Module 4: Protein Function

Myoglobin

• Binds oxygen through ‘simple’ behavior (unlike hemoglobin...)

• Monomer

• First X-ray crystal structure determined

Myoglobin function

1. Improves oxygen solubility to facilitate diffusion in the muscle

2. Prevents heme oxidation

3. Prevents carbon monoxide binding
   1. Distal His blocks CO, which normally binds heme 25,000x stronger than O2

Heme group

• Prosthetic group (permanently attached molecule)
  • NOT proteins
  • Fe (II) with a porphoryin ring

• Binds oxygen reversibly
  • Protein enables this

Oxygen binding curve of myoglobin

P₅₀ is when 50% of Myoglobin binding sites are occupied

• high P₅₀ implies low affinity for oxygen

• low P₅₀ implies high affinity for oxygen

• P₅₀ for myoglobin in blood is 2.8 torr

Myoglobin acts as an oxygen reservoir — it stores O₂ and releases it when tissue pO₂ is low; constant high affinity for oxygen

Ligand binding to protein

• hyperbolic binding observed at multiple biding sites ONLY WHEN binding at each site is independent of binding at other sites

• K_d is concentration at which 50% binding sites are occupied by ligand

Cooperativity

the binding of a ligand to one site affects the binding of additional ligands to additional sites

• requires multiple subunits, so myoglobin's O2-binding is NOT sigmoidal (has rectangular hyperbola)

  • Myoglobin cannot transport O2 because it has a high O2 affinity and would NOT release O2 in the tissues

  • Threshold effect in hemoglobin allows it to release more O2 in tissues that work "harder" and have lower O2

Hemoglobin

• Dimer of dimers
  • Two αβ dimers
  • α2β2 tetramer
  • Each subunit binds one heme
  • Thus, Hb binds 4 hemes!

alpha and beta subunits

The globin fold
• Sequences only ~18% identical at amino acid level
• Important principle: sequences diverge much more rapidly than
structures
• Some residues are absolutely conserved among Mb, Hb a, and Hb

Oxygen binding curve of hemoglobin p50

the oxygen pressure at which hemoglobin is 50% saturated

• hemoglobin has a p50 of 26 torr, meaning hemoglobin is half-saturated with oxygen at a partial pressure of 26 torr

• A lower p₅₀ means less oxygen is needed to reach 50% saturation ⇒ oxygen binds more easily (higher affinity for oxygen)

• A higher p₅₀ means more oxygen is needed to reach 50% saturation ⇒ oxygen binds less easily (lower affinity for oxygen)

  • this causes oxygen release

Fractional saturation (Y)

Y = (pO₂) / (p₅₀) + (pO₂)

Hill plot

• How do we determine n and p50 for hemoglobin?
• Ratio of oxy-Hb (Y) to deoxy-Hb (1-Y)
• Re-arrange Hill equation and take log to get linear equation
log (Yo2/1-Yo2)= nlogpO2 – nlogp50

Structural basis of functional hemoglobin

• Deoxy-hemoglobin: Tense state (T)

• Oxy-hemoglobin: Relaxed state (R)

• Oxygen binding drives structural changes T to R

• One heme bound to oxygen triggers the other three subunits into the R state (allostery)

Structural Basis of Hemoglobin

Step 1
Oxygen pulls iron into heme plane, which pulls on proximal His
F8, which pulls entire Helix F
Step 2
As a result, Tertiary structure changes between subunits
1. At the a1—b2 and a2—b1 interfaces
2. Ion pairs ‘break’ at C-terminus of a and b subunits

How to lower affinity of Hemoglobin for oxygen

important to facilitate R —> T transition
• increase H+ (Bohr effect)
• increase CO2 (Bohr effect part 1 and part 2)
• increase Bisphosphoglycerate (BPG)

Bohr Effect for protons

• as pH goes down (e.g. from increasing CO2), [H+] goes up.

• Protonation (deoxygenation/T-state) is favorable

• T-state ionic interactions are REFORMED

• O2 affinity decreases, causing a shift of the O2 binding curve to the right

• O2 binding is therefore pH dependent

  • lower pH promotes T state (oxygen release)

  • higher pH promotes R state (oxygen binding)