Handout 5 - Intro to Oxygen Binding

Is protein structure static?

No!
- Proteins are dynamic molecules
- Association with other molecules often involves changes in conformation

What other molecule do proteins often associate themselves with?

Ligands

What is one way ligands can bind to proteins?

Reversibly

What may ligands be?

small molecules, ions, other proteins/macromolecules, etc.

Where does binding occur?

specific 3D locations or binding sites/domains on the target protein

What do the binding sites/domains need to be to the ligand? Why?

Complementary

• allows it to discriminate among 1000’s of molecules present in the environment!

What aspects are important in complementarity?

size, shape, charge, hydrophobic/hydrophilic character

The interaction between the ligand and the binding domain needs to have a high degree of what?

Specificity

• so the domain only attracts the correct ligand(s) among the myriad molecules in the cell.

For different ligands, what might the protein have?

Separate binding sites

Binding may involve what element?

“Induced fit”

What is “induced fit”?

The idea that ligand binding can cause changes in the conformation or shape the protein, and this influences the function of the target protein

In multi-subunit proteins, if a conformational change occurs on one subunit, what happens?

Changes in the conformation of the other subunits will occur

What family are myoglobin and hemoglobin in?

the “Globin Family” of proteins

What example of proteins are myoglobin and hemoglobin?

Conjugated proteins

• nonpeptide units absolutely needed for function, tightly associated with the protein, so they have prosthetic units (heme!)

What are protein families?

Proteins with similar primary sequence and/or 3D structure and function

What type of protein (structurally) is Myoglobin (Mb)?

• Monomeric protein

  • single polypeptide chain

  • 3D structure is highly helical (75% alpha-helix, no Beta pleated sheets)

Where is Myoglobin found in the body?

• Inside muscle tissue

  • skeletal and also heart

What critical role does Myoglobin play in our bodies?

• It binds oxygen!

• Important in oxygen storage!

• Helping the tissue to take oxygen from the circulatory system and hold on to it until the muscle cells need the oxygen when the oxygen pressure/tension inside the muscle tissue runs low due to exertion.

What does Mb indicate?

De-oxy Myoglobin

What does Mb O2 indicate?

Oxygenated Myoglobin

What does each Myoglobin molecule have?

• A single O2 binding domain

• This allows it to reversibly bind a single oxygen molecule.

What type of protein (structurally) is Hemoglobin (Hb)?

• Tetrameric = 4 subunits

  • (alpha2, Beta2)

  • 2 alpha subunits have identical structure

  • 2 Beta subunits have identical structure

  • alphas and betas are very similar in their primary, secondary, and tertiary structures

Where is hemoglobin found in the body?

• It is the major cytosolic protein in red blood cells (RBC’s).

  • Its the red color of hemoglobin that gives these cells their characteristic red color.

What are the jobs of hemoglobin?

• Oxygen transport

  • Because hemoglobin is in RBC’s, and these move through the circulatory system, we have a perfect way to move oxygen from the lungs — where de-oxy hemoglobin picks up oxygen to become oxy hemoglobin — and have this move through the tissues where it releases oxygen to be picked up by myoglobin.

  • Oxy hemoglobin is turned back into de-oxy hemoglobin and then the red cells make the transition back to the lungs for another pick up.

  • De-oxy Hb —> Oxy Hb

How many subunits are on a single hemoglobin? What does this mean?

• 4 subunits

• Each one has a separate O2 binding domain (or heme), so while myoglobin only picks up one O2, hemoglobin picks up 4.

What is hemoglobin in the transport of oxygen?

A key molecule!

What type of protein (figuratively) is myoglobin?

A storage protein!

What is Dr. B’s favorite cell?

Red Blood Cells (which contain hemoglobin)

What are the 3 reasons we need proteins to associate with O2 to transport it in the body and to store it in the tissues?

1. Poor diffusion in tissues

2. Need to regulate transport (i.e - respond to changes in the environment or stress)

3. Greatly increases O2 solubility in water

How much does the oxygen solubility increase when using Hemoglobin as the transporter instead of using water?

~100 fold

Does O2 bind to the protein itself?

No, it binds to a prosthetic unit.

What is a prosthetic group or unit?

• Nonpeptide unit

• tightly associated with the protein

• absolutely required for function

How do we see prosthetic groups in Mb and Hb?

Globin: the protein part

Heme: the prosthetic group

Why do we need Heme?

None of the amino acids are suited to bind oxygen

What do all of the species (structures of myoglobin and the beta subunit of hemoglobin) have?

The classic “globin fold”

What exactly is the classic “globin fold?”

• 8 alpha helices, 7 bends, 75% alpha helical

• Very compact (not a lot of free space in the core)

• Interior nearly all Hydrophobic amino acids [except for 2 interior histidine residues (critical for function)]

• The single polypeptide chain of Mb and each (4) subunit of Hb have a heme

  • Located in nonpolar crevices of the protein

What communication occurs within Myoglobin molecules?

Each Mb molecule acts independently (no communication between hemes of different Mb molecules)

What does a fully oxygenated hemoglobin have?

4 oxygen molecules bound

What communication occurs within Hemoglobin molecules?

Between the hemes on the different subunits of a Hb tetramer

What two things are similar within the globin proteins?

• Similar subunit structure (globin fold) and heme association

What is hemoglobin said to be?

• A “dimer of dimers”

• The linkages between the different subunits are not exactly the same.

What do you mean by “dimer or dimers”?

• alpha1beta1 and alpha2beta2: subunits in these dimers are tightly linked; the 4 subunits can “communicate” with each other through subunit contacts

What does O2 binding at hemes cause?

Causes changes at the alpha1Beta1 and alpha2beta2 subunit interfaces which changes O2 binding affinity

What is Heme?

A complex organic ring structure based on a porphyrin

• porphyrin in heme = protoporphyrin 9

Structurally, what is a porphyrin?

• An organic molecule that has 4 pyrrole rings linked by methene bridges

• Different porphyrins have different substituents at the X positions

• Nonpolar; fits in a nonpolar crevice in the protein

• Ring system is somewhat planar

What is the heme in Mb. Hb (oxygen carriers)?

• A coordination compound with a bound iron atom

What must the oxidation state of the iron bound to oxygen carriers be?

2+

• Must be ferrous! “ferroHb”

  • Color will be orange-red

• If not, the heme is no longer able to reversibly bind oxygen, it’ll bind water instead

  • Oxidation state = 3+ (ferric) ; color will be brown; “ferriHb”

What are 4 of the coordination sites on the iron occupied by? What about the 5th and 6th site?

1. By the pyrrole Nitrogen

2. The 5th site = linkage to a nearby histidine (proximal histidine); covalent bond

3. The 6th site = O2 binds to the heme and thus to the oxygen binding protein

What does O2 binding to 6th coordination site of iron cause?

Binding causes iron to move into plane of porphyrin ring

What other ligands other than oxygen can serve as Lewis-Bases to bind to the electron poor iron?

CO, NO, CN-, etc.)

What does the proximal histidine on heme do?

Creates a 6th link to iron

What does the distal histidine on heme do?

• In a position just above where the oxygen binds

When no oxygen is bound, what happens to the iron in the heme?

Iron is actually displaced slightly (0.3 Å) above the plane of the porphyrin when no ligand (oxygen) is bound at the 6th coordination site.

What roles does the proximal histidine have?

Covalent link to heme (when the iron moves, so does the proximal histidine)

What roles does the distal histidine have?

Prevents strong CO binding

• other roles too (i.e - H-bond to O2)

What’s an additional role for globin? (in terms of binding with O2)

Involves the ability of another ligand to compete with O2 for binding: carbon monoxide

How much tighter can carbon monoxide bind to the iron as opposed to oxygen?

CO binding to isolated heme: 20,000 x tighter than O2

CO binding to heme in Hb: 200 x tighter than O2

What is it about the globin that decreases the CO binding affinity to heme?

• O2 may bind in preferred bent geometry (optimal binding affinity)

• CO may not bind in preferred linear geometry (thus lowered binding affinity)

What’s another role of globin?

To prevent oxidation of the heme iron

How can we ensure that the tendency of the iron to become oxidized is very limited?

1. Provide an enzyme that will reduce metHb when it forms the enzyme metHb reductase

2. Cleverly engineer the structure of the globin so that iron is much less likely to oxidize

Overall, what’s our premise regarding globin? How can we look at this?

1. It helps to inhibit oxidation of the iron

2. To see this, we would need to look at heme molecules in the absence of any globin.

In order for oxidation to occur, what needs to be formed?

A “sandwich dimer” intermediate

What is a “sandwich dimer” intermediate?

• Two hemes must get close enough (without obstruction)

• Two hemes bridged by an oxygen molecule (so heme is the bread, oxygen is the filling in the sandwich)

What are the synthetic Heme molecules that were designed to prevent iron oxidation called?

“Picket fence” Porphyrin

• Porphyrin ring

• 4 pyrrole Nitrogen forming the 4 coordination links to the iron atom

• Imidazole molecule forming the 5th coordination link (mimicking R-group of histidine)

• Ring had big bulky substituents built into the structure

What did the researchers find out about their “picket fence” porphyrin? Did it prevent or allow O2 binding?

O2 binding is similar to that in Mb

What did the researchers find out about their “picket fence” porphyrin? Did it allow or prevent the iron from being oxidized?

Iron remains in reduced form (not true for hemes without the picket fence!)

If a protein (P) interacted temporarily with another molecule (L) what would that give?

A reversible, transient chemical equilibrium

What is the molecule that binds to the protein called?

Ligand (typically a small molecule)

What is the region in the protein where the ligand binds called?

The binding site

How does the ligand bind to the protein?

Via the same noncovalent interactions that dictate protein structure

• This allows the interactions to be transient and reversible

How can the process in which a ligand (L) binds reversibly to a site in a protein (P) be quantitatively described?

1. By the association rate constant (ka)

2. By the dissociation rate constant (kd)

After some time, the process will reach the equilibrium where the association and dissociation rates are equal

What is the equilibrium composition characterized by?

The equilibrium association constant (ka) or the equilibrium dissociation constant (kd)

The smaller the Kd (or the larger the Ka)…

The tighter/greater the binding affinity will be