Download Chemical Biology 03 BLOOD

Chemical Biology 03
BLOOD
Biomolecular Structure
Myoglobin and Hemoglobin
9/28-30/09
www.optics.rochester.edu/.../image007.gif
Chemical Biology 03
BLOOD
Biomolecular Structure
Myoglobin and Hemoglobin
Lecture 9 and 10: 9/28-30/09
The biochemistry of O2 binding to Hb
Each of the four subunits of Hb has a central helix which binds a heme with
binds an iron ion (Fe(II) which in turn binds O2.
O2 exchanges from Hemoglobin (Hb) to
Myoglobin (Mb) in the tissue
tissue
lungs
lungs
•Oxygen binding to Hb decreases as
oxygen pressure decreases.
•Oxygen is released at the tissue, and
myoglobin grabs it up.
pO2 in air ~100
torrs
O2 Exchanges from Hb to Mb
• HEMOGLOBIN
• MYOGLOBIN
•Crystal structure is very complicated.
•Crystal structure is very simple
•Hb protein is four subunits, four heme
groups, and seems to behave
differently when all together as
compared with monomers.
•Oxygen saturation curve is “sigmoidal”
complicated mathematical formula.
O2
•Mb protein is one subunit, one
heme, and behaves simply
•Oxygen saturation curve is
hyperbolic, which mathematically is
quite simple y= x/(a+x)
So, let’s first try to understand Mb!!!
Myoglobin Structure: O2 Binding
• Mb is the oxygen storage
protein in muscle
• 153 amino acids
• 17,000 g/mole
• Protein has 8 helices A-H
• Heme Fe(II) is bound through
side group N of His F8
(proximal his)
• His E7 is close to the other
side of heme, but doesn’t
coordinate (distal his)
Myoglobin Structure: O2 Binding
Proximal His
Distal His
• Fe(II), iron is bound to four N
atoms within heme
• 5th coordination is N of
Histidine amino acid, F8 of helix
F.
• 6th coordination site is open for
O2 to bind.
• deoxyMb, deoxyHb, heme ring
is puckered in absence of 6th
ligand; Fe(II) out of plane.
• oxyMb, oxyHb ring is flat with
sixth ligand bound to Fe(II),
metal is in heme plane.
Myoglobin Structure: O2 Binding
Heme group is a really special molecule
made up of carbon, hydrogen, and
nitrogen, with a big fat iron atom sitting
in the center waiting to bind to the O2
The biochemistry of oxygen binding
Here’s a cartoon of what happens when the O2 binds to the Fe in Myoglobin
Myoglobin Structure: O2 Binding
N from distal His
N from proximal His
– Effect of sixth site coordination on the color of myoglobin.
– Oxygen does not bind straight on, the N from the distal His amino acid
side group in the way.
Myoglobin Structure: O2 Binding
• Space filling model of
myoglobin with His F8
coordinating and His E7
poised nearby
• See how little room
oxygen has to snuggle in
and bind to the Iron.
• Heme is bound in a
hydrophobic crevice with
propionic acid groups
projecting into solution
orienting the heme.
A Tale of Two Binding Curves
Myoglobin
•getting the dissociation constant from
the saturation curve
Hemoglobin
• sigmoidal saturation curve
• two state model (T and R)
• O2 binding is cooperative
Myoglobin: O2 Binding
•
What really happens in the binding
(association) and unbinding (disassociation)
of O2 to Mb?
Mb-O2 ⇌ Mb + O2
equilibrium described by the extent to
which the dissociation occurs, measured
by a Kd
•
We can write an equation for this
equilibrium:
Kd = [Mb]free [O2] / [Mb-O2]
Kd = [Mb]free [pO2 ]/[Mb-O2]
[Mb]free = [Mb]T - [Mb-O2]
Kd = ([Mb] - [Mb-O2])(pO2 )
[Mb-O ]
T
2
Myoglobin: O2 Binding
Mb-O2 ⇌ Mb + O2 equilibrium
Kd = ([Mb]T - [Mb-O2])[pO2 ]
[Mb-O ]
2
Now, just allow one more substitution
and a rearrangement, and we’ll get
someplace really great!
Y = fractional saturation
Y = [Mb-O2] / [Mb]T
Y = pO2 / (Kd + pO2)
WOW, that’s a lot simpler!
Let’s EXCEL
together
Conclusions from excel about O2 binding to Mb
Hemoglobin: O2 Binding
• Like Mb, Hb’s saturation
decreases as pO2 decreases.
Y = O2/ (Kd+ O2)
Y = O2n/ (Kd+O2n)
• Unlike Mb, Hb at the same
pO2, say 10 torr, Hb is much
less saturated. If O2 was bound
it would come off.
• Unlike Mb, Hb binding curve
is not hyperbolic, but
sigmoidal.
• Sigmoidal shape suggest two
states for Hb (more on this in a
minute)
Let’s EXCEL
together
Conclusions from excel about O2 binding to Hb
Hb Structure: (ab)2
– Hemoglobin is a dimer of dimers, a1 b1 dimer a2 b2
– see http://www.umass.edu/microbio/chime/hemoglob/2frmcont.htm
Hemoglobin: Two states for O2 Binding
http://www.search.com/reference/Hemoglobin
R state relaxed
T state tense
high affinity for O2
Low affinity for O2
Hemoglobin: Two states for O2 Binding
http://www.search.com/reference/Hemoglobin
R state relaxed
T state tense
high affinity for O2
Low affinity for O2
Hb: Describing O2 Binding
Sigmoidal binding suggests
• Two state model: Hb can be in
either
• high O2 affinity (R state)
• low O2 affinity (T state)
• Binding of O2 to Hb is
cooperative: binding of first
ligand affects the affinity of the
remaining sites for ligand.
Hemoglobin Cooperativity
T state deoxy
NOTHING
R state oxy
ALL
Hemoglobin: ribbon structure