Hemoglobin
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| - | ==Red Blood, Blue Blood== | ||
| - | [[Image:2dhb_goodsell.gif|right]] | ||
| - | Ever wondered why blood vessels appear blue? Oxygenated blood is bright red: when you are cut, the blood you see is brilliant red oxygenated blood. Deoxygenated blood is deep purple: when you donate blood or give a blood sample at the doctor's office, it is drawn into a storage tube away from oxygen, so you can see this dark purple color. However, deep purple deoxygenated blood appears blue as it flows through our veins, especially in people with fair skin. This is due to the way that different colors of light travel through skin: blue light is reflected in the surface layers of the skin, whereas red light penetrates more deeply. The dark blood in the vein absorbs most of this red light (as well as any blue light that makes it in that far), so what we see is the blue light that is reflected at the skin's surface. Some organisms like snails and crabs, on the other hand, use copper to transport oxygen, so they truly have blue blood. | ||
| - | Hemoglobin is the protein that makes blood red. It is composed of four protein chains, two α chains and two β chains, each with a ring-like heme group containing an iron atom. Oxygen binds reversibly to these iron atoms and is transported through blood. Each of the protein chains is similar in structure to [[myoglobin]] (presented in the January 2000 Molecule of the Month), the protein used to store oxygen in muscles and other tissues. However, the four chains of hemoglobin give it some extra advantages, as described on the next page. | ||
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| - | ==Use and Abuse of Hemoglobin== | ||
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| - | Aside from oxygen transport, hemoglobin can bind and transport other molecules like nitric oxide and carbon monoxide. Nitric oxide affects the walls of blood vessels, causing them to relax. This in turn reduces the blood pressure. Recent studies have shown that nitric oxide can bind to specific cysteine residues in hemoglobin and also to the irons in the heme groups, as shown in PDB entry [[1buw]]. Thus, hemoglobin contributes to the regulation of blood pressure by distributing nitric oxide through blood. Carbon monoxide, on the other hand, is a toxic gas. It readily replaces oxygen at the heme groups, as seen in PDB entry [[2hco]] and many others, forming stable complexes that are difficult to remove. This abuse of the heme groups blocks normal oxygen binding and transport, suffocating the surrounding cells. | ||
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| - | ==Artificial Blood== | ||
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| - | Blood transfusions have saved countless lives. However, the need for matching blood type, the short life of stored blood, and the possibility of contamination are still major concerns. An understanding of how hemoglobin works, based on decades of biochemical study and many crystallographic structures, has prompted a search for blood substitutes and artificial blood. The most obvious approach is to use a solution of pure hemoglobin to replace lost blood. The main challenge is keeping the four protein chains of hemoglobin together. In the absence of the protective casing of the red blood cell, the four chains rapidly fall apart. To avoid this problem, novel hemoglobin molecules have been designed where the two of the four chains are physically linked together, as shown in PDB entry [[1c7d]]. In that structure, two additional glycine residues form a link between two of the chains, preventing their separation in solution. | ||
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| - | ==Hemoglobin Cousins== | ||
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| - | Looking through the PDB, you will find many different hemoglobin molecules. You can find Max Perutz's groundbreaking structure of horse hemoglobin in entry [[2dhb]], shown in the picture here. There are structures of human hemoglobins, both adult and fetal. You can also find unusual hemoglobins like leghemoglobin, which is found in legumes. It is thought to protect the oxygen-sensitive bacteria that fix nitrogen in leguminous plant roots. In the past few years some hemoglobin cousins called the "truncated hemoglobins" have been identified, such as the hemoglobin in PDB entry [[1idr]], which have several portions of the classic structure edited out. The only feature that is absolutely conserved in this subfamily of proteins is the histidine amino acid that binds to the heme iron. | ||
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| - | ==Cooperation Makes It Easier== | ||
| - | [[Image:Hb-animation.gif|right]] | ||
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| - | Hemoglobin is a remarkable molecular machine that uses motion and small structural changes to regulate its action. Oxygen binding at the four heme sites in hemoglobin does not happen simultaneously. Once the first heme binds oxygen, it introduces small changes in the structure of the corresponding protein chain. These changes nudge the neighboring chains into a different shape, making them bind oxygen more easily. Thus, it is difficult to add the first oxygen molecule, but binding the second, third and fourth oxygen molecules gets progressively easier and easier. This provides a great advantage in hemoglobin function. When blood is in the lungs, where oxygen is plentiful, oxygen easily binds to the first subunit and then quickly fills up the remaining ones. Then, as blood circulates through the body, the oxygen level drops while that of carbon dioxide increases. In this environment, hemoglobin releases its bound oxygen. As soon as the first oxygen molecule drops off, the protein starts changing its shape. This promotes the remaining three oxygens to be quickly released. In this way, hemoglobin picks up the largest possible load of oxygen in the lungs, and delivers all of it where and when needed. | ||
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| - | In this animated figure, the heme group of one subunit, shown in the little circular window, is kept in one place so that you can see how the protein moves around it when oxygen binds. The oxygen molecule is shown in blue green. As it binds to the iron atom in the center of the heme, it pulls a histidine amino acid upwards on the bottom side of the heme. This shifts the position of an entire α helix, shown here in orange below the heme. This motion is propagated throughout the protein chain and on to the other chains, ultimately causing the large rocking motion of the two subunits shown in blue. The two structures shown are PDB entries [[2hhb]] and | ||
| - | [[1hho]]. | ||
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| - | ==Troubled Hemoglobins== | ||
| - | [[Image:2hbs-fiber.gif|right]] | ||
| - | The genes for the protein chains of hemoglobin show small differences within different human populations, so the amino acid sequence of hemoglobin is slightly different from person to person. In most cases the changes do not affect protein function and are often not even noticed. However, in some cases these different amino acids lead to major structural changes. One such example is that of the sickle cell hemoglobin, where glutamate 6 in the β chain is mutated to valine. This change allows the deoxygenated form of the hemoglobin to stick to each other, as seen in PDB entry [[2hbs]], producing stiff fibers of hemoglobin inside red blood cells. This in turn deforms the red blood cell, which is normally a smooth disk shape, into a C or sickle shape. The distorted cells are fragile and often rupture, leading to loss of hemoglobin. This may seem like a uniformly terrible thing, but in one circumstance, it is actually an advantage. The parasites that cause the tropical disease malaria, which spend part of their life cycle inside red blood cells, cannot live in the fiber-filled sickle cells. Thus people with sickle cell hemoglobin are somewhat resistant to malaria. Other circumstances leading to troubled hemoglobins arise from a mismatch in the production of the α and β proteins. The structure obviously requires equal production of both proteins. However, if one of these proteins is missing, it leads to conditions called Thalassemia. | ||
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| - | ==Exploring the Structure== | ||
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| - | You can look at the binding of oxygen up close in two structures of human hemoglobin. PDB entry [[2hhb]] shows hemoglobin with no oxygen bound. In this picture, the heme is seen edge-on with the iron atom colored in gold. [[Image:Hb-rasmol.gif|left|400px]]You can see the key histidine reaching up on the bottom side to bind to the iron atom. In PDB entry 1hho, oxygen has bound to the iron, pulling it upwards. This in turn, pulls on the histidine below, which then shifts the location of the entire protein chain. These changes are transmitted throughout the protein, ultimately causing the big shift in shape that changes the binding strength of the neighboring sites. | ||
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| - | This picture was created with Rasmol. Note that the PDB entry [[1hho]] only contains two of the four chains in the hemoglobin structure. | ||
==Hemoglobin subunit binding O<sub>2</sub>== | ==Hemoglobin subunit binding O<sub>2</sub>== | ||
Revision as of 14:40, 30 June 2008
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Hemoglobin is an oxygen-transport protein. Hemoglobin is an allosteric protein. It is a tetramer composed of two types of subunits designated α and β, with stoichiometry . The of hemoglobin sit roughly at the corners of a tetrahedron, facing each other across a at the center of the molecule. Each of the subunits prosthetic group. The give hemoglobin its red color.
Each individual molecule contains one atom. In the lungs, where oxygen is abundant, an binds to the ferrous iron atom of the heme molecule and is later released in tissues needing oxygen. The heme group binds oxygen while still attached to the . The spacefill view of the hemoglobin polypeptide subunit with an oxygenated heme group shows how the within the polypeptide.
is facilitated by a histidine nitrogen that binds to the iron. A second histidine is near the bound oxygen. The "arms" (propanoate groups) of the heme are hydrophilic and face the surface of the protein while the hydrophobic portions of the heme are buried among the hydrophobic amino acids of the protein.
Perhaps the most well-known disease caused by a mutation in the hemoglobin protein is sickle-cell anemia. It results from a mutation of the sixth residue in the β hemoglobin monomer from . This hemoglobin variant is termed 'hemoglobin S' (2hbs).
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Hemoglobin subunit binding O2
For hemoglobin, its function as an oxygen-carrier in the blood is fundamentally linked to the equilibrium between the two main states of its quaternary structure, the unliganded "deoxy" or "T state" versus the liganded "oxy" or "R state". The unliganded (deoxy) form is called the "T" (for "tense") state because it contains extra stabilizing interactions between the subunits. In the high-affinity R-state conformation the interactions which oppose oxygen binding and stabilize the tetramer are somewhat weaker or "relaxed". In some organisms this difference is so pronounced that their Hb molecules dissociate into dimers in the oxygenated form. Structural changes that occur during this transition can illuminate how such changes result in important functional properties, such as cooperativity of oxygen binding and allosteric control by pH and anions. Hemoglobin is definitely not a pure two-state system, but the T to R transition provides the major, first-level explanation of its function.
The hemoglobin molecule (or "Hb") is a tetramer of two α and two β chains, of 141 and 146 residues in human. They are different but homologous, with a "globin fold" structure similar to myoglobin.
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Here we see a single of hemoglobin, starting with an overview of the subunit. The 6 major and 2 short α-helices that make up the structure of a Hb subunit (the "globin fold") are , which is the traditional naming scheme. For example, the proximal histidine (the tightest protein Fe ligand) is often called , since it is residue 9 on helix F (it is residue 87 in the human α chain). The helices form an approximately-cylindrical bundle, with the heme and its central Fe atom bound in a .
For this image the oxy and deoxy α1 heme groups were superimposed on each other, to give a local comparison at this site, a closeup around the heme O2-binding site. The heme is quite domed in the blue T-state (deoxy) form, with the 5-coordinate, high-spin Fe (yellow ball) out of the plane. In the pink R-state form a CO molecule is bound at the left; the Fe, now 6-coordinate low-spin, has moved into the heme plane, which has flattenened. The proximal His (at right) connects the Fe to helices on the proximal side, making the Fe position sensitive to changes in the globin structure and vice versa. Remember that this scene shows a subunit in the all-unliganded versus the all-liganded states of Hb; when oxygen binds to just one subunit, then its internal structure undergoes some but not all of these changes, depending on conditions.
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O2 binds in the same place as CO, with similar effects on the structure; however, for O2 the outer atom is angled rather than straight. The equilibrium between free and bound O2 is very rapid, with on and off rates that are sensitive to protein conformation. Both CO and NO dissociate from the Fe atom very slowly, so that these gases act as respiratory poisons. The α and β chains differ somewhat in their rates and relative affinities for O2 and other ligands, by virtue of heme-pocket differences, but the differences between affinities in the R vs T quaternary states are much larger.
Both α and β chains of Hb resemble myoglobin (the single-chain O2-binder in muscle), both in overall tertiary structure and in using an Fe atom centered in a heme group as the site where oxygen is reversibly bound. The heme is surrounded by a hydrophobic pocket, which is necessary in order for it to bind oxygen reversibly without undergoing oxidation or other undesirable reactions.
Here we see some of the hydrophobic sidechains that form the heme pocket. They actually surround the binding site so thoroughly that O2 cannot get in or out without parts of the protein moving out of the way a bit, so that its dynamic properties are essential to have any O2 binding at all; this restrictive process also increases the specificity of ligand binding.
The shift between R and T state requires subunit interactions and does not occur in myoglobin, or in isolated α or β chain monomers. These monomers bind O2 quite tightly, which would work well for loading O2 in the lungs but would not allow unloading it for delivery to the tissues. Therefore, the central critical feature of hemoglobin function is how it achieves, uses, and allosterically controls cooperativity between the 4 binding sites in the tetramer to tune O2 binding for satisfying physiological needs.
Linkage of the heme Fe through the proximal His results in tertiary-structure changes that can then transmit their effects to other subunits in the tetrameric assemblage. This allows O2 binding in one subunit to indirectly affect the affinitiy of other subunits. Briefly, inside the α chains the R/T equilibrium is reflected in changes in Fe spin state and position as it moves in or out of the heme plane; the proximal His changes distance and angle relative to the heme; the F helix shifts; Tyr 140 moves and its H-bond to backbone weakens; and both the C-terminus of the chain and Arg 141 move significantly at the interface. Changes at the subunit interface (coupled with changes at the Fe, as we have seen) alter the equilibrium between the deoxy and oxy quaternary structures, and conversely a change of quaternary structure alters the balance between the two states inside a given subunit. Each O2 that binds increases the likelihood of switching the tetramer into the oxy state, and once it switches, the O2 affinity at all sites increases because the local structure changes have either already occurred or are easier to make.
Click here to show the α1 subunit, but centered for the whole tetramer.
The Hb tetramer T -> R transition
The central cavity, is wider in the deoxy state, forming phosphate sites; quaternary structure change as rigid rotations of α-β dimers; α1-β2 contact overview; "ratchet" vs "hinge" at the a1b2 interface; α1-α2 salt bridges; charged groups at the C-terminus of β2 which stabilize the deoxy form; and finally a summary overview. (from PDB files bio3HHB and bio1HCO)
Look down one of the approximate 2-fold axes, with α subunits at the top and β subunits at the bottom. Notice that the hemes are quite far apart, so that their interactions must be mediated by the protein. For a view down the exact crystallographic 2-fold axis from the β1- β2 end, click here: The yellowtint crosses are phosphate sites present in deoxy but not oxy Hb. In oxy Hb, the β subunits move closer together, squeezing out phosphates (such as 2,3 DPG), and allowing the N- and C-termini to interact. DPG and other phosphates bind much more strongly to the deoxy quaternary structure; therefore they necessarily push the equilibrium toward deoxy Hb, and because of that they decrease O2 affinity. Such regulatory phosphate molecules are useful in the blood, because their concentrations can be controlled to shift the Hb O2-binding curve so that it is working across the steepest and most efficient part under conditions in the lungs and tissues. For instance, at high altitude the body makes more DPG, to unload O2 more effectively in the muscles.
Like the PFK, to the first approximation the Hb molecule consists of two "dimers" (α1-β1 and α2-β2), which rotate relative to each other as rigid bodies in the R-T transition. The α1-β1 unit undergoes relatively little internal rearrangement, but its overall rotation with respect to the α2-β2 unit is considerable. The net rotation of the two dimers alters their interactions with one another, most notably at the allosteric effector site between β1 and β2 (PO4 binding) and at the important α1-β2 interface, where mutations have the largest effect on Hb allosteric properties. Although the symmetry is not exact, similar parts of the subunits contact each other: the C helix, and the "FG corner" between helices F and G.
Have a look at a closeup that emphasizes the ratchet contact between the C helix of α1 and the FG corner of β2; His 97 of the β2 FG corner makes a large jump against Thr 38 and Thr 41 of the α1 C helix. In a closeup of the hinge contact, the motions are mainly rotations without much shift, between the α1 FG corner and the β2 C helix. Labels help identify these parts. Since this is a complex motion orchestrated between the fit of two quite different sets of contacts in the two states, this interface is critical to making Hb allostery work, and mutations of residues in this interface have been found to be especially likely to influence cooperativity and allostery.
There are salt links between α1 and α2, which stabilize the deoxy form. Here’s an overview down the exact 2-fold axis between the subunits, showing that there are two equivalent sets of interactions, on either side of the twofold.
Salt links at the C-terminus of β2 stabilize the deoxy T form and make a large contribution to the pH dependence of Hb oxygen binding, known as the Bohr Effect. In the making and breaking of these interactions, His β 146 moves a great deal, disrupting the salt link (charged H-bond) to Asp β 94 that is formed in the T state. Since His titrates near physiological pH, this interaction is quite pH sensitive. At low pH, when more protons are present, the His ring N is more likely to be protonated and positive; this strengthens its H-bond with Asp 94, thus favoring the T state and decreasing O2 affinity. The pH effect, or Bohr Effect, can be considered as allosteric regulation by the binding of protons. It is important biologically, because it promotes oxygen unloading in the tissues where proton concentrations are elevated, for instance by the production of lactic acid in muscle.
References, for further information on Hemoglobin
To the structures used here:
- Baldwin (1980) "The crystal structure of human carbonmonoxy haemoglobin at 2.7A resolution", J. Mol. Biol. 136: 103. (1hco) PMID: 7373648
- Fermi, Perutz, Shaanan, & Fourme (1984) "The crystal structure of human deoxy haemoglobin at 1.74A resolution", J. Mol. Biol. 175: 159. (3hhb)
General treatments of Hb allostery:
- Perutz (1970) "Stereochemistry of cooperative effects in haemoglobin", Nature 228: 726
- Baldwin & Chothia (1979) "Haemoglobin. The structural changes related to ligand binding and its allosteric mechanism", J. Mol. Biol. 129: 175. link
- Dickerson & Geis (1983) "Hemoglobin: Structure, Function, and Pathology", Benjamin/Cummings Publ., Menlo Park, CA
- Perutz (1989) "Mechanisms of cooperativity and allosteric regulation in proteins", Quarterly Rev. of Biophys. 22: 139-236
- Ackers, Doyle, Myers, & Daugherty (1992) "Molecular code for cooperativity in hemoglobin", Science 255: 54
- Perutz, Fermi, Poyart, Pagnier, & Kister (1993) "A novel allosteric mechanism in haemoglobin: Structure of bovine deoxyhaemoglobin, absence of specific chloride binding sites, and origin of the chloride-linked Bohr Effect in bovine and human haemoglobin", J. Mol. Biol. 233: 536
Hb structures in other quaternary states or intermediates:
- Silva, Rogers, & Arnone (1992) "A third quaternary structure of human hemoglobin A at 1.7A resolution", J. Biol. Chem. 267: 17248
- Smith, Lattman, & Carter (1991) "The mutation β99 Asp-Tyr stabilizes Y - A new, composite quaternary state of human hemoglobin", Proteins: Struct., Funct., Genet. 10: 81
- Liddington, Derewenda, Dodson, Hubbard, & Dodson (1992) "High resolution crystal structures and comparisons of T state deoxyhaemoglobin and two liganded T-state haemoglobins: T(α-oxy)haemoglobin and T(met)Haemoglobin", J. Mol. Biol. 228: 551
More information on hemoglobin
- Perutz, M.F. (1978) Hemoglobin Structure and Respiratory Transport, Scientific American, volume 239, number 6.
- Squires, J.E. (2002) Artificial Blood, Science 295, 1002.
- Vichinsky, E. (2002) New therapies in sickle cell disease. Lancet 24, 629.
Content Donators
Currently (June 22, 2008) most all of the content of this page comes from three main sources of generously donated content. Their work has been imported into this page. In their order of appearance on the page:
- Content adapted with permission from Eric Martz's http://www.umass.edu/molvis/tutorials/hemoglobin/
- Content adapted with permission from David S. Goodsell and Shuchismita Dutta's Molecule of the Month on Hemoglobin http://mgl.scripps.edu/people/goodsell/pdb/pdb41/pdb41_1.html
- Content adapted with permission from Jane S. and David C. Richardson's http://kinemage.biochem.duke.edu/
Proteopedia Page Contributors and Editors (what is this?)
Eran Hodis, Michal Harel, Joel L. Sussman, Alexander Berchansky, Jaime Prilusky, Karsten Theis, Eric Martz, Karl Oberholser, Tihitina Y Aytenfisu, Mark Hoelzer, Marc Gillespie, Ann Taylor, Manisha Chawda, Hannah Campbell
