9- Hemoglobin and Cooperativity: The Quaternary Shift

What is cooperativity?

When binding at one site affects binding at another site

What is a quaternary shift?

A change in the arrangement of a protein’s subunits relative to one another.

Quaternary structure

The arrangement and interactions of multiple protein subunits

Why does hemoglobin’s structure matter for oxygen binding?

Structural changes alter how strongly its binding sites bind oxygen.

What shape is myoglobin’s oxygen-binding curve?

Hyperbolic

What shape is hemoglobin’s oxygen-binding curve?

Sigmoidal, or S-shaped.

Why would a higher p50 make myoglobin less effective at storing oxygen?

It indicates lower oxygen affinity, so myoglobin would hold oxygen less strongly

How are cooperativity and the quaternary shift related in Hb?

The structural shift helps increase the affinity of hemoglobin's remaining sites for oxygen

Why are hemoglobin's binding sites not completely independent?

Binding oxygen at one site can change the protein's structure and affect the other sites.

What does n represent in a reaction?

The number of oxygen molecules bound to hemoglobin

Does positive cooperativity mean the oxygen molecules physically attract one another?

No. The effect is caused by changes in hemoglobin's structure.

What does the Hill coefficient measure?

The degree of cooperativity in a binding system

What does a Hill coefficient of 1 indicate?

No cooperativity

What does a Hill coefficient greater than 1 indicate?

Positive cooperativity

Does a Hill coefficient of 3 mean hemoglobin has three binding sites?

No. Hemoglobin has four binding sites; the coefficient measures cooperativity.

Why must hemoglobin both bind and release oxygen?

It must collect oxygen in the lungs and deliver it to tissues

Why does hemoglobin load oxygen in the lungs?

Oxygen partial pressure is high there, favoring oxygen binding.

Why does hemoglobin release oxygen in tissues?

Oxygen partial pressure is lower in tissues, favoring oxygen unloading

How does the environment affect hemoglobin's oxygen binding?

It can promote structural changes that alter oxygen affinity

What is hemoglobin's oxygen affinity in the lungs?

High affinity, associated with a lower p50

What is hemoglobin's oxygen affinity in tissues?

Lower affinity is associated with a higher p50.

How does p50 relate to oxygen affinity?

They are inversely related: lower p50 means higher affinity

What changes hemoglobin's oxygen affinity?

Structural changes in hemoglobin

Which hemoglobin state has higher oxygen affinity?

The R state

Which hemoglobin state has lower oxygen affinity?

The T state

What happens to Hb’s affinity for oxygen as oxygen binds?

As each oxygen molecule binds, it changes hemoglobin's shape in a way that makes it easier for the next one to attach

• More oxygen binding —> affinity increases

What is hemoglobin's subunit composition?

Two alpha and two beta subunits, written as α2β2

What is a tetramer?

A structure made of four subunits.

What is a dimer?

A structure made of two subunits.

• Hb can be thought of as two αβ diamers!

What interactions help hold hemoglobin's subunits together?

Hydrophobic interactions, ionic interactions, and hydrogen bonds.

How can pH affect hemoglobin's structure?

It can change charges on groups and disrupt ionic interactions and some hydrogen bonds

What is heme?

A ring-shaped group containing iron that binds oxygen.

What is the role of Fe2+ in heme?

It is the iron ion that directly binds oxygen.

What is the proximal histidine and its function?

His F8, which coordinates with the iron and connects it to the protein

What is the distal histidine and its function?

His E7, which hydrogen-bonds to bound oxygen to help stabilize it within the heme pocket

How many and what ligands coordinate with the iron when oxygen is bound?

Six: four porphyrin nitrogens, one nitrogen from His F8, and oxygen.

When does Fe2+ change to Fe3+?

In excess oxygen

What is the role of Val E11 in the heme pocket?

Helps control the space around oxygen

• Affects how easily oxygen enters the pocket and reaches the heme iron, where oxygen binds.

What is the role of Phe CD1 in the heme pocket?

Helps form the walls of the pocket around the heme

• shape and position influence how much space is available for oxygen and other small molecules

What conformation change happens to the iron when oxygen binds?

It moves into the plane of the heme ring.

How does the iron's movement during a conformational change affect His F8?

he iron pulls His F8 along with it.

How does His F8 affect the F helix during a conformational change?

Because His F8 is connected to the F helix, its movement causes the helix to move.

Why are conformational changes important for cooperativity?

It helps trigger a structural change that increases the affinity of other oxygen-binding sites.

What is the F Helix?

A group of amino acids connected to the heme iron, with His F8 being the one directly connected

What are hemoglobin's two major structural states?

The T state and the R state.

Can oxygen bind to the T state?

Yes. Oxygen can bind to both T and R, but it binds more readily to R.

How does oxygen binding affect the T-to-R transition?

Oxygen binding helps shift hemoglobin toward the higher-affinity R state.

What state of hemoglobin is in the T state?

Deoxyhemoglobin

• Oxygen not loaded

What state of hemoglobin is in the R state?

Oxyhemoglobin

• Oxygen is loaded

What is the significance of 15° in the T to R transition?

The 15° rotation refers to the approximate rotation of the two αβ dimers in hemoglobin during the transition from the T state —> R state

What evidence of structural change occurs when deoxy-Hb is exposed to oxygen?

Deoxygenated hemoglobin crystals shatter when exposed to oxygen.

How much does the iron move during a conformational change?

Approximately 0.029 nm

What stabilizes the T state?

Ion pairs at the interface between the two alpha-beta dimers

• pair of oppositely charged amino acid side chains that attract each other and are located where two protein subunits meet.

Is the the T-state simply a shape that is taken up?

The T state is not simply a shape hemoglobin happens to take

• It is stabilized by interactions within the protein, and those interactions must change for the protein to shift toward R.

How does oxygen binding initiate T —> R transition?

Oxygen initiates movement within hemoglobin that changes the point of contact for subunits, disrupting interactions that stabilize the T state.

What is predominantly affected during a conformational shift?

The movement predominantly affects the quaternary structure of the αβ protomers (at the α1β1 and α2β2 interface)

What is the trigger for hemoglobin's structural change?

Oxygen binding to the heme iron

Does Mb undergo significant conformational changes? Why or why not?

No.

• Mb as only one protein subunit and one heme group. Oxygen binding still causes local structural changes, including movement of the iron and proximal histidine, but Mb does not undergo the same quaternary structural transition because it has no other subunits to rearrange

What are sliding contacts?

Contacts between protein subunits that change as the subunits shift relative to one another

• Some contacts are broken, formed, or rearranged as dimers move

How many interacting residues change their contacts during the conformational change of Hb?

19

What causes sliding contacts between a1-b2,a2-b1?

Oxygen binding to iron, which causes the movement of the F helix

• REMEMBER THAT THE F HELIX IS A CHAIN OF AMINO ACIDS

Why do sliding contacts at the interfaces matter?

They help produce the quaternary shift toward the R state

Where does the F helix shift towards when oxygen binds?

The F helix shifts, changing its position relative to the opposite dimer's C-terminal region

What is the C-terminus?

The end of a protein chain

Why can the iron not move independently of His F8?

His F8 coordinates with the iron, so iron movement requires His F8 to reorient.

Why does His F8 movement affect the F helix?

His F8 is part of the F helix, so its movement requires the helix to shift or reorient.

Where does the translation of the F Helix produce quaternary shifts?

a 1C-b2FG contacts

What is a hydrophobic pocket?

A protein region where nonpolar groups are packed together.

Which interactions remain mostly unchanged during the T → R transition?

The interactions within the α1β1 pair and the α2β2 pair

What changes during the T → R transition if the interactions within each pair remain unchanged?

The two αβ pairs rotate and shift relative to each other

Why can oxygen binding at one site cause the whole hemoglobin molecule to change state?

Because hemoglobin's subunits are mechanically connected ( α1β1 pair and the α2β2 pair), a local change at one heme can trigger a rearrangement of the entire molecule

What is the oxygen pressure for venous and arterial blood?

About 30 torr for venous blood and 100 torr for arterial blood.

Overall p50 values for Mb and Hb?

Myoglobin: 2.8 torr

Hemoglobin: 26 torr

Why does the first oxygen bind less readil

Hemoglobin initially favors the lower-affinity T state

What happens during the second and third oxygen-binding event in Hb?

The interface becomes destabilized enough for the T-to-R transition to occur

• Binding becomes easier as you go from second to third binding

Why does the fourth oxygen bind readily in Hb?

Hemoglobin is in the higher-affinity R state.

Why must the T--> R transition occur simultaneously at the interface between the αβ protomers?

The two αβ dimers are connected, and the interactions at their interface determine their relative positions

What is a protomer?

protomer is a structural unit of a larger protein complex

Why do C-terminal ion pairs break during the T → R transition?

Structural changes reposition charged groups, weakening interactions that stabilize the T state.