Download Hemoglobin Myoglobin Lecture

Hemoglobin Myoglobin Lecture
Objectives
1) Describe the structure of the globin fold
a. It is dominated by alpha helices and is connected to the heme group via histidines
b. The interior of the protein is nonpolar (remember O2 isnt very soluble in water); the
outside of the protein is dominated by polar and charged amino acids so they can interact
with water
c. The heme group sits in a crevice in the molecule, which is lined with nonpolar amino
acids (except histadines which are polar and basic at neutral pH)
d. One proximal histadine binds to iron of heme, and other distal histadine binds to the O2
and helps stabilize the binding of iron to the oxygen
e. This portion of the myoglobin creates a special microenvironment for the eme that
permits the reversible binding of one oxygen molecule
2) Describe the major structural features of the prosthetic group of myoglobin and hemoglobin
a. Prosthetic group: small organic molecule that permanently associates with an
enzyme/protein
b. Heme is a complex of Fe2+(ferrous) and protporphyrin IX; The Fe2+ is held in the center
of the heme molecule by bonds to the four nitrogens of the porphyrin ring; The heme
Fe2+ can form two more bonds, one on each side of the planar porphrin ring; one of these
is coordinated to the side chain of a histidine residue of the globin mlecule, whereas the
other position is available to bind oxygen
3) Compare the structures of hemoglobin and myoglobin
a. Myoglobin consists of a single polypeptide chain that is structurally similar to the
individual subunit polypeptide chains of the hemoglobin molecule
b. Hemoglobin is a tetramer of 2 alpha-beta dimmers which each are similar to myoglobin
4) Write the equation that relates fractional saturation (Y) of myoglobin to oxygen pressure.
Which term is related to the affinity of Myoglobin for oxygen?
a. In General, the Fractional Saturation (Y): Y=[ligand]/(Kd+[ligand])
b. For Myoglobin and O2: Y=[O2]/(Kd+[O2])
c. Kd, the dissociation constant, dictates the affinity of Myoglobin for O2. The lower the
Kd (([P]+[L])/[PL]) the higher the affinity.
d. When O2 = 0, Y=0 which means the fractional saturation of Mb is zero, because no Mb
can be saturated if there is no ligand (O2) to saturate it
e. When O2=Kd, Y=0.5; this is the concentration that gives you half saturation
f. When O2>>Kd, Y approaches 1
g. Kd=1 Torr (1 mm Hg)
5) Write the Hill equation. What do the terms K and n indicate about ligand binding?
a. Y=[L]n/(Kn + [L]n or Y=[L]n /(K +[L]n); both are equivalent
b. K indicates the affinity of the hemoglobin for oxygen
c. N indicates the degree of cooperativity involved in ligand binding; could also be saying
the number of binding sites
6) Given values for n, Kd, and O2, calculate the value of Y
a. Non-linear form: Y=[02]n /(Kdn + [O2]n)
b. Linear form used to make Hill Plot / /: where slope = n coefiicient (so Hb has steeper
slope than Mb because Hb has higher n=2.8 vs n=1)
7) What can be deduced from the values of the Hill coefficient?
a. Determination of n gives measure of cooperativity
i. No cooperativity when n=1 (because think of only one binding site like in Mb)
ii. Positive cooperativity when n > 1
iii. Negative cooperativity when n < 1
8) Write the Scatchard equation. What parameters can be obtained from the slope and
intercepts of the plot? Scatchard and Double Reciprocal are ways of analyzing non-linear
graphs in a linear method and was used to derive Kd from [PL] values found by varying [L]
a. ([PL]/[L])=(-1/Kd)x[PL]+([Pt]/Kd)
b. Slope: -1/Kd
c. Y-intercept: [Pt]/Kd or [(Mb]+[MbO2])/Kd
9) Write the double reciprocal equation. What parameters can be obtained from the slope and
y-intercept?
a. (1/[PL])=Kd/[Pt]x(1/[L])+(1/[Pt])
b. Slope: Kd/[Pt]
c. Y-intercept: (1/[Pt])
d. Negative x-intercept: (-1/Kd)
10) Compare the binding isotherms of hemoglobin and myoglobin over the physiological range
of oxygen concentration. How is this difference in behavior related to their physiological
functions?
a. Over the range of physiological oxygen concentration myoglobin shows a high affinity
for oxygen even in low oxygen concentration while hemoglobin shows low affinity for
oxygen at a low concentration of oxygen and high affinity for oxygen in areas of high
concentration of oxygen. Physiologically this makes sense because hemoglobin is acting
to take oxygen from the lungs (high oxygen concentration) and deliver that oxygen to the
tissues (low oxygen concentration) and so needs a mechanism where by it can
consistently release the oxygen in the low oxygen concentration area; Additionally Mb
takes oxygen from Hb in the muscles and so must be able to hold oxygen in areas of low
oxygen concentration in order to do this;
11) What is the role of Hb in the transport of O2, CO2, and H+
a. O2: hemoglobin can bind 4 O2 groups, one at each of its four heme groups; Hb serves to
take O2 from the lungs and deliver it to the tissues
b. CO2: Most of the carbon dioxide produced in metabolism is hydrated and transported as
bicarbonate ion. However, some CO2 is carried as carbamate bound to the uncharged
alpha amino groups of hemoglobin; the binding of CO2 stabilizes the taught (deoxy)
form of hemoglobin, resulting in a decrease in its affinity for oxygen; In the lung CO2
dissociates from the hemoglobin, and is released in the breath
c. H+: The Bohr effect relects the fact that the deoxy form of hemoglobin has a greater
affinity for protons than does oxyhemoglobin. This effect is caused by ionizable groups,
such as the N-terminal alpha amino groups, and specific histidine side chains that have
higher pKas n deoxyhemoglobin than in oxyhemoglobin; Therefore an increase in the
concentration of protons (decrease in pH) causes these groups to become protonated
(charged) and able to form ionic bonds/salt bridges; These bonds preferentially stabilize
the deoxy form of hemoglobin, producing a decrease in oxygen affinity
12) Describe the bonds made to Fe2+ in hemoglobin
a. 4 bonds from Fe to N groups of porrphoryn rings
b. Fe bonded to oxygen and histidine (proximal) as well
13) Describe how O2 binding is linked to salt bridge disruption
a. O2 binding to hemoglobin causes some change in conformation
b. When O2 binds to Fe, Fe moves 0.06nm into the plane of heme; heme goes from being
domed shaped and non-planar to being planar when Hb is oxygenated
c. His on F8 Helix moves with the iron
d. C=O of Val FG5 loses H-bonding contact with Tyr HC2
e. The C-terminus is displaced
f. There are diminished subunit interactions (many contacts are lost; salt bridges)
i. Ionic interactions stabilize the T-state and these interactions are disrupted in the R
state
g. These structural changes (TR) result in the cooperative nature of O2 binding
14) Draw the structure of 2,3-biphosphoglycerate (BPG)
15) What is the stoichiometry of BPG binding to hemoglobin? What is the nature of the BPG
binding site?
16) Compare the O2 affinity of HbA, HbF, Mb, and cytochromes. What is the significance of
their different affinities for oxygen?
17) Explain why 50% saturation of Hb with CO can be fatal but 50% loss of Hb in anemia
generally is not
18) What are the functions of the conserved residues in hemoglobin?
19) List three properties of proteins that exhibit positive cooperativity according to the Monod
model
20) Compare the Koshland and Monod models for cooperativity
a. Monod Concerted Model for Positive cooperativity
i. There are two conformational states: R (relaxed, high affinity) and T(taught, low
affinity)
ii. The conformers are symmetric (meaning all subunits are either in T state or R state)
1. This symmetry is due to subunit contact
iii. Hb exists in an equilibrium of T state and R state, with all 4 of its subunits acting
symmetrical in whichever state the protein is in
iv. 3D view can see a cavity in the T-state model
v. T state still binds O2 but just to a lesser degree than the R state
vi. Conversion of TR is not caused by O2 however O2 can stabilize a Hb in the R
state
1. There are salt bridge contacts that exist during the T-state and hold the protein in
this conformation. However when O2 is bound in hemoglobin in the R state these
contacts are lost and O2 binding shifs the TR state distribution towards R
state (which is the high affinity state cooperative binding because as one O2
binds the Hb it favors/stabilizes the R state which is high affinity and can pick up
remaining O2s more quickly)
vii. T State and O2 release is favored when O2 is low; low oxygen tension favors T state
1. If O2 is released from R state Hb, then interconversion between R and T is again
more likely, allowing for a discontinuance of the cooperativite binding
viii. O2 affinity increases with Y (fractional saturation)
1. Occupation of last 2 sites gives rise to cooperation because low concentrations of
Hb dissociate to alpha and beta dimmers which don’t show cooperativity
b. Koshland Sequential Model for Cooperativity
i. Without ligand, protein is in one conformation
ii. Equilibrium of 2 states:
1. T (low affinity)
2. R (high affinity)
iii. Symmetry is not required (RT states occur; protein could have both R and T
subunits)
iv. Sequential change in conformation (Ligand binding induces TR trasition); this is
different from Monod’s model
v. A ligated subunit can influence the conformation of a neighboring subunit (differet
from monod’s) and its affinity for ligand; affinity can increase or decrease (positive
or negative cooperativity)
vi. Can explain positive AND negative cooperativity (Monod’s can’t)
c. In Monod’s all subunits are R or T, and so when a couple subunits have bound to O2 the
O2 stabilizes the R state of the entire protein meaning all 4 subunits will be in the R state
and will have a higher affinity for oxygen. In Koshlands’s when a subunit binds to
oxygen, oxygen directly induces the conformational change from TR and even
raises/lowers the affinity of its neighbor or changes its conformation so that it can bind
Oxygen tighter, then when it does bind Oxygen it changes from TR etc.
d. General model for cooperativity includes features of both concerted and sequential
models; both indicate that O2 binding is necessary for cooperativty
21) List the factors that enhance O2 delivery in peripheral tissues
a. Low pH
b. High pCO2
c. 2,3-biposphoglycerate (2,3-BPG)
22) Describe the effect the defect in Hb S has on its properties and how this defect leads to
sickling. How do Hb S and Hb C differ?
23) What results in irreverisible sickling of red blood cells in sickle cell disease?
24) What is the nature of the binding site for CO2 binding to Hb?
a. CO2 binds to N-terminus of deoxyhemoglobin Alpha subunit forming a salt bridge
between carbamino valine (on the alpha subunit) and Arg141 stabilizing the T-state
b. N-terminus of B-subunit is also carbamylated and can also carry CO2
c. How many CO2s are carried by one hemoglobin
25) What is responsible for irreversible sickling of red blood cells in individuals with sickle cell
disease?
26) List several ways in which N and C terminal residues of Hb are important in regulating O2
binding
27) How does the binding isotherm of hemoglobin change with temperature and what is the
physiological significance of this property?
28) Explain why the cooperative nature of oxygen binding sites is essential for the physiological
function of hemoglobin
a. Because it allows a high oxygen conctration to affect binding affinity of Hb; where there
is a high concentration of O2 there will be increased binding affinity and where there is a
low concentration of O2 there will be increased delivery, such as in the tissues;
29) What is the Bohr effect and what is the physiological important of this phenomenon?
a. At low pH (high H+ concentration), protons can act as effectors (bind to Hb and affect
affinity of O2 binding) by creating salt bridges that stabilize the T-statedecreasing
Oxygen affinity, releasing it into the tissues; It is physiologically important because the
Bohr effect facilitates the delivery of Oxygen to the tissues; graph shows that at lower pH
values Hb has lower Y or saturation of Oxygen; These salt bridges that are formed are
lost at higher pHs again affecting conformation change, but this time from TR so Hb
can pick up more Oxygen in the lungs (where pH is high)
b. Salt bridge when His146 is protonated and stabilizes the T-state (in tissues); when it is
unprotonated there is no saltbridge and this is indicative of the protein being in the Rstate (in lungs)
30) What amino acid residues mediate the Bohr effect and what is the molar mechanism of this
phenomenon?
a. His146 mediates the Bohr effect; When in low pH, the Nitrogen on His146 becomes
protonated, and when in low pH the hydrogen is removed from His146
31) What is the function of carbonic anhydrase in the release of CO2 in the lungs? Why is this
enzyme essential for this process?
32) What contribution did the following individuals make to our understanding of ligand binding
and hemoglobin structure and function? Christian Bohr, Linus Pauling, Harvey Itano,
Jacques Monod, Daniel Koshland
Key Concepts
1) Hemoglobin is a multifunctional protein that carries O2 and CO2
2) Simple equations describe the binding of ligands to proteins
3) Cooperative behavior of proteins facilitates their physiological function
4) Ligands linked to oxidative metabolism regulate O2 binding and release by binding to
effector sistes
5) Point mutations in hemoglovin have effects on function that can result in disease
Hemoglobin, Myoglobin Summary
 Structure of heme and its association with hemoglobin and myoglobin
 Hemoglobin and myoglobin structure
 Analysis of ligand binding and cooperativity
 Models to explain cooperative ligand binding
 Molecular origins of cooperative binding in hemoglobin
 Physiological Regulation of oxygen transport and release
 Aberrant Hemoglobins – HbS, HbC