Hemoglobin & Myoglobin PDF

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The comparison of structures and functions between hemoglobin and myoglobin....

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MBB 222 Study Guide

Lecture 18

Hemoglobin & Myoglobin 1. Structure of myoglobin and hemoglobin • Both are heme-utilizing oxygen binding proteins

- O2 reversibly binds to the heme prosthetic group (Fe2+ and porphyrin complex) - Heme is needed because no amino acid side chains are ideally suited for the reversible binding of oxygen

- Oxygen will only bind to heme when iron is in the +2 oxidation state - Heme is bound in a pocket of myoglobin/hemoglobin, which can prevent oxidation of the iron into +3 state which would reduce oxygen binding ability • Both myoglobin and the α and β subunits share the same globing fold with 8 α-helices

• Hemoglobin: oxygen transport

- Transports heme-bound O2 from the lungs to other tissues through the circulatory system - Contains 4 polypeptides: 2 α subunits & 2 β subunits, each with its own heme group (total 4) - Hemoglobin is a heterotetramer —> a dimer of heterodimers • Myoglobin: oxygen storage

- Concentrated in muscle tissue, functions as a storage depot for oxygen - Contains only a single polypeptide chain and 1 heme group

MBB 222 Study Guide

Lecture 18

2. Heme structure (in both hemoglobin and myoglobin) • Consists of a protoporphyrin and Fe2+ • Iron (Fe) can form 6 coordination bonds: 4 with nitrogen in the plane of the porphyrin ring + 2 above and below the plane of the ring

- The +2 charge means the iron can form 2 more bonds —> 1 with the imidazole nitrogen on the proximal histidine (His F8) + 1 with oxygen (O=O)

- The bond with proximal histidine is below the plane; the bond with oxygen is above the plane

- Myoglobin can only bind to 1 oxygen molecule (1 heme) while hemoglobin can bind to 4 (4 hemes)

- In the absence of oxygen, Fe2+ is not in the plane of the porphyrin (iron is too large) —> puckered heme • Proximal histidine (His F8) residue coordinates with the Fe2+ at one of the 6 positions

- When myoglobin and hemoglobin becomes oxygenated, oxygen is bound through the 6th coordination bond above the plane

- Upon O2 binding, the shared electrons result in a slightly smaller atomic radius of the iron, allowing it to move into the plane of the porphyrin ring —> planar heme

- His F8 is pulled closer to heme, pulling the F helix polypeptide backbone with it • Distal histidine (His E7) forms a hydrogen bond with the oxygen and stabilizes its interaction with the heme group

- When the diatomic oxygen molecule is bound to the Fe2+, the oxygens pull the electrons on the iron toward itself (oxygen is more electronegative), which results in a negatively-charged superoxide oxygen ion that needs to be stabilized by partially-positive hydrogen • The affinity of carbon monoxide for the Fe2+ is 200 times higher than that of oxygen 3. Oxygen-binding mechanism • Oxygen binding to both hemoglobin and myoglobin is reversible • Oxygen binding curve:

- Fractional saturation: what fraction of the total number of binding sites are saturated - Concentration of oxygen is commonly expressed as the partial pressure of oxygen (pO2) • The backward of the oxygen binding curve is the oxygen dissociation curve

MBB 222 Study Guide

Lecture 18

• Hemoglobin: oxygen binding produces a sigmoidal curve

- Result of cooperative binding of oxygen —> the 4 heme groups can interact with each other - The binding of oxygen onto 1 heme group makes the 3 other unoccupied hemes more likely to bind oxygen

- The release of oxygen from 1 heme group makes the 3 other oxygen-bound hemes more likely to release oxygen (it is either fully loaded or fully unloaded —> no intermediate)

- Binds to oxygen less strongly with a lower affinity than myoglobin - By looking at the oxygen dissociation curve —> it releases oxygen more readily than myoglobin - Physiological significance: hemoglobin is good for oxygen transport, it can bind oxygen in the lungs cooperatively and unload oxygen in other tissues relatively well • Myoglobin: oxygen binding produces a hyperbolic curve

- When pO2 is high (ie. resting muscles), myoglobin stores oxygen —> myoglobin O2 binding sites are almost saturated

- When pO2 decrease (ie. active muscles), myoglobin releases oxygen —> fewer myoglobin O2 binding sites are occupied

- A small increase in pO2 results in a sharp increase in the fractional saturation —> myoglobin binds to oxygen quickly with very strong affinity

- By looking at the oxygen dissociation curve (backward of the binding curve) —> myoglobin does not release oxygen until the pO2 drops to a very low quantity, when the pO2 does drop to a low level, almost all myoglobin releases their oxygens together —> sharp decrease

- Physiological significance: myoglobin is good for oxygen storage inside muscle cells, it only releases oxygen when pO2 drops to a low level

- Why? - Because myoglobin only has 1 heme group, it does not bind oxygen cooperatively

Go backward (dissociation curve)

Go backward (dissociation curve)

Hyperbolic

Myoglobin

Hemoglobin

MBB 222 Study Guide

Lecture 18

4. Allosteric control of oxygen-binding to hemoglobin & ligands • Ligand: a molecule that binds to another (usually larger) molecule • Ligand-protein interactions:

- Ligand binding is reversible, involves non-covalent interactions - Ligand binding induces or stabilizes structural conformations in target proteins - The equilibrium between ligand-bound protein and ligand-free protein can be altered by the binding of effector molecules, which induce conformational changes in the protein that increase or decrease ligand affinity • Oxygen: acts as a positive allosteric effector to hemoglobin

- T-state (tense): deoxyhemoglobin - R-state (relaxed): oxyhemoglobin • Oxygen binds to the iron from the top —> electron density of iron is drawn towards oxygen (higher E.N.) —> iron has a smaller radius and can be pulled into the plane of the porphyrin ring —> iron pulls the proximal histidine (His F8) —> histidine pulls the F-helix —> conformational change, α1β1 dimer is rotated 15° with respect to the α2β2 dimer —> transition from T-state to R-state, molecular basis for the cooperativity. • Homotropic allostery —> binding of oxygen affects binding of the same molecule (oxygen) • 2,3-bisphosphoglycerate (2,3-BPG): acts as negative allosteric effector to hemoglobin

- Negatively charged BPG interacts with positively charged group (His 143, His 2, Lys 82) on hemoglobin • Only binds to the central pocket of the T-state hemoglobin molecule and stabilizes the T-state —> reduces oxygen affinity of hemoglobin —> promotes oxygen unload and prevents oxygen rebinding —> the presence of BPG makes hemoglobin more efficient at unloading oxygen into tissues • Heterotropic allostery —> binding of BPG affects binding of oxygen (different molecules) • BPG levels are elevated during pregnancy

- Fetal hemoglobin is consisted of 2 α subunits and 2 γ subunits - γ subunits are 75% identical in amino acid sequence with β subunits, but His 143 is replaced with serine —> fetal hemoglobin has lower affinity for BPG —> higher affinity for oxygen

- Fetal hemoglobin could effectively pick up oxygen released by maternal hemoglobin - At the same pO2 level, fetal hemoglobin is more saturated with oxygen than maternal Hb

MBB 222 Study Guide

Lecture 18

• Carbon dioxide & H+ : both act as negative allosteric effectors to hemoglobin • The Bohr Effect: pH and CO2 dependence of O2 binding to hemoglobin

- Most of the carbon dioxide is hydrated by carbonic anhydrase to form highly soluble bicarbonate (HCO3-), which also produces H+ —> decrease in pH

- Some of the key residues of hemoglobin can be protonated - β1 His146 can be protonated at the amino group —> N-H bond —> H will bear a partially positive charge due to the high E.N of nitrogen

- The positively charged hydrogen will interact with the negatively charged oxygen on the nearby β1 Asp94, forming a salt bridge —> decreased net charge in the localized region —> T-state is stabilized

- Bicarbonate generates a negatively charged carbamate group on the N-terminal Val of all 4 subunits —> can form salt bridges with positively charged residues —> stabilizes the Tstate —> lowers oxygen affinity

- At low pH and high [CO2] (ie. in active tissues), oxygen affinity of hemoglobin decreases —> more oxygen will be unloaded

MBB 222 Study Guide

Lecture 18

5. Diseases associated with hemoglobin • Porphyria

- Inability of the body to produce heme - Medical explanation for vampires • Sickle-cell anemia

- Autosomal recessive mutation in the gene that encodes the β subunit - Single amino acid mutation: Glu6 —> Val6 - Valine is non-polar, it lies at the surface of the T-state hemoglobin —> hydrophobic effects cause the β chains to stick together and form fibres that cause the RBCs to elongate

- Aggregation only takes place in the T-state (deoxyhemoglobin). In oxyhemoglobin, the Glu6/Val6 is found inside the hemoglobin molecule and will not interact with the outer surface.

- Mutated RBCs can get jammed in capillaries and clog them - Mutated RBCs also have a shorter lifespan —> short supply of RBC —> anemia...