myoglobin and hemoglobin

Write mathematical expression for K_d and relate this parameter to binding affinities

Kd = [L], halfl of the ligand-binding sites are occupied

small Kd value → tight ligand binding

large Kd value → weak ligand binding

Explain why myoglobin is a good O₂ storage protein

present in muscles

has high affinity for oxygen, which allows it to bind oxygen tightly and hold it until it’s needed 

high affinity because of structure, which lacks the cooperative binding mechanism of hemoglobin, meaning it remains fully started at lower oxygen pressures and only releases oxygen under high demand 

O2 binds to heme, F-helix moves

myoglobin is a __

monomer

myoglobin binding curve is ___

hyperbolic

what graph does this represent

myoglobin

Explain why hemoglobin is a good O₂ transport protein

cooperative binding, reversible binding, and iron-containing heme groups 

as one oxygen molecule binds, the hemoglobin’s shape changes to make it easier to bond more oxygen, ensuring efficient uptake in the lungs

O2 binds to heme, F-helix moves 

present in red blood cells

hemoglobin is a ___

tetramer

this graph represents 

hemoglobin 

hemoglobin binding curve is __

sigmoidal

how does pH regulate hemoglobin O2 affinity

pH regulates hemoglobin’s oxgen affinity through the Bohr effect

an increase in pH increases hemoglobin’s affinity for oxygen, allowing it to bind more oxygen in the lungs → hydrogen ions form lower pH bind to hemoglobin, causing a conformational change that favors release of oxygen

how does CO2 regulate hemoglobin O2 affinity 

bicarbonate can bind to the amino-terminus of each hemoglobin subunit 

carbaminohemoglobin favors the T state 

when CO2 is bound, another H+ is released which further contributes to the Bohr effect

CO2 is produced in peripheral tissues and need to be released by the lungs; high CO2 production leads to a decrease in local pH 

carbonic anhydrase

an enzyme that hydrates CO2 to form bicarbonate and H+

Bohr effect

hemoglobin binds O2 less tightly (higher Kd) at lower pH values

how does BPG regulate hemoglobin O2 affinity

2,3-bisphosphoglycerate (BPG) is a side product of glycolysis and acts as a heterotrophic regulator of hemoglobin O2 binding

binds to a positive patch or residues between the B subunits

when BPG is bound, O2 affinity decreases (favors T state)

humans can change BPG levels to tune hemoglobin’s O2 affinity 

• pO2 is higher at sea level than at high altitude 

• humans who live near sea level ~ 5 mM BPG in their blood cells 

• ~ 8 mM BPG for living above 4000 m 

• higher BPG concentration results in more efficient O2 delivery to peripheral tissues 

Describe the biochemical mechanisms involved in fetal hemoglobin O₂binding

fetuses must acquire O2 from maternal hemoglobin

gene expression of hemoglobin subunits changes during fetal development

fetal hemoglobin has a lower affinity for BPG than adult hemoglobin

therefore, fetal hemoglobin has a higher affinity for O2 than adult hemoglobin

fetal hemoglobin gene expression stop at ~6 months of age

Describe the biochemical mechanisms involved in sickle cell disease

surface glutamate residue on a B subunits is changed to valine → if both B subunits contain valine, deoxyhemoglobins form polymers based on hydrophobic interactions the aggregate in cells

complexes that contain one B subunit with valine and one B subunit with glutamate do not polymerize

aggregated hemoglobin fibers distort red blood cell morphology → sickle shaped cells cannot easily pass through some vessels and peripheral tissues do not receive sufficient O2

Describe the mechanism of CO poisoning and treating the poisoning

CO binds competitively with O2 

exposure can be fatal because O2 cannot be delivered to peripheral tissues and respiratory complexes cannot make ATP 

CO binds to the heme irons in hemoglobin and competes with oxygen for binding 

CO is rapidly transferred from carboxyhemoglobin to Ngb-H64Q, which is a mutant neurogloibin protein that binds CO 500 times more tightly than does hemoglobin 

O2 transport in humans

red blood cells pick ip O2 in the lungs and deliver it to tissues throughout the body

proteins in red blood cells need to bind O2 tightly, but not so tightly that they can’t give it away

how does O2 act as a ligand

the concentration of O2 is expressed as a partial pressure, pO2

instead of using Kd, p50 is the pO2 value in which half of the ligand-binding sites are occupied

how does O2 bind to heme 

the heme iron is Fe2+ (red) in myoglobin/hemoglobin 

Fe2+ is slowly oxidized to Fe3+ (brown) in dead cells 

Fe2+ in heme has octahedral geometry 

four equatorial ligands are provided by the porphyrin

O2 binding causes the protein to move, the F-helix moves by about 1 A which changes the geometry of the heme 

when O2 is absent, the heme is puckered or domed; when it is bound, the heme is planar 

porphyrin 

planar organic structure that contains four pyrrole rings linked by methylene groups 

heme

a porphoryin with a coordinating iron ion

proximal histidine residue

one axial ligand

distal histidine residue

one axial ligand is O2 which forms a hydrogen bond

allosteric protein 

binding of a ligand to one site affects the binding affinity of a different ligand binding site on the same protein 

positive allostery

second ligand binds with higher affinity

negative allostery

second ligand binds with lower affinity

homotropic regulator 

affects the binding properties of the protein’s normal ligand 

heterotrophic regulator

affects the binding properties of a different ligand

cooperatively

positive homotropic regulation

how is hemoglobin regulated by O2

deoxyhemoglobin (T state) has no cooperatively

oxyhemoglobin (R state) has strong cooperatively

binding of O2 in one subunit makes the subunits more likely to bind O2

tense state (T)

more subunit-subunit interactions lead to more stable quaternary structure

lower affinity for O2

likely to be deoxyhemoglobin

relaxed state (R)

fewer subunit-subunit interactions lead to more flexible quaternary structure

higher affinity for O2

likely to be oxyhemoglobin

concerted model 

overall assembly can exist in the T state or the R state 

the binding of ligand shifts the equilibrium between these two states 

T→ R transition: as oxygen binds, more hemoglobin tetramers are converted from the T state to teh R state, increasing the oxygen affinity of the available binding sites 

sequential model

the binding of a ligand to one site in an assembly increases the binding affinity of neighboring sites without inducing a full conversation from the T into the R state

why do the model’s not filly account the hemoglobin’s behavior

concerted: the tetramer with three sites occupied by oxygen is almost always in the quaternary structure associated with the R state

sequential: hemoglobin with oxygen bound to only one of four sites remains primarily in the T state

myoglobin and hemoglobin evolution

subunits are structurally very similar

gene duplication events have resulted in multiple global proteins

globin variants can be beneficial or deleterious