Lecture 6 - Hemoglobin II

Intersubunit contacts

1. a1-b1 and a2-b2 (strongest, cannot alter)

2. a1-b2 and a2-b1 (strong but CAN be altered, important)

3. a1-a2 and b1-b2 (few salt bridge contacts)

a1-b1 and a2-b2 contacts

35; VERY strong and cannot alter

a1-b2 and a2-b1 contacts

19; strong but CAN be altered (important for deoxy to oxy shift)

a1-a2 and b1-b2 contacts

Few salt bridges; weak, stabilize deoxy form

What occurs during the shift from deoxy to oxy Hb?

Change in quaternary structure: a1b1 dimer rotates relative to a2b2

Cavity in the middle of Hb

Water-filled; gets smaller after shift to oxy-Hb

What allows the a2-b2 dimer to rotate about a1-b1?

The flexible interactions between a1-b2 and a2-b1 contacts

Important Interactions

Hydrophobic effect, H-bond, and salt bridges between FG corners and C helices

NOTE that some interactions change and stay the same between deoxy and oxy Hb

Deoxy to oxy transition

O₂ covalently binds to Fe on heme, requiring Hb to flatten heme

Flattening is the KEY to Hb cooperativity

Importance of heme flattening

Basis of cooperativity through a chain reaction for all four subunits

Process of Conformation Change

1. O₂ binds to Heme Fe

2. Fe moves into the porphyrin plane with proximal His

3. F helix of b2 moves towards the porphyrin ring

4. b2 FG moves relative to a1C

   1. Intersubunit H-bonds break/form

   2. Salt bridges disrupt

What is required for all subunits to change conformation?

Deoxy to oxy shift must occur simultaneously at a1b2 and a2b1 interfaces because those interactions are flexible

Structural Changes Upon O₂ Binding

• Quaternary structure change

• All 4 hemes adopt R (oxy, high affinity) state

  • MUST exist this way, not just 1 heme (positive cooperativity)

What two forms can Hb exist in?

T or R

T = deoxy

R = oxy

Which subunits communicate → R state?

• a1 → b1

• a1 → b2

• b1 → a2

Sigmoidal Curve related to conformational changes

1. De-oxy state: all subunits in T state, low affinity

2. One molecule of O₂ binds, inducing change in all others

3. Other 3 binding sites occupied

4. Fully in R state, high affinity

In what two ways in Hb regulated?

1. Heterotropically (by CO₂ and 2,3-BPG)

2. Homotropically (by O₂)

What is heterotrophic regulation?

Protein is regulated by something it doesn’t usually bind

Hb’s heterotrophic regulators

1. 2,3-BPG

2. CO₂

CO₂ Regulation of Hb (Bohr effect)

CO₂ lowers pH, causing His to protonate, stabilizing deoxy form with a salt bridge

2,3-BPG Regulation of Hb

Fits into cavity of deoxy-Hb ONLY, stabilizing it with a salt bridge (low affinity for O₂)

Fetal vs. Maternal Hb

Fetal Hb has higher affinity for oxygen because instead of having a Histidine, it has Serine so BPG has one less spot to bind and stabilize deoxy-Hb

Why would fetuses need higher affinity Hb?

They are competing for oxygen with mother

Sickle Cell Anemia Cause

Mutation in gene coding for Hb-b, instead of glutamate there is a valine (Polar charged → Nonpolar)

What does Glu → Val switch do?

Creates sticky patch and hydrophobic effect, causing fibers

What do fibers in Hb lead to?

Sickling of RBCs (wrong shape), cannot transport oxygen and causes pain due to getting stuck in vessels