Hemoglobins: Structure and Properties | Biochemistry

In this article we will discuss about the structure and properties of hemoglobins. 

Structure of Hemoglobins:

As indicated by their name, hemoglobins consist of a prosthetic group; the heme (4%) and a protein part: the globin (96%).

A. Heme:

This is the prosthetic group common to various hemoglobins (while globin varies in different hemoglobins). It contains one molecule of protoporphyrin and one iron atom.

a) The Protoporphyrin:

Four pyrrole rings, linked by methenyl bridges = CH — between their α and α’ carbon atoms form the porphin (see fig. 1-27).

We must note the alternation of the double bonds which are all conjugated.

Porphyrins are simply derivatives of porphin where the 8 β and β’ carbon atoms are carriers of different substituents. It is clear that there are pos­sibilities of isomerism; we will study these, taking the example of uroporphyrins where the 8β and β’ carbon atoms carry 4 acetic radicals (A) and 4 propionic radicals (P); figure 1-28 shows that with 2 different substituents there are 4 possible arrangements; among natural pigments, mostly type III and some­times type I, are found.

Protoporphyrin of hemoglobins is of type III; as maybe seen in figure 1-29, it derives from uroporphyrin III by decarboxylation of the 4 acetic radicals (into methyl groups) and decarboxylation and then dehydrogenation of two of the four propionic radicals (which are thus transformed into vinyl groups).

This is denoted protoporphyrin IX by certain authors, because with 3 dif­ferent substituents there are actually 15 possible isomers (and not 4) and the comparison with different porphyrins obtained by synthesis has shown the identity of natural protoporphyrin with the type IX of synthetic isomers. Protoporphyrin therefore contains 4 methyl groups in 1, 3, 5 and 8; 2 propionic radicals in 6 and 7; and 2 vinyl groups in 2 and 4.

b) Linkage of Iron and Protoporphyrin:

Porphyrins have the property of binding, by their pyrrole nitrogen atoms, metals like Fe, Mn, Ni, Co, Mg; metalloporphyrins containing ferrous iron (Fe⁺⁺) are called hemes; in the particular case of protoporphyrin, the ferroporphyrin is called protoheme (the term “heme” is used by abbreviation). Metalloporphyrins containing ferric iron (Fe^{+ ++}) are called hematins (the one corresponding to protoporphyrin is protohematin).

In the protoheme, the iron atom replaces the 2 hydrogen atoms carried by two of the four nitrogen atoms but it is linked with the 4 nitrogen atoms by coordination. Besides, Fe⁺⁺ can still exchange 2 coordination linkages with nitrogen bases for example, forming a hexacoordinated complex (as in ferrocyanide). These two linkages are perpendicular to the plane of the heme.

c) Important Properties of the Heme:

We have seen that the heme is a ferrous compound; under the action of oxidants (like alkaline ferricyanides, for example), it can be oxidised into hematin (where iron is ferric) which can be again reduced into heme (by sodium hydrosulphite, for example).

This reversible reaction forms the basis of the participation of ferropor- phyrins, like the cytochromes, in the oxidation — reduction processes (but it does not take place during the conversion hemoglobin ⇋ oxyhemoglobin).

As will be seen below, the heme can link with globin. It can also link with other nitrogen bases (pyridine, nicotins) or with the nitrogen groups of proteins, to form homochromogens.

Similarly, hematin can link with different proteins to form parahematins, often having catalase or peroxidase properties.

B. Globin:

This is the protein part of the chromoprotein; it is easily obtained by treating hemoglobin with acetone containing 5% HCl (globin precipitates). Globin is the specific part of hemoglobin which varies according to age, species, and in certain diseases.

In myoglobins there is a single polypeptide chain (combined with one heme) whose primary, secondary and tertiary structure is known, at least in certain species (see figure 1-19). Molecular weight is about 17 000. Histidine content is comparatively high (6 to 10%).

Blood hemoglobins are tetramers; they contain 4 polypeptide chains (each combined with one heme) and their molecular weight is about 68 000.

The 4 chains are united by linkages which are easy to break; in the case of adult human hemoglobin (HbA), 2 types of chains (α and β) are obtained; these are combined in pairs in the original molecule; this is expressed by writing HbA = α₂^A β₂^A (A means “Adult”). The α chain contains 141 amino acids and the β chain 146; the sequences are fully known.

The foetal hemoglobin also contains 4 chains, 2 α chains and 2 γ chains (the latter have about ten amino acids different from those present in β chains). We can write HbF = α₂^A γ₂^F.

The pathological hemoglobins differ from HbA, either by anomalies in the distribution of chains (there are hemoglobins having 4 β chains or 4 γ chains), or by anomalies in the sequence of the α chain or β chain (for example, in sickle-cell anaemia, glutamic acid normally present in position 6 of the β chain is replaced by a valine, and just this difference is sufficient to alter the physiological properties of hemoglobin).

C. Union of Heme and Globin:

In the myoglobin of the sperm whale, which was thoroughly studied from the structural point of view, the heme is lodged in a fold of the tertiary structure of globin (see fig. 1-19).

In all these ferroporphyrin — protein complexes, the iron atom has an oc­tahedral structure of chemical type d²sp³ (double pyramid with square base formed by the 4 pyrrolic nitrogen atoms).

The 5th coordination position of the iron is occupied by the imidazole nitrogen N₃ of the histidyl residue in position 93 (F8 or 8th residue of the helix F in the Perutz nomenclature). The 6th coordination position is free in the non-oxygenated or Deoxy state, but it is occupied by oxygen in the oxygenated or Oxy state (or by the carbon of carbon monoxide, competitive inhibitor of oxygen).

In the case of the α or β chains of hemoglobin, we have exactly the same Deoxy and Oxy structures (fig. 1-30): iron is linked with the nitrogen N₃ of histidine F8 called proximal (position 87 for α and 92 for β).

If the iron atom is oxidized to the ferric state, its 6th coordination position cannot any more be occupied by an oxygen molecule, it is occupied by the oxygen atom of a water molecule, one proton of which is attached by hydrogen bond to the nitrogen N₃ of another histidine called distal and which occupies the position E7 in the Perutz nomenclature (7th residue of the helix E, respec­tive positions 64, 63 and 58 in myoglobin, chain β and chain α).

This form called metmyoglobin or methemoglobin is a stable Deoxy state in which hexacoordinated ferric iron can no longer play the role of oxygen fixing agent, contrary to the previous case.

It is observed that in the Deoxy state, the external electron layer of the ferrous iron atom contains 16 electrons of which 2 are unpaired. The ferroporphyrin-protein complex is then paramagnetic. In the Oxy state, this external layer is saturated with 18 electrons all paired, and the complex is therefore diamagnetic.

In the metmyoglobin or methemoglobin form, the external layer has 17 electrons of which 1 is unpaired, and the complex is paramagnetic. This form can be stabilized by a CN^– cyanide ion which brings 3 electrons to the ferric ion, the external layer of which is saturated with 18 electrons. We then obtain a diamagnetic stable Oxy form where oxygen cannot displace the CN^– ion.

Beside this primary linkage between Fe and N³ of the proximal histidine, two saline linkages (between 2 propionyl radicals of heme and 2 basic groups of lysine or arginine) and Van der Waals type linkages (between hydrophobic groups of heme and globin) maintain the stability of the structure.

It must be noted that if we have a non-denatured globin, it is possible to effect in vitro the combination heme + globin; we obtain a hemoglobin having the properties of the chromoprotein which was used for the isolation of globin (whatever the origin of the heme). Inversely, the combination of a heme with diverse globins produces chromoproteins which differ according to the origin of the globin used.

Properties of Hemoglobins:

When we centrifuge fresh blood or blood made incoagulable, the red cells sediment. Bringing them back in suspension in a hypotonic medium (for example distilled water) is sufficient to make them burst (hemolysis) and, after elimination of the cellular debris by centrifugation, we obtain a red coloured solution.

The solution is dark red for hemoglobin and bright red for oxyhemoglobin (due to differences in the absorption spectra); there is there­fore an oxidized form of hemoglobin whose physiological role — as mentioned in the introduction – is to transport oxygen after forming with this gas an easily dissociable combination. We will now examine the most important property of hemoglobins namely their capacity to reversibly bind certain gases particularly oxygen.

A. Combinations of Hemoglobins with Gases:

a) Combination with Oxygen:

For all hemoglobins, the maximum quan­tity of oxygen which can be bound is a function of the quantity of iron: one atom-gram of iron combines with one molecule-gram of oxygen (or one molecule gram of carbon monoxide).

Since there are 4 hemes (i.e. 4 iron atoms) per molecule of hemoglobin, we can write:

(but very often, oxyhemoglobin is represented by HbO₂). This is an oxygenation and not an oxidation because iron remains in the ferrous state, 17 000 g Hb (corresponding to the monomer which contains 1 iron atom) can therefore combine with 32 g of O₂ and since 1 mole of oxygen occupies 22.4 1, it may be derived that 1 g Hb can bind 1.34 ml O₂. But, 100 ml of blood contains in average 15gHb, so that it can bind 1.34 x 15, i.e. about 20ml O₂; this represents complete saturation in O₂.

The equation of oxygenation reaction shows that oxygen pressure is an important factor controlling this equilibrium. In the air that we breathe where mean pressure is 760 mm Hg and oxygen concentration about 20%, the partial pressure of oxygen is 760/5, i.e. about 150 mm.

In arterial blood, oxygen pressure is about 80 mm, so that (see fig. 1-31) 19 ml of O₂ can be bound for 100 ml blood (95% saturation). In the capillaries, partial pressure of oxygen falls to 40 mm or even 20 mm, therefore O₂ fixation can be only 77% and 40% of saturation respectively.

Figure 1-31 shows that this stage corresponds to the portion of the curve where a comparatively small decrease of oxygen pressure causes a comparatively major liberation of oxygen; when pressure falls from 40 to 20 mm, the quantity of oxygen which can be present in blood falls from 20 x 77/100 i.e. 15.4 ml O₂/100 ml to 20 X 40/100 i.e. 8 ml O₂/100 ml; in other words there is liberation of 7.4 ml O₂/100 ml of blood in favour of the tissues.

It is seen in figure 1-31 that the saturation curve of myoglobin is a branch of hyperbola while that of hemoglobin is a sigmoid (S-shaped). This difference is due to the fact that hemoglobin is a tetramer whose 4 hemes are not in­dependent, because the oxygenation of one of them favours that of others (which thus requires less energy).

This interaction does not take place directly because the hemes are too distant from one another (about 30 Å); it takes place through the quaternary structure of hemoglobin; to obtain this cooperative effect between hemes, the quaternary structure must be intact, and a pathological hemoglobin (by anomaly of the distribution of chains) or a denatured hemoglobin behaves like the myoglobin; furthermore, X-ray studies have actually shown – during the transformation [Hb]₄ → [HbO₂]₄ or inversely — a change of the conformation of the molecule, called allosteric transition, which takes place only with a hemoglobin whose quaternary structure is native.

This is an example of relationship be­tween quaternary structure and biological activity; we will come across other such cases while studying enzymes and we will see that the presence of al­losteric enzymes ensures a regulation of cellular metabolism.

Lastly, it should be noted that oxyhemoglobin, because of changes of confor­mation, has a larger number of dissociated acid groups.

Therefore, H⁺ ion concentration also has an effect on the equilibrium which may be written:

In the tissues, there is production of CO₂ which will lead to an increase of H⁺ concentration and favour liberation of oxygen. In the capillaries, this liberation is therefore caused, mostly by the decrease of O₂ pressure, but also by the increase of CO₂ pressure-, figure 1-31 shows that for a given O₂ pressure, the quantity of oxygen which may be combined is smaller when CO₂ pressure is higher (curve no. 3).

On the contrary, in the lungs, the O₂ pressure is high and CO₂ pressure falls (because CO₂ is eliminated) and this decreases acidity; these two factors — particularly, the former — favour the formation of oxyhemoglobin.

b) Combination with Carbon Monoxide:

This is very similar to the combination with oxygen; there is binding of one molecule-gram CO per atom-gram iron, but the stability of the combination (Hb-CO)₄ called carbonyl- hemoglobin (or carboxyhemoglobin) is about 200 times greater; the dissocia­tion is very difficult; this explains the toxicity of the gas, and the fact that high concentrations of oxygen (oxygenotherapy) are required in the treatment of intoxication by carbon monoxide.

c) Combination with Other Gases:

Carbon dioxide can combine with hemoglobin to give carbhemoglobin, but the heme plays no part, because CO₂ binds to the basic groups of globin; it also binds to those of other proteins (but it should be remembered that hemoglobin represents about 75% of the total blood proteins).

About one fourth of the CO₂ carried by blood travels in the form of carbaminoproteins:

It should be of interest to examine here, how, in general, CO₂ is transported from the tissues where it is formed to the lungs where it is eliminated. This transport must prevent, on the one hand, a progressive acidification of blood as it flows through the tissues liberating CO₂, and on the other hand, the formation of CO₂ bubbles, because the solubility of this gas is comparatively limited.

These difficulties are overcome by the buffer capacity of blood proteins which can bind H⁺ ions as mentioned earlier and so, more H₂CO₃ can be ionized and therefore more CO₂ can be dissolved (see fig. 1-32). On the contrary, when blood reaches the lungs, CO₂ is liberated and the reactions of figure 1-32 take place in the opposite direction; the proteins are again ready to bind H⁺ ions.

It must be noted that the system H₂ CO₃ ↔ HCO₃^– is a buffer system and that it is largely responsible (together with the system H₂PO₄^– ↔ HPO₄⁼) for protecting blood from excesses of acid or base.

B. Reversible Oxidation of Hemoglobin and Oxyhemoglobin:

Hemoglobin treated by a mild oxidising agent like potassium ferricyanide is oxidized into methemoglobin. Iron changes from Fe⁺⁺ state to Fe^{+ + +} state, so that the heme is oxidized into hematin; this oxidation is accompanied by a modification of the absorption spectrum and the loss of the property of the prosthetic group to combine with O₂ or CO.

If oxyhemoglobin or carboxyhemoglobin is subjected to this oxidation, we also observe the formation of methemoglobin accompanied by the liberation of the gas combined, which incidentally permits the titration of the gas combined with hemoglobin.

The formation of met hemoglobin in vivo, either as a result of a serious intoxication or after a metabolic disease, has grave consequences because oxygen transport is made impossible. However, methemoglobin can be again reduced into hemoglobin in vitro as well as in vivo in normal red blood cells, by the action of reducing agents. These inter-conversions may be summarized in a diagram (see fig. 1-33).