Hemoglobin 2 - deoxy vs oxy Hb structure 09/11 Traish Flashcards
T/F binding of O2 is accompanied by changes in the structure of Hb
true
oxyHb corresponds to the __ state
R (relaxed)
deoxy Hb corresponds to the __ state
T (taut)
R state corresponds to __Hb
oxyHb
T state corresponds to __Hb
deoxyHb
this technique was used to observe structural changes in Hb due to O2 binding
x-ray crystallography
(crystalline atoms cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.)
what is x-ray crystallography
(crystalline atoms cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.)
in terms of subunit orientation to one another, describe the structural change in Hb upon oxygenation
O2 binding causes one a-b dimer to rotate ~15 degrees and slide with respect to the other (think Traish’s apples); this movement brings b-chains closer together and narrows the central cavity which binds 2,3-bisphosphoglycerate. this narrowing releases 23BPG and increases Hb affinity for O2
in 2 bullet points, describe the structural change in Hb upon oxygenation with regard to subunit orientation
- a-b dimers rotate ~15 degrees and slide with respect to one another
- this narrows the central cavity and releases 23BPG (2,3-bisphosphoglycerate)
T/F 23BPG binding increases Hb affinity for O2
false - it decreases Hb affinity for O2, by stabilizing the open window, taught form
T/F partial oxygenation favors transition to the R state
true - if one subunit binds O2, conformational changes will induce the R state in other subunits and increase affinity for O2
this residue is the C-terminus of b-Hb
his146
this residue is the C-terminus of a-Hb
arg141
in T state, what is the position of Fe2+?
0.6 angstroms out of the heme plane - because of the doming of the porphyrin group towards his F8
in deoxyHb what is the position of his F8 with respect to the heme plane?
8 degrees from perpendicular
in oxyHb, what is the position of his F8 with respect to the heme plane?
perpendicular
this is a residue neighboring his F8 which, upon O2 binding ot heme, participates in the steric interactions that cause a rearrangement in structure, especially at a-b interfaces
valFG5
describe the role of valFG5 in the T to R shift in Hb
binding of O2 pulls iron 0.6 angstroms back into heme, flattening it. because the epsilon hydrogen of hisF8 and the neighboring valFG5 are pulled sterically too close to the heme, this produces a lever effect that shifts hisF8 from 8 degrees to perpendicular and a corresponding distortion and weakening of the whole complex of H bonds and salt bridges that connect subunits, especially at alpha-beta interfaces
in general terms, how does O2 binding cause the conformational shift from T to R state?
binding of O2 pulls iron 0.6 angstroms back into heme, flattening it. because the epsilon hydrogen of hisF8 and the neighboring valFG5 are pulled sterically too close to the heme, this produces a lever effect that shifts hisF8 from 8 degrees to perpendicular and a corresponding distortion and weakening of the whole complex of H bonds and salt bridges that connect subunits, especially at alpha-beta interfaces
in which state, T or R, is the porphyrin complex slightly domed?
T state
in which state, T or R, is the porphyrin complex flat?
R state
what are 2 key residues that, upon O2 binding, sterically induce conformational shift from T to R state?
valFG5 and hisF8 - when Fe is pulled 0.6 angstroms back into porphyrin complex, valFG5 provides a steric lever to shift hisF8 8 degrees to perpendicular and relieve hisF8 epsilon H strain, resulting in distortion and weakening of the whole complex of H bonds and salt bridges that connect subunits, especially at alpha-beta interfaces
how much does each bound O2 increase the rate of O2 binding in other subunits?
1st bind = x
2nd bind = 9x faster
3rd bind = 38x faster
4th bind = >100x faster
a-helix C is made from which a-Hb residues?
36-42
a-Hb residues 36-42 compose which a-helix?
a-helix C
in Hb: beta 1 interacts mainly with... beta 2 interacts mainly with... alpha 1 interacts mainly with... alpha 2 interacts mainly with... 2,3BPG interacts mainly with...
beta 1 - with 2,3BPG and alpha 2 beta 2 - with 2,3BPG and alpha 1 alpha 1 - with beta 2 and alpha 2 alpha 2 - with beta 1 and alpha 1 2,3BPG - with beta 1 and beta 2
beta subunit C-termini stabilize deoxy Hb through these residue interactions:
beta His146 - beta Asp94 (R-R)
beta His146 - alpha Lys40(C5) (C-R)
(x2, beta reacting with self or opposite alpha)
alpha subunit C-termini stabilize deoxy Hb through these residue interactions:
alpha Arg141 - Cl- - alpha Nterm (R-N)
alpha Arg141 - alpha Asp126 (R-R)
alpha Arg141 - alpha Lys127 (C-R)
( x2, alpha reacting with opposite alpha)
deoxy Hb is stabilized through these non-terminus intra-subunit residue interactions:
alpha Tyr140 - alpha Val93 (R-C H-bond)
beta Tyr145 - beta Val98 (R-C H-bond)
(x2, alphas & betas reacting with selves)
deoxy Hb is stabilized through this non-terminus inter-subunit residue interaction:
beta His97(FG4) - alpha Thr41(C6) (x2, betas reacting with opposite alphas)
deoxy Hb is stabilized through what residue interactions with 2,3BPG?
beta N terminus, His2, Lys82, His143
x2, betas reacting with 2,3BPG
in the Hb conformation shift from T to R, which bonds stabilizing deoxy HB are broken?
- bonds involving beta C-termini
- bonds involving alpha C-termini
- intra-subunit H-bonds
- beta subunit bonds with 2,3 BPG
in the Hb conformation shift from T to R, which bonds stabilizing deoxy Hb are shifted?
beta His97(FG4) - alpha Thr41(C6) (T) beta His97(FG4) - alpha Thr38(C3) (R) (x2, betas reacting with opposite alpha)
why is oxyHb called the relaxed (R) state?
bonds have been broken in the deoxy-oxy transition and the oxy form is “looser”
why is the R state energetically favorable when O2 binds?
O2 binding pays the energy price for the steric breaking of many the other bonds
why is the T state energetically favorable when O2 is released?
the extra bonds and tighter conformation of the T state is more stable than the R state in the absence of an O2-heme bond to pay the energy price for breaking all of these bonds in the R state
name 3 theories proposed to describe allosteric transitions
- sequential model
- concerted model
- multistate model
sequential model of allosteric transitions
each subunit can change one at a time, so molecules can have some T and some R mixed
concerted model of allosteric transitions
entire molecule exists in one of two forms, T or R, no mixed intermediates
multistate model of allosteric transitions
combines sequential and concerted models. involves concept of concerted switch and also more than 2 states of molecule. changes in tertiary structure can be tolerated up to a certain point before T-R switch occurs. specifically, when one site is occupied on each a-b dimer the molecule as a whole switches to R
when does the T-R switch occur in Hb as a whole?
when one site is occupied on each a-b dimer, the molecule as a whole switches to R
does Hb obey the sequential or concerted model of allosteric transitions?
neither completely – obeys a multistate model that combines features of both. sequential until one site on each a-b dimer causes concerted switch of entire molecule
the Bohr effect
- higher [H+] causes increased release of O2 from Hb in the capillaries
- higher [O2] causes increased release of H+ from Hb in the lungs, which results in H+ and HCO3- combining to form CO2 and H2O, which is exhaled
equation for the bicarbonate buffer system
CO2 + H2O H2CO3 H+ + CO3-
equation for Bohr effect
Hb(O2)n + nH+ Hb(H+)n + nO2
what is the beta Hb residue involved in the Bohr effect?
beta His146 (beta C-termini)
- in the T state, beta His146 forms a salt bridge with beta Asp94, which stabilizes its protonated form with a higher pKa
- in the R state the salt bridge is sterically disrupted and His146 pKa drops back to ~6.5, which is largely deprotonated at physiologic pH
what is the alpha Hb residue involved in the Bohr effect?
N-termini of alpha chains
- protonated at high [H+], stabilizing T
- deprotonated at low [H+], stabilizing R
what are two Hb residues involved in the Bohr effect?
beta His146 (beta C-termini) alpha N-termini
does Mb display Bohr effect?
no - there are no allosteric interactions in Mb. the Bohr effect is due to stabilized protons in the T state versus destabilized protons in the R state
will Mb structure and function be affected by pH changes?
- not small pH changes (no allosteric interactions for Bohr effect like Hb)
- but large pH changes will affect the structure and function of any protein
in what 2 ways does release of carbon dioxide by respiring tissues lower oxygen affinity of hemoglobin?
-some CO2 becomes HCO3-, releasing H+ to contribute to the Bohr effect
(CO2 + H2O H2CO3 HCO3- + H+)
-some CO2 binds to N-termini of Hb chains, stabilizing salt bridges characteristic of T Hb while releasing more H+ to contribute to the Bohr effect
(R-NH3+ + HCO3- R-NH-COO- + H+ + H2O)
how does Hb transport CO2?
at high pCO2 in tissues:
-H+ binds to beta His146 and alpha N-termini
-HCO3- forms carbamates with N-termini
at high pO2 in lungs:
-O2 binding causes Hb switch to R and release of H+ & HCO3- to be expired as CO2 & H2O
name 4 allosteric effectors of Hb O2 binding
- other Hb subunits (cooperativity)
- H+
- CO2
- 2,3-bisphosphoglycerate
what is a carbamate?
R-NH-COO-
by what reaction does CO2 combine with Hb?
R-NH3+ + HCO3- R-NH-COO- + H+ + H2O
how does 2,3-bisphosphoglycerate binding affect the oxygen affinity of hemoglobin?
lowers it
where does BPG bind Hb?
in the cavity between beta subunits
beta N-terminus, His2, Lys82, His143
does BPG bind oxyHb?
no - the opening between beta subunits is too small
how does smoking affect BPG levels?
increases BPG levels - inhibits toxic binding of CO and increases release of O2 to tissues
CO poisoning occurs because…
- CO competes with Hb O2 binding, blocking respiration
- Hb affinity for CO is 300x higher than for O2 and not readily reversible, so CO binding will raise Hb affinity for O2 above physiologic affinity and inhibit O2 release to tissues
how does CO affect P50 of Hb binding to O2?
decreases P50 (increases affinity)
does CO binding shift the Hb-O2 binding curve left or right?
left - decreases P50, increases affinity
how will a low dose of CO affect the Hb-O2 binding curve?
will shift the binding curve left, decreasing P50 and increasing affinity, while dropping the horizontal asymptote (curve behaves as though only 3 O2 binding sites exist and more likely in R state because one is occupied by CO)
how will a high dose of CO affect the Hb-O2 binding curve?
will change the sigmoidal curve to hyperbolic (like Mb), while shifting P50 right and dropping the asymptote to 1/4 its healthy value (curve behaves as though only 1 O2 binding site exists and only in R state because 3 sites are occupied by CO)
how is fetal blood functionally different from adult blood?
fetal blood must have higher O2 affinity to obtain oxygen from mother’s blood through placental exchange
how is fetal hemoglobin structurally different from adult Hb?
beta chains replaced by gamma chains
what is the tetrameric structure of HbF
a2g2
what is the tetrameric structure of HbA
a2b2
T/F the intrinsic oxygen affinity of HbF is greater than that of HbA
false - the intrinsic O2 affinity of HbF is very similar to that of HbA, but HbF has a much lower affinity for BPG
how does HbF achieve a higher O2 affinity than HbA?
HbF has a lower affinity for BPG
what is the key difference in HbF that gives it lower affinity for BPG?
His143 in HbA is changed to Ser143 in HbF
His143(+) interacts with BPG(-) in HbA
His143 vs Ser143 is the key difference between what two Hb subunits?
beta vs gamma
(HbA vs HbF)
Ser143 does not bind (-)BPG as well
how do the [BPG] in maternal and fetal blood compare?
roughly equivalent
therefore, since HbF has less affinity for BPG, it will bind less and have higher O2 affinity
how does high altitude alter O2 transport by Hb?
high altitudes raise BPG levels to compensate for reduced pO2 (more O2 released to tissues, not more O2 extracted from air)
how does pulmonary emphysema alter O2 transport by Hb?
raise BPG levels to compensate for reduced pO2 (more O2 released to tissues, not more O2 extracted from air)
how will chronic anemia alter O2 transport by Hb?
raise BPG levels to compensate for lesser degree of O2 transport (more O2 dropped off at tissues)
*also RBC apparently metabolize BPG, so fewer RBCs, more BPG
how may BPG levels need to be adjusted in stored blood prior to transfusion?
BPG levels may need to be reconstituted (raised) in stored blood prior to transfusion because stored RBCs metabolize BPG
4 examples of physiologic conditions that raise BPG include:
- high altitude
- CO from smoking
- emphysema
- chronic anemia
what is the structure of BPG?
2,3-bisphosphoglycerate
COO-
|
H---C---O---PO3 2-
|
H---C---O---PO3 2-
|
Hif blood is stripped of CO2 and BPG, what is the effect on hemoglobin?
what happens when blood reconstituted with CO2 and BPG?
- Hb reverts to high affinity oxyHb
- Hb restored to intermediate binding properties
T/F the allosteric effects of CO2, H+, and BPG upon Hb are additive
true - each allosteric effector interacts with Hb at a different site
stripping blood of CO2 and BPG will shift the Hb-O2 binding curve which direction?
left
which has a greater allosteric effect on Hb, CO2 or BPG?
BPG
why, without cooperativity, does Mb bind more strongly than Hb?
binding affinity is just an intrinsic property of the molecule. it’s aa sequence and folding results in stronger affinity than that of Hb
how does NO interact with Hb?
NO is a physiologic modulator produced by endothelial cells that relaxes smooth muscle, it is also sometimes administered therapeutically to treat pulmonary hypertension. However, a side effect of this treatment is that NO can fit into the heme cleft and oxidize Fe II to Fe III (methemoglobin), which cannot bind oxygen
T/F transport of CO2 from tissues to lungs requires the co-transport of Cl- and HCO3- to maintain electroneutrality in the RBC
true - Cl- and HCO3- are pumped together in cotransport (symport or antiport) to get HCO3- out of RBC in the lungs
what is the effect of a hisF8gly mutation in Hb?
The cooperativity of oxygen binding to this new Hb variant is largely lost and oxygen
binding is not significantly affected by CO2, BPG or a small decrease in pH
