Transcript Lehninger Principles of Biochemistry 5/e

Protein Function
Myoglobin & Haemoglobin
© 2008 W. H. Freeman and Company
Spectroscopic Detection of Oxygen Binding to Myoglobin
The heme group is a strong chromophore that absorbs both in ultraviolet and
visible range
Ferrous form (Fe2+ ) without oxygen has an intense Soret band at 429 nm
Oxygen binding alters the electronic properties of the heme, and shifts the position
of the Soret band to 414 nm
Binding of oxygen can be monitored by UV-Vis spectrophotometry
Deoxymyoglobin (in venous blood) appears purplish in color and oxymyoglobin (in
arterial blood) is red
Binding: Quantitative Description
Consider a process in which a ligand (L)
binds reversibly to a site in the protein (P)
P
+
ka
L
The kinetics of such a process is described by:
association rate constant ka
kb
PL
the
the dissociation rate constant kd
After some time, the process will reach the equilibrium where
the association and dissociation rates are equal
The equilibrium composition is characterized by the the
equilibrium constant Ka
ka [P] [L]  kd [PL]
ka
[PL]
Ka 

[ P ]  [ L] k d
Binding: Analysis in Terms of the Bound Fraction
• In practice, we can often determine the
fraction of occupied binding sites

[P L]
[P L] [P]
• Substituting [PL] with Ka[L][P], we’ll
eliminate [PL]

K a [L][P]
K a [L][P]  [P]
• Eliminating [P] and rearranging gives
the result in terms of equilibrium
association constant:
• In terms of the more commonly used
equilibrium dissociation constant:

[ L]
[ L] 

1
Ka
[L]
[L]  K d
Binding: Graphical Analysis
The fraction of bound sites depends on the free ligand
concentration and Kd
In a typical experiment, ligand concentration is the known
independent variable
Kd can be determined graphically or via least-squares
regression

[L]
[L]  K d
[L]  [L]total
Binding: Thermodynamic Connections
Interaction strength can be expressed as:
– association (binding) constant Ka, units M-1
– dissociation constant Kd, units M, Kd = 1/Ka
– interaction (binding) free energy Go, units: kJ/mol
Definitions:
– Go = Ho -TSo : enthalpy and entropy
– Ka = [PL]/[P][L]
Kd=[P][L]/[PL]
Relationships:
– Go = -RT ln Ka = RT ln Kd
Magnitudes
– Strong binding: Kd < 10 nM
– Weak binding: Kd > 10 M
(RT at 25 oC is 2.48 kJ/mol)
Salt bridges and Hydrogen Bonds in Hemoglobin
All these interactions abolished in transition from deoxy to oxy Hb
Mutant Hemoglobin s
What can be learned from experiments of nature?
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Mutant hemoglob ins provide unique opportunities to probe structurefunction relations in a protein.
There are nearly 500 known mutant hemoglobin s and >95% represent
single amino acid substitutions.
About 5% of the population carries a variant hemoglobin.
Some mutant hemoglobin s cause serious illness.
The structure of hemoglobin is so delicately balanced that small changes can
render the mutant protein nonfunctional.
Mutations that lead to loss of heme
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Heme prosthetic group can be dislodged by mutations in heme binding
pocket.
In Hb Hammersmith [Phe(42)Ser] the normal Phe in HbA at position
42 in  chain is converted to Ser.
In HbA, Phe blocks access of water to the heme pocket,
but smaller, polar Ser in Hb Hammersmith allows water to enter heme
pocket.
This causes heme to be dislodged from pocket,
producing a nonfunctional protein.
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In Hb Kansas [Asn(102)Thr] a critical hydrogen bond in the 1-2
interface between 2-Asn(102) and 1-Asp(94) that stabilizes the R state
is lost.
Thus the R-T equilibriu m is shifted toward the T state.
Hb Kansas has low O2 affinity (P50 = 70 mm Hg = about 9.3 kPa).
Little cooperativity in O2 binding (Hill coefficient = 1.3).
1 subnit is red and 2 subunit is green
stabilizes the Fe+3 state
protects the iron from becoming oxidized, has been replaced by Glu, which
This is Hb Milwaukee [His(87)Glu] in which the distal His, which normally
Crocodile Hemoglobin
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Crocodiles can remain under water for up to one hour and usually kill
their prey by drowning them.
How do crocodile's tissues get O2 while submerged ?
Unlike deep diving whales, which use myoglobin to store O2 in muscle,
crocodiles rely on a unique allosteric effector of Hb to ensure delivery of
O2 to tissues.
While submerged, metabolism produces CO2, which is converted to
HCO3 and it is the HCO3 that acts as the allosteric effector by binding
to residues at the 12 interface in deoxy Hb and stabilizing the deoxy
Hb.
This unique allosteric effect is only found in crocodile Hb.
Crocodile Hb does not bind BPG due to mutations in the BPG-binding
pocket