View source for Hemoglobin

<StructureSection load='1gzx' size='350' side='right' caption="Human Hemoglobin α chain (grey and pink) β chain (green and yellow) with bound O2 [[1gzx]]" scene="Hemoglobin/Foursubunits/5" >
== Function ==
'''Hemoglobin''' is an oxygen-transport protein.  Hemoglobin is an allosteric protein.  It is a <jmol>
  <jmolLink>
    <script> script /scripts/32/32/Subunits_1hho/1.spt; 
             center visible;</script>
    <text>tetramer</text>
  </jmolLink>
</jmol> composed of two types of subunits designated α and β, with stoichiometry <scene name='Hemoglobin/Alpha2beta2/7'>α2β2</scene>. The  <scene name='Hemoglobin/Foursubunits/5'>four subunits</scene> of hemoglobin sit roughly at the corners of a tetrahedron, facing each other across a <scene name='Hemoglobin/Cavity/9'>cavity</scene> at the center of the molecule. Each of the subunits <scene name='Hemoglobin/Bbsubunitswithheme/5'>contains a heme</scene> prosthetic group. The <scene name='Hemoglobin/4heme/3'>heme molecules</scene> give hemoglobin its red color.

Each individual <scene name='Hemoglobin/Deoxyheme/8'>heme</scene> molecule contains one <scene name='Hemoglobin/Deoxyheme_fe/9'>Fe2+</scene> atom. In the lungs, where oxygen is abundant, an <scene name='Hemoglobin/Oxyheme_fe/7'>oxygen molecule</scene> 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 <scene name='Hemoglobin/Oxysubunit/8'>hemoglobin monomer</scene>. The spacefill view of the hemoglobin polypeptide subunit with an oxygenated heme group shows how the <scene name='Hemoglobin/Oxysubunitsf/4'>oxygenated heme group is held</scene> within the polypeptide. 

<scene name='Hemoglobin/Anchortrace/5'>Anchoring of the heme</scene> 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 <scene name='32/32/Hemoglobins_1hho/9'>glutamic acid to a valine</scene>.  This hemoglobin variant is termed 'hemoglobin S' ([[2hbs]]).
*'''mini hemoglobin''' found in neural tissue and contains 109 residues<ref>PMID:9642264</ref> .
*'''giant hemoglobin''' are sulfur-binding 400kDa hemoglobin found in mouthless and gutless marine animals which get their nutrition by symbiosis with sulfur-oxidizing bacteria<ref>PMID:16204001</ref> .
*'''truncated hemoglobin''' found in bacteria and plants.  They are 20-40 residues shorter than other Hb and have 2-on-2 alpha helical sandwich structure vs the 3-on-3 of other Hbs<ref>PMID:11696555</ref> .
*'''methemoglobin''' contains Fe+3 rather than Fe+2 can cause the lethal disease methemoglobinemia<ref>PMID:30726002</ref> .
*'''leghemoglobin''' found in roots of legumes<ref>PMID:29642729</ref> .
*'''flavohemoglobin''' is flavin-binding.  It binds NO and acts in its catabolism<ref>PMID:18379989</ref> .

==Hemoglobin subunit binding O<sub>2</sub>==
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]].   

<!-- <applet load='3hhb' size='400' frame='true' align='right' caption='Human deoxyhemoglobin (PDB code [[3hhb]])'/> -->

Here we see a single <scene name='Hemoglobin/3hhb_chaina_rainbow/4'>α chain</scene> 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 <scene name='Hemoglobin/3hhb_chaina_heliceslabeled/4'>labeled A through H</scene>, which is the traditional naming scheme.  For example, the proximal histidine (the tightest protein Fe ligand) is often called <scene name='32/32/Hisf9/1'>His F9</scene>, 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 <scene name='Hemoglobin/3hhb_chaina_efpocket/4'>hydrophobic pocket between the E and F helices</scene>.  

<scene name='32/32/Cv/2'>In the present animation scene</scene> the <span style="color:pink;background-color:black;font-weight:bold;">oxy (in pink)</span> and <span style="color:deepskyblue;background-color:black;font-weight:bold;">deoxy (in deepskyblue)</span> 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 <span style="color:deepskyblue;background-color:black;font-weight:bold;">deepskyblue T-state (deoxy) form</span>, with the 5-coordinate, high-spin <span style="color:orange;background-color:black;font-weight:bold;">Fe (orange ball)</span> out of the plane.  In the <span style="color:pink;background-color:black;font-weight:bold;">pink R-state form</span> a CO molecule is bound at the right <span style="color:lime;background-color:black;font-weight:bold;">(C in green</span>,<font color='red'><b>O in red</b></font>); the Fe, now 6-coordinate low-spin, has moved into the heme plane, which has flattenened.  The proximal His (at left) 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.   <jmol><jmolButton>
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script>
<text>Toggle Animation</text>
</jmolButton></jmol>


<!-- kinemage not supported
{|
|<kinemage align="left" width="420" height="300" file="HbAllo.kin"/>
|}
-->
O<sub>2</sub> 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 O<sub>2</sub> 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 O<sub>2</sub> 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.  

The heme binding pocket contains mostly <scene name='32/32/Heme_binding_pocket_apo/1'>hydrophobic residues</scene>, shown in grey. 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.  These movements are animated at this [[User:Jaime_Prilusky/How_do_we_get_the_oxygen_we_breathe|page]].  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 (PO<sub>4</sub> 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.

==Truncated hemoglobins==

<!-- <StructureSection load='Jbic8.pdb' size='500' side='right' scene='Journal:JBIC:8/Cv/1' caption=''> -->
<scene name='Journal:JBIC:8/Trhb/1'>Truncated hemoglobins</scene>, also known as 2/2 hemoglobins, can be further classified into three different groups (I, II, and III). Genomic sequences of bacteria, cyanobacteria, and plants indicate that trHbs are rather common. Group I, Group II, and Group III trHbs have distinct phylogenetic trees and show different ligand-binding properties. The Group I trHb of the ciliated protozoan ''Tetrahymena pyriformis'' (''Tp'' trHb) was first discovered by Keilin and Ryley in 1953.

It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein.

The three-dimensional structure of an <scene name='Journal:JBIC:8/Trhb/2'>Fe(II)-O2 complex of Tp trHb</scene> was determined at 1.73 Å resolution ([[3aq9]]). <scene name='Journal:JBIC:8/Trhb/3'>Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound dioxygen molecule</scene>. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, <scene name='Journal:JBIC:8/Trhb/4'>Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues</scene>.

The O<sub>2</sub> association and dissociation rate constants of ''T. pyriformis'' trHb were 5.5 μM<sup>-1</sup> s<sup>-1</sup>, and 0.18 s<sup>-1</sup>, respectively. The oxygen affinity was determined to be 33 nM. The autooxidation rate constant was 3.8 x 10<sup>-3</sup> h<sup>-1</sup>. These values are similar to those of <scene name='Journal:JBIC:8/Hbn/3'>HbN from Mycobacterium tuberculosis</scene>.

'''Mutations:'''
 
*Mutation at Tyr25: <scene name='43/435485/As/6'>Wildtype Y25 and mutant Y25F together</scene> and <scene name='43/435485/As/5'>animation of this scene</scene>. <jmol><jmolButton>
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script>
<text>Toggle Animation</text>
</jmolButton></jmol>
*Mutation at Gln46: <scene name='43/435485/Ad/4'>Wildtype Q46 and mutant Q46E together (animation)</scene> <jmol><jmolButton>
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script>
<text>Toggle Animation</text>
</jmolButton></jmol> 
*Mutation at <scene name='Journal:JBIC:8/Trhb/11'>Gln50</scene> increased the O<sub>2</sub> dissociation and autooxidation rate constants, and partly disrupted the hydrogen-bonding network.

An <scene name='Journal:JBIC:8/Ag/4'>Fe(III)-H2O complex of Tp trHb was formed following reaction of the Fe(II)-O<sub>2</sub> complex of Tp trHb</scene>, in a crystal state, with nitric oxide. This suggests that ''Tp'' trHb functions in nitric oxide detoxification.

==3D Printed Physical Model of Hemoglobin at The MSOE Center for BioMolecular Modeling==

[[Image:CbmUniversityLogo.jpg | left | 150px]]

Shown below is a 3D printed physical model of Hemoglobin, based on the structure [http://proteopedia.org/wiki/index.php/1a3n 1a3n.pdb]. The two alpha-globin chains are colored light red, the two beta globin chains are colored dark red, and the four heme groups are colored yellow. It has been designed with precisely embedded magnets that allow the four chains to pull apart into individual pieces.

[[Image:Cbm_hemoglobin1.jpg|450px]]
[[Image:Cbm_hemoglobin2.jpg|550px]]

==Additional Resources==
*[[Tutorial:How do we get the oxygen we breathe]]
*Hemoglobin structure tutorial at [http://molviz.org MolviZ.Org]. Includes smooth animations of deoxy↔oxy transitions (morphs), a 16-molecule polymer of sickle hemoglobin
*[[User:Eric Martz/Hemoglobin Quiz|Practice Hemoglobin Quiz]] (immediate feedback)
*[[Ann Taylor/Hemoglobin]]
*[[Glycated hemoglobin]]
*[[Molecular Playground/Hemoglobin-Haptoglobin Complex]]
*[http://hemoglobin.molviz.org Hemoglobin Molecular Structure] including smooth animations of deoxy&harr;oxy transitions (morphs) and a 16-molecule polymer of sickle hemoglobin.
*[[Porphyrin]]
*[[Student Projects for UMass Chemistry 423 Spring 2012-8]]
*[[Hemoglobin (Hebrew)]]

==Hemoglobin 3D structures==
See [[Hemoglobin 3D structures]].

</StructureSection>


==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]]) [http://www.ncbi.nlm.nih.gov/pubmed/7373648 PMID: 7373648]
*Fermi, Perutz, Shaanan, & Fourme (1984) "The crystal structure of human deoxy haemoglobin at 1.74A resolution", J. Mol. Biol. 175: 159.  ([[3hhb]])
*Jotaro Igarashi, Kazuo Kobayashi and Ariki Matsuoka (2011) "A hydrogen-bonding network formed by the B10-E7-E11 residues of a truncated hemoglobin from Tetrahymena pyriformis is critical for stability of bound oxygen and nitric oxide detoxification", J. Biol. Inorg. Chem. 16(4):599-609 ([[3aq9]]) [http://www.ncbi.nlm.nih.gov/pubmed/21298303 PMID: 21298303]
 

'''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. [http://www.nature.com/nature/journal/v228/n5273/abs/228726a0.html 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.
<references/>

{{Clear}}

=='''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 hemoglobin tutorial at http://molviz.org'''
# '''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/'''

[[he: Hemoglobin (Hebrew)]]
[[Category:Topic Page]]