Human Adult Deoxyhemoglobin
Created by Megan Stangeby
Human adult deoxyhemoglobin (PDP ID = 2HHB) from Homo sapiens is an allosteric protein used for oxygen transport. Hemoglobin is an extensively studied protein due to its physiological importance and the fact that there is an abundant supply of it and it can easily be crystallized. It is the main component of the red blood cells and is responsible for the transfer of oxygen from the lungs to tissues and the transfer of carbon dioxide from tissues back to the lungs (4). Hemoglobin has a shift in the equilibrium between the two quaternary structures. It shifts between the low-affinity deoxygenated T (tense) state to the high-affinity oxygenated R (relaxed) state (9). The molecular weight of unligated deoxyhemoglobin (just the alpha and beta chains) is 61,933.10 Da and its isoelectric point is 8.13. Its molecular mass is around 64,450 Da when taking into account the heme groups (3).
Deoxyhemoglobin is a compact globular protein (6.4 x 5.5 x 5.0 nm) and an α2β2-tetramer consisting of four subunits arranged in a tetrahedral assortment. There are two dimeric halves, an α1β1-subunit pair and an α2β2-subunit pair (3). It has two identical alpha subunits, each containing 141 amino acid residues, and two identical beta subunits, each containing 146 amino acid residues (7). These four chains are called globin (4). Deoxyhemoglobin has two associated ligands: protoporphyrin IX containing Fe, otherwise known as a heme group, and a phosphate ion. The phosphate ion decreases the affinity of deoxyhemoglobin for oxygen and is only bound to the two beta subunits. Each subunit contains a heme group, allowing deoxyhemoglobin to have a total of four heme groups. The heme, a prosthetic group, consists of an iron (II) atom at the center of a porphyrin molecule (4). This iron ion has six electrons in the d atomic orbital and can accommodate five ligands: the four nitrogen atoms of the porphyrin ring system and a histidine amino acid residue. In this state the iron is paramagnetic and in the high-spin state, where it has a spin value of S=2, making it more sterically hindered (3). The iron atom can only take up oxygen when the heme is in combination with globin.
The secondary structure of the alpha subunits is 76% helical with the rest being random coils. The alpha helices of the alpha subunits are comprised of 108 residues. The secondary structure of the beta subunits is 79% helical with the rest being random coils. The alpha helices in the beta subunits are comprised of 116 residues (11). Both the alpha and the beta subunits are composed of eight alpha helices which are denoted by the letters A through H. The short regions that connect the alpha helixes are named by the segments they connect, as in the AB region is the region in-between the A and B alpha helices (3). Despite having different residues in the alpha and beta sequences, the subunits have very similar tertiary structure.
There are many proteins with tertiary structural similarities to deoxyhemoglobin, as demonstrated by the Dali Structural Alignment test. The following proteins all have a Z score of 30.6 with an rmsd value of 0.1 (5). The first protein is ferrous carbonmonoxyhemoglobin (PDB ID = 1COH). The secondary structure of ferrous carbonmonoxyhemoglobin is 76% alpha helices for the alpha chain and 78% alpha helices for the beta chain with the rest consisting of random coils. Since, ferrous carbonmonoxyhemoglobin and deoxyhemoglobin share the same sequence for their alpha and beta subunits, the differences in their tertiary structures originate from the different binding ligands. The second protein is hemoglobin catonsville (PDB ID = 1BZ0). The secondary structure of hemoglobin catonsville is 75% alpha helices for the alpha chain and 80% alpha helices for the beta chain with the rest consisting of random coils. A single point mutation accounts for the slight differences in the tertiary structure, but this mutation does not have a large impact. The third protein is deoxy recombinant hemoglobin (PDB ID = 1DXU). The secondary structure of deoxy recombinant hemoglobin is 75% alpha helices for the alpha chain and 80% alpha helices for the beta chain with the rest consisting of random coils. It also has similar tertiary structure but its differences can be attributed to the addition of a methionine residue at the amino-terminus of the beta subunits (11). All three of these proteins have nearly identical tertiary structures with four subunits in a spherical shape despite having differences in their sequences.
The deoxygenated form of hemoglobin resists oxygenation because it is stabilized by hydrogen bonds and salt bridges, or ion-pair bonds. The carboxyl-termini of the four subunits are restrained in the T state, while these carboxyl-termini are free to rotate in the R state. When hemoglobin is in its T state, oxygen is only accessible to the heme groups on the alpha subunits. The heme groups on the beta subunits have too much steric hindrance from the amino acid residues that make up the E helix, so they are only accessible after the conformation change to the R state (3). The beta subunit heme is specifically blocked by Val-β67 on the E helix (11). There is nothing specifically blocking the heme groups on the alpha subunits but they are stabilized by certain other stereochemical factors, decreasing their affinity for oxygen.
The subunit interactions are mainly between the alpha and beta subunits, not between the similar subunits. More specifically, the α1β1-interface and α2β2-interface contacts involve helices B, G, H, and the GH corner. When hemoglobin changes from its deoxygenated to oxygenated form, human oxyhemoglobin (PDB ID = 1HHO) these contacts remain unchanged (3). These intermolecular contacts distort the molecular structure and are correlated with molecular asymmetry. This distortion of the molecule is a highly localized effect (11). In other words, an asymmetric area with many intermolecular contacts is bounded by regions with small asymmetry. The α1β2-interface and α2β1-interface contacts involve the C, G, and FG corner and are called sliding contacts. These contacts are altered when deoxyhemoglobin undergoes a conformational change. Each αβ-dimer moves as a rigid body and slides past one another when deoxyhemoglobin undergoes its conformational change upon oxygenation (3). The alpha and beta F helices shift one angstrom relative to the heme, while the beta E helix shifts about two angstroms relative to the heme. There is little effect on the alpha E helix (11).
The non-covalent, electrostatic salt bridge interactions are disrupted upon oxygenation. For example, the interactions between His-β146 and Lys-α40 and Arg-α141 and Val-β34, which stabilize the alpha and beta chains, are broken in the conformation change. Other interactions are not disrupted upon oxygenation and are there to stabilize their respective subunits. His-β146 and Asp-β94 and Val-β78 and Tyr-β145 help to stabilize the beta subunits, while Arg-α141 and Val-α1, Val-α93 and Tyr-α140, Arg-α141 and Asp-α126, and Arg-α41 and Lys-α127 help to stabilize the alpha subunits (3).
The heme groups have different characteristics in the alpha subunits and beta subunits. His-α87 attaches a heme group to the alpha subunits and His-β92 attaches a heme group to the beta subunits. The histidine amino acid residue forms a bond with one of the nitrogen atoms in the heme group in both cases (3). In deoxyhemoglobin, the heme iron atom is in its high-spin paramagnetic form (7). In the alpha subunit, the iron is about 0.40 angstroms from the mean plane of porphyrin and the iron-nitrogen bond length is about 2.12 angstroms. In the beta subunit, the iron is about 0.36 angstroms from the mean plane of porphyrin and the iron-nitrogen bond length is about 2.06 angstroms. The iron atom is forced to lie out of the porphyrin plane due to steric repulsion between the histidine residue and nitrogen atom along with electrostatic repulsion between the electrons of the iron atom and the pi electrons in the porphyrin group. So, the hemes are domed toward the proximal side in the deoxygenated state. In the alpha hemes the plane between the nitrogen and carbon of the porphyrin are titled uniformly toward the heme center by about three degrees relative to the heme normal which is perpendicular to the plane. There is additional folding of about four degrees of the heme about an axis running between the methene carbons that are between the pyrrole rings. This folding is not observed in the beta hemes, but the second and fourth pyrroles are tilted by about eight degrees relative to the heme normal which is perpendicular to the plane (11). The iron on the alpha heme is further out from the heme plane than the iron on the beta heme due to the doming of the alpha and beta hemes, not the difference in the bond distances.
The heme groups are between the E and F alpha helices and are exposed at the surface of the molecule. When a molecule of oxygen binds to a heme, the heme iron ion is drawn into the plane of the porphyrin ring in its respective subunit. This causes the same conformational change in the other three subunits, increasing the affinity of their heme groups for oxygen. Following oxygenation, the iron ion is free to move closer to the plane by about 0.039 nm. As the iron atom moves closer to the plane, it drags the histidines along with it, causing the F, EF corner, and FG corner to also follow, leading to the rupture of interchain salt links (3). So, the interactions that once stabilized the deoxygenated form are broken to stabilize the new conformation.
The binding of oxygen to hemoglobin is altered by protons, chloride ions, and inorganic phosphate. These factors stabilize the T state and aid in the release of oxygen (9). In fact, deoxyhemoglobin has a higher affinity for protons than oxyhemoglobin and as pH decreases, the deoxygenated state is enhanced (3). The oxygen affinity in the deoxygenated state is about 70 times lower than that in the oxygenated state (6). As previously mentioned, His-α87 and His-β92 are the residues which are bound to the hemes in each subunit. These proximal histidines are fully (doubly) protonated when bound to the heme groups in deoxyhemoglobin. Two distal histidines: His-α58 and His-β63 are also fully protonated. These positively charged distal histidine residues contradict what occurs in the R state since full protonation cannot occur in the R state. Thus, the pKa values for these two distal residues are much higher in deoxyhemoglobin than in liganded hemoglobin, indicating they play a role in the T state Bohr effect of hemoglobin (9). The Bohr effect states that protons inhibit oxygen binding to hemoglobin (1). The release of protons associated with quaternary structural transition from the T state to the R state and the release of protons associated with oxygen binding by the T state are the two factors that affect the T state Bohr effect of hemoglobin (9). The amino-termini of the two alpha subunits and the His-β146 residues also play a major role in the Bohr effect. The carboxylate groups of neighboring Asp-β94 residues help to stabilize the protonated state of the His-β146 imidazoles in deoxyhemoglobin (3). Furthermore, His-α103, His-α122, and His-β116 are all doubly protonated and aid in connecting the alpha and beta subunits (9).
His-α103 acts as a hydrogen bond donor and forms a hydrogen bond to the side subunit carbonyl of Gln-β131. Additionally, it forms a water-mediated hydrogen bonding network connecting the hydroxyl group of Tyr-β36 and the carboxyl group of Asp-α126 and the carboxyl group of His-α122. Overall, 10 of the 19 histidines in one set of alpha and beta subunits are protonated in deoxyhemoglobin. His-α122 similarly forms a water-mediated hydrogen bond with the hydroxyl group of Tyr-β25. His-β116 forms a hydrogen bond to the carbonyl Pro-α114 (9). These buried histidines greatly contribute to the α1β1-interface stabilization.
Deoxyhemoglobin binds better to carbon dioxide, chloride ions, and 2,3-bisphosphoglycerate (BPG) than does oxyhemoglobin. As a result, these compounds favor the release of oxygen and the conversion from oxyhemoglobin to deoxyhemoglobin. Carbonic anhydrase promotes the hydration of carbon dioxide into carbonic acid. A proton and bicarbonate are then formed upon ionization of carbonic acid. Ultimately, carbon dioxide dissolves in the blood and forms protons. As oxygen dissociates, deoxyhemoglobin picks up these protons. The binding of BPG to hemoglobin promotes the release of oxygen. The binding site for BPG is found within the central cavity formed by the association of the four subunits. BPG is a very negative molecule and strongly interacts with the positively charged functional groups of Lys-β82, His-β2, His-β143, and the amino-terminus of each beta subunit (3). BPG essentially indirectly links the two beta subunits because it forms ionic bonds between both beta chains and helps stabilize the deoxygenated conformation of hemoglobin, thus inhibiting the binding of oxygen.
Another inorganic phosphate, inositol hexaphosphate (IHP), also binds to the central cavity of deoxyhemoglobin. Like BPG, IHP also lowers oxygen affinity of hemoglobin. IHP binds at the His-β2, Lys-β82, Asn-β139, and His-β143 amino acid residues (7). So, it also promotes the release of oxygen by stabilizing the two beta subunits.
The Bohr effect is very physiologically significant. Actively metabolizing tissues produce acid, thus promoting oxygen release where it is most needed. After the oxygen is released, deoxyhemoglobin takes up two protons. These protons lower the oxygen affinity of deoxyhemoglobin and allow it to travel back to the lungs. Once deoxyhemoglobin is back at the lungs it is oxygenated and the protons are released. The protons react with the bicarbonate and reform carbonic acid, liberating carbon dioxide so it can be exhaled from the body as a gas (3). With deoxygenation, the intracellular pH rises by 0.1-0.15 (8).
Mutant human carbonmonoxyhemoglobin C (PDBID = 1K1K) from Homo sapiens has very similar sequence similarity to deoxyhemoglobin. The results of DALI (Z=27.6, rmsd=0.7) and protein BLAST searches (E=5e-85) show that the carbonmonoxy-liganded R-state form of hemoglobin has both primary and tertiary similarities to deoxyhemoglobin, with approximately 50% sequence similarity (5). Carbonmonoxyhemoglobin contains the same alpha subunit of 141 amino acid residues and the same beta subunit of 146 amino acid residues as deoxyhemoglobin, but it is a dimer and only contains one of each. The secondary structure of the alpha subunit is 73% helical, while that of the beta subunit is 78% helical. The rest of the subunits are composed of random coils. Carbonmonoxyhemoglobin has two associated ligands: carbon monoxide and protoporphyrin IX containing Fe. Like deoxyhemoglobin, carbonmonoxyhemoglobin has a heme group attached to both subunits, but the heme group binds to carbon monoxide rather than oxygen. There is a fundamental difference between the alpha and beta subunits affinity for oxygen compared to their affinity for carbon monoxide (2). Even though carbonmonoxyhemoglobin and deoxyhemoglobin have approximately a 50% match for sequence similarity and tertiary similarities, they have different functions. In fact, carbonmonoxyhemoglobin is deadly in the bloodstream because it binds more tightly to hemoglobin than oxygen, preventing the transportation of oxygen to tissues. In both deoxygenated and carbonmonoxy forms of hemoglobin, the amide bonds of most amino acids are rigid on the fast time scale. The carboxy-terminal His-β146 amino acid residue is rigid in deoxyhemoglobin; however, it is free from restrictions to its backbone motions in the carbonmonoxy form. In addition, Arg-α31 and Thr-β123 exhibit stiffening upon carbon monoxide bonding, and there is considerable flexibility in the α1β1-interface (7). The carbonmonoxyhemoglobin exhibits very similar characteristics to that of oxyhemoglobin. Even though it is only a dimer it has the characteristics of hemoglobin liganded to oxygen.
Cyanomet RHB 1.1, or recombinant hemoglobin (PDP ID = 1ABY), from Homo sapiens also has very similar primary and tertiary similarities as evidenced by the results of DALI (Z=27.5, rmsd=0.5) and protein BLAST searches (E=2e-154). Recombinant hemoglobin has approximately 50% sequence similarity (5). It has two associated ligands: cyanide ion and protoporphyrin IX containing Fe. The secondary structure is 73% helical for its alpha subunit and 75% helical for its beta subunits. Recombinant hemoglobin contains one alpha subunit, known as the a chain, of 283 amino acid residues and two beta subunits, known as the b and d chains, of 146 amino acid residues. The Asnβ-108 of deoxyhemoglobin is replaced with Lys-β108 in recombinant hemoglobin. This mutation is the cause of recombinant hemoglobin’s reduced oxygen affinity. In addition, dimerization of the alpha chain is prevented because there is an insertion of a glycine residue between the sequences of the normal alpha chains, producing one covalently continuous di-alpha chain (11).
All in all, deoxyhemoglobin resists oxygenation because it is stabilized by hydrogen bonds and salt bridges in its terse state and oxygenation breaks these stabilizing interactions.