Haemoglobin and Myoglobin

a) What are haemoglobin and myoglobin?

b) State their biofunctions and indicate the difference in their structures.

c) What metal ion is present at the active sites of these proteins and what is its oxidation state?

2+4+2

a) Haemoglobin (Hb) and myoglobin (Mb) are dioxygen carrier metalloproteins, responsible for the
transport and storage of oxygen in vertebrates.

b) Hb transports oxygen from its source (viz. lungs, skin and gills) to the site of its use in the muscle cells,
where oxygen is transferred to Mb for use in mitochondrial oxidation (i.e. respiration).

The structure of an oxygen carrier molecule should be such that the oxygen molecule is held with sufficient
strength, but not so strongly as to irreversibly oxidize the active site Fe (II) to Fe (III), in order that oxygen
can be released and delivered at a rapid rate at the proper site. Both haemoglobin and myoglobin have a
structural unit heme, i.e. Fe (II) bound with protoporphyrin, with extensive delocalisation of dπ orbital
electrons of Fe (II) into the π orbital network of the ring.

Structure:

As the Fe heme is enclosed in a hydrophobic pocket of the associated protein molecule globin, water
molecule or any polar solvent cannot occupy the sixth coordination position. It is free to be occupied by non-
polar O2. thus the hydrophobic globin chain inhibits the displacement of O2 by water molecule, present in
blood.

In the absence of globin Fe (II) heme is irreversibly oxidized to Fe (III) heme hydroxide (haematin), which
cannot bind to O2. Oxy-Hb and Oxy-Mb remain stable because the globin chain helps keep the haeme groups
apart, inhibiting the formation of µ -oxo Fe (III) heme. There is formation of stable σ bonding between sp2
lone pair of O2 with sixth d2sp3 hybrid orbital of Fe and interaction between filled dπ orbitals of Fe(II) (dxy,
∗
dyz) with π (pz – pz) orbital of O2.

In Hb or Mb, Fe(II) is in high spin state, and is 0. 8 Å above the plane of the porphyrin ring nitrogens. When
O2 gets bound to the Fe(II) at the vacant 6th postion, the metal ion is pulled down to the centre of the plane
of the macrocyclic ring. Fe(II)-N distance is reduced to 2 Å, a strong field is created, Fe(II) goes to a low
spin state.
 Structure of Haemoglobin Structure of Myoglobin

Hb transports O2 in blood, taking dioxygen from air in Mb is the O2 binding protein found principally in the
the lungs and delivering it to Mb in tissues. muscle tissues of vertebrates.

Hb is a tetramer (MW ≈ 64450 daltons) with two α It is a monomeric protein (MW ≈ 17500 daltons)
and two β polypeptide chains. consisting of a single polypeptide chain of 153 amino
acids called globin made up of seven α -helical and
six non-helical segments.

In each chain there is an iron protoporphyrin IX group Attached to the chain by coordination to the
held by a proximal histidine imidazole residue as in Mb. imidazole ring of a histidine residue is the dioxygen-
binding prosthetic group iron (II) protoporphyrin IX.

In deoxy Hb the iron liew 0.36 to 0.40 Å out of the De-oxy Mb has a pentacoordinate iron (II) centre in
porphyrin ring plane but moves within ± 0.12 Å of the which the metal atom lies 0.42 Å out of the plane of
plane upon binding of dioxygen. The two α and two β the four pyrrole-ring donor nitrogen atoms. It is

polypeptide chains are interlinked through long-range displaced towards the bonded imidazole group which
electrostatic (COO- …. NH4+) or H-bonded interaction is called the proximal side of heme. Mb has no
(O—H ….O-H). Due to these bondings there is structural constraints and can take up O2 at a lower
constraint on the structure of Hb. Hence the overall rate pressure in the muscles.
of binding of O2 with Hb is less than with Mb.
Consequently Hb can take up O2 at a higher pressure, a
condition available in the lungs (higher animals) gills
(fishes) or skin (smaller organisms).
Downward movement of Fe (II) is accompanied by Upon binding of dioxygen the iron atom moves
coordinated movement of histidine towards porphyrin towards the FeN4 planes.
ring. This brings a conformational change throughout Myoglobin, with no constraint in structure, is
the protein chain, resulting in the rupture of several converted to MbO at low oxygen pressure (pO ).
2 2
- +
COO …. NH4 interactions. Thus the constrained (No cooperativity here). Hence, in tissues, where
(tensed T form) Hb becomes relaxed on binding of O2 pO is low, the transfer of oxygen from oxy-Hb to
2
with one haeme centre and in the relaxed (R) form the Mb is easy.
remaining haeme centres in the tetramer become
In oxy Mb the dioxygen molecule is bonded end-on
favourably exposed to O2 and bind with it easily
to iron forming a bent structure with a Fe—O—O
(homotropic allosteric cooperativity).
bond angle of 115o.

As the oxygenation of haemoglobin involves the There is no cooperative interaction in Mb

breaking of COO- …. NH4+ salt bridges, oxygenation is oxygenation and hence, no Bohr effect.
pH dependent. With the increase in pH, more bonds
break and O2 uptake is favored. At low pH, electrostatic
salt bridges are strengthened and O2 uptake is lowered.
Thus deoxygenation is favored at low pH. This is called
Bohr effect.

c) The metal ion present at the active sites of these proteins is iron, in its +2 oxidation state.