Transcript No Slide Title

Bio 98 - Lecture 7
Oxygen Binding Proteins
Myoglobin and Hemoglobin
(Mb)
(Hb)
Tetrapyrrole ring system
Protophorphyrin IX
Octahedral coordination
Heme biosynthesis
Octahedral coordination
Werewolves?
Erythropoietic porphyrias are associated with accumulation of porphyrins in
erythrocytes and are rare. The rarest is congenital erythropoetic porphyria (CEP)
otherwise known as Gunther’s disease. The signs may present from birth and
include severe photosensitivity, brown teeth that fluoresce in ultraviolet light due to
deposition of type I porphyrins and later hypertrichosis. Hemolytic anemia usually
develops.
Recent use: the comic book Ultimate Comics Avengers Vol. 3 #1 (October 2010), in
which writer Mark Millar employed porphyria as the explanation for vampires in
that fictional universe.
C
His64
His93
N
I. Similarities and differences
Similarities
• polypeptide length (~140 residues) & sequence (27 residues /
20% identical) are similar
• presence of heme; mechanism of O2 binding
Differences
Location:
Subunits:
Function:
O2 source:
O2 destination:
O2 binding:
Mb
muscle
1
O2 reserve
blood
mitochondria
simple
hyperbolic
Hb
blood
4
O2 transport
lung
periphery
complex
sigmoidal
(cooperativity)
II. O2 binding curves
A. O2 binding curve for myoglobin (Mb)
1.0
Y
(fraction of
Mb with O2
bound, aka
Mb:O2)
0.5
0
[O2]0.5
[O2]
How does the availability of oxygen or [O2] influence
the amount of Mb that has oxygen bound to it?
Y = fraction of ligand binding site occupied (Mb:O2)
[Mb:O2]
(1) Y = ——————— =
[Mb:O2] + [Mb]
bound Mb
—————
total Mb
An equation of the form f(x) = x/(x + z) describes a hyperbola
(2) binding rxn: Mb:O2
Mb + O2
[Mb] [O2]
Kd = ————— = dissociation constant
[Mb:O2]
(just like the Ka of a weak acid)
Solve eq. 2 for [Mb:O2], then substitute into eq. 1 ...
[Mb] [O2]/Kd
(1) Y =
—————
[Mb] [O2]/Kd + [Mb]
[O2]
(3) Y = —————
Kd + [O2]
Mb:O2
[Mb] [O2]
=
—————
[Mb] (Kd + [O2])
Another name for Kd is [O2]0.5
Mb + O2
Kd = [O2] when Mb is half-saturated (Y = ½),
i.e. when [Mb] = [Mb:O2].
At this condition, Kd = [O2], often written as [O2]0.5
Use pO2 (partial pressure) in place of [O2]
[O2]
(3) Y = —————
[O2]0.5 + [O2]
Y
pO2
Y = —————
P50 + pO2
mitochondria
Buffering of O2: Mb:O2
Mb + O2
blood
B. O2 binding curve for hemoglobin
Hb has a different function - transport, not buffering
1. Hb needs to pick up O2 efficiently in the lung where
pO2 is high (100 mm Hg or Torr = 13.3 kPa).
(1 Torr = 0.133 kPa)
2. Hb needs to drop off O2 efficiently in the tissues
where pO2 is lower (30 mm Hg = 4 kPa).
3. Normal or “hyperbolic” binding won’t suffice; not
enough difference in saturation when pO2 drops to
only ~1/3 the maximal value (30 vs. 100 mm Hg).
Simple (hyperbolic) myoglobin-like binding
does not suit a transport protein well.
1
8%
P50 = 2.8 mm Hg
0.8
P50 = 26 mm Hg
25%
0.6
[mm Hg]
pO2 in
lung >
Y
q
0.4
< pO2
in
tissues
0.2
0
0
20
40
60
80
pO2 [mm Hg]
100
120
The state with a higher affinity for O2 has a lower P50 value (aka
[O2]0.5) compared to the P50 value of the lower affinity state.
Sigmoidal (cooperative) binding does suit a transport
protein well (Hb, magenta curve)
1
R-form
0.8
55 %
0.6
Y
q
T-form
0.4
0.2
pO2 in
lung >
< pO2 in
tissues
0
0
20
40
60
80
p O2 [mm Hg]
100
120
Change in conformation of heme
as Hb goes from T -> R state
His93
His93
Val68
His93
Val68
T (Tense): lower affinity for O2 (deoxy state)
R (Relaxed): higher affinity for O2 (oxy state)
Val68
Change in conformation of subunits in
tetramer as Hb goes from T -> R state
Hb is an allosteric protein whose properties are affected by changes in
quaternary structure, which are mediated by interactions with small
molecules: i.e. O2 (and other effectors such as pH, see last two slides)
III. Models of Cooperativity
A. A simple 2-subunit model
B. 4-subunit models (Hb)
1. Concerted model
2. Sequential model
A simple 2-subunit model of cooperative ligand binding
B.1: Concerted model of the cooperative
transition of Hb subunits: symmetry
T (Tense): lower affinity for O2 (deoxy state)
R (Relaxed): higher affinity for O2 (oxy state)
Monod, Wyman, Changeux
B.2: Sequential model
of the cooperative
transition of Hb subunits:
induced fit
T (Tense): lower affinity for O2 (deoxy state)
R (Relaxed): higher affinity for O2 (oxy state)
Some oxygen bound. Each
binding of an oxygen molecule
favors the transition of adjacent
subunits to the strong-binding
state and promotes their
binding of oxygen
More oxygen bound. More and
more subunits next to oxygenoccupied sites are switching to the
strong-binding state.
Koshland, Nemethy, Filmer
Bohr Effect
blood pH alters affinity of Hb for O2 in a useful way
tissue
70 %
Y
40 %
lung
In the lungs, [CO2]
is reduced (pH 7.6)
compared to tissues
(pH 7.2)
CO2 + H2O
H+ + HCO3-
H+ and CO2 both bind to
Hb and stabilize the T
(deoxy) state:
H+ and CO2 are
allosteric effectors
BPG (2,3 bisphosphoglycerate)
helps us adapt when climbing Mt. Whitney!
BPG binds to positively charged
groups stabilizing the T (deoxy)
state.
Y
Increasing [BPG] lowers [O2]
affinity, i.e. increases the P50.
As you climb to higher altitude
your body adapts to lower pO2 by
increasing [BPG] from 5 to 8 mM.