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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