The protein that lets you hold your breath

the artwork for myoglobin

Think about those times when you've been playing or running around and, suddenly, you feel like you need to stop and catch your breath. That's your body's way of saying, "Wait a minute, I need more oxygen!" All cells in our body need oxygen to function. Whether exercising or not, this oxygen is used to break down glucose, creating fuel for our muscles—that is, adenosine triphosphate, or ATP, the energy source that keeps our entire body going at all times.

Now, take a look at this stunning image of a majestic snow leopard. These magnificent creatures are like athletes in the wild and can travel a super long distance of 25 miles in just one night. Some can even jump up to 9 metres, six times their body size! Whether it's us running or a snow leopard jumping, we all need oxygen to keep our bodies going.

Inside the muscles of snow leopards, humans and almost all other mammals, there's a special protein called Myoglobin. It grabs onto oxygen and acts like a small storage for oxygen, so muscles get a quick supply when they need it, especially during intense activities. It also acts as a scavenger of free radicals and other reactive oxygen species, avoiding cell damage due to oxidative stress. 

Sea mammals such as dolphins and whales live in the water, and yet still breathe air into their lungs, just like we do. Evolution has allowed them to hold their breath for far longer than we can. The secret lies in the abundance of myoglobin in their bodies. They have 30 % more myoglobin than animals that live on land.  Protein tends to stick together in high amounts, but in these marine mammals, it doesn’t. Notably, marine mammals' myoglobin possesses additional positively charged amino acids on its surface. This special adaptation prevents protein aggregation at high concentrations, as like charges repel each other. 

Structure and oxygen binding 

Myoglobin is a globular protein consisting of two main components: a single polypeptide chain of 154 amino acids that folds into eight α-helices (named A through H) connected by loops, and a ligand heme that enables reversible binding to gaseous ligands such as Oxygen.  The protein's exterior has polar residues, and the interior is primarily non-polar, except for two polar histidines. The hydrophobic core of the protein houses most of the heme, with only a partial exposure to the surface.

Heme is a large, aromatic porphyrin ring with four pyrrole nitrogen bound to a ferrous (Fe2+) ion at the centre. This iron is the attachment point for oxygen, making heme primarily responsible for transporting oxygen (Figure 1). The ferrous ion has six coordination sites: four surrounding the flat surface of the heme ring, occupied by pyrrole nitrogens, one beneath the flat surface taken up by a histidine residue (His-93), and the last one, located above the flat surface, available for binding with the oxygen molecule. Another histidine residue (His-64) is situated above the flat surface on the same side of the heme group as the bound oxygen. Although this histidine is not directly bound to the iron or any part of the heme group, it acts as a gate that opens and closes as oxygen enters the hydrophobic pocket to bind to the heme. The presence of His-64 introduced some bulkiness or clashes, preventing oxygen from binding at its preferred (90°) angle to the heme plane.

 

Figure 1: Oxygen binding site of Myoglobin (PDB 1A6M): Heme’s ferrous ion has six coordination sites; the pyrrole nitrogens occupy four, histidine HIS93 occupies one, and the final site reversibly binds with an O2 molecule via HIS64. Other oxygen-interacting residues include VAL68 and PHE43. [Source:/pdbe/entry/pdb/1a6m/bound/OXY]
Figure 1: Oxygen binding site of Myoglobin (PDB 1A6M): Heme’s ferrous ion has six coordination sites; the pyrrole nitrogens occupy four, histidine HIS93 occupies one, and the final site reversibly binds with an O2 molecule via HIS64. Other oxygen-interacting residues include VAL68 and PHE43. [Source:/pdbe/entry/pdb/1a6m/bound/OXY]

 

Why does oxygen have imperfect binding to the heme group?

Heme can bind more than one molecule, including oxygen and carbon monoxide. Free heme exhibits a much greater affinity for carbon monoxide (CO) than oxygen�25,000 times stronger. When carbon monoxide is compelled to bind at an angle in myoglobin due to steric hindrance by His-64 (Figure 2), its advantage over oxygen decreases significantly by two orders of magnitude. This is a protective mechanism, so small amounts of carbon monoxide produced in our bodies don't take up all the spaces meant for oxygen. Even though too much carbon monoxide is harmful, it's essential for our bodies that oxygen doesn't stick too perfectly to the heme. This way, oxygen can be carried where it's needed and released when necessary.

Figure 2: Oxygen and carbon monoxide binding to the hem group of myoglobin: The presence of the His-64 forces oxygen to bind at 123° angle (PDB 1a6m) or carbon monoxide to bind at 171° angle (PDB 1a6g) instead of the preferred 90°. The angle between the iron, the nearer ligand atom, and the farther ligand atom is shown angle to the heme plane.
Figure 2: Oxygen and carbon monoxide binding to the hem group of myoglobin: The presence of the His-64 forces oxygen to bind at 123° angle (PDB 1a6m) or carbon monoxide to bind at 171° angle (PDB 1a6g) instead of the preferred 90°. The angle between the iron, the nearer ligand atom, and the farther ligand atom is shown angle to the heme plane.

 

Impact of heme or oxygen binding

The presence of the heme significantly affects the conformation of the polypeptide. The apoprotein, which is the isolated polypeptide chain without heme, lacks tight folding. Conversely, the holoprotein, which has a heme bound showcases a folded helix where HIS93 is responsible for holding the heme in place (Figure 3). 

 

Figure 3: Ligand superposition view on the PDBe-KB aggregated view for Myoglobin, (UniProt accession: P02185) The magenta-coloured ligand heme (HEM) is partially exposed to the surface, primarily situated inside the protein. The apoprotein (cluster 1 shown in green), is not as tightly folded as the complete molecule (cluster 2 shown in orange) [Source: /pdbe/pdbe-kb/proteins/P02185/ligands].
Figure 3: Ligand superposition view on the PDBe-KB aggregated view for Myoglobin, (UniProt accession: P02185) The magenta-coloured ligand heme (HEM) is partially exposed to the surface, primarily situated inside the protein. The apoprotein (cluster 1 shown in green), is not as tightly folded as the complete molecule (cluster 2 shown in orange) [Source: /pdbe/pdbe-kb/proteins/P02185/ligands].

 

When comparing the oxygenated and deoxygenated forms of myoglobin, the binding of oxygen induces significant structural changes at the iron centre of heme, causing it to shrink in radius and move into the centre of the N4 pocket. In the oxygenated state, the iron is in the plane of the heme ring, while in the deoxygenated state (no oxygen), the iron is slightly above the plane of the heme (Figure 4).

 

Figure 4. Superposed oxygenated and deoxygenated Myoglobin (PDB 1a6m and PDB 1a6n, respectively) are custom-selected, with all other chains hidden from the 3D view shown in Figure 3. The inset shows the oxygenated state (yellow-coloured HEM), where the iron is in the plane of the heme ring, while in the deoxygenated state (no oxygen, magenta colour), the iron is slightly above the plane of the heme.
Figure 4. Superposed oxygenated and deoxygenated Myoglobin (PDB 1a6m and PDB 1a6n, respectively) are custom-selected, with all other chains hidden from the 3D view shown in Figure 3. The inset shows the oxygenated state (yellow-coloured HEM), where the iron is in the plane of the heme ring, while in the deoxygenated state (no oxygen, magenta colour), the iron is slightly above the plane of the heme.

 

Muscle Injury

In humans, myoglobin is only found in the bloodstream following heart or skeletal muscle injury. The kidneys filter myoglobin from the blood and release it into the urine. Elevated myoglobin levels, particularly due to severe trauma or muscle injuries, can be harmful to the kidneys, potentially causing kidney failure. In 2019, Myoglobinopathy, the first disease caused by a myoglobin mutation was identified, resulting in progressive muscle weakness and advanced-stage effects on respiratory musculature and the heart, attributed to alterations in heme pocket shape and biochemical functions.

First Protein Structure 

Myoglobin was the first protein to have its three-dimensional structure revealed by X-ray crystallography. This achievement was reported in 1958 by John Kendrew and associates. For this discovery, Kendrew shared the 1962 Nobel Prize in chemistry with Max Perutz. More information on the history of this discovery can be found in our previous article at  /pdbe/news/it-all-started-myoglobin.

Preeti Choudhary

About the artwork

Seisenbay Sabina (Year 10) from Nazarbayev Intellectual School, Taraz, Kazakhstan draws a powerful connection between myoglobin and Kazakhstan's cultural identity. Inspired by the country's diverse landscapes, the piece symbolises Kazakhstan's strength and rapid development. Using the snow leopard as a metaphor for myoglobin's role in storing energy, the artwork portrays Kazakhstan as a resilient and fast-developing nation. Through skilled artistry with pencils, pens, and calligraphy, Sabina captures the essence of myoglobin, celebrating the symbiotic relationship between nature, science, and national aspirations in a concise yet impactful manner.

 

View the artwork in the digital 2024 PDBe Calendar.

You can also check out the 2023 PDB art exhibition here

Structures mentioned in this article

Oxy-myoglobin, Atomic Resolution

Deoxy-myoglobin, Atomic Resolution

Carbonmonoxy-myoglobin, Atomic Resolution

Crystal Structure of apo-myoglobin

Sources