In the ferrous form (deoxymyoglobin), the iron is five-coordinate and high spin. Because high-spin Fe²⁺ is too large to fit into the “hole” in the center of the porphyrin, it is about 60 pm above the plane of the porphyrin. When O₂ binds to deoxymyoglobin to form oxymyoglobin, the iron is converted from five-coordinate (high spin) to six-coordinate . Because low-spin Fe²⁺ and Fe³⁺ are smaller than high-spin Fe²⁺, the iron atom moves into the plane of the porphyrin ring to form an octahedral complex. The O₂ pressure at which half of the molecules in a solution of myoglobin are bound to O₂ (P_1/2) is about 1 mm Hg (1.3 × 10⁻³ atm).

A vacant coordination site at a metal center in a protein usually indicates that a small molecule will bind to the metal ion, whereas a coordinatively saturated metal center is usually involved in electron transfer.

Hemoglobin consists of two subunits of 141 amino acids and two subunits of 146 amino acids, both similar to myoglobin; it is called a tetramer because of its four subunits. Because hemoglobin has very different O₂-binding properties, however, it is not simply a “super myoglobin” that can carry four O₂ molecules simultaneously (one per heme group). The shape of the O₂-binding curve of myoglobin (Mb; Figure 1.2) can be described mathematically by the following equilibrium:

In contrast, the O₂-binding curve of hemoglobin is S shaped (Figure 1.3)). As shown in the curves, at low oxygen pressures, the affinity of deoxyhemoglobin for O₂ is substantially lower than that of myoglobin, whereas at high O₂ pressures the two proteins have comparable O₂ affinities. The physiological consequences of the unusual S-shaped O₂-binding curve of hemoglobin are enormous. In the lungs, where O₂ pressure is highest, the high oxygen affinity of deoxyhemoglobin allows it to be completely loaded with O₂, giving four O₂ molecules per hemoglobin. In the tissues, however, where the oxygen pressure is much lower, the decreased oxygen affinity of hemoglobin allows it to release O₂, resulting in a net transfer of oxygen to myoglobin.

Oxygen is not unique in its ability to bind to a ferrous heme complex; small molecules such as CO and NO bind to deoxymyoglobin even more tightly than does O₂. The interaction of the heme iron with oxygen and other diatomic molecules involves the transfer of electron density from the filled t_2g orbitals of the low-spin d⁶ Fe²⁺ ion to the empty π* orbitals of the ligand. In the case of the Fe²⁺–O₂ interaction, the transfer of electron density is so great that the Fe–O₂ unit can be described as containing low-spin Fe³⁺ (d⁵) and O₂⁻. We can therefore represent the binding of O₂ to deoxyhemoglobin and its release as a reversible redox reaction:

As shown in Figure 1.4 ,the Fe–O₂ unit is bent, with an Fe–O–O angle of about 130°. Because the π* orbitals in CO are empty and those in NO are singly occupied, these ligands interact more strongly with Fe²⁺ than does O₂, in which the π* orbitals of the neutral ligand are doubly occupied.

Although CO has a much greater affinity for a ferrous heme than does O₂ (by a factor of about 25,000), the affinity of CO for deoxyhemoglobin is only about 200 times greater than that of O₂, which suggests that something in the protein is decreasing its affinity for CO by a factor of about 100. Both CO and NO bind to ferrous hemes in a linear fashion, with an Fe–C(N)–O angle of about 180°, and the difference in the preferred geometry of O₂ and CO provides a plausible explanation for the difference in affinities. As shown in Figure 1.4 , the imidazole group of the distal histidine is located precisely where the oxygen atom of bound CO would be if the Fe–C–O unit were linear. Consequently, CO cannot bind to the heme in a linear fashion; instead, it is forced to bind in a bent mode that is similar to the preferred structure for the Fe–O₂ unit. This results in a significant decrease in the affinity of the heme for CO, while leaving the O₂ affinity unchanged, which is important because carbon monoxide is produced continuously in the body by degradation of the porphyrin ligand (even in nonsmokers). Under normal conditions, CO occupies approximately 1% of the heme sites in hemoglobin and myoglobin. If the affinity of hemoglobin and myoglobin for CO were 100 times greater (due to the absence of the distal histidine), essentially 100% of the heme sites would be occupied by CO, and no oxygen could be transported to the tissues. Severe carbon-monoxide poisoning, which is frequently fatal, has exactly the same effect. Thus the primary function of the distal histidine appears to be to decrease the CO affinity of hemoglobin and myoglobin to avoid self-poisoning by CO.