RGI.7 Haemoproteins I: Structures and oxygenation equilibria

Haemoglobin and Myoglobin

• Haemoglobin:
  • Located in red blood cells.
  • Responsible for oxygen transport in higher animals.
  • Contains approximately 65% of the iron in the human body.
  • Relative molecular mass (RMM) is approximately 66,000 (66 kDa).
• Myoglobin:
  • Located in muscle cells.
  • Stores oxygen and transports it across muscle cells.
  • Contains approximately 6% of the iron in the human body.
  • RMM is approximately 17,800 (17.8 kDa).

Myoglobin Structure

• Composed of:
  • Haem group: iron(II) – protoporphyrin IX.
  • Globin chain: 153–160 amino acid residues.
• Primary Structure: Linear sequence of amino acids held together by covalent peptide bonds.
• Secondary Structure: Regular local sub-structures like alpha helices and beta strands/sheets, defined by hydrogen bonds between main-chain peptide groups.
• Tertiary Structure: Three-dimensional structure of the protein; alpha-helices and beta-pleated sheets fold into a compact globular structure. Interactions include salt bridges, hydrogen bonds, tight packing of side chains, and disulfide bonds.
• Quaternary Structure: Three-dimensional structure of a multi-subunit protein and how the subunits fit together, stabilized by non-covalent interactions and disulfide bonds (same as tertiary).

Iron – Protoporphyrin IX

• Porphyrins: Family of tetradentate, planar ligands.
  • Contain 4 nitrogen donor atoms.
  • Heteroaromatic.
  • Composed of 4 pyrrole-like rings joined by CH groups.
  • Have peripheral substituents.

Myoglobin Molecule

• Compact shape with the protein chain folded around the haem group.
• Globin chain is linked to the haem group through a proximal histidine residue.
• Features:
  • 8 major helical regions.
  • Non-helical regions or loops.
  • Non-helical chain termini.
  • Hydrophobic interior.
  • Hydrophilic exterior.
  • Only two hydrophilic residues inside, both histidines, essential for biological activity.
• The haem group resides in a non-polar (hydrophobic) crevice to prevent oxidation of Fe^{2+} in the presence of O2 and H2O.
  • If oxidation occurs, it would form Fe^{3+} which cannot bind (carry) O_2.

Coordination Sphere of Haem Iron

• The Fe^{2+} is coordinatively unsaturated (CN = 5) because it has fewer ligands than its maximum coordination number.
• It has a vacant coordination site which can be used to complex with O_2.

Forms of Myoglobin (Mb)

• Myoglobin: Fe^{2+}, coordinated to Histidine (His), purple-red.
• Oxymyoglobin: Fe^{2+}, coordinated to His & O_2, bright red.
• Metmyoglobin: Fe^{3+}, coordinated to His & H2O, brownish-red; does not bind O2.

Colour of Red Meat

• Myoglobin
• Oxymyoglobin
• Denatured myoglobin
• Metmyoglobin

Spin States of Iron(II) in Myoglobin and Oxymyoglobin

• Energies of metal ion d orbitals in an octahedral complex depend on the ligands surrounding the metal ion.
• Orbitals are split into two sets (axial and inter-axial).
• Consider a d6 Ion (Fe^{2+}):
  • High spin, paramagnetic (e.g., Fe^{2+} in myoglobin, haemoglobin).
  • Low spin, diamagnetic (e.g., Fe^{2+} in oxymyoglobin & oxyhaemoglobin).

Metal Ion in Myoglobin

• Myoglobin:
  • Fe^{2+}, d6, is high spin, lies out of the porphyrin plane and has one empty coordination site.
• Oxymyoglobin:
  • Fe^{2+} is low spin and smaller in size, can now fit into the porphyrin plane, and is coordinatively saturated (CN = 6).

Myoglobin and Oxymyoglobin

• Myoglobin
  • Fe^{2+}
  • C.N. = 5
  • High Spin
  • Paramagnetic
  • Out of plane of ring
• Oxymyoglobin
  • Fe^{2+}
  • C.N. = 6
  • Low Spin
  • Diamagnetic
  • In plane of ring

Haemoglobin Structure

• Structure solved by Perutz (Cambridge, 1959) using X-ray diffraction.
• Nearly spherical shape.
• Haems in non-polar crevices.
• 3D structure of each globin chain in Hb very similar to that in Mb, although amino acids are identical at only 24 positions.
• 4 subunits – each Mb-like:
  • 2α and 2β globin chains
  • Salt linkages between chains.

Principal Adult Human Haemoglobin (HBA)

• A tetrameric protein (α2β2).
  • α chain – 141 a.a.
  • β chain – 146 a.a.
• Each α is in contact with both β chains.
• Very few interactions between the two α chains or between the two β chains.

Subunit Interactions

• Most important interactions between subunits are salt bridge linkages.
• A salt bridge in proteins is an interaction between oppositely charged amino acid side chains.
• Terminal backbone groups can also participate in salt bridge interactions.

Subunit Haem Groups

• Each haem group is like the Mb haem.
• The Fe^{2+} is coordinatively unsaturated (CN = 5).
• Vacant coordination site can be used to complex with O_2.
• Fe^{2+} is d6 high spin with 4 unpaired e-.
• Fe^{2+} lies out of the plane of the porphyrin ring.

Haemoglobin and Myoglobin

• Primary structure of each globin chain in haemoglobin is very different from that of myoglobin, but the secondary and tertiary structures are very similar.
• Haemoglobin has a quaternary structure; myoglobin does not.

Changes Upon Oxygenation of Haemoglobin

• Hb + 4O2 ightharpoonup Hb(O2)_4
• Fe^{2+} Changes:
  • CN = 5 (coordinatively unsaturated) → CN = 6 (coordinatively saturated)
  • High spin, paramagnetic → low spin, diamagnetic
  • Iron out of porphyrin plane → iron in porphyrin plane
• Haemoglobin (or deoxyhaemoglobin) converts to oxyhaemoglobin upon oxygenation.

Oxygenation

• Oxygenation of Haemoglobin is reversible: Hb + 4O2 ightharpoonup Hb(O2)_4
• Haemoglobin Oxyhaemoglobin