Principles of Protein Structure - H. Schultz 1982

Protein-ligand interactions
Heme-binding sites
Heme ligands as trigger systems for structural changes in mammalian hemoglobin

Deoxyhemoglobin contains a high-spin Fe²⁺ ion, whereas oxyhemoglobin contains a low-spin Fe²⁺ ion. Disagreements still persist regarding the conformational changes that occur during the redox cycle of cytochrome c [459, 509, 564]. Therefore, we will restrict our Structure/133.html">Discussion to the well-characterized case of mammalian tetrameric Hemoglobin [60, 662, 666].

Crystallographic studies have demonstrated that the quaternary structures of the oxy and deoxy forms of hemoglobin differ significantly. These differences are summarized in Table 10.1. Specifically, upon oxidation, the four subunits shift relative to one another, resulting in a 6.5 Å decrease in the distance between the iron atoms of the ß-chains. This substantial alteration in quaternary structure is likely rooted in the purely Chemical aspects of iron-porphyrin interactions [637, 663, 664]. Two Types of d-electron distribution are possible within the iron-porphyrin complex. The high-spin configuration found in deoxyhemoglobin features a greater number of orbitals with unpaired electrons than the low-spin configuration of oxyhemoglobin. One consequence of this is that the high-spin iron atom is too large to remain in the plane of the porphyrin ring and is displaced from it by 0.5 Å [550] (Fig. 10.3).

The Mechanism of O₂ binding by hemoglobin has been elucidated at the atomic level. Let us now trace The process of oxygenation of deoxyhemoglobin [60, 550]. Molecular oxygen approaches the high-spin iron atom of one a-subunit (in the ß-subunits of deoxyhemoglobin, the sixth coordination site is blocked by the Val-67 residue and is inaccessible to O₂) and converts it to the low-spin configuration. As a result, the iron atom shifts by 0.5 Å and returns to the plane of the porphyrin ring. The imidazole ring of the proximal Histidine, which coordinates the iron atom, moves by the same distance—or perhaps even by 0.7 Å—since the iron–axial Ligand bond shortens by 0.1 to 0.2 Å upon the transition of the iron atom from the high-spin to the low-spin ferrous state.

Class="center">Table 10.1 Differences between deoxyhemoglobin and oxyhemoglobin [550, 666]

Helix F, to which the proximal His is attached, approaches helix H so closely that the side chain of the penultimate Tyr residue can no longer fit between the two helices and is forced out, pulling along the C-terminal residue that was anchored by a salt bridge to the other a-chain. This disrupts the primary bond stabilizing the deoxy form (Fig. 10.3, b). The binding of O₂ to the next subunit exerts a similar effect on the intersubunit bonds, further amplifying the quaternary structural change. One consequence of this shift is the withdrawal of the Val-67 residue from the ligand pocket of the ß-subunits, thereby making their iron atoms accessible to O₂ as well. The energetic aspects of this transition have been discussed by several authors [60, 268, 634].

The example of deoxyhemoglobin illustrates why it can be difficult to identify the chemical interaction that triggers critical changes in protein quaternary or tertiary structure: the "triggering" event must be structurally subtle. In the case of hemoglobin, many years elapsed before it was established that the trigger mechanism originates at the iron atom, which sets the hemoglobin subunits in motion [666].

In tetrameric hemoglobin, the affinity of the heme for O₂ is regulated by the concentrations of O₂, CO₂, H⁺, and 2,3-diphosphoglycerate. What was gained through the evolution of a monomeric, Myoglobin-like hemoglobin into the complex mammalian hemoglobin? The primary advantage lies in the enhanced physiological adaptability of the tetrameric protein, achieved by bringing the oxygen affinity of the binding site under the control of external influences [276, 549, 667] (Fig. 10.4).

One such advantage is an improved variant of the Haldane effect [668, 669], which involves the release of protons upon the binding of molecular oxygen to hemoglobin, and vice versa. This effect is crucial for The transport of molecular oxygen from the Lungs to oxygen-consuming Tissues, as well as for the transport of protons from these tissues back to the lungs. Furthermore, by utilizing Different types of hemoglobin with varying sensitivities to hydrogen ion concentration, certain fish species can harness O₂ for two independently controlled Functions: filling the swim bladder and directing molecular oxygen into metabolic pathways [136]. Also of great significance is The ability to use specialized organic phosphoryl compounds—such as 2,3-diphosphoglycerate (DPG) or Inositol hexaphosphate [670, 671]—as allosteric effectors that favor the deoxy conformation. These effectors play a decisive role in O₂ transfer from maternal to fetal Blood [274, 671], in high-altitude adaptation [672], and in pathologies where arterial blood is only partially saturated with oxygen [85, 673] (Fig. 10.4).

Subunit cooperativity facilitates the complex task of O₂ transport. Another essential factor is the so-called heme–heme interaction—namely, The Effect of O₂ binding at one heme on the O₂ affinity of heme groups in other subunits [634]. This effect enables hemoglobin to bind O₂ in the lungs at a free O₂ concentration of 130 µM and release approximately 30% of that O₂ in the capillaries of metabolizing tissues (Fig. 10.4). Here, the free O₂ concentration must remain at about 50 µM to ensure oxygen diffusion into tissue Cells. The fraction of released oxygen (30%) is relatively insensitive to decreases in lung O₂ concentration and can be enhanced by shortening the average O₂ diffusion path in tissues. Conversely, a carrier with independent O₂ binding sites would release less than 20% of the transported oxygen under favorable conditions and would lack adaptative capacity (Fig. 10.4). Thus, oligomeric cooperativity provides superior functional adaptability.

Fig. 10.4. O₂ binding curves for Hemoglobins [85].

The dependence of hemoglobin (Hb) oxygen saturation at pH 7.2 on free oxygen concentration is shown. The O₂ concentrations in the lung capillaries (125 µM) and in the capillaries of oxygen-consuming tissues (50 µM) are confined to narrow ranges. Curve a: in the absence of diphosphoglycerate (DPG), hemoglobin is saturated with O₂ in the lungs but fails to deliver it to the tissues. Curve b: at physiological DPG levels (4.5 mM), approximately 30% of the O₂ taken up in the lungs is released in the tissues (arrow I). Curves b and c: because fetal hemoglobin (curve c) has a lower DPG affinity than maternal hemoglobin, molecular oxygen released from maternal blood can be captured by fetal hemoglobin (arrow III). Curve d: high DPG concentration (8 mM) leads to enhanced oxygen delivery to tissues (arrows I and II). Curve e: in the absence of cooperativity between hemoglobin subunits, less O₂ would be transported from the lungs to the tissues. A dissociation constant of 38 µM was assumed for the hypothetical binding curve (d) of the HbO₂ complex.

Many concepts regarding protein action and interaction emerged from studies on hemoglobin. Numerous ideas and models concerning protein–ligand and protein–Protein Interactions were developed through investigations of hemoglobin; these include sigmoidal binding [674–676], the Hill coefficient [677], sequential ligand-binding constants in Oligomeric Proteins [678], cooperativity based on conformational changes [679, 680], and allosteric protein regulation [92, 681, 682]. It is worth noting that many of these concepts were introduced and mathematically formalized before The structure of any protein was known. Consequently, the ongoing relevance and utility of these concepts must be subject to constant re-evaluation. The example of diphosphoglycerate—whose influence on hemoglobin function and structure was overlooked for decades—underscores the potential hazards of rigid theoretical formulations in biology.