Myoglobin is a small globular monomeric protein, expressed in the red muscles of vertebrates, where it serves as molecular oxygen storage and delivery and as nitric oxide scavenger.

It can reversibly bind oxygen thanks to the presence of a prosthetic group, the haem, which is hosted in a fairly hydrophobic crevice, within the protein matrix. The iron is responsible of keeping the haem inside the protein being coordinated to the so‐called proximal histidine. On the sixth coordination site, Fe(II) is able to combine with high affinity, but reversibly, with O2 and several other ligands.

The iron–ligand bond is broken by light; therefore, Mb has also been employed for exploring the very first relaxation events after photodissociation by fast and ultra‐fast kinetic methods.

Mb has also been a molecule of choice in unveiling the role of molecular motions in controlling function and stability of proteins.

Key Concepts

  • Myoglobin (Mb) is a water‐soluble globular protein of molecular weight 17 000 Da, expressed in the skeletal muscles and the heart.
  • The α‐helical organisation of the polypeptide chain yields a unique topology called the ‘globin fold’.
  • Its red colour is due to one molecule of Fe(II) haem, bound in a crevice of the globin in between helices E and F.
  • Mb's function is intracellular oxygen transport/storage, and nitric oxide scavenging.
  • Mb has been the paradigm for the discovery and the studies of protein plasticity and dynamics.
  • The photolability of the iron–ligand bond has been exploited for laser‐activated transient relaxation.
  • Many of the advanced biophysical techniques now available have been developed and tested on Mb.
  • Mb has been a benchmark for bioinformatics topological annotation and molecular dynamics simulations.
  • Globins paved the way to the concepts of molecular clock and molecular evolution, as they are expressed in all phyla.
  • Early studies on the structure–function relationships in Mb have represented a proof‐of‐principle of this powerful approach.

Keywords: globin fold; haem; oxygen storage; functional and structural dynamics; evolution

Figure 1. The structure of myoglobin. (a) Ball‐and‐stick representation of the haem bound to the active site of sperm whale oxymyoglobin (Protein Data Bank id: 1MBO). Key residues on the distal and proximal haem pocket are also shown, labelled following the topological annotation. Note the hydrogen bond between the Nϵ of the distal His E7 and the oxygen molecule, which is bound at an angle of about 130°. Neutron diffraction, NMR and computational chemistry have shown that in MbO2 this amino acid is protonated only on Nϵ. It may be noticed that in the CO derivative protonation also occurs on Nδ, which faces the bulk water, thereby favouring a more perpendicular geometry. (b) Ribbon representation of sperm whale myoglobin, highlighting the 3/3 globin fold. Side chains have been omitted for clarity. Helices are indicated with capital letters (A through H) from the N‐terminus to the C‐terminus. The haem group is in gold stick representation.
Figure 2. Oxygen reactivity curves of haemoglobin (Hb), Mb and cytochrome oxidase (COX) expressed as percentage of protein oxygenation at pH 7.4 as a function of oxygen concentration in mol/L (M). It may be noticed that the gradient of oxygen affinities of the three proteins correlates with the increasing local concentration of the gas in going from mitochondria to blood (RBC).
Figure 3. Ligand migration pathway within the protein matrix. The combination of structural dynamics, mainly by picosecond laser photolysis coupled with Laue crystallography, and static structure determination in the presence of 30 atm of Xenon demonstrated the importance of the small solvent excluded internal cavities in the modulation of ligand binding. The structure of the main chain is represented in ribbon coloured from red to blue starting from the N‐terminus. The haem, His F8 and His E7 are in stick representation with the atoms coloured by their canonical code (C green, O red, N blue, Fe grey). CO is in van der Waals representation; the cavities occupied by Xe atoms (PDB: 4NXA) are in light grey. (a) The structure of Mb–CO (PDB: 1MYZ); notice the CO bound to the haem iron which is flat. (b) The first photolytic intermediate trapped at cryogenic temperature (20 K in liquid helium). The photolyzed CO, which lies in the distal pocket parallel to the haem (PDB: 1DXD), may either rebind directly to the haem iron in a reaction called geminate recombination, or migrate in the protein matrix populating the Xe binding cavities. (c) The CO is momentarily trapped in the so‐called Xe4 cavity (PDB: 1DWS). Subsequently, the CO is observed inside the proximal Xe1 cavity (PDB: 1DWT) as shown in (d). The dynamics of these events, which range from femtoseconds to 100 nanoseconds, is best illustrated in the movie available as Supplementary video 1. We express our appreciation to A. Di Nola (‘Sapienza’ University of Rome) and A. Amadei (University of Rome ‘Tor Vergata’) for the MD calculations and to G. Giardina (‘Sapienza’ University of Rome) for the final wrap‐up of the movie.
Figure 4. The evolution of vertebrate globins. A phylogenetic tree has been built based on amino acid sequence similarities between the globins belonging to the superfamily. The y‐axis indicates the time in millions of years, from today backwards to about 1 billion years. Branching from a common ancestor has first seen the divergence of neuroglobin from cellular globins. Neuroglobin is found in the brain and has a role in the protection from injuries due to reactive oxygen and nitrogen species. The second branch saw the separation of monomeric from polymeric globins. About 500 millions years ago, cytoglobin (found in the liver) and Mb (in the muscles) diverged. Subsequently, the separation between the α and β chains of haemoglobin has been mapped about 400 million years ago. In primates, neuroglobin, cytoglobin, Mb and the clusters of the α and the β chains of haemoglobin are on five different chromosomes (as boxed on the top of the figure).


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Further Reading

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Brunori, Maurizio, and Miele, Adriana E(Aug 2015) Myoglobin. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000656.pub2]