Myoglobin and Hemoglobin

1
Myoglobin and Hemoglobin

• Myoglobin structure - a monomer
• Iron complexed by Heme, surrounded by protein
• Hemoglobin structure - a tetramer
• A dimer of (ab) heterodimers
• Oxygen binding leads to structural changes
• Oxygen binding
• Myoglobin binds oxygen with a hyperbolic
  saturation curve
• Cooperativity in O2 binding to hemoglobin
• Binding gt Conformational changesgt Affinity
  changes
• Hemoglobin variants

2
Myoglobin structure

• Globin
• 8 helices
• A (blue) to H (red)
• Protein segments (including sidechains)
  completely surround the Heme.

3
Heme

• A porphyrin
• Held noncovalently
• Iron (II) ligated by 4 pyrrole nitrogens
• Histidine (below), the 5th ligand
• O2 binds above
• Color change on O2 binding

4
Oxygen Binding

• Heme ligation state is reflected by color change
• Oxygenation turns the heme red
• Oxygen saturation can be monitored by absorption
  of visible light
• A580 for Oxy myoglobin gt A580 for Deoxy Mb

5
Figure 7 Absorption spectra of O. bicornis Mb
Biochemical Journal www.biochemj.org
Biochem. J. (2005) 389, 497-505
6
Dissociation constants and concentrations

• A dissociation constant (Kd) governs the oxygen
  dissociation reaction
• MbO2 ltgt Mb O2
• Kd products/ reactants MbO2/MbO2
• Concentrations can be recast in terms of Kd
• Mb KdMbO2 /O2
• Dissociation constant ligand concentration at
  half saturation
• When Mb MbO2 Mb is 50 saturated
• Kd O2 p50
• O2 is proportional to partial pressure
• Chemical potentials are equal in the gas and
  liquid phases

7
Saturation Curves

• The fractional saturation (Q) is a hyperbolic
  function of O2
• Q oxygenated subunits /total subunits
• Q MbO2 / (Mb MbO2)
• Substituting KMbO2 /O2 for Mb
• Q MbO2 / (KMbO2 /O2 MbO2)
• Q 1 / (K /O2 1)
• Q O2 / (K O2)

8
Hemoglobin structure

• A dimer of heterodimers (ab)
• Oxygen binding leads to structural changes
• T state corresponds to deoxy form
• R state corresponds to oxygenated form
• Tertiary changes transmitted from heme gt His gt
  helix F
• Tertiary changes lead to quaternary shifts
  between a1b1 and a2b2

9
Tertiary structure changes
10
(No Transcript)
11
Cooperativity

• Binding of one ligand affects the affinity for
  the next.
• Positive cooperativity
• If subsequent binding steps are very strong
  relative to the first binding steps, ligand
  saturation is essentially concerted.
• Hb nO2 gt Hb(O2)n
• How many ligands bind in one event?

12
Hill plots and cooperativity estimates

• Assuming that binding is all or none, how many
  ligands bind (or dissociate) at once?
• For the reaction Hb(O2)n gt Hb n O2
• Kd (Hb O2n) / Hb(O2)n
• Y Hb(O2)n / HbT
• HbT Hb Hb(O2)n
• Rearranging the equilibrium expression
• Hb(O2)n /Hb O2n / Kd
• Substituting for Hb
• Hb(O2)n /(HbT - Hb(O2)n ) O2n / Kd
• Dividing by HbT gives
• Q / (1 - Q) O2n / Kd
• Log (Q / (1 - Q) ) n log O2 - log Kd

13
Hill plot of O2 binding to myoglobin and
hemoglobin
14
Cooperativity models
The sequential (Koshland)model Each subunit
undergoes 3 changes upon ligand binding R states
interact better with Rs
Monod-Wyman-Changeaux T and R are 4 states all
or none R states bind tighter
15
Allostery

• Allosteric proteins can exist in 2 conformational
  states (often denoted T and R)
• States differ in ligand affinity or enzymatic
  activity
• Conformational changes in tertiary structure and
  can also be in quaternary contacts
• Effector binding shifts the distribution of
  states
• Homotropic effectors (binding of one ligand or
  substrate affects the binding of the next of the
  same type)
• Heterotropic effectors (different than the ligand
  or substrate)

16
Allosteric Effectors
Small molecule binding can affect the energetics
of the T to R transition Increasing pH increases
O2 affinitiy The Bohr effect CO2 decreases pH BPG
binds to deoxy hemoglobin decreases O2 affinity
17
Hemoglobin physiology
High H
Low H
18
Hemoglobin variants

• 1 in 20 people has a variant hemoglobin
• Most are silent, single amino acid changes
• Some can cause real problems
• Thallesemias
• Sickle cell anemia
• Deoxy form polymerizes at low pH
• Sickled erythrocytes dont flow very well
• Adaptation to malaria

19
A single amino acid change is responsible for
sickle cell anemia