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bchm 310 > lecture 37-41 - myoglobin/hemoglobin > Flashcards

lecture 37-41 - myoglobin/hemoglobin Flashcards

1
Q

do o2 binding proteins bind reversibly or irreversibly?

A

reversible binding

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2
Q

what are o2 binding proteins involved in? what are they required for?

A
  • required for cellular respiration

- involved in transport, delivery and storage of o2

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3
Q

does o2 have low or high solubility?

A

low

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4
Q

what are the two proteins were looking at in the globin family? what are their jobs?

A
  • myoglobin - o2 storage

- hemoglobin - o2 transport/delivery and co2 transport

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5
Q

what are common features of myoglobin and hemoglobin?

A
  • share similar sequence and structure (homologues)

- contain prosthetic group heme (binds o2)

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6
Q

describe the similar sequences/structures of myoglobin and hemoglobin

A
  • ~150 aa residues/polypeptide

- 8 alpha helices (A-H)

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7
Q

describe the unique features of myoglobin

A
  • storage - high affinity for o2

- 4 structure = monomer

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8
Q

describe the unique features of hemoglobin

A
  • transport/delivery - variable affinity (higher in the lungs and lower in peripheral tissues)
  • 4 structure = heterotetramer (2 alpha, 2 beta)
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9
Q

explain the process of o2 binding

A
  • use Fe2+ to coordinate o2 binding
  • associated to protoporphyrin (within heme group)
  • proximal his from helix F forms coordinate bond to the Fe
  • distal his from helix E forms h-bond with the o2 which coordinates to the Fe ion
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10
Q

describe protoporphyrins in terms of o2-binding

A
  • four planar hydrophobic N rings (which comprise protoporphyrin) are bonded to an Fe ion in the middle via coordiante bonds, forming a cyclical structure
  • functions to keep Fe in the reduced (ferrous state)
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11
Q

what comprises the heme group?

A

4 protoporphyrins + Fe

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12
Q

Fe bound to oxygen has 6 coordinating atoms, what are they?

A

-4 N from heme (protoporphyrin)
-1 N from proximal his
-1 O from bound o2
(without o2 theres only 5)

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13
Q

where does heme sit in the strutcure? what is the affinity

A
  • sits deep in the hydrophobic cleft

- bound very tightly

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14
Q

where does o2 enter to bind with Fe? is movement of the protein required for this to occur? what does this process highlight?

A
  • enters the cleft
  • some movement’s required
  • highlights dynamic nature of protein structures
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15
Q

what function does the cleft serve?

A

helps protect Fe2+ from oxidation

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16
Q

what is oxygenation

A

o2 binding to heme

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17
Q

how many heme’s per globin structure? what does this mean for oxygen binding?

A
  • 1 heme/globin

- only 1 o2/globin

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18
Q

discuss diferences in structure for oxygenated heme vs deoxygenated heme

A

deoxy:
-Fe has 5 coordinating atoms
-slightly puckered - Fe out of the plane of protoporphyrin
oxy:
-Fe has 6 coordinating atoms
-flat, planar - Fe in the plane of protoporphyrin

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19
Q

what adjustments must be made to the heme structure when o2 binds?

A
  • proximal his must move when o2 binds to maintain coordinate bond with Fe
  • causes small conformatinal change to helix F’s backbone
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20
Q

where is myoglobin predominantly found?

A

heart and skeletal muscles

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21
Q

why is myoglobin important?

A

helps avoid anaerobic respiration

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22
Q

discuss the realtionship between the quaternary structure of myoglobin and how it binds o2

A
  • monomeric (1 polypeptide, 1 heme, 1 o2)

- displays simplistic reversible o2 binding

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23
Q

what is the Kd for the Mb + o2 Mbo2 reaction?

A

Kd = [Mb][o2] / [Mbo2]

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24
Q

what is the fractional saturation for the Mb + o2 Mbo2 reaction? do we use this? why or why not?

A

theta = [o2] / [o2] + Kd

  • [o2] is difficult to measure bc o2 has low solubility
  • use partial pressure of o2 instead (po2)
25
Q

describe the po2 and what it means for the fractional saturation of the Mb + o2 Mbo2 reaction.

A
  • po2 is proportianl to [o2]
  • P50 is proportional to the Kd
  • therefore, theta = po2 / po2 + P50
26
Q

what is the P50?

A
  • the point of half saturation

- the po2 when theta = 0.5

27
Q

is the P50 for Mb high or low? what does this mean in terms of the function of Mb?

A
  • P50 for Mb = very low = ~0.13 kPa
  • means high affinity (of Mb for o2)
  • helps maintain aerobic respiration - won’t give up o2 unless surrounding concentration is very low
28
Q

where is hemoglobin predominantly found?

A

in rbcs (1/3 the mass of rbcs)

29
Q

discuss the quaternary structure of Hb. How does this structure contribute to its physiological role?

A
  • 2 alpha globin units and 2 beta globin units

- allows for more complex binding behaviour then Mb (required for its physiological role)

30
Q

describe the o2 saturation of Hb in arterial and venous blood.
what does this indicate?

A
  • arterial - 96% saturated (12 kPa)
  • venous - 64% saturated (4 kPa)
  • indicates that Hb gives up 1/3 of its o2 cargo under normal conditions
31
Q

compare the o2 binding hyperbolas for Mb and Hb

A
  • Mb = rectangular hyperbola

- Hb = sigmoidal hyperbola (bc cooperative binding)

32
Q

discuss the interactions that hold the Hb heterotetramer together (2)

A

non-covalent interactions:

  • hydrophobic contacts
  • salt bridges (ionic)
33
Q

describe the Hb heterotetramer (3)

A
  • rigid
  • contacts btwn alph1 and beta1 / alpha 2 and beta 2 are somewhat dynamic
  • has 2 conformational states
34
Q

what are the two conformational states of Hb? describe them

A

(1) tense state (T) - no o2 bound
- numerous electrostatic reactions btwn beta subunits
(2) relaxed state (R) - o2 bound
- fewer interactions btwn beta subunits

35
Q

can o2 bind both conformation states of Hb?

A

yes

36
Q

does o2 bind to both conformtaional states of Hb with the same affinity?

A

no - binds w a much higher affinity to R-state Hb

37
Q

explain the mechanism for positive cooperativity in Hb

A
  • start at low po2 –> T-state = some o2 binding and releasing
  • as po2 increases, fractional saturation at each heme will increase (lifetime of o2 bound at each site increases as po2 increases)
  • at a certain po2, o2 is bound long enough to trigger conformational change in one subunit, and for that change to be transmitted to the neighbouring subunits
  • at a high enough po2: conformational change from T–>R
  • individual subunits dont change very much - there is a movement btwn alpha and beta subunits = narrowing the pocket btwn the 2 beta subunits

-causes Hb to switch from low affinity to high affinity

38
Q

describe the conformational change that occurs in the T-state

A
  • have Fe bound to proximal his of helix F via coordinate interactions
  • when add o2 - Fe now also bound to distal his of helix E
  • ie helix F shifts when o2 is bound
  • results in a change in the number of electrostatic interactions btwn subunits
39
Q

what is the result of the conformational change that occurs in the T-state during cooperativity

A

the result is increased o2 affinity - one binding site is sensing o2 binding at another site w/in the same Hb molecule

40
Q

define allostery and provide an example

A
  • binding of one ligand to a site affects the binding properties of other sites in the same molecule
  • binding of o2 enhances the affinity for o2 at other sites
41
Q

discuss the hill equation

A

-reacll: Kd = [P][L] / [PL], theta = [L] / [L] + Kd
-for Hb: theta = [po2] / [po2] + P5
-linearize to get:
log(theta / 1 - theta) = n log(po2) - n log(P50)
-plot where log(theta/1-theta) is on the y axis and log(po2) is on the x axis
-see S curve for Hb - where bottom is T-state, top is R-state and and middle is increasing relative to binding affinity

42
Q

using the Hill equation, what are the likely log(po2) values for the T-state and the R-state?

A
  • T: po2 = 4 kPa

- R: po2 = 0.04 kPa

43
Q

what conclusions can be made from the Hill equation? (3)

A

(1) at very low po2: Hb has low affinity,
- po2 at theta=0.05 is 4 kPa (T-state)
(2) at very high po2: Hb has high affinity
- po2 at theta=0.05 is 0.04 kPa (R-state)
- therefore R-state has 100x more affinity than T-state and has a higher affinity than Mb
(3) at an intermediate po2: nH=3
- range of po2 where Hb undergoes conversion from T to R-state

44
Q

discuss limitations to the Hill equatin

A
  • Hill equation was derrived assuming a perfectly cooperative system - never observed irl
  • why nH is < the # of binding sites (why nH for increasing portion doesn’t = 4)
45
Q

what is the Bohr effect?

A
  • effect of pH and co2 on o2 affinity
  • recall: co2 + h20 <=> h+ + hco3-
  • h+ and co2 are byproducts of respiration - Hb transports these from tissues to the lungs
46
Q

describe the bohr effect in peripheral tissues

A
  • low po2/pH and high co2/[h+] leads to low affinity for o2
  • h+/co2 is taken up by Hb, causing equation to shift to produce more hco3-, decreasing pH
  • ie Hb = unloading o2 in tissues
47
Q

describe the bohr effect in the lungs

A
  • high po2/pH and low co2 = high o2 affinity
  • co2 is exhaled = release h+ = increased pH
  • equilibrium shifts to produce more co2
  • ie Hb = loading o2 in lungs
48
Q

why do H+ and co2 have these effects on o2 binding?

A
  • h+ and co2 bind at sites uniques from o2 binding sites

- h+ and co2 are negative allosteric effectors of o2 binding to Hb

49
Q

describe salt bridge formation of Hb in the T-state

when is this processes favoured?

A
  • his 146 and asp 94 within the same beta subunits form a salt bridge
  • pkr of his is increased by asp so that its close to physcial pH (which favours T-state)
  • this is favoured at a low pH (peripheral tissues)
50
Q

describe the salt bridge in peripheral tissues

A
  • low pH - ~7.2
  • salt bridge can only form in the T-state
  • therefore low pH stabilizes the T-state & promotes o2 release
51
Q

describe the salt bridge in lungs

A
  • higher pH = ~7.6
  • his is deprotonated - salt bridge not formed
  • R-state is stabilized increasing o2 affinity
52
Q

does the P50 change depending on pH?

A
  • yes - bohr effect

- higher pH = lower P50

53
Q

do the effects of po2 and pH on o2 affinity occur simultaneously or singly?

A

simultaneously

54
Q

describe the interactions between co2 and Hb and how these interactions contribute to the bohr effect

A
  • most co2 = transported to lungs as dissolved hco3-
  • therefore ~15-20% of co2 is transported by Hb
  • co2 interacts w n-term amino group to generate a carboxylic acid and 2 h+ molecules
  • extra h+ generated further contributes to bohr effect
55
Q

what is BPG?

A
  • highly negatively charged molcule
  • neg allosteric effector of Hb binding o2
  • binds (reversibly) the cavity btwn 2 beta subunits of Hb (1 BPG/tetramer)
  • acts as a wedge btwn beta subunit narrowing - preventing transition into the R-state
  • ie stabilizes the T-state
  • decreases o2 affinity
56
Q

describe the use of BPG at different altidudes

A
  • concentration of BPG is increased at high elevations (loe [o2])
  • bc it shifts the curve to the right, allowing Hb to release the same amount of o2 despite the difference in po2
57
Q

describe the role of BPG in fetal development

A
  • maternal Hb = alpha2beta2, fetal Hb = alpha2gamma2
  • numerous differences btwn beta and gamma: fewer pos charged res in the cleft (what BPG binds)
  • therefore: decreased BPG affinity in gamma = decreased T-state = increased R-state = increased o2 affinity
  • therefore fetal Hb can bind o2 tighter than maternal Hb
  • this optimizes o2 transfer from mother to fetus
58
Q

what is sickle cell anemia?

A

-inherited disease
-mutation in a single aa res in the B-sununit:
Glu6 –>Val (neg –> hydrophobic)
-normally this wouldnt be important but is is bc of the high [ ] of Hb in the body
-caused Hb to be “sticky” resulting in lower solubility of Hb when in T-state (still soluble in R-state)
-causes the formation of large fibrils of Hb (deformity of RBCs) causing them to become fragile and rupture easily
-not as many RBCs available (anemia)

bchm 310 (41 decks)

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