Chemistry 340 Exam 1 Lectures 8-9 Myoglobin and Hemoglobin |
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Major concepts:
1. What is Kd for a protein (P) that reversibly binds a ligand (L), and what does it indicate about the PL complex?
2. What is Θ, and what type of curve is produced by graphing Θ as a function of [L] for myoglobi
3. What type of curve is produced by graphing Θ as a function of [L] for hemoglobin, and what causes the difference from myoglobin?
4. What part of hemoglobin (heme or globin) is affected by changing the concentration of CO2, H+, or BPG (1,3-bis-phosphoglycerate)? How does the change affect O2-binding?
5. What does carbonic anhydrase (in tissues and lungs) do that affects hemoglobin function?
Core knowledge:
1. What is the function of myoglobin, and how are both heme and globin involved?
2. How do the structure and function of hemoglobin resemble and differ from those of myoglobin? (That is, compare and contrast the two proteins.)
3. To what specific part of hemoglobin does CO2 bind, and what change occurs as a result?
4. To what specific part of hemoglobin does H+ bind, and what change occurs as a result?
5. To what specific part of hemoglobin does BPG bind, and what change occurs as a result?
6. How is sickle cell Hb different from normal Hb, and how is that related to the disease?
Protein binding of a ligand (reversibly): P + L ↔ PL (a complex, not a compound)
Kd = ([P] [L])/[PL] and Ka = [PL]/([P] [L]) – usually use Kd
Myoglobin (Mb): function = binding O2 using heme
structure of Mb:
heme (porphyrin) = protoporphyrin ring + Fe2+ coordinated to 4 N's in the ring – Fig. 5-1
globin = protein
primary structure = 153 residues
secondary structure = 8 (A-H) α-helices
tertiary structure = relationship of 8 helices and the bends connecting them (AB, BC, etc.)
heme Fe is coordinated to His F8 = His 93 – Fig. 5-2 and Fig. 5-3
Quantitative and graphical evaluation of O2 (L) binding by Mb (P)
For a population of Mb molecules, some have O2 bound (= PL) and some do not (P)
The % of binding sites that are occupied = Θ = [PL]/([PL] + [P])
By substituting and rearranging terms, Θ = [L]/(Kd + [L])
Graphing an equation in this form → hyperbolic curve – Fig. 5-4
At [L] = Kd , 50% of binding sites are occupied.
Kd indicates the affinity of a protein for a ligand. Table 5-1
For experimental observations of O2 binding by Mb, change [O2 ] to pO2 and Kd to P50
P50 = pO2 at which 50% of Mb sites are occupied.
Θ = pO2 /(pO2 + P50)
Effect of globin on ligand (O2 and CO) binding by heme – Fig. 5-5
1. Prevents Fe2+ oxidation to Fe3+
2. Reduces affinity of CO binding, although CO Kd is still smaller than O2 Kd
3. Globin movement (breathing) required for reversible binding of O2 .
Hemoglobin (Hb): function = transport of O2
structure of Hb: like Mb but with four subunits for globin, each with its own heme
adult Hb = 2 α chains + 2 β chains (α2β2)
primary structures = 141 (α) and 146 (β) residues – Fig. 5-7
secondary structures = α helices for both types of chains, also labeled A-H
Note that the α chain does not have a D helix
tertiary structure = relationship of helices in one subunit – very much like Mb – Fig. 5-6
Quaternary structure = relationship of subunits to each other
two αβ pairs = α1β1 and α2β2,
with many interactions between α and β subunits
Quantitative and graphical evaluation of O2 binding by Hb
Equation for binding: P + nL ↔ PLn
Kd = ([P] [L]n)/[PLn] and Θ = [L]n/([L]n + Kd )
Rearranging and taking log of both sides gives
Hill equation: log [ Θ/(1-Θ )] = n log [L] – log Kd
Hill plot is graph of log [ Θ/(1-Θ )] as a function of log [L], which should have slope n.
Remember that for O2, [L] = pO2 and Kd = P50, so log [ Θ/(1-Θ )] = n log pO2 – n log Pn50.
Actual graph does not have slope = number of binding sites, but
slope = affinity of binding sites for O2 – Fig. 5-14
so slope is designated nH (Hill coefficient)
For Mb, nH = 1. No cooperativity
For Hb, nH = 1 when log pO2 is low and when log pO2 is high. No cooperativity
At intermediate log pO2 , nH = 3, indicating cooperative binding of O2 by Hb.
Hb changes conformation upon binding O2 :
T (tense) state has low affinity for O2
stabilized by salt bridges (ionic interactions) between α1β2 and α2β1 interfaces – Fig. 5-9
R (relaxed) state has high affinity for O2 .
Heme changes from slightly curved (T) to flat (R), with change inHis F8 → F helix change
Change in overall structure – Fig. 5-10
Advantage to cooperative binding
High affinity in lungs → efficient O2 binding and 96% saturation
Low affinity in tissues → efficient O2 release – Fig. 5-12
Sigmoidal graph indicates cooperative binding
only occurs in proteins with multiple binding sites (multiple protein subunits)
Hb is a model of an allosteric protein that has cooperative binding of a ligand.
Allosteric proteinHas two shapes (R and T).
Changing shapes is influenced by modulator binding to the protein.
Homotropic modulator = a molecule whose binding at one site influences the binding of
another molecule of the same type at another site
For Hb, O2 is a homotropic modulator
Heterotropic modulator = a molecule whose binding at one site influences the binding of
a different type of molecule at another site
Activating modulator (A) = a molecule that increases binding affinity for a ligand (L)
Inhibiting modulator (I) = a molecule that decreases binding affinity for a ligand
Models for cooperative binding – Fig. 5-15
MWC (Monod-Wyman-Changeux) = concerted model
All protein subunits are similar and tend to change conformation together.
T4 ↔ R4, where T is unlikely to bind L, and R is likely to bind L.
Sequential (Koshland) model
Protein subunits can change conformation separately,
change in one subunit increases the probability that another will change.
Binding L stabilizes the R conformation.
Hemoglobin modulators
O2 is a positive homotropic modulator (stabilizes R conformation)
Binding O2 to one subunit promotes binding of O2 by another subunit.
Negative modulators (stabilize T conformation):
H+ binds side chains, especially His HC3 of the β subunits → salt bridge with Asp
stabilizes T conformation = Bohr effect – Fig. 5-16
formed by the reaction catalyzed by carbonic anhydrase: H2O + CO2 ↔ HCO3- + H+
CO2 binds amine terminus: 
also forms salt bridges that stabilize the T conformation
BPG (2,3-bis-phosphoglycerate) binds positively-charged side chains