Structure of haemoglobin (1980)

in this film all the molecular models are drawn by computer for three-dimensional stereoscopic viewing by the red-green anaglyph system look through the glasses provided with the red filter to your left eye in the pictures of the hemoglobin structures the left eye view of the molecule will be drawn in red and so will be invisible to your right eye hear the word left shows only to your left eye as it is in red the word right should be invisible to your left eye try with your right eye this time the word left should be invisible in this way the glasses separate the two perspectives one for each eye that are printed together on every frame of the film looking with both eyes together you will fuse the two perspectives to give a nearly white image of the structure hanging in space in front of the screen this is a simplified 3d image of a human hemoglobin molecule only the alpha carbon atoms of the constituent amino acids are drawn and these are linked together to show the shape of the polypeptide backbone of the protein if you have fused the two stereo images you may see that the molecule is a symmetrical grouping of four subunits the nearest two are in the upper left and lower right quadrant and the other two at the rear are a symmetrical pair on the other diagonal each subunit is a globin chain that encloses one specialized group the heme group that contains one atom of ferrous iron in the top left hand quadrant you can see one of the four heme groups looking rather like a cartwheel this is embedded in one of the two forms of globin chain the Alpha with 141 a minor acid residues the globins surrounding the heme at the top right is also an alpha chain the heme group coming into view at the bottom right is embedded in a beta chain made up of 146 residues the fourth subunit at the bottom left is also a beta chain oxygen binds to the atoms of ferrous iron and the strengths of the iron oxygen bonds are controlled by the surrounding protein each iron atom is held in the center of this symmetrical porphyrin structure which together with the iron forms the heme group the nitrogen atoms are marked with radial lines the oxygen atoms have the lines of longitude and the carbon atoms are the unshaded spheres the oxygen molecule is nearest to us with the further of its two atoms directly in front of the central iron hemoglobin functions as a carrier of oxygen but the way in which it does this is by no means obvious we shall explain it by working out from this site where the oxygen molecule is bound examining as we go the organization of the molecule and the relations between its structure organization and function each oxygen molecule is bound to the iron at an octahedral ligand site the binding does not break the oxygen molecule and the iron atom is not oxidized there are five more of these octahedral ligand sites for lie in a plane and these are satisfied by the electron pairs from the nitrogen atoms of the four coplanar parole rings of the porphyrin the whole team group is almost perfectly flat it's aromatic nature enables easy transmission of electronic forces between the iron atom and the edges of the heme group the two phases and three of the edges are hydrophobic they have a preference for a non aqueous environment and will be buried deep within the protein this fourth edge of the heme group has two propionic acid side chains which you can see clearly projecting outwards from the heme group these side chains are hydrophilic and they will project from the molecule there is one remaining ligand site on the iron atom and it lies opposite to the oxygen molecule it is satisfied by an electron pair from a ring nitrogen atom of a histidine residue on the protein chain this histidine one of eight in the chain is called the proximal histidine meaning that it is attached to and therefore nearest to the iron atom of the heme it forms the only strong bond between the heme group and the surrounding protein each protein subunit is formed from lengths of alpha helix folded in such a way as to form a pocket for the heme we are now looking down the rigid helical tube called the G helix it is connected by a non helical segment G H to the next helix the H helix the chain ends at the c terminal so-called because the last amino acid has a free carboxyl group the chain starts at the N terminal with its free amino group and we shall reach this by growing the chain outwards from the proximal histidine along the F II d c e and a hella sees for each chain type each amino acid shown here only by its alpha carbon atom is identified in two ways by its position in a segment of the chain and by its number along the whole chain both counting away from the n-terminal for example the proximal histidine shown here has the notation F 887 alpha this folding of the protein chain is known as the tertiary structure of the subunit in this view you can see the sides of the heme pocket formed by the F helix to the right and the e helix to the left the back formed by the G helix with the seedy section at the bottom front and the FG section at the lower right it is the amino acid side chains of those residues which are close to the heme group that form the lining of the heme pocket most of the residues are hydrophobic and keep charged molecules out of the pocket if these reach the heme the iron may be oxidized and cannot then bind oxygen reversibly these residues have an affinity for parts of the heme group and are of such a size and shape that they keep the heme in position and shaped the binding site for oxygen we can examine where some of these shaping residues are for example this phenyl alanine C D 143 alpha has its aromatic ring almost parallel to the heme group so that there can be electronic coupling between the two further towards the front there is a nearby histidine residue CD 345 alpha this histidine has a hydrogen atom on its nitrogen atom nearest to the oxygen of the lower of the propionic acid side chains with which it forms a hydrogen bond we are now looking at one subunit from outside the molecule and you can see that the propionic acid residues are pointing towards us and that we are looking down the side of the heme group with its iron atom in the center of the planar structure to the left of it you can see the two atoms of the oxygen molecule in a little cleft opposite to the proximal histidine this residue is a valine FG 593 alpha and this residue is a leucine FG 391 alpha and there is a second leucine f 786 alpha nearby all these are hydrophobic and helped to form a pocket for the hem on the far side there is a valine 1162 alpha which is not in Van der Waals contact with the heme but helps to shape the site where the oxygen is bound these are only some of the 16 residues which form the principal contacts between the globin and it's him except for the one strong covalent bond to the proximal histidine and the hydrogen bonds the heme is kept firm in its pocket by relatively weak nonpolar interactions with molecular oxygen bound to it the iron is very nearly in the plane of the porphyrin ring if the iron moves out of this plane towards the proximal histidine the iron oxygen bond is weakened the heme is held firm in its pocket and the iron is connected directly to the globin chain so any movement of the iron relative to its porphyrin ring is transmitted to other parts of the chain this combination of sections of alpha helix linked by less rigid non helical sections acts like a set of rods and hinges that transmits motion at the iron atom to parts of the surfaces of the subunit this transmission works both ways distortions of the same regions of the surface express themselves as tensions or shifts near the iron atom here we are showing the globin subunit making the transition between two of its tertiary confirmations deoxy as seen in the fully deoxygenated hemoglobin with the iron out of the plane of the porphyrin ring and oxy as seen in a fully oxygenated hemoglobin with the iron in the plane there is an equilibrium between these confirmations which can be influenced by constraints as well as by the presence of oxygen we have represented a constraint that favors the deoxy state by making the transition to that state much faster than the relaxation to the oxy form indicating a shift in equilibrium towards the deoxy form if the subunit can be constrained to hold the iron atom out of the plane of the heme even in the presence of oxygen the iron will bind oxygen less strongly because the most stable configuration with oxygen bound for the iron cannot now be so easily reached this ability of the confirmation of the globin to affect the oxygen affinity of the iron atom is the key to the way in which hemoglobin works and the mechanism of applying constraint lies in the manner in which the subunits are arranged in the whole molecule the two different globin chains alpha and beta are arranged so that the relative position of each iron atom to its porphyrin ring is in some way communicated to the other heme groups the precise mechanism of this cooperation is still under discussion but its principal features are clear the alpha subunit that we have built has a large hydrophobic area on its surface formed by the side chains of the residues on the h G and B hella C's which interacts strongly with a complementary hydrophobic region covering the b g and h hella C's on the surface of a second the beta subunit we are looking at the alpha 1 beta 1 contact from outside with the Alpha chain at the top-right and the beta chain bottom-left this contact is very strong and the residues that for the alpha 1 beta 1 contact do not move relative to each other as the hems change their states except to show that as the molecule goes from oxy to deoxy these H G and B helical structures stay in a fixed relationship to each other we shall not examine this contact in much more detail we must now look at the other side of this alpha beta dimer there are two further hydrophobic areas one on each chain on the surface coming round from the back this is really the inside surface of the molecule it can bind with complementary areas of a second identical dimer alpha to beta 2 symmetrically located with respect to the first we rotate the molecule to leave the original alpha alpha 1 top left and the new beta beta 2 bottom right this is the alpha 1 beta 2 region and you notice that this is more widely spaced than the alpha 1 beta 1 region the area of this contact is smaller and more mobile than the alpha 1 beta 1 contact all the same it contains 13 residues from the alpha chain that can interact with 12 from the beta chain one of the most important differences between this contact and the alpha 1 beta 1 contact is the large amount of relative motion between the 2 subunits during oxygenation and deoxygenation as the subunits move particular residues come into or out of bonding range so that in effect 2 different contacts form one for oxygenated hemoglobin and one for deoxygenated hemoglobin looking at the oxy contact in detail we concede that the carbonyl oxygen marked by the segments of the valine FG 593 alpha is close to the hydrogen sites on the side chain of the tryptophan C 337 beta note that the methyl group of the valine points towards the heme so that while the main chain of this residue is part of the alpha 1 beta 2 subunit contact the sidechain is an important heme contact another pair of residues that make close contact in the oxy form are the threonine C 338 alpha and the backbone of the valine FG 598 beta this contact is for the beta 2 heme what the previous one was for alpha 1 the valine backbones form notches into which the projecting sea side chains of the opposite chain may fit another very important contact is the hydrogen bond between the oxygen of the aspartic acid G 194 alpha and the nitrogen of the asparagine g4 on o2 beta in this way we could build up the contact until all 30 or more residues involved were included the importance of this alpha 1 beta to contact is that it changes as the hems release their oxygens this is easier to see if we take the residues one at a time here all the motions are referred to the alpha one heme group to show the relative motions more clearly the Alpha 1 and beta 2 subunits slide past each other and so form new bonds as the gap between the Alpha aspartic acid and the beta asparagine widens so the distance between the tyrosine C 742 alpha and the aspartic acid G 199 beta decreases the two residues on the beta chain pointing into the contact are three residues apart this interval of three is characteristic of the helix and residues 99 and 100 and 2 are adjacent residues on the same side the outside of this beta G helix this threonine C 641 alpha is again three residues away from the threonine C 338 alpha the valine near the beta heme moves between these two as the state changes these are just three of the many contacts in the alpha 1 beta 2 region that change when the state of oxygenation of the hems is changed there are some 12 significant contacts so far identified here you can see the arrangement of the four subunits to form the hemoglobin molecule this arrangement is called the quaternary structure of the molecule there are two possible quaternary confirmations available to the hemoglobin depending on which of the two types of alpha 1 beta 2 contact is formed when the alpha 1 beta 2 contact for deoxygenated hemoglobin is formed and the whole molecule is in its quaternary deoxy conformation certain residues on the outside of the molecule are brought close enough together to form ionic bonds these bonds are called salt bridges the c-terminal arginine 141 alpha of each alpha chain forms two salt bridges with the other alpha chain similarly the c-terminal histidine of each beta chain forms a salt bridge across the alpha 1 beta 2 subunit contact with lysine c 540 alpha it also forms an intra subunit salt bridge with aspartic acid FG 194 beta of the same beta chain these salt bridges stabilized a quaternary deoxy structure and they together with the deoxy alpha 1 beta 2 subunit contact tighten up the tertiary conformation of the individual subunits making it more difficult for the iron atom to move on oxygen binding this ensures a lower oxygen affinity for the subunits than they would have if they were free the bonds between the dimers of the quaternary structure constrain each subunit to retain the conformation appropriate to the structure of the whole molecule for example if one heme picks up an oxygen molecule with the hemoglobin in the quaternary deoxy state that entire subunit is held within a quaternary deoxy structure although its tertiary structure moves towards it's free oxy form such an oxygenated subunit in a mainly deoxy tetramer would have an intermediate tertiary structure mismatch between tertiary and quaternary structure causes strain at some point in the oxygenation of the subunits the strains on the quaternary structure are so great that the whole molecule changes to the quaternary oxy conformation because this becomes the more stable of the two here we've shown the molecule changing to its quaternary oxy structure as the second heme binds an oxygen molecule the constraints on the hems previously exerted by the subunits interfaces and the salt bridges no longer exist these subunits therefore have a higher oxygen affinity than they did in the quaternary deoxy conformation and therefore very rapidly become oxygenated the structure of intermediates is not known so we have represented oxy and deoxy subunits as they appear in fully oxy or fully deoxy tetramers that consequence of these strained intermediates is dramatically to reduce the range of oxygen pressures over which the hemoglobin is partially loaded with oxygen this is expressed in the characteristically sigmoid shape of the hemoglobin oxygen equilibrium curve the physiological consequence of this is the increasing ease with which hemoglobin delivers its oxygen to the tissues as the partial pressure of oxygen Falls