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2. Contents:
1. Brief Introduction
2. Myoglobin and Hemoglobin Basics
3. Structure of Heme prosthetic group and oxygen
binding
4. Graphs related to Hemoglobin and Myoglobin
5. Explanation of Graphs
6. Structure of Hemoglobin and Myoglobin Active
sites
7. Selective Binding of Dioxygen with Hemoglobin
and Myoglobin
8. Functions of Hemoglobin and Myoglobin
9. Comparison between hemoglobin and myglobin
3
5
7
9
10
11
13
14
15
Page
No.
3. Brief Introduction
• Iron is the most abundant transition metal found in biological system with a percentage of weight
in human body approximately 5 x 10-3% Hence, this is no surprising fact that there is a myriad of
Iron-containing proteins and enzyme in biological systems
Iron Containing species can be
categorized in two categories
Non Porphyrin Ligand System
Non-Heme, Iron containing Proteins
Porphyrin ligand system--- An Iron
bearing Heme moiety.
**Hemoglobin and
Myoglobin Falls
under this Category
4. • Myoglobin(Mb) and Hemoglobin(Hb) is an essential part in maintaining the biological functions.
Dioxygen(O2) solubility in water is as low as 6.6cm3/Liter or 3 x 10-4M but myoglobin and
hemoglobin increases the solubility of dioxygen by 30 folds making it 200 c.m3/Liter.
• The Myoglobin and Hemoglobin forms complex with Iron through prosthetic heme group, a planar
Four-coordinated porphyrin ligand. The protein side chain, ε-nitrogen(Imidazole Nitrogen) atoms of
histidine completes iron’s ligand coordination sphere in Mb and Hb.
The Heme Group found in Hemoglobin and
Myoglobin
5. Myoglobin and Hemoglobin Basics
• Myoglobin(Mb) is globular monomeric, protein containing a single polypeptide chain of 160 Amino
acid residues made of six α-Helical segments and six nonhelical segments
• Myoglobin heme prosthetic group center contains an iron ion complexed by a porphyrin called
protoporphyrin IX.
• The Iron [Fe(II)] protoporphyrin cofactor is held in place in the protein by noncovalent hydrophobic
interactions of about 80 residues generally Leucine, Isoleucine, Valine and phenylalanine and one
covalent linkage at the proximal Histidine residue (Also known as his93).
• Myoglobin stores Oxygen in muscles and other cellular tissue binding one oxygen atom per protein
subunit.
• Hemoglobin a tetramer of four globular protein subunits, transports oxygen through the blood
plasma. Hemoglobin’s Four subunits comprises two α-Chains of 141 residues and two β-Chains of 146
residues making up a total molecular weight of 64.5KDa.
• Hemoglobin Binds to O2 in a Cooperative Manner- That is, Once a O2 molecule attaches to the
enzyme then the 2nd, 3rd, 4th O2 attaches themselves more readily. For attachment of O2 in both the
Hemoglobin and Myoglobin it is prerequisite that the Iron should be present in it’s reduced state i.e.
Fe(II) state. The terms oxy- and deoxy- in hemoglobin and myoglobin is used to show oxygenated and
deoxygenated Hemoglobin or Myoglobin with Fe(II) in both. While the Met- refers to oxygenated
heme proteins having Fe(III) centers.
6. O2 + 4H + 4e- 2H2O E0= +0.82V
Hb(Fe3+) + e- Hb(Fe2+) E0= +0.17V
*The comparison of reduction potential in above reaction shows that dioxygen should oxidize Fe(II)
Circumstances favoring heme-O2 Stability:-
1. Placement of heme in hydrophobic pocket with enzymes that is inaccessible to water
molecules and protons
2. A bent binding mode for O2 favoured by the prosthetic group’s pocket shape that prevent
μ-oxo dimerization.
3. σ-Bonding donation from an sp2-hybridized superoxide ion to empty d2
z Fe(II) orbital
facilitated by bent orientation of bound O2 and formation of π-back bonds through
interaction of a half filled dxz orbital of Fe(II) with a half filled π* orbital of superoxide ion.
7. Structure of Heme Prosthetic Group and
Oxygen Binding
• Deoxymyoglobin and deoxyhemoglobin contain pentacoordinate Fe(II) center in which the
metal ion lies out of plane of porphyrin’s four pyrrole-nitrogen donor ligands.
Perutz has called this state as the T or Tense state, The T state is a term which describes the
quaternary structure of Hb teramer which is one of low oxygen affinity in which protein is
restrained by binding of Proximal Histidine. In the T state the Fe(II) center is held at
approximately 0.55A (Angstrom) outside of the porphyrin plane and no Hb subunit out of four
posses dioxygen ligand atom.
• The porphyrin ring is also anchored at the active site by iron’s coordination to proximal
histidine’s Imidazole Nitrogen.
T-State
R-State
• In R-State or relaxed quaternary state dioxygen is bound to iron on the so called distal side of the
porphyrin ring.
• Switching from T-State to R-State in Hb tetramer takes place during or after binding of approx. two
dioxygen molecules, this binding of dioxygen relieves the constrains within the surrounding protein
matrix allowing the Iron atom to move back into the porphyrin plane.
8. • Binding of sixth ligand dioxygen and consequent movement of the Fe atom back into the plane of
porphyrin includes a tertiary structural change as the proximal Histidine changes its bond angle with iron
atom. The F-Helix which contains the proximal histidine also changes position
• In hemoglobin these factors in turn change he quaternary structure of Hb tetramer and influence the
affinity of the hemes for dioxygen.
• The metal ion movement into the porphyrin plane is accompanied by a spin change of high (S=2) to low
spin (S=1/2) and Fe(II) to Fe(III), Fe ion become smaller hence better fits into the cavity. The Dioxygen
molecule is guided by the protein pocket surrounding it to attach in a bent structure with an Fe-O-O bond
angle of 115o.In oxidation of Fe(II) to Fe(III) the dioxygen becomes superoxide.
Pictures showing transition from T-State to R-State
10. (a) A curve describing the binding of oxygen to myoglobin. The partial pressure of O2 in the air above the solution is
expressed in kilopascals (kPa). Oxygen binds tightly to myoglobin, with a P50 of only 0.26 kPa. The equation which
describes the curve is:-
(b) A sigmoid (cooperative) binding curve. A sigmoid binding curve can be viewed as a hybrid curve reflecting a
transition from a low-affinity to a high-affinity state. Cooperative binding, as manifested by a sigmoid binding curve,
renders hemoglobin more sensitive to the small differences in O2 concentration between the tissues and the lungs,
allowing hemoglobin to bind oxygen in the lungs (where pO2 is high) and release it in the tissues (where pO2 is low).
(c) Effect of pH on the binding of oxygen to hemoglobin. The pH of blood is 7.6 in the lungs and 7.2 in the tissues.
Experimental measurements on hemoglobin binding are often performed at pH 7.4
(d) Effect of BPG on the binding of oxygen to hemoglobin. The BPG concentration in normal human blood is about
5 mM at sea level and about 8 mM at high altitudes. Note that haemoglobin binds to oxygen quite tightly when BPG
is entirely absent, and the binding curve appears to be hyperbolic. In reality, the measured Hill coefficient for O2-
binding cooperativity decreases only slightly (from 3 to about 2.5) when BPG is removed from hemoglobin, but the
rising part of the sigmoid curve is confined to a very small region close to the origin. At sea level, hemoglobin is
nearly saturated with O2 in the lungs, but only 60% saturated in the tissues, so the amount of oxygen released in
the tissues is close to 40% of the maximum that can be carried in the blood. At high altitudes, O2 delivery declines
by about one-fourth, to 30% of maximum. An increase in BPG concentration, however, decreases the affinity of
hemoglobin for O2, so nearly 40% of what can be carried is again delivered to the tissues.
Explanation of Graphs
11. Structure of Hemoglobin and Myoglobin Active
Site
• First X-Ray crystallographic studies regarding eluciding structure of Mb and Hb was done in 1966 and
1975 respectively. The scientists have also studued the carbon monoxide bound moieties of MbCO and
HbCO as well as MbNO.
• Site directed mutagenesis of residues near the active site of Hb and Mb have yielded info on the exact
nature of the O2, CO, NO binding and the small molecule’s orientation at heme site.
• These studies were confirmed by many other analytical techniques and a clear picture of
metalloprotein’s active site emerged out.
• The active site of Hb/Mb contains an Fe(II) proporphyrin IX encapsulated in a water resistant pocket
and bound to protein through single coordination bond between Imidazole Nitrogen of proximal
Histidine(his93, F8 for Mb) and Fe(II). Additionally, Leucine, Isoleucine, Valine, Phenylalanine interact
with heme, holding it in place through hydrophobic interactions.
• The five coordinate Fe(II) can add O2 in its sixth vacant coordination site, and also a variety of other
small ligands (CO,NO,RCN). When O2 is bound, it is stabilized by hydrogen bond interactions through
the distal Histidine (his64, E7). The hydrogen bond interaction may affect O2 affinity and inhibit
pathways leading to further oxidation and μ-oxo dimerization.
12. • Despite the many model compounds that have been prepared, picket fence porphyrin, fist reported by
Collman research group in 1974, remains the only porphyrin type ligand system yielding crystallographic data
comparable to Mb oxyheme stereochemistry.
Distal histidine hydrogen bonding structure for hemoglobin (left) and a heme model
(right)
13. Selective Binding of O2 over CO in Hemoglobin
and Myoglobin
The Selective binding of O2 rather CO in wild type biological systems is complicated by the fact that
naturally occurring metalloprotein include Hemoglobin and Myoglobin produce CO during degradation
process. Therefore, hemes must be able to carry out their oxygen transport and storage functions in
presence of significant concentration of CO. In addition, CO binds to Mb and HB with affinity of respectively
25 and 200 times those of O2. The discrimination (As suggested by Collmen) is based on steric hindrance
constrains imposed by amino acids residues on distal side of porphyrin and by selective Hydrogen bond
favoring O2 over CO. O2 is capable of bent geometry when bound to heme facilitating the Hydrogen bond
to distal histidine, whereas CO preferred linear binding mode not only prevents hydrogen bond formation
but also results in steric clashes with neighbouring amino acids. For synthetic system of picket-fence,
Pocket, capped porphyrin, CO affinity exceeds that for O2 sometimes by many orders of magnitude.
Also for small-molecules, metal-carbon monoxide complexes, the carbon monoxide ligand is almost always
in a linear conformation and perpendicular to metal. If one assumed bonding of CO to Hb or Mb in its
normal linear perpendicular mode then steric conflicts would occur and hence CO binding would be less
favoured.
14. Functions of Myoglobin and Hemoglobin
• Hemoglobin transports oxygen from lungs to tissues, lungs are the loading point of oxygen in hemoglobin
because partial pressure of dioxygen here is more and unloading point is tissue because dioxygen partial
pressure is less here. Also, it has a essential role as carrier because the solubility product of dioxyegn is
quite low in water based plasma and hemoglobin makes increases the solublity my many folds, therefore
acting as a efficient carrier
• Hemoglobin also transports Carbon Dioxide back to lungs and also maintains acid base balance in blood (As
a buffer).
• The hemoglobin gives the blood its characteristic Red colouration. The hemoglobin acts as a physiologically
active catabolite, It plays a crucial role in erythrocyte metabolism
• Myoglobin has a strong affinity for binding for oxygen, which makes it able to store it effectively in muscles.
• Myoglobin helps body at the starving situation of oxygen, especially in the anaerobic situation.
• Myoglobin helps in regulating body temperature.
16. References:-
1. Lippard, Principles of Bioinorganic Chemsitry, 1st Edition
2. Leninger, Principles of Biochemistry, 3rd Edition
3. Rossette, Bioinorganic Chemistry-A short course, Wiley, 2nd Edition
4. Britannica.com, Hemoglobin and Myoglobin
5. Wiley Online Library, Foundation course in Biochemistry
**Slide Backgrounds, Cover Image and illustrations used are and shall remain property of their
respective owners.
