Hemoglobin (Part 1) Flashcards
Heme (General Information):
- a prosthetic group (non-protein molecule required for biologic activity of some enzymes)
- tightly, but not covalently bonded to hemoglobin or myoglobin (does not dissociate)
- iron-containing molecule
- a functional oygen carrier
Heme synonyms:
- heme
- hemin
- protoporphyrin IX
Heme is bound to a hemoglobin molecule via:
- a histidine residue
Heme structure:
- protoporphyrin ring ligated to an iron (in center)
- iron binds to four nitrogens of the protoporphyrin ring, a histidine sidechain on the bottom, and an oxygen on top
Iron attaches to the protoporphyrin ring of heme via:
- 4 nitrogen bonds
Hb and Mb themselves are protected from degradation by:
- sequestration in red blood cells (Hb) and muscle (Mb).
Myoglobin (Mb) Structure and Location:
- monomeric protein (single polypeptide chain) with one heme group.
- found in muscle only
- binds oxygen tightly until an oxygen-depleted state induces its release for metabolic oxidation
Mb amino acid make-up and secondary structure:
- 153 amino acids in 8 alpha-helical segments connected by beta-bends.
The purpose of the heme group and surrounding polypeptide chain in Mb is:
- to keep ferrous iron from being oxidized to the ferric state (which would prevent it from binding oxygen).
- Mb does this by providing a hydrophobic pocket to bind heme, while allowing a channel for oxygen access.
Mb secondary structure is dominated by:
- alpha-helice segments (8)
- hydrophobic core
Only the ____ form of heme is capable of reversibly binding oxygen.
- ferrous (+2)
- reduced
- “ferrohemoglobin”
The form of heme/hemoglobin not capable of reversibly binding oxygen is:
- ferric (+3)
- oxidized
- methemoglobin
- non-functional
CO forms a particularly strong bond with:
- ferrous iron in hemoglobin
- favored 200-fold over oxygen.
- In the presence of CO, oxygen is displaced and hemoglobin can no longer function as an oxygen carrier.
Function of polypeptide chain (Hb or Mb) surrounding a heme group:
- prevents aggregating
- prevents rust (going from ferric to ferrous - oxidizing)
- destabilize CO bonding to heme/active site
Are heme groups hydrophobic or hydrophilic?
hydrophobic
- reason for aggregating in solution when not bound to protective protein such as Hb or Mb
How does Hb and Mb destabilize CO binding to heme?
- second histidine (E7) coming from the E-helix forces CO into a bent conformation which is energetically unfavorable.
- shifts the equilibrium between O and CO binding from a 25,000 fold affinity difference to a 200 fold difference.
Hemoglobin (Hb) Structure:
- tetrameric protein (four polypeptide chains)
- two alpha-globins
- two beta-globins
- four heme groups
Hb location and general function:
- high concentration in red blood cells
- transports oxygen from the lungs throughout the body/tissues
How are the four subunits of Hb held together?
- series of ion pairs (salt bridges)
- between oppositely charged amino acids on adjacent domains
Despite low amino acid sequence similarity between Hb and Mb, the two show:
- remarkably similar tertiary structure
- residues that surround the heme binding site are well conserved in the primary sequence
Most of the intramolecular interactions between the four different subunits of Hb are between:
- alpha and beta chains
- NOT between alpha-alpha or beta-beta
In a fully oxygenated Hb, between what residues do the salt bridges form on the alpha-helices?
- Arg 141 (+) and Asp 126 (-)
- Carboxy-terminus 141 (-) and Lys 127 (+)
Mb general function:
- storage molecule, acting as a reserve supply of oxygen
- in muscle
- has higher oxygen affinity than Hb so that blood flow does not deplete these stores through regular circulation
Partial pressure of oxygen is directly related to:
- concentration.
- higher the concentration, greater its partial pressure.
P50 is a term used to describe:
- oxygen pressure at which Hb or Mb is 50% saturated.
P50 of Hb:
26 torr
P50 of Mb:
1 torr
The oxygen binding curve for Mb is:
- hyperbolic
- suggests a simple equilibrium between oxygenated and deoxygenated states (i.e. no cooperativity)
The higher the P50 =
- the lower the oxygen affinity
The oxygen binding curve for Hb is:
- sigmoidal (S-shaped)
- indicates allosteric interactions
- subunits in the tetramer cooperate (interact) during the binding event.
As oxygen molecules bind to hemoglobin:
- the affinity for oxygen increases
- due to cooperativity of the subgroups
- reason why most Hb molecules are either found with no oxygen (deoxyHb) or with all four oxygens (oxyHb)
Why is histidine critical in the structure of Hb and Mb?
- one histidine holds heme in place
- another histidine protects us from CO poisoning
- through making the CO-heme bond sterically/energetically unfavorable
Myoglobin is held together via:
hydrophobic forces
Hemoglobin is held together via:
four salt bridges
Hill coefficient (n):
- tells you extent of cooperativity of subunits.
- 1 = no cooperativity
- 4 = perfect cooperativity
- 2.8 = somewhere in between (the Hill coefficient of hemoglobin). Not perfect cooperativity, but pretty good. Leads to sigmoidal curve.
Hill coefficient of Mb:
1 (no cooperativity)
leads to hyperbolic curve
Hill coefficient of Hb:
2.8 (moderate cooperativity)
leads to sigmoidal curve
As the Hill Coefficient (n) increases, what happens to the binding curve?
it becomes more sigmoidal (due to increasing cooperativity)
Allostery allows Hb to:
- bind and release more oxygen over physiological oxygen concentrations.
Allostery =
- action at a distance
- binding at one site affects the activity of an enzyme (or a transport protein) at another site.
After the first oxygen binds to Hb, the change in conformation is about:
- ½ angstrom
- very subtle change that causes the cooperativity
The two forms of Hb:
- T-form (taut)
- R-form (relaxed)
T-form of Hb:
- tense/taut (closed fist)
- deoxygenated
- low affinity for oxygen
R-form of Hb:
- relaxed (open hand)
- oxygenated
- high oxygen affinity
In a solution of Hb, what is in equilibrium with one another?
T-form and R-form
What form of Hb has more salt-bridges: T-form or R-form?
T-form
this is why it is more tense/taut than the R-form
Difference between T-form and R-form of Hb:
- T-form: iron and heme ring different planes
- forces heme into unfavorable state for oxygen binding
- R-form: iron and heme same plane
- oxygen binding favorable
Overall model for allosteric regulation of oxygen binding in Hb:
- No oxygen bound: all four subunits in T-form (low affinity)
- One oxygen bound: induces strain in the T-form, forces other subunits to take up the R-form, oxygen affinity is increased.
Physiological factors affecting Hb oxygen affinity:
- pH (Bohr Effect)
- BPG
- CO2
Bohr Effect:
- lower pH, lower Hb oxygen affinity
- lowering the pH stabilizes the T-form of Hb
As you acidify the blood, you:
- push Hb back into the T-state, forcing oxygen out of Hb and lower the affinity of oxygen.
- dumps more oxygen to the tissues
Bohr Effect mechanism:
- As Hb changes conformation from T-state to R-state, some protons are ejected into solution, increasing the acidity of the solution.
- Asp94-beta and His146-beta are responsible for this (become protonated and deprotonated)
T-form of Hb and Bohr Effect:
- Asp94beta and His146beta are right next to each other and the histidine wants to have a higher pKa and hold on to a proton.
- Push oxygen off Hb, push on protons
R-form of Hb and Bohr Effect:
- Asp94beta and His146beta are far from each other and the histidine wants to have a lower pKa and lose a proton.
- Push oxygen on Hb, push off protons.
What two amino acids in Hb drive the Bohr Effect?
- Asp94-beta
- His146-beta
- lowering the pH stabilizes the T-form
The two effects of CO2 on Hb:
- CO2 dissolves in blood, in equilibrium with carbonic acid, acidifies blood
- stabilizes T-form
- Bohr Effect - lowers Hb oxygen affinity
- CO2 forms carbamates with N-terminus of the Hb beta chain at valine
- stabilizes T-form
- how CO2 is transported out of the body
Increasing the acidity of the blood has what effect on Hb?
- stabilizes the T-state
- more oxygen is let go in tissues since oxygen affinity is now lower
- raises the P50 of hemoglobin
BPG is a normal constituent of red blood cells. Its synthesis may be upregulated in situations where:
- greater oxygen delivery to tissue is needed
- BPG decreases Hb affinity for oxygen (Hb delivers more oxygen to tissue)
- stabilizes the T-state
BPG is generated as a product of:
glycolysis
BPG only binds to what state of Hb?
- T-form
- stabilizes the T-form, lowers Hb oxygen affinity, delivers more oxygen to tissue
BPG binding site in deoxyHb is where?
- the pore between the four chains.
- Interacting sidechains include:
- His 143-beta
- His 2-beta
- Lys 82-beta
- beta-chain amino terminus.
Nitric oxide is an allosteric regulator of:
- bloodflow
- vasodilator, increases blood flow to certain tissues, thereby increasing oxygen delivery
Nitric Oxide binding to Hb:
- binds to a cysteine on oxyHb (in lungs)
- released in deoxyHb (in tissues)
Hb T-form stabilized by:
- low pH (Bohr Effect)
- BPG
- CO2
Hb R-form stabilized via:
- high pH
- NO
- oxygen
Does myoglobin experience any allosteric regulation?
No. It has no cooperativity.
Allostery needs cooperativity.
Methemoglobin:
- when iron atom of the heme group is in the ferric (3+) charge state.
- cannot bind oxygen
- mutations in cytochrome b5 or cytochrome b5 reductase can lead to increases in metHb
- very high affinity for cyanide
What can methemoglobin be used as a treatment for?
- cyanide poisoining
- metHb has very high affinity for cyanide poisoining
Methemoglobinemia (excess metHb accumulation) can be caused by:
- mutations in cytochrome b5 or cyt b5 reductase
- mutations in the hydrophobic pocket of Hb
- exposure to oxidizing agents
Methemoglbin aka:
- ferrihemoglobin
- when the heme iron is in the ferric state (3+)
- cannot bind oxygen
- high affinity for cyanide
