lecture 11- Myoglobin and Hemoglobin

Ligand

Molecule that binds to protein reversibly

PL

Protein ligand complex

Bonding site

Location on the protein molecules that interacts w/ ligand

Cooperativity

Binding of one ligand on one subunit affects binding on other subunits

Negative cooperativity

Binding of one ligand negatively affects /hinders the binding of another protein

How does ligand binding affect a protein with multiple subunits

When a ligand binds it often induces a conformational change that can change the binding site of the other subunits. By either making it so that ligand can”t bind or that it can.

What is Kd

Dissociation constant  Kd=[P][L]/[PL] The concentration of ligand when 50% of protein would be in PL complex while the other 50% would be a free protien. Smaller value means higher affinity. (more PL than free ligands and proteins)

Myoglobin

• Oxygen storage protein (binds to O₂₎

• _{found in mammalian muscle tissue}

• _{Diagnostic of muscle injury}

• _{first protien sturtcure ever determined}

Hemoglobin

• Oxygen transport protein found in red blood cells

• Binds to O₂ in lungs and releases it in tissues

• Job is to transport O₂ from our lungs to tissue

• Composed of four subunits, allowing cooperative binding

• Plays a role in carbon dioxide transport and H⁺ transport

• Quaternary structure 

How does Hemoglobin and Myglobin bind to O₂ ?

Hemoglobin and Myoglobin use a prosthetic group called Heme that is very good at binding to O_{2 since amino groups are not good at it} 

Heme, What is it, what is it composed of, what can it do, and how is it affected by binding to myoglobin

• A prosthetic group that helps hemoglobin and myoglobin bind to oxygen

• 2 parts of heme:

  • Protoporphyrin ring

  • Fe²⁺

• Really good at binding diatomic molecules like O_₂ 

• They are even better at CO_₂, since it binds straight up, while oxygen binds at an angle

• Myoglobin structure affects the specificity of ligand binding (heme).

  • Heme by itself has a much higher affinity for CO_₂ than oxygen 

  • But because distal His forms an interaction with O₂ that it doesn’t with CO₂, it only binds to O₂. 

Structure of Heme

• Planar ring called protoporphyrin with Fe²⁺ In the center connected to 4 nitrogens 

• Fe²⁺ can connect to 6 binding sites, but the nitrogens that are part of the protoporphyrin ring only connect to 4 (in the plane of heme), leaving 2 open.

• A proximal histidine is occupying one of the 2 open binding sites

• Leaving one open for O₂

Myoglobin structure

• Monomeric 

• small—153 153 amino acids

• 8 alpha helix

• Has a single oxygen-binding site 

• tertiary structure

Nomenclature of Myoglobin and Hemoglobin

• 1st α-helix (N-terminus) is A, all the way to the (8th a-helix) C-terminus in alphabetical order

• letter tells you what helix it is in

• number tells you its position within the a-helix

• Example His F8

Hemoglobin structure

• Tetramic

• Quartenary

• 2 alpha subunites

• 2 Beta subunits 

• 4 heme groups( one heme for each subunit)

Hemoglobin interfaces

Hemoglobin has a large number of intersubunit contact between different subunits (so huge interface between the alpha 1 subunit and the beta 2 subunit )

so if a subunit of hemoglobin binds to oxygen, it will cause a conformational shift within the other subunits.

Oxygen binding induces a structural transition in hemoglobin (what are the two states )

• When there is a lack of oxygen, the hemoglobin is in T-state (tense/deoxyhemoglobin) because the tense state has a low affinity(releases it more) for O₂ and predominates when O₂ is not bound

• When oxygen concentration is added, it begins to shift the structure of the hemoglobin into the R-state. (relaxed state/oxyhemoglobin) with high concentrations of O₂, hemoglobin now has a high affinity (holds it tighter) for oxygen and predominates when O₂ is not bound.

• T and R state are reversible

What stabilizes T-state?

• T state is stabilized by a number of ionic interactionsThe interaction

• “The chain of interactions inside hemoglobin connects the oxygen-binding site to the places where the subunits meet, allowing oxygen binding to change the overall shape of the protein.”(cooperative binding)

• These ionic chains between interfaces protect T-state from binding to O2

What are the rearrangements of the heme that drive the T-to-R transition?

• Heme is a planar ring-shaped molecule that holds Fe²⁺ in the center

• However, in the T state (puckered heme, no O₂ bound), the iron does not sit perfectly flat on the ring

• When it binds to O_₂ and turns into the R state (planar heme), the O₂ pulls the iron into the plane of the ring. (downwards)

• The heme becomes planar (unpuckered).

• This small flattening movement tugs on the attached proximal histidine F8 and helix (to maintain coordination), which helps trigger the T → R conformational change in hemoglobin.

True or false in hemoglobin all 4 subunits can either be T or R You CANNOT have a mix. Since a change in one subunit causes a change in the rest.

True

How does cooperative ligand binding work for hemoglobin?

• Hemoglobin is positive cooperativity in its ligand binding

• As O₂ binds at the lungs, in order to pick up as much oxygen as possible, subunits transition from the T state to the R state (the entire tetramer converts). to the high affinity state

• As O₂ is released at the tissues in order to release as much oxygen as possible, subunits transition from R state to the T state (low affinity), and the entire tetramer converts

Why does the conformational change in Heme trigger T to R state transition

• In the T state there are ionic bonds that resist the binding ofbinding of oxygen

• When O₂ binds to the Fe²⁺ ino it moves down the plan of the protoporphyrin ring that pulls te F8 His residue wihc initiaes breaking of ionic bonds that were in T state which causes the conformational change.