Lecture 7 - Proteins in Action - Haemoglobin & Myoglobin (Similarities & Differences) Flashcards
Myoglobin (muscle globin)
Protein Primary structure
~ 150 amino acids
Myoglobin (muscle globin)
Protein secondary structure
Eight a-helices
A-H and connecting loops (AB, BC, etc.)
Myoglobin (muscle globin)
Tertiary structure
globin fold with a Hydrophobic pocket (Val E11 and Phe CD1) to bind a haem group.
Myoglobin (muscle globin)
Quaternary structure
monomeric (a single polypeptide chain)
Haem binds to
His F8 (the eighth amino acid in helix F, histidine) in globin protein.
bound in deep pocket
Haem (heme) is a
prosthetic group, or cofactor.
Haem has 4
pyrrole rings linked together (a protoporphyrin) in a plane
How many co-ordinate bonds does Iron have in heme?
6 coordinate bonds –
4 to N of haem,
1 to N of histidine F8 the globin,
1 to O2
what colour does Electronic molecular orbitals of protoporphyrin give?
red
Is Binding of oxygen to the Fe2+ is a reversible interaction?
yes
Spectroscopy
quantifies dissolved molecules.
Spectroscopy
Higher concentration =
less transmitted light
higher absorbance
Beer-Lambert Law converts
from absorbance to concentration
Spectroscopy of globins measures
oxygen binding
Spectroscopy of globins
Shape of spectrum differs with
colour and with chemical nature of solute.
Protein is colourless but has
UV absorbance
Haem has
visible absorbance and therefore colour
Hb bright red
HbO2 dull red
oxyhaemoglobin (Hb) colour
bright red
deoxyhaemoglobin (HbO2) colour
dull red
What does Myoglobin do?
Stores Oxygen
in the tissue and increases the amount that can be there above its natural solubility
Globin Fold provides a
hydrophobic pocket (Val E11 and Phe CD1) to bind a haem group.
Different wavelengths are
absorbed more
less efficiency
Myoglobin - Haem interaction with oxygen
(incl where heme sits, O binds to, heme held by what bond and between what, hydrophobic interactions, Iron, His E7, O affinity)
Haem sits between His F8 and His E7.
Oxygen binds on His E7
Haem held by a covalent bond between His F8 and Fe2+.
Hydrophobic interactions with Phe CD1 and Val E11.
Leaves the 6th position of iron to bind to oxygen
Large shift of His E7 when oxygen binds there
Reduces O binding affinity to myoglobin, makes easier to release O to muscle cell.
Oxygen binding changes
haemoglobin’s shape.
Deoxyhaemoglobin heme shape
dished haem.
oxyhaemoglobin heme shape
what happens
O flattens haem
pulls histidine F8 and helix F toward the binding site.
Anything that keeps helix F away will weaken oxygen binding.
Myoglobin shows
‘allosteric control’ of oxygen affinity
how does Lactate effect myoglobin?
decreases myoglobin’s affinity for oxygen.
Does not bind where oxygen binds.
Lactate build up in muscles promotes
O release from myoglobin,
increase O2 availability for respiration.
‘Allosteric’
“without overlapping”.
Not binding in the same place as oxygen.
For enzymes away from the active site.
For carrier proteins (haemoglobin and Myoglobin) away from the binding site for its ligand (oxygen).
‘allosteric’ builds on
‘steric hindrance’,
the impossibility of two atoms occupying the same space.
Myoglobin binds
quite tightly to oxygen
lactate binding to myoglobin
releases more oxygen
When muscle is short of oxygen
it generates lactate.
what curve shows myoglobin bind to O?
Hyperbolic curve
To get myoglobin to release O need to go into
highly active tissue with oxygen pressure very low
Binding curve / saturation curve.
Myoglobin
O2 saturated at low pO2
only releasing O2 to muscle cells when pO2 is very low.
function “back-up” store of O2 in muscle cells.
partial pressure of oxygen in
lungs, or pO2,
~100 Torr
partial pressure of oxygen in resting muscle or pO2,
~ 20 Torr.
haemoglobin functioning as an O2 transporter in blood evolved a much weaker binding affinity for oxygen. Why?
curve is ‘sigmoidal’.
The availability of O2 to cellular proteins depends on:
- The pO2 in the local environment
- The binding affinity of O2 to myoglobin or haemoglobin
Haemoglobin in RBC in the blood needs to
- Bind O2 in lungs where pO2 is ~100 Torr
- Release O2 in peripheral tissues where pO2 is ~20 Torr
Haemoglobin evolved to bind O2
less tightly.
Haemoglobin
tetramer –
4 globin proteins together non-covalently
Each globin protein contains 1 haem and each can bind 1 O2
Myoglobin
monomer
acts as O2 store in muscle
Two interchangeable crystal morphologies reveal two…
hemoglobin conformations
Aerobic exposed to Oxygen crystal morphologies…
Interface of air and protein solution
Needle-shaped crystals
Anaerobic zone no oxygen crystal morphologies…
Hexagonal, plate-shaped crystals
In both Haemoglobin and Myoglobin
Oxygen binds to iron of haem.
Shift from dull to bright red allows monitoring O2 binding.
Affinity for oxygen is altered by molecules (e.g. lactate to myoglobin) binding elsewhere (allosteric control).
a-helices forms a hydrophobic cleft that holds the haem where oxygen binds
Myoglobin versus haemoglobin
Monomer versus tetramer
Tighter, hyperbolic (in myoglobin) versus weaker, sigmoidal binding curve (haemoglobin)
Store in tissue versus transport molecule
