lecture 37-41 - myoglobin/hemoglobin Flashcards
do o2 binding proteins bind reversibly or irreversibly?
reversible binding
what are o2 binding proteins involved in? what are they required for?
- required for cellular respiration
- involved in transport, delivery and storage of o2
does o2 have low or high solubility?
low
what are the two proteins were looking at in the globin family? what are their jobs?
- myoglobin - o2 storage
- hemoglobin - o2 transport/delivery and co2 transport
what are common features of myoglobin and hemoglobin?
- share similar sequence and structure (homologues)
- contain prosthetic group heme (binds o2)
describe the similar sequences/structures of myoglobin and hemoglobin
- ~150 aa residues/polypeptide
- 8 alpha helices (A-H)
describe the unique features of myoglobin
- storage - high affinity for o2
- 4 structure = monomer
describe the unique features of hemoglobin
- transport/delivery - variable affinity (higher in the lungs and lower in peripheral tissues)
- 4 structure = heterotetramer (2 alpha, 2 beta)
explain the process of o2 binding
- 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
describe protoporphyrins in terms of o2-binding
- 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)
what comprises the heme group?
4 protoporphyrins + Fe
Fe bound to oxygen has 6 coordinating atoms, what are they?
-4 N from heme (protoporphyrin)
-1 N from proximal his
-1 O from bound o2
(without o2 theres only 5)
where does heme sit in the strutcure? what is the affinity
- sits deep in the hydrophobic cleft
- bound very tightly
where does o2 enter to bind with Fe? is movement of the protein required for this to occur? what does this process highlight?
- enters the cleft
- some movement’s required
- highlights dynamic nature of protein structures
what function does the cleft serve?
helps protect Fe2+ from oxidation
what is oxygenation
o2 binding to heme
how many heme’s per globin structure? what does this mean for oxygen binding?
- 1 heme/globin
- only 1 o2/globin
discuss diferences in structure for oxygenated heme vs deoxygenated heme
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
what adjustments must be made to the heme structure when o2 binds?
- proximal his must move when o2 binds to maintain coordinate bond with Fe
- causes small conformatinal change to helix F’s backbone
where is myoglobin predominantly found?
heart and skeletal muscles
why is myoglobin important?
helps avoid anaerobic respiration
discuss the realtionship between the quaternary structure of myoglobin and how it binds o2
- monomeric (1 polypeptide, 1 heme, 1 o2)
- displays simplistic reversible o2 binding
what is the Kd for the Mb + o2 Mbo2 reaction?
Kd = [Mb][o2] / [Mbo2]
what is the fractional saturation for the Mb + o2 Mbo2 reaction? do we use this? why or why not?
theta = [o2] / [o2] + Kd
- [o2] is difficult to measure bc o2 has low solubility
- use partial pressure of o2 instead (po2)
describe the po2 and what it means for the fractional saturation of the Mb + o2 Mbo2 reaction.
- po2 is proportianl to [o2]
- P50 is proportional to the Kd
- therefore, theta = po2 / po2 + P50
what is the P50?
- the point of half saturation
- the po2 when theta = 0.5
is the P50 for Mb high or low? what does this mean in terms of the function of Mb?
- 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
where is hemoglobin predominantly found?
in rbcs (1/3 the mass of rbcs)
discuss the quaternary structure of Hb. How does this structure contribute to its physiological role?
- 2 alpha globin units and 2 beta globin units
- allows for more complex binding behaviour then Mb (required for its physiological role)
describe the o2 saturation of Hb in arterial and venous blood.
what does this indicate?
- arterial - 96% saturated (12 kPa)
- venous - 64% saturated (4 kPa)
- indicates that Hb gives up 1/3 of its o2 cargo under normal conditions
compare the o2 binding hyperbolas for Mb and Hb
- Mb = rectangular hyperbola
- Hb = sigmoidal hyperbola (bc cooperative binding)
discuss the interactions that hold the Hb heterotetramer together (2)
non-covalent interactions:
- hydrophobic contacts
- salt bridges (ionic)
describe the Hb heterotetramer (3)
- rigid
- contacts btwn alph1 and beta1 / alpha 2 and beta 2 are somewhat dynamic
- has 2 conformational states
what are the two conformational states of Hb? describe them
(1) tense state (T) - no o2 bound
- numerous electrostatic reactions btwn beta subunits
(2) relaxed state (R) - o2 bound
- fewer interactions btwn beta subunits
can o2 bind both conformation states of Hb?
yes
does o2 bind to both conformtaional states of Hb with the same affinity?
no - binds w a much higher affinity to R-state Hb
explain the mechanism for positive cooperativity in Hb
- 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
describe the conformational change that occurs in the T-state
- 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
what is the result of the conformational change that occurs in the T-state during cooperativity
the result is increased o2 affinity - one binding site is sensing o2 binding at another site w/in the same Hb molecule
define allostery and provide an example
- 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
discuss the hill equation
-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
using the Hill equation, what are the likely log(po2) values for the T-state and the R-state?
- T: po2 = 4 kPa
- R: po2 = 0.04 kPa
what conclusions can be made from the Hill equation? (3)
(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
discuss limitations to the Hill equatin
- 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)
what is the Bohr effect?
- 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
describe the bohr effect in peripheral tissues
- 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
describe the bohr effect in the lungs
- 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
why do H+ and co2 have these effects on o2 binding?
- h+ and co2 bind at sites uniques from o2 binding sites
- h+ and co2 are negative allosteric effectors of o2 binding to Hb
describe salt bridge formation of Hb in the T-state
when is this processes favoured?
- 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)
describe the salt bridge in peripheral tissues
- low pH - ~7.2
- salt bridge can only form in the T-state
- therefore low pH stabilizes the T-state & promotes o2 release
describe the salt bridge in lungs
- higher pH = ~7.6
- his is deprotonated - salt bridge not formed
- R-state is stabilized increasing o2 affinity
does the P50 change depending on pH?
- yes - bohr effect
- higher pH = lower P50
do the effects of po2 and pH on o2 affinity occur simultaneously or singly?
simultaneously
describe the interactions between co2 and Hb and how these interactions contribute to the bohr effect
- 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
what is BPG?
- 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
describe the use of BPG at different altidudes
- 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
describe the role of BPG in fetal development
- 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
what is sickle cell anemia?
-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)
