Molecular Structure and Types of Hemoglobin
The hemoglobin molecule has a tetrameric structure, consisting of four polypeptide subunits. It always contains two $\alpha$-chains, while the second pair of chains varies depending on the developmental stage:
- Embryonic hemoglobin ($2\alpha, 2\epsilon$) — synthesized during early prenatal development.
- Fetal hemoglobin, Hb F ($2\alpha, 2\gamma$) — replaces embryonic hemoglobin, peaking around the 6th month of gestation.
- Adult hemoglobin, Hb A ($2\alpha, 2\beta$) — the primary postpartum hemoglobin, accounting for about 98%.
The second pair of chains determines how a specific form of hemoglobin functions and binds gases.
Heme Structure and the Hydrophobic Pocket
Heme is a non-protein prosthetic group. Its chemical core is a tetrapyrrole protoporphyrin IX ring. An iron atom ($Fe^{2+}$) coordinated with four nitrogen atoms of the pyrrole rings sits at the center. Substituents around the ring include 4 methyl groups, 2 vinyl groups, and 2 propionic acid side chains.
Heme is embedded in a specialized hydrophobic pocket between the F and E $\alpha$-helices. Two histidine residues play a critical role:
- Proximal histidine (His F8): Forms a coordination bond with $Fe^{2+}$, firmly anchoring the heme within the protein.
- Distal histidine (His E7): Located on the oxygen-binding side. It ensures proper orientation for the $O_2$ molecule and sterically hinders carbon monoxide (CO) binding.
Protective function: The hydrophobic environment of the pocket keeps water out. If water reached the iron atom, it would oxidize to $Fe^{3+}$ (forming methemoglobin), rendering oxygen transport impossible.
Cooperative Binding
Hemoglobin binds oxygen cooperatively rather than simply summing the efforts of individual subunits. In deoxyhemoglobin, the iron atom sits slightly out of the porphyrin plane toward His F8. When oxygen binds to $Fe^{2+}$ (near His E7), the iron atom is pulled directly into the plane of the heme ring.
This shift pulls the attached proximal histidine (His F8) and the entire protein helix along with it, triggering a chain reaction of conformational changes across the entire molecule. As a result of this transition, the fourth $O_2$ molecule binds to the tetramer 300 times more easily than the first. In tissues, the process works in reverse: unloading one oxygen molecule facilitates the release of the others.
Bohr Effect (Influence of $CO_2$ and $H^+$)
In actively metabolizing tissues (such as skeletal muscle), catabolism generates large amounts of carbon dioxide and water. $CO_2$ diffuses into erythrocytes, where the enzyme carbonic anhydrase converts it into carbonic acid, which dissociates into bicarbonate ($HCO_3^-$) and protons ($H^+$).
Protons bind to histidine residues (specifically His146) on the hemoglobin chains. This allosteric interaction decreases hemoglobin's affinity for oxygen, breaking the bond between $Fe^{2+}$ and $O_2$ and releasing oxygen into the tissues. The higher the metabolic rate, the more $H^+$ is produced, and the more oxygen the protein unloads. In the lungs, the reverse occurs: high $O_2$ partial pressure forces the protein to bind oxygen, displacing protons. Protons then combine with bicarbonate to form $CO_2$, which is exhaled.
Allosteric Regulation by 2,3-BPG
2,3-Bisphosphoglycerate (2,3-BPG) is a strongly negatively charged ligand (-2 to -5 depending on pH, with -5 physiological state) produced in erythrocytes as a glycolysis intermediate.
In deoxyhemoglobin, additional ionic bonds form between protomers, widening the central cavity. 2,3-BPG binds directly into this cavity, interacting with positively charged amino acids of the $\beta$-chains (valine, lysine, and histidine). Its binding alters protein conformation and reduces oxygen affinity, promoting $O_2$ release to peripheral cells. In the lungs, oxygen binding narrows the central cavity, expelling 2,3-BPG from the molecule.