Myoglobin and Haemoglobin Flashcards
When were the structures of Mb and Hb first determined?
Mb : 1958
Hb : 1960
What are the functions of Mb?
increases oxygen solubility in the cytoplasm and facilitates local diffusion
detoxifies No to NO3-
acts as a local store of oxygen in skeletal + cardiac tissues
How do you describe the structure of Mb?
e.g. sperm whale 17.3kDa
1ry : 154 amino acids
2ry : 8 alpha-helices A to H with connecting loops
3ry : ‘globin’ fold
4ry : tightly bound heme prosthetic group 616 Da (C34H32O4N4Fe)
What is the heme group structure?
porphyrin ring bound to iron
Where does the prosthetic group bind in Mb and Hb?
to deep pocket on the molecule with hydrophobic portion to the inside, which is crucial to stability of protein
if prosthetic group is removed, molecule begins to unfold
what is the coordinate geometry of iron bonding?
octahedral: 4 equatorial bonds and 2 axial bonds
what are the 2 axial positions occupied by?
proximal histidine: F8 (residue 8 in helix F)
the place where O2 coordinates to Fe
What is near to the coordination place for O2 at the axial position?
distal histidine: E7 (does not coordinate to Fe but is highly conserved and is close enough to Fe to form a H bond)
How does the distal histidine E7 modulate the relative affinities of CO and O2?
discriminates between the binding of 2 molecules
blocks CO from edge-on binding to Hb, decreasing CO affinity for Hb
his E7 can form H bonds, increasing O2 affinity simultaneously
how do we know that the E7 histidine is crucial for modulating relative affinities of CO and O2?
If the E7 His is changed in alpha subunit of Hb to Gly, oxygen affinity for Hb decreases as H bonds cannot form to stabilise the O2-Hb complex
and CO has 7000 fold higher affinity than O2 for this subunit
where does the O2 bind to Mb? why?
sequestered into the heme-pocket
prevents formation of heme-O2-heme sandwich that would promote Fe oxidation to Fe 3+
allows distal histidine to block CO and promote O2 binding over CO
what is the fractional oxygen saturation (Y) equation for Mb, and the shape of the oxygen loading curve?
Y = [O2]/Kd + [O2]
hyperbolic (Michaelis Menten stylez)
define Kd
the [O2] at which half of the sites are occupied
(known as p50)
does Mb form dimers?
can create a domain-swapped dimer
and the binding cleft will still accept O2,
but the CO binding constant (affinity) doubles since the binding site is expanded and CO cannot be blocked as effectively
what does a lower P50 indicate?
what is Mb’s?
stronger O2 binding
very low
Does Mb bind oxygen tightly?
yes
at high/intermediate pO2 it will be almost completely saturated (y close to 1)
what is required to unbind O2 from Mb?
very low pO2
why does Mb not oligomerize like Hb?
idk bro
what is the brief comparison of structure + function between Hb and Mb?
Mb = monomeric protein w singular binding site buried deep in hydrophobic pocket. acts as store of oxygen in skeletal + cardiac tissues
Hb = 4 subunits and 4 oxygen binding sites that sit in less occluded positions, whose oxygen binding affinity can be affected by the previous binding of O2 molecules
describe the detailed structure of Hb
tetrameric dimer of alpha-beta dimers
each subunit similar in structure + fold to 1 Mb
what is the function of Hb?
binding oxygen in alveolar capillaries and delivering it to areas of high demand in tissues (muscles, kidney, brain)
what happens if you remove a prosthetic group?
Hb and Mb become very unstable and begin to unfold
which model of cooperativity is used to explain Hb binding?
the concerted model explains the actions and structural changes since all subunits appear to change conformation from R to T at the same time
what is the mechanism by which Hb exhibits cooperative binding?
mechanical coupling exists between O2 binding sites
O2 BS on subunit 1 pulls proximal histidine attached to it (1ry), which affects F helix (2ry)
FG turn at quaternary interface with another subunit on a different dimer (4ry) causes the subunit to twist ~15 degrees relative to the other : molecule enters R state
transmits conformational changes across the molecule, increasing binding affinity for subsequent O2 molecules
which state (R or T) has high/low affinity for oxygen?
low affinity = T
high affinity = R
how does the sigmoidal curve arise?
the averaging of 2 hyperbolic curves, 1 shallow (T) and 1 steep like Mb (R)
a low conc the sigmoidal curve tracks the T curve and at high conc [substrate] it tracks the R curve
describe the sequential model of O2 binding?
each ligand-binding event affects the affinity of the neighbouring subunits for O2, making it easier for O2 to bind to the next subunit
[Koshland]
describe the concerted model of O2 binding
high and low affinity states exist in dynamic equilibrium without O2 being present. binding of the ligand stabilises high affinity (R) more than low affinity (T)
the R state therefore becomes more strongly favoured the more O2
describe the concerted model of O2 binding
high and low affinity states exist in dynamic equilibrium without O2 being present. binding of the ligand stabilises high affinity (R) more than low affinity (T)
the R state therefore becomes more strongly favoured the more O2 binds
implies proteins can switch freely between T and R state, with T state predominating in the absence of ligand
[Monod, Wyman, Changeux]
can a ligand bind to an active site on a subunit in either conformation?
yes
but only the conformational change alters the affinity of a binding site for the ligand.
the regulators merely shift the equilibrium toward one state or another.
what is another word allosteric unit
protomer/subunit
how does the concerted model explain the sigmoidal curve/positive cooperativity?
change in concentration of ligand over a small range will lead to more ligand binding
and therefore a large increase in the proportion of molecules in the R state as it is favoured due to high affinity for O2,
and thus will lead to a high association of the ligand to the protein.
how does Hb’s functions determine its oxygen affinity requirements?
requires very sharp variation in oxygen affinity
needs to be able to transport oxygen over long distances, needs high saturation in the lungs and very low saturation in the capillaries
how does Mb’s functions determine its oxygen affinity requirements?
main purpose is short distance release of O2 to muscles during exercise
can have very high percentage O2 saturation even at comparatively low partial pressures to Hb
based on what factors can Hb change its affinity for oxygen? (what influences T/R state equilibrium)
[CO2], pH and 2,3-bisphosphoglycerate
what does Hb’s affinity for O2 heavily depend on?
subunits
e.g. gamma in fetal Hb
what happens in thalassemias?
missing gene for alpha or beta Hb, or impaired RNA synthesis
alpha = some oxygen still transported
beta = very bad
what happens in alpha thalassemia?
1 of alpha genes i misisng
only beta Hb left: homotetramer: stable on its own but binds O2 with very high affinity so doesn’t exhibit cooperativity: transport efficiency lower
what happens in beta thalassemia?
only alpha Hb left: unstable on its own: oxidises and precipitates
why are low levels of free alpha-Hb normally tolerated?
exists bound to alpha-Hb stabilising protein
F-helix disordered, Fe coordinated not with proximal F8 his but distal E7
Heme has O2 bound to other side of the plane
what are the 2 types of allosteric modulators?
homotropic: modulating agent is equivalent to the substrate (O2 binding affects O2 binding)
heterotropic: modulating agent differs from substrate (ligand binding affects O2)
how does a heterotropic modulator work?
has 2 regulatory domains with 2 catalytic domains bound to them
when you bind cAMP to a site not at the interface between regulatory and catalytic domains, causes conformational change that removes pseudo substrate sequence, releases catalytic domain and makes protein active
how can we sense a pH change? over what range?
sense a change in protonation state above or below the physiological pH (~7.2 in active respiration)
use histidines and N termini
what does addition of H+ change? what does this allow us to do?
charge: can remove/introduce electrostatic interactions
under what circumstances does the oxygen dissociation curve of Hb shift left? what does this mean for O2 affinity?
lower pCO2
lower 2,3-BPG
lower temperature
increased pH
[fetal Hb]
leads to increased affinity for O2
under what circumstances does the oxygen dissociation curve of Hb shift right? what does this mean for O2 affinity?
higher pCO2
higher 2,3-BPG
higher temperature
decreased pH
leads to decreased affinity for O2
what happens with the boston mutation in Hb?
promotes methaemoglobin formation
(metHb contains Fe3+ which doesn’t allow for reversible binding)
what histidine is used as the marker for pH? why?
H146
forms ionic bond to Asp94 in FG1 loop in the T state
whether or not the H+ binds to H146 dictates whether H146 is positive and whether this ionic bond formation is favourable (if bound is favourable)
in the R state it flips out to interact and make an ionic bond with lysine
this H146 forms a salt bridge in the T state but in the R state, terminal histidines flip into centre and 2 histidines from beta subunits get close together and favour shift to T state on protonation
how does CO2 affect O2 binding affinity?
- linked to pH of respiring tissue: carbonic acid, H+, decreases pH in RBC as bicarbonate can leave on transporters but H+ cannot
- binds to amino terminus of val1 of alpha subunit, changing the charge from +ve to -ve
in T state, carbamate terminal of Val1 can then interact with +ve arg141 of other alpha subunit to stabilise T state and decrease oxygen affinity
does Hb in RBCs bind more or less tightly to O2 than purified Hb? why?
purified binds much more tightly
due to shift in p50 due to 2,3-BPG
from where does Hb allostery arise? what does this mean?
from inter-subunit interactions
changing subunits can be exploited to change Hb properties, such as in development
what do subunit differences in fetal Hb allow?
the flow of O2 from mother to fetus, by competing with the mother’s Hb
what subunits are present in fetal Hb?
-9 to -6 = zeta, epsilon
-6 to -3 = alpha, gamma
+3 = alpha, beta (delta)
why does fetal gamma Hb have different 2,3-BPG affinity?
the positive charges that exist on the adult Hb that stabilise the negatively charged BPG are replaced in fetal Hb
his 143 becomes serine in the gamma subunit
less efficient BPG binding, curve shifted left, higher affinity than maternal Hb
what is the net charge on 2,3-BPG?
-5
what does 2,3-BPG stand for?
2,3-bisphosphoglyerate
where is 2,3-BPG produced?
produced as an intermediate in glycolysis
how does 2,3-BPG work?
stabilises the T state by binding the centre pocket, allowing it to exist without quickly converting to R state
decreases Hb’s affinity for O2, shifting curve right, allowing Hb to be an effective O2 carrier in the body, unloading ~66% of O2 to exercising tissue rather than 8%
how is 2,3-BPG used in high altitude adaptation?
produce 2x more
curve shifted further right so the curve is still mostly saturated and is there isn’t a big difference in saturation across a large range of PO2 can ‘discount’ the far right
the middle portion of the curve where most of the change happens is now further right, allowing dissociation of O2 from Hb at the respiring tissues
in what protein is negative cooperativity exhibited?
CTP synthetase: catalyses last step in pyrimidine synthesis
can only be explained by sequential model
which contacts between subunits change between R and T state in Hb?
a1/b2 and a2/b1 contacts
a1/b1 and a2/b2 are extensive and don’t change
how do the 2 distinct R and T states arise from ‘ratchet’ interactions?
T state: T42 (alpha) interacts w aspartic acid 99 (beta) and as you slide the subunits in relation to each other you move A99 far enough that it interacts with A94 (alpha) which interacts with asparagine 102 (beta) [R state]
only a limited number of states are stabilised as only a limited number of H bond interactions can happen
describe the differences between sequential and concerted model
S: ligand-free = T , ligand-bound = R; breaks symmetry; accounts for +ve and -ve coop
T: T state predominates when no ligand, R state predominates in presence of ligand; preserves symmetry (all convert between states at once); +ve coop only
draw a Hb/Mb comparison O2 binding affinity graph for lungs vs tissue:
page 3 of Mb notes
or google ig
what is the pO2 of O2 in lungs and peripheral capillaries?
lungs = 100 torr
capillaries = 20 torr
what does the Hb-O2 curve shifting left cause? what causes it?
increased affinity for O2
decreased pCO2
decreased [H+]
decreased 2,3 BPG
decreased temp
feral Hb
how does CO reduce delivery of oxygen to tissues?
binds w 210x more affinity to Hb
blocks the binding of O2 to that subunit but also is similar enough that it causes conformational changes to the R state in other subunits, increasing Hb affinity for O2
significantly reduces delivery to tissues
