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genes + molecules > Myoglobin and Haemoglobin > Flashcards

Myoglobin and Haemoglobin Flashcards

1
Q

When were the structures of Mb and Hb first determined?

A

Mb : 1958
Hb : 1960

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2
Q

What are the functions of Mb?

A

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

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3
Q

How do you describe the structure of Mb?

A

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)

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4
Q

What is the heme group structure?

A

porphyrin ring bound to iron

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5
Q

Where does the prosthetic group bind in Mb and Hb?

A

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

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6
Q

what is the coordinate geometry of iron bonding?

A

octahedral: 4 equatorial bonds and 2 axial bonds

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7
Q

what are the 2 axial positions occupied by?

A

proximal histidine: F8 (residue 8 in helix F)

the place where O2 coordinates to Fe

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8
Q

What is near to the coordination place for O2 at the axial position?

A

distal histidine: E7 (does not coordinate to Fe but is highly conserved and is close enough to Fe to form a H bond)

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9
Q

How does the distal histidine E7 modulate the relative affinities of CO and O2?

A

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

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10
Q

how do we know that the E7 histidine is crucial for modulating relative affinities of CO and O2?

A

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

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11
Q

where does the O2 bind to Mb? why?

A

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

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12
Q

what is the fractional oxygen saturation (Y) equation for Mb, and the shape of the oxygen loading curve?

A

Y = [O2]/Kd + [O2]

hyperbolic (Michaelis Menten stylez)

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13
Q

define Kd

A

the [O2] at which half of the sites are occupied
(known as p50)

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14
Q

does Mb form dimers?

A

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

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15
Q

what does a lower P50 indicate?
what is Mb’s?

A

stronger O2 binding
very low

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16
Q

Does Mb bind oxygen tightly?

A

yes
at high/intermediate pO2 it will be almost completely saturated (y close to 1)

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17
Q

what is required to unbind O2 from Mb?

A

very low pO2

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18
Q

why does Mb not oligomerize like Hb?

A

idk bro

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19
Q

what is the brief comparison of structure + function between Hb and Mb?

A

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

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20
Q

describe the detailed structure of Hb

A

tetrameric dimer of alpha-beta dimers
each subunit similar in structure + fold to 1 Mb

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21
Q

what is the function of Hb?

A

binding oxygen in alveolar capillaries and delivering it to areas of high demand in tissues (muscles, kidney, brain)

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22
Q

what happens if you remove a prosthetic group?

A

Hb and Mb become very unstable and begin to unfold

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23
Q

which model of cooperativity is used to explain Hb binding?

A

the concerted model explains the actions and structural changes since all subunits appear to change conformation from R to T at the same time

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24
Q

what is the mechanism by which Hb exhibits cooperative binding?

A

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

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25
Q

which state (R or T) has high/low affinity for oxygen?

A

low affinity = T
high affinity = R

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26
Q

how does the sigmoidal curve arise?

A

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

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27
Q

describe the sequential model of O2 binding?

A

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]

28
Q

describe the concerted model of O2 binding

A

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

29
Q

describe the concerted model of O2 binding

A

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]

30
Q

can a ligand bind to an active site on a subunit in either conformation?

A

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.

31
Q

what is another word allosteric unit

A

protomer/subunit

32
Q

how does the concerted model explain the sigmoidal curve/positive cooperativity?

A

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.

33
Q

how does Hb’s functions determine its oxygen affinity requirements?

A

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

34
Q

how does Mb’s functions determine its oxygen affinity requirements?

A

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

35
Q

based on what factors can Hb change its affinity for oxygen? (what influences T/R state equilibrium)

A

[CO2], pH and 2,3-bisphosphoglycerate

36
Q

what does Hb’s affinity for O2 heavily depend on?

A

subunits
e.g. gamma in fetal Hb

37
Q

what happens in thalassemias?

A

missing gene for alpha or beta Hb, or impaired RNA synthesis

alpha = some oxygen still transported
beta = very bad

38
Q

what happens in alpha thalassemia?

A

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

39
Q

what happens in beta thalassemia?

A

only alpha Hb left: unstable on its own: oxidises and precipitates

40
Q

why are low levels of free alpha-Hb normally tolerated?

A

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

41
Q

what are the 2 types of allosteric modulators?

A

homotropic: modulating agent is equivalent to the substrate (O2 binding affects O2 binding)

heterotropic: modulating agent differs from substrate (ligand binding affects O2)

42
Q

how does a heterotropic modulator work?

A

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

43
Q

how can we sense a pH change? over what range?

A

sense a change in protonation state above or below the physiological pH (~7.2 in active respiration)
use histidines and N termini

44
Q

what does addition of H+ change? what does this allow us to do?

A

charge: can remove/introduce electrostatic interactions

45
Q

under what circumstances does the oxygen dissociation curve of Hb shift left? what does this mean for O2 affinity?

A

lower pCO2
lower 2,3-BPG
lower temperature
increased pH
[fetal Hb]

leads to increased affinity for O2

46
Q

under what circumstances does the oxygen dissociation curve of Hb shift right? what does this mean for O2 affinity?

A

higher pCO2
higher 2,3-BPG
higher temperature
decreased pH

leads to decreased affinity for O2

47
Q

what happens with the boston mutation in Hb?

A

promotes methaemoglobin formation
(metHb contains Fe3+ which doesn’t allow for reversible binding)

48
Q

what histidine is used as the marker for pH? why?

A

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

49
Q

how does CO2 affect O2 binding affinity?

A
  1. linked to pH of respiring tissue: carbonic acid, H+, decreases pH in RBC as bicarbonate can leave on transporters but H+ cannot
  2. 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

50
Q

does Hb in RBCs bind more or less tightly to O2 than purified Hb? why?

A

purified binds much more tightly
due to shift in p50 due to 2,3-BPG

51
Q

from where does Hb allostery arise? what does this mean?

A

from inter-subunit interactions

changing subunits can be exploited to change Hb properties, such as in development

52
Q

what do subunit differences in fetal Hb allow?

A

the flow of O2 from mother to fetus, by competing with the mother’s Hb

53
Q

what subunits are present in fetal Hb?

A

-9 to -6 = zeta, epsilon
-6 to -3 = alpha, gamma
+3 = alpha, beta (delta)

54
Q

why does fetal gamma Hb have different 2,3-BPG affinity?

A

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

55
Q

what is the net charge on 2,3-BPG?

A

-5

56
Q

what does 2,3-BPG stand for?

A

2,3-bisphosphoglyerate

57
Q

where is 2,3-BPG produced?

A

produced as an intermediate in glycolysis

58
Q

how does 2,3-BPG work?

A

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%

59
Q

how is 2,3-BPG used in high altitude adaptation?

A

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

60
Q

in what protein is negative cooperativity exhibited?

A

CTP synthetase: catalyses last step in pyrimidine synthesis

can only be explained by sequential model

61
Q

which contacts between subunits change between R and T state in Hb?

A

a1/b2 and a2/b1 contacts

a1/b1 and a2/b2 are extensive and don’t change

62
Q

how do the 2 distinct R and T states arise from ‘ratchet’ interactions?

A

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

63
Q

describe the differences between sequential and concerted model

A

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

64
Q

draw a Hb/Mb comparison O2 binding affinity graph for lungs vs tissue:

A

page 3 of Mb notes
or google ig

65
Q

what is the pO2 of O2 in lungs and peripheral capillaries?

A

lungs = 100 torr
capillaries = 20 torr

66
Q

what does the Hb-O2 curve shifting left cause? what causes it?

A

increased affinity for O2

decreased pCO2
decreased [H+]
decreased 2,3 BPG
decreased temp
feral Hb

67
Q

how does CO reduce delivery of oxygen to tissues?

A

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

genes + molecules (7 decks)

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