 3 that a prosthetic group is a compound permanently associated with a protein that contributes to the protein’s function.) Heme is found in many oxygen-transporting proteins, as well as in some proteins, such as the cytochromes, that participate in oxidationreduction (electron-transfer) reactions (Chapter 19). Heme consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous ( ) state (Fig. 5-1). The iron atom has six coordination bonds: four to nitrogen atoms that are part of the ﬂat porphyrin ring system, and two perpendicular to the porphyrin. FIGURE 5-1 Heme. The heme group is present in myoglobin, hemoglobin, and many other proteins, designated heme proteins. Heme consists of a complex organic ring structure, protoporphyrin IX, with a bound iron atom in its ferrous ( ) state. (a) Porphyrins, of which protoporphyrin IX is just one example, consist of four pyrrole rings linked by methene bridges, with substitutions at one or more of the positions denoted X. 619 Coordination bond = a covalent bond (a shared pair of electrons) (b, c) Two representations of heme. The iron atom of heme has six coordination bonds in which four both in the plane of, and bonded to, the flat porphyrin ring system, and (d) two electrons come to it. [(c) Data from PDB ID 1CCR, H. Ochi et al., J. Mol. Biol. 166 407, 1983.] from the perpendicular same atom Iron in the state binds oxygen reversibly; in the state it does not bind oxygen. The structure of heme and the globins to which it is bound represents an evolutionary adaptation to prevent oxidation. Free heme molecules (heme not bound to protein) leave with two “open” coordination bonds. Simultaneous reaction of one with two free heme molecules (or two free irreversible conversion of to molecule ) can result in . The coordinated nitrogen atoms (which have an electron-donating character) help prevent conversion of the heme iron to the ferric state. In heme- containing proteins, this reaction is further prevented by sequestering each heme deep within the protein structure. Thus, access to the two open coordination bonds is restricted. In the globins that will shortly become the focus of our narrative, one of these two coordination bonds is occupied by a side-chain nitrogen of a highly conserved His residue referred to as the proximal His. oxygen The other is the binding site for molecular (Fig. 5-2). When oxygen binds, the electronic properties of heme iron change; this accounts for the change in color from the dark purple of oxygen-depleted venous blood to the bright red of oxygen-rich arterial blood. Some small molecules, such as carbon monoxide (CO) and nitric oxide (NO), coordinate to heme iron with greater afﬁnity than does a molecule of CO is bound to heme, 620 . When is excluded, which is why CO is highly toxic to aerobic organisms (a topic explored in Box 51). By surrounding and sequestering heme, oxygen-binding proteins regulate the access of small molecules to the heme iron. FIGURE 5-2 The heme group viewed from the side. This view shows the two coordination bonds to that are perpendicular to the porphyrin ring system. One is occupied by a His residue called the proximal His, designated His F8 (the eighth residue in in myoglobin, also helix F see Fig. 5-3) the other is the 621 binding site for oxygen. The remaining four coordination bonds are in the plane of, and bonded to, the flat porphyrin ring system. Globins Are a Family of OxygenBinding Proteins The globins are a widespread family of proteins. Globins are commonly found in eukaryotes of all classes, as well as in archaea and bacteria. All evolved from a common ancestral protein. Their tertiary structure is highly conserved, made up of eight -helical segments connected by bends. This folding pattern, illustrated by myoglobin (Fig. 5-3), constitutes a structural motif known as the globin fold. The primary amino acid sequence of globins is less conserved, well reﬂecting both the ancient origins of this protein family and the evolutionary relatedness of species from which globin comparisons might be made. 622 FIGURE 5-3 Myoglobin. The eight -helical segments (shown here as cylinders) are labeled A through H. Nonhelical residues in the bends that connect them are labeled AB, CD, EF, and so forth, indicating the segments they interconnect. A few bends, including BC and DE, are abrupt and do not contain any residues these are sometimes not labeled. The heme is bound in a pocket made up largely of the E and F helices, although amino acid residues from other segments of the protein also participate. [Data from PDB ID 1MBO, S. E. Phillips, J. Mol. Biol. 142 531, 1980.] Most globins function in oxygen transport or storage, although some play a role in the sensing of oxygen, nitric oxide, or carbon monoxide. The simple nematode worm Caenorhabditis elegans has genes encoding 33 different globins. In humans and other mammals, there are at least four kinds of globins. The monomeric myoglobin facilitates oxygen diffusion in muscle tissue. Myoglobin is particularly abundant in the muscles of diving marine mammals such as seals and whales, where it also 623 has an oxygen-storage function for prolonged excursions undersea. The tetrameric hemoglobin is responsible for oxygen transport in the bloodstream. The monomeric neuroglobin is expressed largely in neurons and helps to protect the brain from hypoxia (low oxygen) or ischemia (restricted blood supply). Cytoglobin, another monomeric globin, is found at high concentrations in the walls of blood vessels, where it functions to regulate levels of nitric oxide, a localized signal for muscle relaxation (see Box 12-2). Myoglobin Has a Single Binding Site for Oxygen Any detailed discussion of protein function inevitably involves protein structure. Myoglobin ( 16,700; abbreviated Mb) is a single polypeptide of 153 amino acid residues with one molecule of heme (Fig. 5-3). About 78% of the amino acid residues in the protein are found in the eight named A through H. helices typical of the globin fold, Myoglobin structure ^^ An individual amino acid residue is designated either by its position in the amino acid sequence or by its location in the sequence of a particular -helical segment. For example, the His residue coordinated to the heme in myoglobin — the proximal His — is (the ninety-third residue from the amino-terminal end of the myoglobin polypeptide sequence) and is also called His F8 (the eighth residue in helix F). The bends in the structure are 624 designated AB, CD, EF, FG, and GH, reﬂecting the -helical segments they connect. Protein-Ligand Interactions Can Be Described Quantitatively The function of myoglobin depends on the protein’s ability not only to bind oxygen but also to release it when and where it is needed. Function in biochemistry o en revolves around a reversible protein-ligand interaction of this type. A quantitative description of this interaction is a central part of many biochemical investigations. In general, the reversible binding of a protein (P) to a ligand (L) can be described by a simple equilibrium expression: (5-1) The reaction is characterized by an equilibrium constant, , such that (5-2) 625 where and are rate constants (more on these below). The term is an association constant (not to be confused with the that denotes an acid dissociation constant; p. 57) that describes the equilibrium between the complex and the unbound components of the complex. The association constant provides a measure of the afﬁnity of the ligand L for the protein. units of ; a higher value of has corresponds to a higher afﬁnity of the ligand for the protein. The equilibrium term is also equivalent to the ratio of the rates of the forward (association) and reverse (dissociation) reactions that form the PL complex. The association rate is described by the rate constant the rate constant , and dissociation is described by . As discussed further in the next chapter, rate constants are proportionality constants, describing the fraction of a pool of reactant that reacts in a given amount of time. When the reaction involves one molecule, such as the dissociation reaction PL → P + L, the reaction is ﬁrst order and the rate constant units of reciprocal time has . When the reaction involves two molecules, such as the association reaction P + L → PL, it is called second order, and its rate constant has units of . KEY CONVENTION Equilibrium constants are denoted with a capital K and rate constants are denoted with a lowercase k. 626 A rearrangement of the ﬁrst part of Equation 5-2 shows that the ratio of bound to free protein is directly proportional to the concentration of free ligand: (5-3) When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligand by the protein does not appreciably change the concentration of free (unbound) ligand — that is, [L] remains constant. This condition is broadly applicable to most ligands that bind to proteins in cells, and it simpliﬁes our description of the binding equilibrium. We can now consider the binding equilibrium from the standpoint of the fraction, Y, of ligand-binding sites on the protein that are occupied by ligand: (5-4) Substituting [L][P] for [PL] (see Eqn 5-3) and rearranging terms gives 627 (5-5) The value of can be determined from a plot of Y versus the concentration of free ligand, [L] (Fig. 5-4a). Any equation of the form x = y/(y + z) describes a hyperbola, and Y is thus found to be a hyperbolic function of [L]. The fraction of ligand-binding sites occupied approaches saturation asymptotically as [L] increases. The [L] at which half of the available ligand-binding sites are occupied (that is, Y = 0.5) corresponds to 628 . FIGURE 5-4 Graphical representations of ligand binding. The fraction of ligand-binding sites occupied, Y, is plotted against the concentration of free ligand. Both curves are rectangular hyperbolas. (a) A hypothetical binding curve for a ligand L. The [L] at which half of the available ligand-binding sites are occupied is equivalent to , or . The curve has a horizontal asymptote at Y = 1 and a vertical asymptote (not shown) at (b) A curve describing the binding of oxygen to myoglobin. The partial pressure of in the air above the solution is expressed in kilopascals (kPa). Oxygen binds tightly to myoglobin, with a 629 of only 0.26 kPa. . It is more common (and intuitively simpler), however, to consider the dissociation constant, , which is the reciprocal of and has units of molar concentration (M ). is the equilibrium constant for the release of ligand. The relevant expressions change to (5-6) (5-7) (5-8) When [L] equals , half of the ligand-binding sites are occupied. As [L] falls below , progressively less of the protein has ligand bound to it. For 90% of the available ligand-binding sites to be occupied, [L] must be nine times greater than In practice, . is used much more o en than to express the afﬁnity of a protein for a ligand. Note that a lower 630 value of corresponds to a higher afﬁnity of ligand for the protein. The mathematics can be reduced to simple statements: is equivalent to the molar concentration of ligand at which half of the available ligand-binding sites are occupied. At this point, the protein is said to have reached half-saturation with respect to ligand binding. The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding sites to be occupied, and thus the lower the value of . Some representative dissociation constants are given in Table 5-1; the scale shows typical ranges for dissociation constants found in biological systems. TABLE 5-1 Protein Dissociation Constants: Some Examples and Range Protein Ligand Avidin (egg white) Biotin Insulin receptor (human) Insulin Anti-HIV immunoglobulin (human)b gp41 (HIV-1 surface protein) Nickel-binding protein (E. coli) Calmodulin (rat)c 631 (M)a Color bars indicate the range of dissociation constants typical of various classes of interactions in biological systems. A few interactions, such as that between the protein avidin and the enzyme cofactor biotin, fall outside the normal ranges. The avidin-biotin interaction is so tight it may be considered irreversible. Sequence-specific protein-DNA interactions reflect proteins that bind to a particular sequence of nucleotides in DNA, as opposed to general binding to any DNA site. aA reported dissociation constant is valid only for the particular solution conditions under which it was measured. values for a protein-ligand interaction can be altered, sometimes by several orders of magnitude, by changes in the solution’s salt concentration, pH, or other variables. bThis immunoglobulin was isolated as part of an eﬀort to develop a vaccine against HIV. Immunoglobulins (described later in the chapter) are highly variable, and the reported here should not be considered characteristic of all immunoglobulins. cCalmodulin has four binding sites for calcium. The values shown reflect the highest- and lowest-aﬀinity binding sites observed in one set of measurements. WORKED EXAMPLE 5-1 ReceptorLigand Dissociation Constants Two proteins, A and B, bind to the same ligand, L, with the binding curves shown below. What is the dissociation constant, , for each protein? Which protein (A or B) has a greater afﬁnity for ligand L? 632 SOLUTION: We can determine the dissociation constants by inspecting the graph. Because Y represents the fraction of binding sites occupied by ligand, the concentration of ligand at which half the binding sites are occupied — that is, the point where the binding curve crosses the line where Y = 0.5 — is the dissociation constant. For A, ; for B, . Because A is half-saturated at a lower [L], it has a higher afﬁnity for the ligand. WORKED EXAMPLE 5-2 ProteinLigand Binding 633 A protein has a of 2.0 M. What is the [L] where Y = 0.6? SOLUTION: Solving Equation 5-8 for [L], ﬁrst we obtain Distributing Y on the right side gives Subtracting Y[L] from both sides then factoring out [L] and dividing both sides by 1 − Y gives the ﬁnal expression 634 Substituting the values for Y and given in the problem gives The binding of oxygen to myoglobin follows the patterns discussed above. However, because oxygen is a gas, we must make some minor adjustments to the equations so that laboratory experiments can be carried out more conveniently. We ﬁrst substitute the concentration of dissolved oxygen for [L] in Equation 5-8 to give (5-9) As for any ligand, equals the ligand-binding sites are occupied, or becomes 635 at which half of the available . Equation 5-9 thus (5-10) In experiments using oxygen as a ligand, it is the partial pressure of oxygen in the gas phase above the solution that is varied, because this is easier to measure than the concentration of oxygen dissolved in the solution. The concentration of a volatile substance in solution is always proportional to the local partial pressure of the gas. So, if we deﬁne the partial pressure of oxygen at as , substitution in Equation 5-10 gives (5-11) A binding curve for myoglobin that relates Y to is shown in Figure 5-4b. Protein Structure Aﬀects How Ligands Bind The binding of a ligand to a protein is rarely as simple as the above equations would suggest. The interaction is greatly affected by protein structure and is o en accompanied by conformational changes. For example, the speciﬁcity with which heme binds its various ligands is altered when the heme is a component of myoglobin. For free heme molecules, carbon 636 monoxide binds more than 20,000 times better than does is, the or for CO binding to free heme is more than 20,000 times lower than that for better than (that ), but it binds only about 40 times when the heme is bound in myoglobin. For free heme, the tighter binding by CO reﬂects differences in the way the orbital structures of CO and interact with . Those same orbital structures lead to different binding geometries for CO and when they are bound to heme (Fig. 5-5a, b). The change in relative afﬁnity of CO and for heme when the heme is bound to a globin is mediated by the globin structure. 637 FIGURE 5-5 Steric eﬀects caused by ligand binding to the heme of myoglobin. (a) Oxygen binds to heme with the axis at an angle, a binding conformation readily accommodated by myoglobin. (b) Carbon monoxide binds to free heme with the CO axis perpendicular to the plane of the porphyrin ring. (c) Another view of the heme of myoglobin, showing the arrangement of key amino acid residues around the heme. The bound hydrogen-bonded to the distal His, His E7 ( is ), facilitating the binding of compared with its binding to free heme. [(c) Data from PDB ID 1MBO, S. E. Phillips, J. Mol. Biol. 142 531, 1980.] 638 When heme is bound to myoglobin, its afﬁnity for is selectively increased by the presence of the distal His ( His E7 in myoglobin). The Fe- , or complex is much more polar than the Fe-CO complex. A partial negative charge is distributed across the oxygen atoms in the bound due to partial oxidation of the interacting iron atom. A hydrogen bond between the imidazole side chain of His E7 and the bound stabilizes this polar complex electrostatically (Fig. 5-5c). The afﬁnity of myoglobin for is thus selectively increased by a factor of about 500; there is no such effect for Fe-CO binding in myoglobin. Consequently, the 20,000-fold stronger binding afﬁnity of free heme for CO compared with declines to approximately 40-fold for heme embedded in myoglobin. This favorable electrostatic effect on binding is even more dramatic in some invertebrate hemoglobins, where two groups in the binding pocket can form strong hydrogen bonds with , causing the heme group to bind with greater afﬁnity than CO. This selective enhancement of afﬁnity in globins is physiologically important: it helps prevent poisoning by the CO that is generated in small amounts from metabolism and that is sometimes generated in larger amounts by our industrial-age environment. The binding of to the heme in myoglobin also depends on molecular motions, or “breathing,” in the protein structure. The heme molecule is deeply buried in the folded polypeptide, with limited direct paths for oxygen to move from the surrounding solution to the ligand-binding site. If the protein were rigid, could not readily enter or leave the heme pocket. 639 However, rapid molecular ﬂexing of the amino acid side chains produces transient cavities in the protein structure, and makes its way in and out by moving through these cavities. Computer simulations of rapid structural ﬂuctuations in myoglobin suggest that there are many such pathways. The distal His acts as a gate to control access to one major pocket near the heme iron. Rotation of that His residue to open and close the pocket occurs on a nanosecond ( ) time scale. Thus, subtle conformational changes can be critical for protein activity. Structural changes take on more complexity in the multisubunit hemoglobin, which we consider next. Hemoglobin Transports Oxygen in Blood Nearly all the oxygen carried by whole blood in animals is bound and transported by hemoglobin in erythrocytes (red blood cells). Normal human erythrocytes are small (6 to 9 m in diameter), biconcave disks. They are formed from precursor stem cells called hemocytoblasts. In the maturation process, the stem cell produces daughter cells that form large amounts of hemoglobin and then lose their organelles — nucleus, mitochondria, and endoplasmic reticulum. Erythrocytes are thus incomplete, vestigial cells, unable to reproduce and, in humans, destined to survive for only about 120 days. Their main function is to carry 640 hemoglobin, which is dissolved in the cytosol at a very high concentration (~34% by weight). In arterial blood passing from the lungs through the heart to the peripheral tissues, hemoglobin is about 96% saturated with oxygen. In the venous blood returning to the heart, hemoglobin is only about 64% saturated. Thus, each 100 mL of blood passing through a tissue releases about one-third of the oxygen it carries, or 6.5 mL of gas at atmospheric pressure and body temperature. Myoglobin, with its hyperbolic binding curve for oxygen (Fig. 54b), is relatively insensitive to small changes in the concentration of dissolved oxygen, so it functions well as an oxygen-storage protein. Hemoglobin, with its multiple subunits and -binding sites, is better suited to oxygen transport. As we shall see, interactions between the subunits of a multimeric protein can permit a highly sensitive response to small changes in ligand concentration. Interactions among the subunits in hemoglobin cause conformational changes that alter the afﬁnity of the protein for oxygen. The modulation of oxygen binding allows the -transport protein to respond to changes in oxygen demand by tissues. Hemoglobin Subunits Are Structurally Similar to Myoglobin 641 Hemoglobin ( 64,500; abbreviated Hb) is roughly spherical, with a diameter of nearly 5.5 nm. It is a tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain. Adult hemoglobin contains two types of globin: two chains (141 residues each) and two chains (146 residues each). The three-dimensional structures of the two types of subunits are very similar to each other and to myoglobin (Fig. 5-6), reﬂecting their evolution within the larger globin superfamily. However, fewer than half of the amino acid residues are identical in the polypeptide sequences of the and subunits, and only 27 are identical in the three polypeptides (Fig. 5-7). The helix-naming convention described for myoglobin is also applied to the hemoglobin polypeptides, except that the subunit lacks the short D helix. The heme-binding pocket is made up largely of the E and F helices in each of the subunits. FIGURE 5-6 Comparison of the structures of myoglobin and the subunit of hemoglobin. [Data from (le ) PDB ID 1MBO, S. E. Phillips, J. Mol. Biol. 642 142 531, 1980 (right) PDB ID 1HGA, R. Liddington et al., J. Mol. Biol. 228 551, 1992.] FIGURE 5-7 Comparison of whale myoglobin with the and chains of human hemoglobin. (a) Sequence alignment with dashed lines marking helix boundaries. For optimal alignment, short gaps are introduced into both Hb sequences where a few amino acids are present in the other sequences. With the exception of the missing D helix in the Hb chain (Hb ), this alignment permits the use of the helix lettering convention that emphasizes the common positioning of amino acid residues. Residues conserved in all known globins are shaded black. Residues conserved in the three 643 globins shown here are shaded dark blue. Positions where the amino acid class (hydrophobic, charged, etc.) is conserved are shaded light blue. The common helixletter-and-number designation for amino acids does not necessarily correspond to a common position in the linear sequence. For example, the distal His residue is His E7 in all three structures, but corresponds to , , and in the linear sequences of Mb, Hb , and Hb , respectively. Nonhelical residues at the amino and carboxyl termini are labeled NA and HC, respectively. (b) The location of shaded residues is shown in a ribbon representation of myoglobin structure. (c) Ribbon representations of the three globin structures are overlaid to show structural conservation. [Data from PDB ID 1MBO, S. E. Phillips, J. Mol. Biol. 142 531, 1980 PDB ID 1HGA, R. Liddington et al., J. Mol. Biol. 228 551, 1992.] The quaternary structure of hemoglobin features strong interactions between unlike subunits. The interface (and its counterpart) involves more than 30 residues, and its interaction is sufﬁciently strong that although mild treatment of hemoglobin with urea tends to disassemble the tetramer into dimers, these dimers remain intact. The (and ) interface involves 19 residues (Fig. 5-8). The hydrophobic effect plays the major role in stabilizing these interfaces, but there are also many hydrogen bonds and a few ion pairs (or salt bridges), whose importance is discussed below. 644 FIGURE 5-8 Dominant interactions between hemoglobin subunits. In this representation, subunits are light and subunits are dark. The strongest subunit interactions (highlighted) occur between unlike subunits. When oxygen binds, the change at the contact changes little, but there is a large contact, with several ion pairs broken. [Data from PDB ID 1HGA, R. Liddington et al., J. Mol. Biol. 228 551, 1992.] Hemoglobin Undergoes a Structural Change on Binding Oxygen X-ray analysis has revealed two major conformations of hemoglobin: the R state and the T state. Although oxygen binds to hemoglobin in either state, it has a signiﬁcantly higher afﬁnity for hemoglobin in the R state. Oxygen binding stabilizes the R state. When oxygen is absent experimentally, the T state is more 645 stable and is thus the predominant conformation of deoxyhemoglobin. T and R originally denoted “tense” and “relaxed,” respectively, because the T state is stabilized by a greater number of ion pairs, many of which lie at the ) interface (Fig. 5-9). The binding of (and to a hemoglobin subunit in the T state triggers a change in conformation to the R state. When the entire protein undergoes this transition, the structures of the individual subunits change little, but the subunit pairs slide past each other and rotate, narrowing the pocket between the subunits (Fig. 5-10). In this process, some of the ion pairs that stabilize the T state are broken and some new ones are formed. FIGURE 5-9 Some ion pairs that stabilize the T state of deoxyhemoglobin. Many of the ion pairs stabilizing the T state lie at the (and ) interface. Close-up view of a portion of a deoxyhemoglobin molecule in the T state. Interactions between the ion pairs His HC3 and Asp FG1 of the subunit (blue) and between Lys C5 of the -carboxyl group) of the subunit (gray) and His HC3 (its subunit are shown with dashed lines. (Recall 646 that HC3 is the carboxyl-terminal residue of the subunit.) These examples do not represent the entire network of ion pairs that stabilize the structure. [Data from PDB ID 1HGA, R. Liddington et al., J. Mol. Biol. 228 551, 1992.] FIGURE 5-10 The T → R transition. In these depictions of deoxyhemoglobin, as in Figure 5-9, the subunits are blue and the subunits are gray. Positively charged side chains and chain termini involved in ion pairs are shown in blue, and their negatively charged partners are shown in red. The Lys C5 of each subunit and the Asp FG1 of each subunit are visible but are not labeled (compare Fig. 5-9). Note that the molecule is oriented slightly diﬀerently than in Figure 5-9. The transition from the T state to the R state shi s the subunit pairs substantially, aﬀecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl termini of the subunits, which are involved in ion pairs in the T state, rotate in the R state toward the center of the molecule, where they are no longer in ion pairs. Another dramatic result of the T → R transition is a narrowing of the pocket between the subunits. [T state Data from PDB ID 1HGA, R. Liddington et al., J. Mol. Biol. 228 551, 1992. R state Data from PDB ID 1BBB, M. M. Silva et al., J. Biol. Chem. 267 17,248, 1992.] Max Perutz proposed that the T → R transition is triggered by changes in the positions of key amino acid side chains surrounding the heme. In the T state, the porphyrin is slightly puckered, causing the heme iron to protrude somewhat on the 647 proximal His (His F8) side. The binding of causes the heme to assume a more planar conformation, shi ing the position of the proximal His and the attached F helix (Fig. 5-11). These changes lead to adjustments in the ion pairs at the interface. FIGURE 5-11 Changes in conformation near heme on binding to deoxyhemoglobin. The shi in the position of helix F when heme binds is thought to be one of the adjustments that triggers the T → R transition. [T state Data from PDB ID 1HGA, R. Liddington et al., J. Mol. Biol. 228 551, 1992. R state Data from PDB ID 1BBB, M. M. Silva et al., J. Biol. Chem. 267 17,248, 1992 R state modified to represent instead of CO.] Hemoglobin Binds Oxygen Cooperatively Hemoglobin must bind oxygen efﬁciently in the lungs, where the is about 13.3 kPa, and it must release oxygen in the tissues, where the is about 4 kPa. Myoglobin, or any protein that binds oxygen with a hyperbolic binding curve, would be ill-suited to this function, for the reason illustrated in Figure 5-12. A 648 protein that bound with high afﬁnity would bind it efﬁciently in the lungs but would not release much of it in the tissues. If the protein bound oxygen with a sufﬁciently low afﬁnity to release it in the tissues, it would not pick up much oxygen in the lungs. FIGURE 5-12 A sigmoid (cooperative) binding curve. A sigmoid binding curve can be viewed as a hybrid curve reflecting a transition from a lowaﬀinity state to a high-aﬀinity state. Because of its cooperative binding, as manifested by a sigmoid binding curve, hemoglobin is more sensitive to the small diﬀerences in concentration between the tissues and the lungs, allowing it to bind oxygen in the lungs (where the tissues (where is low). 649 is high) and release it in Hemoglobin solves the problem by undergoing a transition from a low-afﬁnity state (the T state) to a high-afﬁnity state (the R state) as more molecules are bound. As a result, hemoglobin has a hybrid S-shaped, or sigmoid, binding curve for oxygen (Fig. 5-12). A single-subunit protein with a single ligand-binding site cannot produce a sigmoid binding curve — even if binding elicits a conformational change — because each molecule of ligand binds independently and cannot affect ligand binding to another molecule. In contrast, binding to individual subunits of hemoglobin can alter the afﬁnity for ﬁrst molecule of in adjacent subunits. The that interacts with deoxyhemoglobin binds weakly, because it binds to a subunit in the T state. Its binding, however, leads to conformational changes that are communicated to adjacent subunits, making it easier for additional molecules of to bind. In effect, the T → R transition occurs more readily in the second subunit once (fourth) is bound to the ﬁrst subunit. The last molecule binds to a heme in a subunit that is already in the R state, and hence it binds with much higher afﬁnity than the ﬁrst molecule. An allosteric protein is one in which the binding of a ligand to one site affects the binding properties of another site on the same protein. The term “allosteric” derives from the Greek allos, “other,” and stereos, “solid” or “shape.” Allosteric proteins are those having “other shapes,” or conformations, induced by the binding of ligands referred to as modulators. The conformational changes induced by the modulator(s) interconvert more-active and less-active forms of the protein. The 650 modulators for allosteric proteins may be either inhibitors or activators. When the normal ligand and modulator are identical, the interaction is termed homotropic. When the modulator is a molecule other than the normal ligand, the interaction is heterotropic. Some proteins have two or more modulators and therefore can have both homotropic and heterotropic interactions. Cooperative binding of a ligand to a multimeric protein, such as we observe with the binding of to hemoglobin, is a form of allosteric binding. The binding of one ligand affects the afﬁnities of any remaining unﬁlled binding sites, and can be considered as both a ligand and an activating homotropic modulator. There is only one binding site for on each subunit, so the allosteric effects giving rise to cooperativity are mediated by conformational changes transmitted from one subunit to another by subunit-subunit interactions. A sigmoid binding curve is diagnostic of cooperative binding. It permits a much more sensitive response to ligand concentration and is important to the function of many multisubunit proteins. The principle of allostery extends readily to regulatory enzymes, as we shall see in Chapter 6. As is the case with myoglobin, ligands other than oxygen can bind to hemoglobin. An important example is carbon monoxide, which binds to hemoglobin about 250 times better than does oxygen (the critical hydrogen bond between and the distal His is not quite as strong in human hemoglobin as it is in most mammalian 651 myoglobins, so the binding of relative to CO is not augmented quite as much). Human exposure to CO can have tragic consequences (Box 5-1). BOX 5-1 MEDICINE Carbon Monoxide: A Stealthy Killer Summer, 2017. A Florida man drove his late-model SUV into his garage, took his wireless key fob with him inside the house, and went to bed. He was found, days later, dead from carbon monoxide poisoning. The man had assumed that removing the fob was enough to shut down the automobile. Instead, the car had idled in the garage until it ran out of gas, ultimately filling the attached house with CO. Since 2006, several dozen deaths and scores of injuries have resulted from similar incidents. Measures to address the safety issues posed by keyless ignitions, now standard in most new cars, have lagged. Meanwhile, the deaths are a vivid reminder that life depends on the reversibility of proteinligand interactions. Carbon monoxide (CO), a colorless, odorless gas, is one of the most common known causes of death due to poisoning (in countries where such records are kept). CO has an approximately 250-fold greater aﬀinity for hemoglobin than does oxygen. Consequently, relatively low levels of CO can have substantial and tragic eﬀects. When CO combines with hemoglobin, the complex is referred to as carboxyhemoglobin, or COHb. Some CO is produced by natural processes, but locally high levels generally result only from human activities. Engine and furnace exhausts are important sources, as CO is a byproduct of the incomplete combustion of fossil fuels. In the United States alone, nearly 4,000 people succumb to CO poisoning each year, both accidentally and intentionally. Many of the accidental deaths involve undetected CO buildup in enclosed spaces. This might occur when a household furnace malfunctions, or a gas generator is operated indoors during a power outage or shutoﬀ, venting CO into a home. CO poisoning can also occur in open spaces, as unsuspecting people at work or play inhale the exhaust from 652 kPa) is supplied. Thus, rapid treatment by a properly equipped medical team is critical. A home carbon monoxide detector is a simple and inexpensive measure to avoid possible tragedy. A er completing the research for this box, we immediately purchased several new CO detectors for our homes. Cooperative Ligand Binding Can Be Described Quantitatively Cooperative binding of oxygen by hemoglobin was ﬁrst analyzed by Archibald Hill in 1910. From this work came a general approach to the study of cooperative ligand binding to multisubunit proteins. For a protein with n binding sites, the equilibrium of Equation 5-1 becomes (5-12) and the expression for the association constant becomes (5-13) 657 The expression for Y (see Eqn 5-8) is (5-14) Rearranging, then taking the log of both sides, yields (5-15) (5-16) where Equation 5-16 is the Hill equation, and a plot of log [Y/(1 − Y)] versus log [L] is called a Hill plot. Based on the equation, the Hill plot should have a slope of n. However, the experimentally determined slope actually reﬂects not the number of binding sites but the degree of interaction between them. The slope of a Hill plot is therefore denoted by , the Hill coefﬁcient, which is a measure of the degree of cooperativity. If 658 equals 1, ligand binding is not cooperative, a situation that can arise even in a multisubunit protein if the subunits do not communicate. An of greater than 1 indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of others. The theoretical upper limit for is reached when . In this case the binding would be completely cooperative: all binding sites on the protein would bind ligand simultaneously, and no protein molecules partially saturated with ligand would be present under any conditions. This limit is never reached in practice, and the measured value of is always less than the actual number of ligand-binding sites in the protein. An of less than 1 indicates negative cooperativity, in which the binding of one molecule of ligand impedes the binding of others. Well-documented cases of negative cooperativity are rare. To adapt the Hill equation to the binding of oxygen to hemoglobin, we must again substitute for [L] and for : (5-17) Hill plots for myoglobin and hemoglobin are given in Figure 5-13. 659 FIGURE 5-13 Hill plots for oxygen binding to myoglobin and hemoglobin. When , there is no evident cooperativity. The maximum degree of cooperativity observed for hemoglobin corresponds approximately to . Note that while this indicates a high level of cooperativity, less than n, the number of is -binding sites in hemoglobin. This is normal for a protein that exhibits allosteric binding behavior. Two Models Suggest Mechanisms for Cooperative Binding In a multisubunit ligand-binding protein like hemoglobin, how does the T → R transition occur? Two models for the cooperative binding of ligands to proteins with multiple binding sites have 660 greatly inﬂuenced thinking about this problem, deﬁning the two major possibilities. The ﬁrst model was proposed by Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux in 1965, and is called the MWC model or the concerted model (Fig. 5-14a). The concerted model assumes that the subunits of a cooperatively binding protein are functionally identical, that each subunit can exist in (at least) two conformations, and that all subunits undergo the transition from one conformation to the other simultaneously. In this model, no protein has individual subunits in different conformations. The two conformations are in equilibrium. The ligand can bind to either conformation, but it binds much more tightly to the R state. Successive binding of ligand molecules to the low-afﬁnity T state (which is more stable in the absence of ligand) gradually shi s the equilibrium to favor the R state. 661 FIGURE 5-14 Two general models for the interconversion of inactive and active forms of a protein during cooperative ligand binding. Although the models may be applied to any protein — including any enzyme (Chapter 6) — that exhibits 662 cooperative binding, here we show four subunits because the model was originally proposed for hemoglobin. (a) In the concerted, or all-or-none, model (MWC model), all subunits are postulated to be in the same conformation, either all ◯ (low aﬀinity or inactive) or all between ◯ and (high aﬀinity or active). Depending on the equilibrium, , forms, the binding of one or more ligand molecules (L) will pull the equilibrium toward the form. Subunits with bound L are shaded. (b) In the sequential model, each individual subunit can be in either the ◯ form or the form. The equilibrium is altered as additional ligands are bound, progressively favoring the R state. In the second model, the sequential model (Fig. 5-14b), proposed in 1966 by Daniel Koshland and colleagues, ligand binding can induce a change of conformation in an individual subunit. A conformational change in one subunit makes a similar change in an adjacent subunit more likely, and makes the binding of a second ligand molecule more likely as well. There are more potential intermediate states in this model than in the concerted model. The two models are not mutually exclusive; the concerted model may be viewed as the “all-or-none” limiting case of the sequential model. Unfortunately, the two models have proven very difﬁcult to distinguish experimentally. In Chapter 6 we use these models to investigate allosteric enzymes. Hemoglobin Also Transports and In addition to carrying nearly all the oxygen required by cells from the lungs to the tissues, hemoglobin carries two end products of cellular respiration — 663 and — from the tissues to the lungs and the kidneys, where they are excreted. The , produced by oxidation of organic fuels in mitochondria, is hydrated to form bicarbonate: This reaction is catalyzed by carbonic anhydrase, an enzyme particularly abundant in erythrocytes. Carbon dioxide is not very soluble in aqueous solution, and bubbles of would form in the tissues and blood if it were not converted to bicarbonate. As you can see from the reaction catalyzed by carbonic anhydrase, the hydration of results in an increase in the concentration (a decrease in pH) in the tissues. The binding of oxygen by hemoglobin is profoundly inﬂuenced by pH and concentration, so the interconversion of and bicarbonate is of great importance to the regulation of oxygen binding and release in the blood. Hemoglobin transports about 40% of the total of the and 15% to 20% formed in the tissues to the lungs and kidneys. (The remainder of the is absorbed by the plasma’s bicarbonate buffer; the remainder of the and is transported as dissolved .) The structural effects of hemoglobin favor the T state. and binding to Thus, the binding of and is inversely related to the binding of oxygen. At the relatively low pH and high concentration of peripheral tissues, the 664 afﬁnity of hemoglobin for oxygen decreases as bound, and and are is released to the tissues. Conversely, in the capillaries of the lung, as is excreted and the blood pH consequently rises, the afﬁnity of hemoglobin for oxygen increases and the protein binds more for transport to the peripheral tissues. This effect of pH and concentration on the binding and release of oxygen by hemoglobin is called the Bohr effect, a er Christian Bohr, the Danish physiologist (and father of physicist Niels Bohr) who discovered it in 1904. A complete statement of the binding equilibrium for hemoglobin and one molecule of oxygen can be designated by the reaction where denotes a protonated form of hemoglobin. This equation tells us that the inﬂuenced by the -saturation curve of hemoglobin is concentration (Fig. 5-15). Both and are bound by hemoglobin, but with inverse afﬁnity. When the oxygen concentration is high, as in the lungs, hemoglobin binds and releases protons. When the oxygen concentration is low, as in the peripheral tissues, is bound and 665 is released. FIGURE 5-15 Eﬀect of pH on oxygen binding to hemoglobin. The pH of blood is 7.6 in the lungs and 7.2 in the tissues. Experimental measurements on hemoglobin binding are o en performed at pH 7.4. Oxygen and are not bound at the same sites in hemoglobin. Oxygen binds to the iron atoms of the hemes, whereas binds to any of several amino acid residues in the protein. A major contribution to the Bohr effect is made by (His HC3) of the subunits. When protonated, this residue forms one of the ion pairs — to (Asp FG1) — that helps stabilize deoxyhemoglobin in the T state (Fig. 5-9). Protonation of the amino-terminal residues of the subunits, certain other His residues, and perhaps other groups has a similar effect. 666 Hemoglobin also binds , again in a manner inversely related to the binding of oxygen. Carbon dioxide binds as a carbamate group to the -amino group at the amino-terminal end of each globin chain, forming carbaminohemoglobin: This reaction produces , contributing to the Bohr effect. The bound carbamates also form additional salt bridges (not shown in Fig. 5-9) that help to stabilize the T state and promote the release of oxygen. When the concentration of carbon dioxide is high, as in peripheral tissues, some afﬁnity for binds to hemoglobin and the decreases, causing its release. Conversely, when hemoglobin reaches the lungs, the high oxygen concentration promotes binding of and release of . It is the capacity to communicate ligand-binding information from one polypeptide subunit to the others that makes the hemoglobin molecule so beautifully adapted to integrating the transport of by erythrocytes. 667 , , and Oxygen Binding to Hemoglobin Is Regulated by 2,3Bisphosphoglycerate The interaction of 2,3-bisphosphoglycerate (BPG) with hemoglobin molecules further reﬁnes the function of hemoglobin, and provides an example of heterotropic allosteric modulation. BPG binds at a site distant from the oxygen-binding site and regulates the -binding afﬁnity of hemoglobin in relation to the in the lungs. BPG is present in relatively high concentrations in erythrocytes. When hemoglobin is isolated, it contains substantial amounts of bound BPG, which can be difﬁcult to remove completely. In fact, the -binding curves for hemoglobin that we have examined up to this point were obtained 668 in the presence of bound BPG. 2,3-Bisphosphoglycerate greatly reduces the afﬁnity of hemoglobin for oxygen — there is an inverse relationship between the binding of and the binding of BPG. We can therefore describe another binding process for hemoglobin: BPG is important in the physiological adaptation to the lower at high altitudes. For a healthy human at sea level, the binding of to hemoglobin is regulated such that the amount of delivered to the tissues is nearly 40% of the maximum that could be carried by the blood (Fig. 5-16). Imagine that this person is suddenly transported from sea level to an altitude of 4,500 meters, where the is considerably lower. The delivery of to the tissues is now reduced. However, a er just a few hours at the higher altitude, the BPG concentration in the blood has begun to rise, leading to a decrease in the afﬁnity of hemoglobin for oxygen. This adjustment in the BPG level has only a small effect on the binding of on the release of in the lungs, but it has a considerable effect in the tissues. As a result, the delivery of oxygen to the tissues is restored to nearly 40% of the that can be transported by the blood. The situation is reversed when the person returns to sea level. The BPG concentration in erythrocytes also increases in people suffering from hypoxia, lowered oxygenation of peripheral tissues due to inadequate functioning of the lungs or circulatory system. 669 hemoglobin for , so approximately 37% of what can be carried is again delivered to the tissues. The site of BPG binding to hemoglobin is the cavity between the subunits in the T state (Fig. 5-17). This cavity is lined with positively charged amino acid residues that interact with the negatively charged groups of BPG. Unlike , only one molecule of BPG is bound to each hemoglobin tetramer. BPG lowers hemoglobin’s afﬁnity for oxygen by stabilizing the T state. In the absence of BPG, hemoglobin is primarily present in the R state, where it binds efﬁciently in the lungs but fails to release it in the tissues. 671 FIGURE 5-17 Binding of 2,3-bisphosphoglycerate to deoxyhemoglobin. (a) BPG binding stabilizes the T state of deoxyhemoglobin. The negative charges of BPG interact with several positively charged groups (shown in blue in this surface contour image) that surround the pocket between the subunits on the surface of deoxyhemoglobin in the T state. (b) The binding pocket for BPG disappears on oxygenation, following transition to the R state. (Compare with Fig. 5-10.) [Data from (a) PDB ID 1B86, V. Richard et al., J. Mol. Biol. 233 270, 1993 (b) PDB ID 1BBB, M. M. Silva et al., J. Biol. Chem. 267 17,248, 1992.] Regulation of oxygen binding to hemoglobin by BPG has an important role in fetal development. Because a fetus must extract oxygen from its mother’s blood, fetal hemoglobin must have greater afﬁnity than the maternal hemoglobin for fetus synthesizes subunits rather than . The subunits, forming hemoglobin. This tetramer has a much lower afﬁnity for 672 BPG than normal adult hemoglobin, and a correspondingly higher afﬁnity for . Sickle Cell Anemia Is a Molecular Disease of Hemoglobin The hereditary human disease sickle cell anemia demonstrates strikingly the importance of amino acid sequence in determining the secondary, tertiary, and quaternary structures of globular proteins, and thus their biological functions. Almost 500 genetic variants of hemoglobin are known to occur in the human population; all but a few are quite rare. Most variations consist of differences in a single amino acid residue. The effects on hemoglobin structure and function are o en minor but can sometimes be extraordinary. Each hemoglobin variation is the product of an altered gene. Variant genes are called alleles. Because humans generally have two copies of each gene, an individual may have two copies of one allele (thus being homozygous for that gene) or one copy of each of two different alleles (thus heterozygous). Sickle cell anemia occurs in individuals who inherit the allele for sickle cell hemoglobin from both parents. The erythrocytes of these individuals are fewer and also abnormal. In addition to an unusually large number of immature cells, the blood contains many long, thin, sickle-shaped erythrocytes (Fig. 5-18). When hemoglobin from sickle cells (called hemoglobin S, or HbS) is 673 deoxygenated, it becomes insoluble and forms polymers that aggregate into tubular ﬁbers (Fig. 5-19). Normal hemoglobin (hemoglobin A, or HbA) remains soluble upon deoxygenation. The insoluble ﬁbers of deoxygenated HbS cause the deformed, sickle shape of the erythrocytes, and the proportion of sickled cells increases greatly as blood is deoxygenated. FIGURE 5-18 A comparison of (a) uniform, cup-shaped, normal erythrocytes and (b) the variably shaped erythrocytes seen in sickle cell anemia, which range from normal to spiny or sickle-shaped. 674 675 FIGURE 5-19 Normal and sickle cell hemoglobin. (a) Subtle diﬀerences between the conformations of HbA and HbS result from a single amino acid change in the chains. (b) As a result of this change, deoxyHbS has a hydrophobic patch on its surface, which causes the molecules to aggregate into strands that align into insoluble fibers. The altered properties of HbS result from a single amino acid substitution, a Val instead of a Glu residue at position 6 in the two chains. Replacement of the Glu residue by Val creates a “sticky” hydrophobic contact point at position 6 of the chain, which is on the outer surface of the molecule. These sticky spots cause deoxyHbS molecules to associate abnormally with each other, forming the long, ﬁbrous aggregates characteristic of this disorder. Sickle cell anemia is life-threatening and painful. People with this disease suffer repeated crises brought on by physical exertion. They become weak, dizzy, and short of breath, and they also experience heart murmurs and an increased pulse rate. The hemoglobin content of their blood is only about half the normal value of 15 to 16 g/100 mL, because sickled cells are very fragile and rupture easily; this results in anemia (“lack of blood”). An even more serious consequence is that capillaries become blocked by the long, abnormally shaped cells, causing severe pain and interfering with normal organ function — a major factor in the early death of many people with the disease. Without medical treatment, people with sickle cell anemia usually die in childhood. Curiously, the frequency of the sickle 676 cell allele in populations is unusually high in certain parts of Africa. Investigation into this matter led to the ﬁnding that when heterozygous, the allele confers a small but signiﬁcant resistance to lethal forms of malaria. The heterozygous individuals experience a milder condition called sickle cell trait; only about 1% of their erythrocytes become sickled on deoxygenation. These individuals may live completely normal lives if they avoid vigorous exercise and other stresses on the circulatory system. Natural selection has resulted in an allele population that balances the deleterious effects of the homozygous condition against the resistance to malaria afforded by the heterozygous condition. SUMMARY 5.1 Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins Protein function o en entails interactions with other molecules. A protein binds a molecule, known as a ligand, at its binding site. Myoglobin contains a heme prosthetic group, which binds oxygen. Heme consists of a single atom of within a porphyrin. coordinated Globins are a specialized family of transport proteins containing heme; most globins store oxygen. Oxygen binds to myoglobin reversibly. Reversible binding of a ligand to a protein can be described quantitatively by a dissociation constant 677 . For a monomeric protein such as myoglobin, the fraction of binding sites occupied by a ligand is a hyperbolic function of ligand concentration. Proteins may undergo conformational changes when a ligand binds, a process called induced ﬁt. In a multisubunit protein, the binding of a ligand to one subunit may affect ligand binding to other subunits. Ligand binding can be regulated. Hemoglobin transports oxygen in blood. Normal adult hemoglobin has four heme-containing subunits, two and two myoglobin. , similar in structure to each other and to Hemoglobin exists in two interchangeable structural states, T and R. The T state is most stable when oxygen is not bound. Oxygen binding promotes transition to the R state. Oxygen binding to hemoglobin is both allosteric and cooperative. As binds to one binding site, the hemoglobin undergoes conformational changes that affect the

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