Biochemistry

1. Myoglobin and Hemoglobin
By
Mrs Sanchita Choubey
(M.Sc., PGDCR, Pursuing Ph. D)
Assistant Professor of Microbiology
Dr. D Y Patil Arts Commerce and Science College Pimpri, Pune

2. Myoglobin & Hemoglobin
Objectives
 Identify biological functions.
 Identify parts of Mb & their roles in
O2 transport.
 Identify how Hb differs from Mb.

3. Myoglobin
 O2 transport, storage
in cells.
 Two parts: protein
and heme prosthetic
group.
 Protein:
 155 amino acids, ~ 17
kDa.
 Compact, globin fold.
 75% helix.

4. Heme
 Heme is composed of porphyrin and Fe2+.
 Porphyrin is non-polar, except two proprionate groups.
 Porphyrin binds O2.

5. Heme Ligands
 Fe is 6-coordinate.
 Four N of heme group
 One N from proximal His.
 One from H2O or O2.

6. Heme – Protein Interaction
 Heme stabilizes
protein fold.
 Binds through hf
interactions
 Prooprionate groups
on surface.
carboxyl group of
proprionate.

7. Myoglobin
 Protein completely
surrounds heme.
 Function of protein:
 ↑ heme solubility.
 ↓ heme oxidation
(Fe2+ → Fe3+)
 metmyoglobin inactive.
 ↓ CO binding.

8. O2 vs. CO2 binding.
 CO binds
tightly; linear.
 O2 binds less
tightly, bent
structure.
 Distal His forces
bent binding of
both, weakens
CO2 binding.
Distal His
Proximal
His
Fe
C
O
Fe
O
O
Fe
C
O

9. Hemoglobin
 Tetrameric protein
 Dimer of dimers, (ab)2
 a,b chains resemble Mb.
 Each chain contains, heme,
prox. histidine.
 Each binds 1 eq. O2.
 ab dimer interface different
from aa, bb interface.
 Marked by salt bridges that
stabilize the deoxy structure.

10. 0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100 120
pO
2,
mm Hg
Y,
fractional
saturation
4° Structure of Hb alters O2 binding.
 Interactions between
dimers alters oxygen
binding.
 Direct plot shows Hb
has lower affinity than
Mb.
 Sets up delivery
system.
 O2 bound by Hb in
lungs; released in
tissues.
Mb Hb

11. Cooperative regulation
Hb oxygen binding:
 Start binding with given affinity in deoxy state, subsequent binding
enhances affinity.
 Defines positive cooperative regulation.
 Only seen in multi-domain proteins.
 Hill coefficient ~ number of interacting subunits. Advantage: binding
is more sensitive to small changes in [ligand].

12. Molecular Model
 In deoxy state, Fe out
of heme plane;
domed.
 Bind O2, moves Fe.
 This moves proximal
His and its helix.
 Moving helix alters
a/b interface.
 Deoxy = Tense (T)
 Oxy = Relaxed (R)
Fe
N N
N
O
O

Fe
N N
N

0.7A
His-F8
proximal side
distal side
N
N
C
O
Fe
Fe
CO
F Helix.
T
R

13. H146 D94
Bohr Effect
 Oxygen affinity sensitive to pH.
 ↓ pH; ↑ pO2,50 (lowers sensitivity).
 D94 ↔ H146 salt bridge in T
state only.
 Excess H+ forms salt bridge,
favors deoxy state.
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100 120
pH = 7.5
pH = 7.2
pH = 6.8
log pO2
log
Q

14. CO2
 Produced during aerobic
metabolism.
 Reacts with N terminal
amino; carbamylation
reaction.
 Negative charge forms salt
bridge with aR141,
stabilizes deoxy state.
R-NH2 + CO2 R-NH-CO2
- + H+
In tissues,
High CO2
In lungs,
Low CO2
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100 120
pH = 7.5
pH = 7.2
pH = 6.8
log pO2
log
Q

15. CO2 is Coupled to Bohr Effect
In Tissue
 CO2 is bound by Hb or
converted to bicarbonate by
carbonic anhydrase.
 Buffers blood pH.
 Hb binds 2H+ / 4 O2 released, also
buffers (Bohr effect).
In Lungs
 Low pCO2; reaction reverses;
 CO2 and H+ released from Hb,
 pO2,50 decreased (increased oxygen
affinity).
From Lange’s Biochemistry

16. 2,3-BPG
 Side product of
glycolysis.
 indicates active
respiration, need O2.
 Binds cationic region in
T-form.
 Favors deoxy, releases O2
to tissues.
 [2,3-BPG] is high,
responsible for observed
pO2,50 of 27 mm Hg.
 stripped Hb has pO2, 50 ~
8 mm Hg.

17. Another look at 2,3-BPG
 BPG acts as a
“wedge” and drives
the R state to the T
state.
 Forces release of
bound O2 in active
tissue.
 BPG increases at
high altitude.