The human haemoglobin molecule

Evolution
Human hemoglobin belongs to an ancient family of molecules, the ancestor of which emerged some 3000 Mya as a part of the protein equipment present in the last universal common ancestor (LUCA). Thus, molecules belonging to the hemoglobin family are described in unicellular organisms, plants, insects and all animals. The main function of vertebrate hemoglobins is to bind oxygen reversibly for transport and storage. In other species, hemoglobin may play a role in the protection against oxidative stress. ,

Structure of the globin molecule
In vertebrates, hemoglobin is contained in the red blood cells (RBC), where, in man, its concentration is of about 5mM. It is a heterotetramer made by two types of chains (Figure 1). The tertiary structure of each of these chains is characterized by the “myoglobin fold”, which has been described fifty years ago: it is made by a heme group surrounded by 8 helices designed A through H from the N to the C terminal, ,. Helices A, B, C and E are on one side of the heme, named the distal side, and helices F, G and H on the other side, named proximal side. In each helix and inter-helical region, the residues are labeled by their position: as an example the distal and proximal His which are involved in heme binding are respectively at position E7 and F8 (Figure 2). To this structure, a pattern of 37 hydrophobic residues located at conserved, solvent-inaccessible positions has to be added. The 3D nomenclature is useful when comparing chains from different hemoglobins or different species.

Within the tetramer, two chains belong to the α type and two to the β type. In man, α type chains contain 141 residues and are encoded by genes located on chromosome 16 ; β type chains contain 146 residues and are encoded by genes located on chromosome 11. Since the N-terminal methionine is enzymatically removed early in the globin biosynthesis of each of these chains, the classical use is to label as 1 the residue which is at the N-terminal position of the mature protein (and not as “p.2” according to the genetic nomenclature). As a result HbS is β6 Glu>Val (and not β7). Within the tetramer the globin chains are associated into two dimers named α1β1 and α2β2 in which the subunits are in tight contacts.

Both types of chains are independently synthesized but with a slight excess of α chains. Heme binds to the globin chain during its synthesis helping to the correct folding. As soon as synthesized, the free α hemoglobin (α Hb), which are very unstable molecules, are protected by forming a stable complex with a chaperon protein named the alpha hemoglobin stabilizing protein (AHSP). The binding sites in the AHSP/ α Hb complex are located in the G and H helix regions of α Hb explaining why many structural alterations of this part of the molecule are cause of instability (Figure 3). The complex AHSP/ α Hb brings the α subunits in contact with the β one and helps to formation of the α1β1 type dimer. This is explained by a affinity of the β chains higher for α Hb than that of AHSP, leading to a transfer of α Hb with formation of a strong contact between the two type of chains, involving again G and H helix. ,

During ontogenic evolution, several hemoglobins succeed one to another. ,, Three Hbs are specific of the embryonic life: Hb Gower 1 (ζ2ε2), Hb Gower 2 (α2ε2) and Portland 1 (ζ2γ2). During fetal live, HbF is the major Hb component. It is made by α and γ chains. The biosynthesis of the α chains is under the control of two genes α1 and α2 (HBA1 and HBA2) whose products are identical. The biosynthesis of γ chains is also under the control of two genes, Gγ and Aγ (HBG1 and HBG2), but this results into two types of γ chains which differ by the residue at position 136, respectively glycine and alanine. Their relative proportion depends upon the fetal age and the action of some regulatory factors. Normally, HbF disappears progressively after birth and is only present in trace amounts after the age of 6 months. During adult life, HbA is the major Hb component. It is made by two α and two β chains. HbA2 is the minor adult component, which is made by α and δ chains, it normally amounts to ca. 3%. It is important to note that, with the exception of embryonic hemoglobins, the α chain is present in all the other hemoglobins explaining why an α chain abnormality may affect the different hemoglobins.

In addition, in man, other molecules are present which belong to the globin family. They are monomers with a general 3D structure close to that of the globin chains and displaying different amino acid sequences with only identical residues at key positions. The most abundant of those is myoglobin which stores oxygen in the muscle. The other molecules of this family (neuroglobin, cytoglobin and androglobin) are described in some specific tissues where they may play a protective function.

Oxygen binding to the hemoglobin molecule
Oxygen binds reversibly to the ferrous iron of the heme group, on the distal side between, the iron, His E7 and Val E11. It is known since the beginning of last century that oxygen binding to the heterotetramer hemoglobin molecule is described by a sigmoidal curve. , This is explained by the fact that hemoglobin molecule displays two quaternary states with different oxygen binding properties. The first conformation, which corresponds to oxyhemoglobin, is a high oxygen affinity form. This form is a relaxed structure (named R state) in which the contact between the two dimers within the tetramer (named α1β2 contact) is quite loose. In contrast, the second conformation, the deoxygenated one, named the tense form (or T state) displays a low oxygen affinity. In the T structure, the α1β2 contact is tight. The transition from the T to the R state starts with oxygen binding to the heme ferrous iron and triggers a series of structural changes leading to the rupture of the salt bonds that stabilize the T conformation and to a rotation of the α-chains relative to the β-chains. This transition involves a free energy change, reflected as cooperativity between subunits, which is measured by the value of the Hill coefficient (n), normally around a value of 2.7. Deoxyhemoglobin has a high affinity for allosteric effectors such as 2,3-diphosphoglycerate (2,3-DPG) and Bohr protons, both stabilizing the T structure. ,, , , The molecule of 2,3-DPG binds between the two β chains in deoxyhemoglobin, and is ejected when oxygen is fixed. Protons bind to several residues to a greater extent in deoxyhemoglobin than in oxyhemoglobin, this effect is known as alkaline Bohr effect. The allosteric model of Monod, Wyman and Changeux is one of the more adapted descriptions of the oxygen binding mechanism.

In the lungs the hemoglobin is almost completely under its high oxygen affinity form (oxyhemoglobin), while in the peripheral tissues 30-40% of the total hemoglobin is in the low affinity form (deoxyhemoglobin).

Oxygen affinity of hemoglobin is measured by the p50 parameter corresponding to the partial pressure at which the hemoglobin is half-saturated in oxygen (Figure 4). In the blood, at pH 7.4 and 37°C, the p50 amounts to about 27 ± 2 mm Hg. The oxygen dissociation curve of a normal blood shows that, under these conditions, when the pO2 drops from arterial oxygen pressure (ca. 100 mmHg) to capillary venous oxygen pressure (ca. 40 mmHg), 4 to 5 ml oxygen are delivered to the tissues by 100 ml blood. The amount of oxygen delivered may be insufficient in case of anemia but also in case of a too high oxygen affinity of the blood, which hinders correct oxygen delivery. This may be the consequence of an abnormality of the hemoglobin molecule but also from a defect in the 2,3-DPG mediated regulatory mechanism. Another mechanism for increasing oxygen affinity results from binding a ligand other than oxygen (CO or water) to the heme in one hemoglobin subunit. This pushes the hemoglobin tetramer towards the R-state.