Sperm whale myoglobin was the first protein for which the three-dimensional structure was determined. X-ray crystallographic studies pioneered by John Kendrew revealed the structure of this protein in the 1950s (Figure 7.1). Myoglobin consists largely of α helices that are linked to one another by turns to form a globular structure.

Myoglobin can exist in an oxygen-free form called deoxymyoglobin or in a form with an oxygen molecule bound called oxymyoglobin. The ability of myoglobin and hemoglobin to bind oxygen depends on the presence of a heme molecule. As we shall discuss in Chapter 9, heme is one example of a prosthetic group, a molecule that binds tightly to a protein and is essential for its function.

The heme group gives muscle and blood their distinctive red color. It consists of an organic component and a central iron atom. The organic component, called protoporphyrin, is made up of four pyrrole rings linked by methine bridges to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate side chains are attached to the central tetrapyrrole.

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The iron atom lies in the center of the protoporphyrin, bonded to the four pyrrole nitrogen atoms. Although the heme-bound iron can be in either the ferrous (Fe²⁺) or ferric (Fe³⁺) oxidation state, only the Fe²⁺ state is capable of binding oxygen. The iron ion can form two additional bonds, one on each side of the heme plane. These binding sites are called the fifth and sixth coordination sites. In myoglobin, the fifth coordination site is occupied by the imidazole ring of a histidine residue from the protein. This histidine is referred to as the proximal histidine.

Oxygen binding occurs at the sixth coordination site. In deoxymyoglobin, this site remains unoccupied. The iron ion is slightly too large to fit into the well-defined hole within the porphyrin ring; it lies approximately 0.4 Å outside the porphyrin plane (Figure 7.2, left). Binding of the oxygen molecule at the sixth coordination site substantially rearranges the electrons within the iron so that the ion becomes effectively smaller, allowing it to move within the plane of the porphyrin (Figure 7.2, right). Remarkably, the structural changes that take place on oxygen binding were predicted by Linus Pauling, on the basis of magnetic measurements in 1936, nearly 25 years before the three-dimensional structures of myoglobin and hemoglobin were elucidated.

The change in electronic structure that occurs when the iron ion moves into the plane of the porphyrin is paralleled by alterations in the magnetic properties of hemoglobin; these changes are the basis for functional magnetic resonance imaging (fMRI), one of the most powerful methods for examining brain function. Nuclear magnetic resonance techniques detect signals that originate primarily from the protons in water molecules and are altered by the magnetic properties of hemoglobin. With the use of appropriate techniques, images can be generated that reveal differences in the relative amounts of deoxy- and oxyhemoglobin and thus the relative activity of various parts of the brain. When a specific part of the brain is active, blood vessels relax to allow more blood flow to that region. Thus, a more-active region of the brain will be richer in oxyhemoglobin.

These noninvasive methods identify areas of the brain that process sensory information. For example, subjects have been imaged while breathing air that either does or does not contain odorants. When odorants are present, fMRI detects an increase in the level of hemoglobin oxygenation (and, hence, of activity) in several regions of the brain (Figure 7.3). These regions are in the primary olfactory cortex, as well as in areas in which secondary processing of olfactory signals presumably takes place. Further analysis reveals the time course of activation of particular regions. Functional MRI shows tremendous potential for mapping regions and pathways engaged in processing sensory information obtained from all the senses. A seemingly incidental aspect of the biochemistry of hemoglobin has enabled observation of the brain in action.

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Oxygen binding to iron in heme is accompanied by the partial transfer of an electron from the ferrous ion to oxygen. In many ways, the structure is best described as a complex between ferric ion (Fe³⁺) and superoxide anion , as illustrated in Figure 7.4. It is crucial that oxygen, when it is released, leaves as dioxygen rather than superoxide, for two important reasons. First, superoxide and other species generated from it are reactive oxygen species that can be damaging to many biological materials. Second, release of superoxide would leave the iron ion in the ferric state. This species, termed metmyoglobin, does not bind oxygen. Thus, potential oxygen-storage capacity is lost. Features of myoglobin stabilize the oxygen complex such that superoxide is less likely to be released. In particular, the binding pocket of myoglobin includes an additional histidine residue (termed the distal histidine) that donates a hydrogen bond to the bound oxygen molecule (Figure 7.5). The superoxide character of the bound oxygen species strengthens this interaction. Thus, the protein component of myoglobin controls the intrinsic reactivity of heme, making it more suitable for reversible oxygen binding. The distal histidine may also impair access of carbon monoxide to the heme, which binds tightly to the heme iron with dire consequences.

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The three-dimensional structure of hemoglobin from horse heart was solved by Max Perutz shortly after the determination of the myoglobin structure. Since then, the structures of hemoglobins from other species including humans have been determined. Hemoglobin consists of four polypeptide chains, two identical α chains and two identical β chains (Figure 7.6). Each of the subunits consists of a set of α helices in the same arrangement as the α helices in myoglobin (see Figure 6.15 for a comparison of the structures). The recurring structure is called a globin fold. Consistent with this structural similarity, alignment of the amino acid sequences of the α and β chains of human hemoglobin with those of sperm whale myoglobin yields 25% and 24% identity, respectively, and good conservation of key residues such as the proximal and distal histidines. Thus, the α and β chains are related to each other and to myoglobin by divergent evolution (Section 6.2).

The hemoglobin tetramer, referred to as hemoglobin A (HbA), is best described as a pair of identical αβ dimers (α₁β₁ and α₂β₂) that associate to form the tetramer. In deoxyhemoglobin, these αβ dimers are linked by an extensive interface, which includes the carboxyl terminus of each chain. The heme groups are well separated in the tetramer by iron–iron distances ranging from 24 to 40 Å.