Lecture 8 - Proteins in Action - Oxygen transport by Haemoglobin & Myoglobin (Allostery & Cooperatively in Haemoglobin) Flashcards
Haemoglobin shape
Haem and Fe2+ in middle connected to helix by His F8
Deoxyhaemoglobin has dish shape
Oxyhaemoglobin has flat shape
Oxygen flatten haem and pulls His F8 and helix F towards binding site.
Anything that keeps helix F away will weaken oxygen binding.
R state
Relaxed
Oxyhaemo
Active
T state
Taut state
Deoxy
Stretched tight
Not as active
R- and T-states stabilised
by
steric interactions
Alternate side-chain packing in R- and T-states
Helices
F and C move one
turn past one another.
Haemoglobin is under
allosteric control of 2,3-bisphosphoglycerate (BPG)
Helices F and C move one turn past one another which …
Allows one sub unit to communicate with each other
Subunits are shifting relative to each other to get communication between them
2,3 BPG
Product of aerobic metabolism
Signal that a tissue is using a lot of oxygen.
Binds in site in middle of haemoglobin
-‘ve charge molecule, carboxyl group, 2 phosphate groups
BPG
Binds (stabilizes) to deoxy in T state by electrostatic interaction
Reduces oxygen affinity
Produced during respiration in peripheral tissues (conc higher in muscle than in lungs it will dump oxygen in tissue, and pick it up in the lung), promotes oxygen release where it’s needed
Cooperativity allows
efficient ‘unloading’.
Monomeric myoglobin has a
hyperbolic binding curve.
Cooperative, tetrameric haemoglobin has a
sigmoidal binding curve.
YO2 =
fraction of protein bound to O2
MWC, concerted model
For tetramer all subunits in T state or all in R state
Binding each substrate (S) shifts equilibrium in favour of R.
Subunits in a
low-activity, tense (T) or
high-activity, relaxed (R) conformation.
T state deoxyheme
R state oxyheme
monod, wyman, changeux
MWC, concerted model
Inhibitors (BPG) stabilise…
T state
MWC, concerted model Activators stabilise (push towards)...
R state
KNF, sequential model
One substrate binding induces a T to R conformational change in one subunit.
conformational change influences neighbouring
subunits (i.e. cooperativity), making them more likely to
bind substrate. Many conformations possible.
Explains negative cooperativity.
Concerted model describes
haemoglobin
Sometimes weaker binding is
better
The first adaptation to high altitude increase BPG
shifts curve to right as it delivers more oxygen to the tissues.
Reduce Haemoglobin oxygen binding
pH
Works through the binding site of BPG
Drop in pH favours BPG binding which favours oxygen release,
Higher pH in lungs
Lower pH in tissues
H+ bind to haemoglobin releases Oxygen
CO2
reduces O2 affinity, both directly and via lowered pH of blood.
binds to hemoglobin at the extreme N terminus
Influences BPG binding
Bohr effect
Increase co2 and decrease pH (increase H+)
decrease affinity of haemoglobin for O2,
what contributes to stabilising
the deoxy-Hb conformation.?
CO2 bind to extreme N-
terminal amino group
H+ protonate certain amino acid side chains
O2 binding to Hb at lower pH
less tightly
Cooperativity is prominent (important) only in presence of…
Allosteric inhibitors
what Allosteric inhibitors stabilise T-state and unmasks
cooperativity?
BPG
CO2
H+
What state is stripped hemoglobin absence of inhibitors in?
R state
Little cooperativity
Do Foetuses hold oxygen tighter than adults?
yes
Response to high altitude
Increase BPG
Decrease Hemoglobin O binding
Increase O delivery to tissues
Foetus hemoglobin have different isoforms that Replace alpha and beta subunits found in adult hemoglobin
what are they?
ζ (Zeta)
ε (Epsilon)
γ (Gamma)
Better at binding O than adult isoforms
Foetal isoforms
Better at binding O
Pick up O from placenta
Lack +’ve charge amino acids in BPG binding site
Doesn’t bind BPG well
Remains more in R state than T state
Isoforms
Genetically different forms of an enzyme
Methaemoglobin
Oxidation of haem Fe2+ to Fe3+
shifts one subunit to the R state conformation, without oxygen bound.
Methaemoglobin Impairs function two ways:
binds oxygen less well.
The other subunits of the tetramer are shifted to the R-
state, so do not release oxygen in the tissues as they should.
what does enzyme cytochrome b5 reductase do?
regenerates haemoglobin by reducing methaemoglobin back to Fe2+ state with transfer of electrons from NADH.
HbM, or Boston haemoglobin
His E7 mutation to TyrE7
- cause Fe2+ to Fe3+
heme plane moves slightly & Breaks connection on Fe - His F8
what state does HbM remain in?
T-state
low affinity for oxygen
HbS, or sickle cell haemoglobin
‘sickle’ shape RBC get
stuck in blood capillaries
What gain of function mutation does HbS have?
Hb b6 Glu to Val mutation
enables abnormal hydrophobic
interaction between Hb molecules, when in deoxy form,
cause polymerisation of Hb into chains that distort the red blood cells
HbS
Hb β6 Glu to Val mutation
Hydrophobic on adjacent hemoglobin molecule build long fibres of hemoglobin
Long chains of tetramers associated with one another
Mutant val 6 sits within hydrophobic pocket that exists on all Hb molecules
Distorts RBC shape
Haemoglobin function
Shifts tetramer to T state
Oxygen binding weakened allosterically by BPG, CO2
and low pH.
When shifted haemoglobin shows cooperative
binding of oxygen, shown in a sigmoidal binding curve.
Whats shown in sigmoidal binding curve?
When shifted haemoglobin shows cooperative
binding of oxygen
How does R and T state differ in Hemoglobin?
helix F interacts with
haem and helix C, and spacing between H helices.
Physiological effects
- Oxygen affinity is tuned in pregnancy and at high altitude.
- Mutations to haemoglobin impair oxygen transport.
- Sickle-cell anaemia results from haemoglobin polymerisation.
