The structure and metabolism of haem

Haemoglobin is interesting from many points of view and it has probably been studied more extensively than any other protein. Its shape and general characteristics and its relationship to myoglobin have already been described (page 65) but little has been said about the haem group. Myoglobin, which consists of a single polypeptide chain, has one haem group while haemoglobin, which has four globin subunits, correspondingly has four haem groups, each of which can combine with one molecule of oxygen. Globin itself is colourless and contains a greater proportion of basic amino acids than any of the other blood proteins. Although the haemoglobins of all mammals are similar in shape and structure, they show appreciable differences in their amino acid composition, affinity for O₂ and other gases, and in their crystalline form. All these differences are attributable to the globin since the haem group is the same in all species. Haem is an iron–porphyrin compound.

The porphyrin ring is a planar structure formed from four pyrrole rings which are joined into a larger ring by me thine (–CH=) bridges. Four methyl (M), two vinyl (V) and two propionate (P) groups are attached to the ring. The highly polar propionate side chains of the haem are situated on the surface of the molecule and are ionized at physiological pH values. The centrally

+H3N−Tyr−Gly−Gly−Phe−Met−COO−+H3N−Tyr−Gly−Gly−Phe−Leu−COO−

placed iron atom is bound to the four N atoms of the pyrrole rings leaving two of the six coordination valencies on the iron atom free. In both myoglobin and haemoglobin the iron atom of the haem is directly bonded to a histidine residue. This is known as the proximal histidine. Oxygen binding occurs at the sixth coordination position near which the second important or distal histidine is situated. In deoxyhaemoglobin this position is empty.

+H3N−Tyr−Gly−Gly−Phe−Met−COO−+H3N−Tyr−Gly−Gly−Phe−Leu−COO−

Synthesis of haem is readily accomplished from simple precursors. In the first step of porphyrin synthesis succinyl-CoA, which is produced in the citrate cycle and during the metabolism of various amino acids, is condensed with glycine to give δ-aminolaevulinic acid, and it is this reaction which is rate-controlling. Subsequently two molecules of δ-aminolaevulinic acid condense to form porphobilinogen and four molecules of porphobilinogen undergo a series of reactions to produce protoporphyrin which combines with ferrous ions to form haem.

+H3N−Tyr−Gly−Gly−Phe−Met−COO−+H3N−Tyr−Gly−Gly−Phe−Leu−COO−

The functions of haemoglobin and myoglobin

In the haemoglobin molecule, although the four haem groups are separated by appreciable distances, the state of one haem is influenced by that of the other three and these haem–haem interactions are of considerable physiological importance. This can be judged by comparing the properties of haemoglobin and myoglobin. As can be seen from Figure 25.1, the O₂ dissociation curve of myoglobin is a simple rectangular hyperbola while that of haemoglobin is sigmoid. The two pigments are well suited for their respective physiological roles. Thus myoglobin acts as a store of O₂ in muscles that may be deprived of O₂ for relatively long periods. For this purpose it is important that it should take up O₂ when it is readily available and release it only when it cannot be obtained from the atmosphere. From the curve it can be seen that at an O₂ pressure of 20 mmHg myoglobin is 80% saturated with O₂ and only releases significant amounts at O₂ pressures below this. Haemoglobin on the other hand, although it also has storage capacity for O₂, is essentially a carrier and is required alternately to pick up and release O₂ with changes of O₂ pressure operating at a higher range.

The difference in properties between the two proteins, whose tertiary structures are very similar (page 67), is due to the superimposed quaternary structure of haemoglobin and the fact that the ease with which any haem group binds O₂ is determined by the state of the other three. Starting with deoxyhaemoglobin the first O₂ molecule is taken up very slowly, the second and third are taken up more and more readily and the fourth is taken up several hundred times more rapidly than the first; hence the sigmoid shape of the curve.

Changes in haemoglobin in response to the environment

When deoxyhaemoglobin is converted into oxyhaemoglobin or vice versa subtle changes occur in the tertiary structure of the subunits which cause relatively large changes in the quaternary structure.

In deoxyhaemoglobin the iron atom lies about 0·6 Å (0·06 nm) out of the plane of the ring but, on oxygenation, it moves into the plane of the ring and is able to form a strong bond with oxygen (Figure 25.2). This tiny structural change within the subunits is translated into a shift in the relationship between them. One pair of αβ subunits rotates relatively to the other pair causing the β chains to move closer together and the α chains to move slightly away from one another. This change in the quaternary structure on oxygenation and deoxygenation may be regarded as a ‘breathing movement’ at the molecular level.

Not only does haemoglobin have multiple binding sites for oxygen which, as described above, show positive cooperativity, it also combines at different sites with CO₂ and H⁺ ions and also with the substance 2,3-diphosphoglycerate, and a complex relationship exists between the binding of these various ligands. This is physiologically highly advantageous and has the result that when oxygenated blood from the lungs reaches the tissues where H⁺ and CO₂ are being produced, the combined effects of the lower O₂ tension and higher H⁺ and CO₂ concentrations ensure that O₂ is released in appropriate amounts while, at the same time, CO₂ is taken up. The promotion of O₂ dissociation by an increase in CO₂ tension and therefore of acidity is known as the Bohr Effect and is illustrated in Figure 25.1.

The role of haemoglobin in carbon dioxide transport

As already stated when oxygenated blood from the lungs reaches the tissues where acid metabolites are being produced, oxyhaemoglobin tends to lose O₂ and the combined effects of the lower O₂ and higher CO₂ tensions ensure that O₂ is released in appropriate amounts.

Haemoglobin plays a part in the carriage of CO₂ from the tissues to the lungs in two ways:

1. It combines directly with the CO₂ which reacts with free amino groups to form carbamino compounds.

R−NH2+CO2⇌R−NH−COOHCarbamino compound

    This reaction is not peculiar to haemoglobin and all proteins with free amino groups will react in this way. The important point about haemoglobin is that the degree to which the reaction takes place depends almost entirely on its state of oxygenation and hardly at all on the CO₂ tension. Thus deoxyhaemoglobin readily forms carbamino compounds but when it is converted to oxyhaemoglobin most of the CO₂ is released. Although only about 5% of the total CO₂ in venous blood is present in the form of carbaminohaemoglobin it represents a very labile form of CO₂ and may account for as much as 30% of the CO₂ that is taken up in the tissues and released in the lungs.

2. It has a buffering effect. When haemoglobin, which is notably rich in histidine, is oxygenated the resulting conformational changes increase the tendency for specific protonated histidine residues to lose H⁺, i.e. their pK value in oxyhaemoglobin is 7·16 compared with 7·3 in deoxyhaemoglobin. Thus, in the lungs, the proportion of HbO₂ is increased and that of HHb is reduced. Conversely, in the tissues, deoxyhaemoglobin binds H⁺; at the same time the plasma bicarbonate is increased (see the next paragraph). In this way a large proportion of the acid produced in the course of the tissue metabolism is carried as H⁺ by haemoglobin in the corpuscles and as bicarbonate in the plasma.

Formation of carbonic acid from H₂O and CO₂ is a relatively slow process, but within the corpuscles it is speeded up by the presence of the enzyme carbonic anhydrase. As a result the concentration of bicarbonate ions rises more rapidly in the corpuscles than in the plasma and some of them leave the corpuscles in exchange for chloride ions. This chloride-bicarbonate exchange ensures that a large fraction of the acidic CO₂ produced in the tissues is carried as plasma bicarbonate. Consequently there is very little increase in the acidity of venous blood.

It can be calculated that, if the respiratory quotient is 0·7 (i.e. 1 volume of O₂ is utilized for every 0·7 volume of CO₂ produced), all the H⁺ resulting from the conversion of can be buffered by haemoglobin without any alteration in pH. However, since the respiratory quotient is usually greater than 0·7, other blood buffer systems, notably the Na₂HPO₄/NaH₂PO₄ system, also operate to keep the pH change within narrow limits.

The overall process of CO₂ uptake and O₂ release can be represented as follows:

R−NH2+CO2⇌R−NH−COOHCarbamino compound

The role of the plasma bicarbonate in acid–base regulation

In addition to the 13 mol or so of CO₂ produced each day most of which is buffered by haemoglobin, small amounts of certain non-volatile acids are produced. These usually account for rather less than 0·1 mol of H⁺ per day and, since they cannot be eliminated through the lungs, they must be excreted via the kidneys. The non-volatile acids include sulphuric acid, formed by oxidation of the sulphur present in cysteine and methionine, phosphoric acid from phospholipids and phosphoproteins, and lactic acid produced during severe exercise. Appreciable amounts of acetoacetic acid and β-hydroxybutyric acid are released into the blood in ketosis. All these acids are buffered by the plasma bicarbonate according to the following reaction:

HA+NaHCO3→NaA+H2CO3↓H2O+CO2→Lungs

The carbonic acid which is produced causes a fall in the NaHCO₃/H₂CO₃ ratio and a slight drop in the pH of the blood. The overall effect is to stimulate the respiratory centre of the brain causing an increase in pulmonary ventilation so that the extra CO₂ is lost from the lungs and the normal NaHCO₃/H₂CO₃ ratio is restored. However, in the course of the reaction some of the plasma bicarbonate has been used up so that, although the NaHCO₃/H₂CO₃ ratio and pH are normal, the plasma bicarbonate is reduced. The responsibility for regulating the absolute amount of bicarbonate in the plasma belongs to the kidney (page 395).

Haemoglobin derivatives

A large number of haemoglobin derivatives are known, some of which occur naturally. The following are among the most important.

Plasma proteins

Normal human plasma contains a very complex mixture of proteins amounting to about 7·0–7·5 g/100 ml. Structural and physicochemical data are available on 22 distinct human plasma proteins. A host of others are known to be present in low concentrations and to have important biological functions, but they have not yet been isolated.

For clinical investigations of the plasma proteins serum is commonly used instead of plasma since fibrinogen and fibrin tend to interfere with some of the tests. The serum proteins are usually separated on a variety of materials when five distinct fractions are produced. These are albumin and the α₁, α₂, β and γ globulin fractions. Typical electrophoretic patterns for normal serum are shown in Figure 25.3 and the percentage composition of the total plasma proteins in Table 25.1.