Lecture 7 Flashcards
Myoglobin and Hemoglobin
Big Picture Items
• Myoglobin contains a heme group where an Fe(II) binds O
• Hemoglobin contains four myoglobin-like subunits
• Myoglobin’s oxygen binding curve is hyperbolic, while hemoglobin’s is S-shaped
• Conformational changes occur in hemoglobin upon
oxygen binding
• BPG increases oxygen delivery at high altitude by decreasing hemoglobin’s affinity for oxygen
• Hemocyanins perform the same function as hemoglobin through a very different mechanism
• Sickle cell anemia arises from a mutation in hemoglobin that causes formation of fibers.
Myoglobin
Waiting in muscle cells for oxygen to arrive
Heme group in red with spherical Fe(II) ion in center.
- The eight helices are labeled A to H.
- Helix-connecting loops are AB, BC, etc
Oxy-myoglobin’s heme group with Fe(II) binding oxygen
Histidine F8 interacts with the Fe(II), as do four nitrogens of the heme group.
• In Oxy-Mb one oxygen atom of O2 also binds to the Fe(II).
• The Fe(II) in oxy-Mb is octahedrally coordinated by six ligands.
The key features shown of oxygen binding and heme group are the same in myoglobin and all four hemoglobin chains.
In hemoglobin and myoglobin, residue “F8” means: the 8th amino acid in helix F.
Myoglobin – the “Heme complex”
Heme group is packed between hydrophobic side chains Val E11 and Phe CD1
• When O2 is bound it also interacts with Nε2 of the imidazole of His E7.
• The oxygen binding pocket is quite tight
(several additional residues are not shown for clarity).
Oxygen Binding Curve of Myoglobin
General: The fractional saturation Y of a protein by ligand L, is the fraction of binding sites occupied by L relative to the total available sites.
Specific: The fractional oxygen saturation YO2 , is the fraction of O2-binding sites occupied by O2
pO2 is the partial pressure of O2.
(pO2 in venous blood is ~30 torr; 760 torr = 760 mm Hg = 1 atmosphere)
Hemoglobin
The oxygen transporter in the blood of most animals
It is usually embedded in red blood cells.
Hemoglobin (Hb) is much more sophisticated than myoglobin:
• Hb has four myoglobin-like subunits
• Two Hb chains are called α chains, the other two β chains
• The two α chains have the same amino acid sequence
• The two β chains have also the same sequence
• The α chain and β chain are ~57% different (in humans)
• As a result of cross-talk between these four subunits, the O2 binding curve of Hb is NOT hyperbolic but “S” (sigmoidal) shaped
• This is called cooperativity
• Increased efficiency of oxygen transport from lung to other organs.
• Its oxygen-affinity is pH-dependent.
• Its oxygen-affinity can be regulated “allosterically” by BPG.
Deoxy versus Oxy Hemoglobin
Shift in structure in the T R transition involves both tertiary and quaternary structure changes.
Deoxy-Hb “T-state”
Oxy-Hb “R-state”
- Note the change in size of the central cavity.
- Salt bridges are an important component of the interactions in Deoxy-Hb
O2 binding to heme in Hemoglobin
• Fe moves into the plane of the heme when O2 binds.
(The reason for that is fascinating - but you do not have to know)
• The heme becomes planar when O2 binds.
• No oxidation or reduction of Fe(II) occurs.
• Movement of His F8 acts like a lever and moves helix F
• Salt bridges are changed between the subunits.
• The subunits move relative to one another.
• The shift in pKa of the groups in the salt bridges causes proton release.
Changes in the heme upon O2 binding: the “trigger”
Upon oxygenation: the movement of the Fe2+ into the plane of the heme group, and the greater planarity of the heme after this move, is the “trigger” of the Tstate (Deoxy-Hb) to R-state (Oxy-Hb) transition
Oxygen-binding curves in Mb and Hb
The shapes of the curves are drastically different
The Mb curve is hyperbolic; the Hb curve is S-shaped (sigmoidal)
(The dotted curve is a hyperbolic O2-binding curve with the same P50 as Hb)
Hb picks up O2 in lungs and delivers O2 to muscle
with MUCH greater efficiency than Mb would be able to do:
Hb saturation varies between ~ 96% in the lung and 35 – 65% in the tissue.
(The dotted curve is a hyperbolic O2-binding curve with the same P50 as Hb)
The S-shaped curve of Hb-O2 binding can be described by the Hill equation.
The larger the Hill coefficient, the steeper the S-shaped oxygen binding Hill plot.
(Except for the name, you do not need to know specifics of the Hill plot for this course)
Allosteric Proteins – Symmetric model
Allosteric Effect:
• The binding of one ligand at one site affects the binding of another ligand at another site.
• This often, but not necessarily always, requires interactions between subunits of oligomeric proteins.
Four models of allosteric transitions in hemoglobin
• Sequential model
• Symmetry (“concerted”) model
• Multistate model: complex, combining features of sequential and symmetry models
• Dynamic model: changes due to dynamic properties rather than conformational changes per se
We will only consider the symmetry model of
hemoglobin allostery.
Allosteric Proteins – Symmetric model
The symmetric model assumes:
• An oligomer of symmetrically related subunits
• Each subunit can exist in two states, designated R and T;
• The ligand can bind to either conformation
• Molecular symmetry is maintained, i.e. the tetramer is either all squares or all spheres.
The key point is that hemoglobin cannot adopt an intermediate conformation.
The tetramer is either in the deoxy (T) state or in the oxy (R) state.
Allosteric Proteins – Sequential model
The sequential model assumes:
• Ligand binding induces conformational change in subunit to which it binds
• These conformational changes alter the neighboring subunits and their affinity for the ligand: cooperative interactions
• Subunits can adopt multiple conformations
• Symmetry needs not be maintained during ligand binding.
BPG : our friend at high altitudes
At high altitudes the [BPG] in human red blood cells (RBCs) increases quickly from ~ 4 mM to ~ 8 mM.
(Animals living at high altitudes, such as llama’s, have an other solution: they have hemoglobin variants with higher O2-binding affinities than their sea-level cousins.)
BPG binds at one mole BPG per mole deoxy-Hb tetramer
(Thereby decreasing the O2 affinity of Hb)
- BPG binds in the central cavity between the β-chains in deoxy Hb
- BPG does not bind to oxy-Hb.
- [BPG] is increased in short term altitude adaptation.
- The net effect of increased [BPG] is to decrease Hb’s affinity for O2.
- The net effect of increased [BPG] at high altitude is that the delivery of O2 from lung to tissue is almost as efficient as before at sea level!
BPG only binds to deoxyHb
Hb – oxygen binding after short-term altitude adaptation
O2 delivery depends on:
ΔY = Ylung – Ytissue
[BPG] is ~ 4 mM in sea level red blood cells (RBCs) and increases to ~ 8 mM after high
altitude adaptation.
With this increase of [BPG],
the p50 of Hb increases from 26 to 31 torr.
As a result, the number of O2 molecules per Hb subunit traveling from lung to tissue is about the same at sea level as
at high altitude:
• 0.38 at sea level
• 0.37 at high altitude!
Hemocyanins
The oxygen binding protein in blue blood of mollusks (snails) and arthropods (lobsters, crabs, spiders, etc.)
Hemocyanins are entirely different from hemoglobins even though the function of the two molecules is the same.
Molecular weights
• hemocyanins: 500-10000 kDa
• tetramer hemoglobins: ~80 kDa
Hemocyanins come in two entirely different architectures:
• Mollusk hemocyanins: gigantic cylinders with typically 10 or 20 subunits per cylinder and with many domains per subunit.
• Arthropod hemocyanins: hexamers with D3 dihedral symmetry – that is six subunits per hexamer.
The oxygen binding centers of these two classes of hemocyanins are nevertheless very similar and are based on Cu.
Global Architecture of a small Hemocyanin
A hexamer with dihedral D3 symmetry
View along the three-fold axis.
The three 2-fold axes run in between the
upper and lower trimer of subunits.
Each “peanut” is a subunit of ~ 75 kDa
View along a two-fold axis,
which runs perpendicular to the three-fold axis
Architecture of hexameric hemocyanin from
the spiny lobster Panulirus interruptus.
This architecture is prototypical for all arthropodan hemocyanins.
e.g. the structure of horse shoe crab Limulus polyphemus hemocyanin is essentially the same
The oxygen binding center in Hemocyanin
Hemocyanin from the horse shoe crab Limulus polyphemus.
Oxygen (red) bound using two Cu(I) ions (purple) liganded by six histidines.
And all embedded in a totally different protein environment than in hemoglobin.
Typical biology: an entirely different solution for the same problem!
(Yes: in spite of the name “hemocyanin” there is no heme in hemocyanins whatsoever
Hemoglobin and Sickle Cell Anemia
Normal RBC with HbA These RBCs can be squeezed though capillaries quite readily
(HbA is normal hemoglobin)
Sickled RBC with HbS
These RBCs are more rigid and cannot easily pass through blood capillaries.
This leads to a painful, debilitating and
often fatal disease
Importantly:
Heterozygotes, with about 40% HbS and 60% HbA, have quite normal RBCs.
What is the cause of the sickling in HbS patients and why it is so common in Africa?
Sickle Cell Anemia
What is the basis of sickle-cell anemia? Why is HbS bad?
- The prime event is the point mutation from Glu6 in HbA to Val6 in the HbS β-chain.
- In HbS there is a new hydrophobic patch at the surface of the protein.
- It so happens that such a patch on β2 interacts favorably with a hydrophobic pocket on the surface of a β1 subunit of a HbS tetramer.
The critical difference between HbA and HbS: β: Glu6 → Val
Sickle Cell Anemia 2
The result of this new β1↔β2 interaction is disastrous:
• Deoxy HbS tetramers start forming long fibers in an uncontrolled manner
• Under deoxy conditions HbS-RBCs are becoming too rigid to pass through capillaries
• This can be excruciatingly painful for patients
Sickle Cell Anemia and Malaria
It appears that the mixed HbA:HbS RBCs have properties which protect heterozygotes against malaria parasites – but how is still not yet fully understood.
• Perhaps, by possibly quicker removal of infected cells.
• Or, perhaps, because infected red blood cells with HbA:HbS damage the parasites.
In endemic malaria regions, the HbA:HbS heterozygotes confer an advantage over HbA and HbS homozygotes and survive. But at a terrible cost:
• The HbA homozygotes succumb to malaria,
• The HbS homozygotes die early because of sickle cell anemia.
The price of communities in the affected regions for HbS-protection against malaria can be horrible: a considerable percentage of the population in some areas in the past
