Myoglobin and hemoglobin

What is the structure and function of myoglobin? What type of secondary structure(s) does it have? Where is it found?

Myoglobin stores oxygen in muscle cells and can be used as an O2 source during intense exercise. Myoglobin is a monomer with irregular loops and 8 alpha helices.

It has a heme prosthetic group that can bind one O2

What is Kd?

The dissociation constant for a ligand-protein complex. It is the concentration of ligand at which the protein is at 50% saturation. The lower the more affinity a protein has for its ligand.

How do ligands bind to proteins?

Via non-covalent bonds and in a reversible manner

Bind with specific orientation.

What is the structure of heme? Hydrophobic and hydrophilic portions? how does it fit into myoglobin?

Heme is a nearly planar, porphyrin ring with 4 nitrogens that form coordination bonds with Ferrous iron (2+). It is amphipathic because it also contains 2 propionyl groups that are polar. 

It binds to myoglobin (and hemoglobin) in a hydrophobic pocket between helices E and F. The polar groups face outwards

What are the 6 coordination bonds with Fe2+ and which is the most important for securing heme as a prosthetic group?

1 - 4 with the nitrogens in heme

*5 with the proximal histidine (F8) below the plane of heme

6 with Oxygen molecule above the plane

#5 is very important for attaching Heme securely to the globin chain

What are the 2 requirements for a prosthetic group?

1. it is a non-protein

2. permanently tightly bound to the protein’s tertiary structure

How is Zinc 2+ held in a zinc finger motif

It is coordinated by 2 histidine residues and 2 cysteine residues

What is a coordination bond?

a covalent bond in which both electrons come from one atom

What is the proximal histidine?

His F8

What is the distal histidine? what is its function?

His E7

function = to stabilize the binding of O2 by forming an H-bond with it and to lower the affinity of CO binding

Why can’t heme function alone/why be bound to a globin chain? (2 reasons)

1. because Iron will oxidize to fe3+

2. When not bound to globin, CO can bind to Fe straight on with 20,000x the affinity of oxygen. When heme is bound to globin, CO is forced to bind at an angle and thus only has 200x affinity.

What is the shape of a plot for a ligand with constant affinity?

hyperbolic

When does myoglobin offload oxygen?

during periods of intense exercise that cause the ppO2 to drop in the muscle tissue

What protein supplies O2 to resting muscle?

hemoglobin

What are apoproteins and holoproteins?

Apoproteins are proteins without their prosthetic group. A Holoprotein is a protein with its prosthetic group.

Describe the structure of hemoglobin. How many oxygens can it bind?

It is a tetramer containing 2 beta globin chains and 2 alpha globin chains. It contains 4 heme groups therefore it can bind a total of 4 oxygen molecules.

Each chain has irregular loops and 8 alpha helices

What are critical and conservative substitutions?

Conservative amino substitutions have a minor effect on protein structure and therefore don’t alter function

Critical amino substitutions can change structure depending on their location and thus can alter protein function.

Hemeglobin and myoglobin are _________ proteins. Differences between their primary sequences are ________ substitutions.

homologous

conservative

What is cooperative binding? what kind of curve is it represented by? Can monomers have cooperative binding?

Binding of one ligand changes the binding affinity of subsequent ligands (usually facilitates it) = sigmoidal curve

Monomers usually have 1 binding site therefore cooperative binding is rare.

Hb and Mb must have different affinities between ____ and _____ ppO2

20 and 40 (resting tissue)

Differentiate between the T and R state of hemoglobin

T is deoxyhemoglobin:

• tense and low affinity

• larger central cavity

• salt bridges in centre stabilise it

• found in tissues/muscle capillaries

R state is oxyhemoglobin:

• smaller central cavity 

• Relaxed and higher O2 affinity

• found in the lungs

Where would hemoglobin be in the R state?

the Lungs because it wants to pick up oxygen

What is allostery? What is an allosteric effector?

allostery is when the binding of a molecule at one site on a protein causes a change in ligand binding affinity at a different site

What is homoallostery? Heteroallostery?

Homo= a molecule affects further binding of the same molecule

Hetero = a molecule binding at an allosteric site affects the binding of a different molecule. can be an activator or an inhibitor

How does hemoglobin switch from the T to the R state?

the first oxygen binds causing a conformational change

How does binding of 1 O2 activate hemoglobin?

the oxygen binds at the 6th coordination position and lifts Fe2+ into the plane of the porphyrin heme ring. This pulls up the proximal histidine (F8), which pulls on F helix, which pulls on the globin subunit, which causes all the globin subunits to shift into the R state.

What are the heteroallosteric inhibitors of hemoglobin?

CO2

Biphosphoglycerate

Protons

How do BPG and pH cooperate to stabilize the T state?

High pH (low H+ concentration) meaning histidines in the central cavity will be deprotonated/neutral and will not bind BPG.

Low pH (Many protons) causes histidine to protonate and take on a positive charge. Then it can form salt bridges with BPG and stabilize T state.

where is CO2 low/pH higher?

In the lungs since CO2 is exhaled

How many negative charges does BPG have? How does it bind in the central cavity?

5

binds via formation of salt bridges with 4 histidine residues in the central cavity of T state hemoglobin.

What is the Bohr affect?

The affects of [H+] on the binding of O2 to hemoglobin. Involves switch from T to R and from R to T

What are the direct and indirect effects of CO2 on O2 binding?

Indirect = pH

Direct = binds to hemoglobin and prevents O2 binding

What kind of disorder is sickle cell anemia? What substitution causes it? What is the result of the mutation on RBC shape?

Substitution from Glu to Valine in the beta chain

How do fetuses have a higher affinity for O2? What is the structure of their hemoglobins?

has 2 gamma subunits instead of Beta subunits. These 2 subunits lack the central cavity histidine and instead have a serine which cannot make a salt-bridge with BPG. Therefore, fetal hemoglobin has a higher O2 affinity.