Hemoglobin

Lisa Natzke, '98

References:

 Liddington, R. et al. 1992. High resolution crystal structures and comparisons of T state deoxyhemoglobin and two liganded T-state hemoglobins. J. Mol. Biol. 228: 551.

 Perutz, M. F. 1970. Stereoschemistry of cooperative effects in haemoglobin. Nature. 228: 726-734.

 Shaanan, B. 1982. The iron-oxygen bond in human oxyhaemoglobin. Nature. 296: 683.

 Shaanan, B. 1983. Structure of human oxyhaemoglobin at 2.1 resolution. J. Mol. Biol. 171: 31.

I. Introduction

     Approximately one third of the mass of a mammalian red blood cell is hemoglobin. Its major function is to carry oxygen from the lungs through the arteries to the tissues and help to carry carbon dioxide through the veins back to the lungs. The process whereby hemoglobin performs this essential physiological role is characterized by a cooperative interaction among its constituent subunits. Hemoglobin has thus assumed the role of a model system whose study acquires ramifications extending far beyond its own function as an oxygen transport system.
 
 

II. Protein Structure

     The hemoglobin molecule is made up of four polypeptide chains: two alpha chains < >of 141 amino acid residues each and two beta chains < > of 146 amino acid residues each. The alpha and beta chains have different sequences of amino acids, but fold up to form similar three-dimensional structures. The four chains are held together by noncovalent interactions. There are four binding sites for oxygen on the hemoglobin molecule, because each chain contains one heme group < >. In the alpha chain, the 87th residue is histidine F8 < >and in the beta chain the 92nd residue is histidine F8 >. A heme group is attached to each of the four histidines. The heme consists of an organic part and an iron atom < >. The iron atom in heme binds to the four nitrogens in the center of the protoporphyrin ring. The hemoglobin molecule is nearly spherical, with a diameter of 55 angstroms . The four chains are packed together to form a tetramer. The heme groups are located in crevices near the exterior of the molecule, one in each subunit. Each alpha chain is in contact with both beta chains< >. However, there are few interactions between the two alpha chains or between the two beta chains >.

      Each polypeptide chain is made up of eight or nine alpha-helical segments < >and an equal number of nonhelical ones placed at the corners between them and at the ends of the chain. The helices are named A-H, starting from the amino acid terminus, and the nonhelical segments that lie between the helices are named AB, BC, CD, etc. The nonhelical segments at the ends of the chain are called NA at the amino terminus and HC at the carboxyl terminus.

      To form the tetramer < >, each of the subunits is joined to its partner around a twofold symmetry axis, so that a rotation of 180 degrees brings one subunit into congruence with its partner. One pair of chains is then inverted and placed on top of the other pair so that the four chains lie at the corners of a tetrahedron. The four subunits are held together mainly by nonpolar interactions and hydrogen bonds. There are no covalent bonds between subunits. The twofold symmetry axis that relates the pairs of alpha and beta chains runs through a water-filled cavity >at the center of the molecule. This cavity widens upon transition form the R structure to the T structure to form a receptor site for the allosteric effector DPG (2,3 diphosphoglycerate) between the two beta chains. The heme group is wedged into a pocket of the globin with its hydrocarbon side chains interior and its polar propionate side chains exterior.

      There are nine positions in the amino acid sequence that contain the same amino acid in all or nearly all species studied thus far. These conserved positions are especially important for the function of the hemoglobin molecule. Several of them, such as histidines F8 (His87)< > and E7 (His63)< >, are directly involved in the oxygen-binding site< > . Phenylalanine CD1 (Phe43) < > and leucine F4 (Leu83) < > are also in direct contact with the heme group< >. Tyrosine HC2 (Tyr140) < >stabilizes the molecule by forming a hydrogen bond between the H< > and F helices< >. Glycine B6 (Gly25)< >is conserved because of its small size: a side chain larger than a hydrogen atom would not allow theB< > and E helices< > to approach each other as closely as they do. Proline C2 (Pro37)< > is important because it terminates the C helix. Threonine C4 (Thr39) and lysine H10 (Lys127) are also conserved residues, but their roles are uncertain.
 
 

III. Transition from the T Structure to the R Structure

     There are two kinds of contact regions between the alpha and beta chains: the alpha1beta1 and the alpha1beta2 contacts. Upon transmission from the deoxy (T) structure to the oxy (R) structure, the alpha1beta2 dimer rotates relative to the other by 15 degrees. Some atoms at this interface shift by as much as 6 angstroms . The alpha1beta2 contact region is designed to act as a switch between two alternative structures. The T structure is constrained by additional bonds between the subunits, which oppose the changes in tertiary structure needed to flatten the hemes upon combination with oxygen. These bonds take the form of salt bridges.

      Transition from the T structure< > to the R structure< > is triggered by stereochemical changes at the hemes. In deoxyhemoglobin, the iron atom is about 0.6 angstroms out of the heme plane because of steric repulsion between the proximal histidine and the nitrogen atoms of the porphyrin. The heme group and proximal histidine make intimate contact with some fifteen side chains and so the structures of the F helix, the EF corner, and the FG corner change on oxygenation. These changes are then transmitted to the subunit interfaces. The expulsion of the tyrosine HC2 from the pocket between the F and H helices leads to the rupture of interchain salt bridges. Consequently, the equilibrium between the two quaternary structures is shifted to the R form on oxygenation.
 
 

IV. Cooperative Binding of Oxygen

     The binding of oxygen to the heme group of one subunit has the effect of increasing the affinity of a neighboring subunit (on the same molecule) for oxygen< >. Deoxyhemoglobin is a taut moleucule, contrained by its eight salt links between the four subunits. Oxygenation cannot occur unless some of these salt links are broken so that the iron atom can move into the plane of the heme group. The number of salt links that need to be broken for the binding of an oxygen molecule depends on whether it is the first, second, third, or fourth to be bound. More salt links must be broken to permit the entry of the first oxygen molecule than of subsequent ones. Because energy is required to break salt links, the binding of the first oxygen molecule is energetically less favorable than that of subsequent oxygen molecules.

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