Allosteric Proteins: Myoglobin and Hemoglobin - Oxygen Transport in Living Organisms - Pro | Study notes Biochemistry

Allosteric Proteins: Myoglobin and Hemoglobin

The transition from anaerobic to aerobic life was a major step in evolution because it uncovered a rich

reservoir of energy. Fifteen times as much energy is extracted from glucose in the presence of oxygen.

Vertebrates have evolved two principal mechanisms for supplying their cells with a continuous and

adequate flow of oxygen.

1. Circulatory system actively delivers oxygen to cells.

2. Oxygen carrying molecules to overcome the limitation imposed by the low solubility of

oxygen in water.

a. hemoglobin serves as the oxygen carrier in blood and also plays an important

role in the transport of CO2 and H+

b. myoglobin, located in muscle, provides oxygen storage in diving mammals

and facilitates diffusion of O2 from hemoglobin to the tissues

Hemoglobin and myoglobin are the best researched globular proteins. They illustrate many important

principles of protein conformation, dynamics, and function. Their 3-dimensional structures, known in

atomic detail, reveal much about how proteins fold, bind other molecules, and integrate information.

Hemoglobin is the best understood allosteric protein. The discovery of mutant hemoglobins first revealed

that disease can result from a change in a single amino acid in a chain. Hemoglobin has been a rich source

of insight into the molecular basis of evolution.

Both contain the heme prosthetic group. The heme consists of an organic part, Protoporpyhrin IX, and an

iron atom. The properties of the heme group are dependent on the Fe(II) that is bound to the heme group.

Fe(II) has 6 coordination sites, four are bound to the N’s on the heme group, one is bound to a proximal

histidine, and the 6th coordination site is for reversible binding to O2.

Myoglobin

Found in high concentration in muscle tissue

Storage in diving mammals and off-loading in others

Is a single polypeptide chain that contains 8 sections of -helix

Extremely compact

Heme group is located in a hydrophobic crevice in the myoglobin molecule. The highly polar

propionate side chains of the heme are on the surface of the molecule. The rest of the heme is

inside the molecule where it is surrounded by nonpolar residues except for two histidines. One is

the proximal histidine which is bonded to the Fe, the other is the distal histidine which is near but

not bonded. The protein provides the protective environment for Fe to bind O2.

Three distinct forms possible

Form Oxidation state 5th Position 6th position

Deoxymyoglobin Fe+2 His F8 empty

Oxymyoglobin Fe+2 His F8 O2

Ferrimyoglobin Fe+3 His F8 H2O

If the Fe is not protected, it is oxidized to the +3 state which cannot bind oxygen. Even though the

oxygen-binding site comprises only a small fraction of the volume of the molecule, the large

protein part is necessary to protect the Fe from oxidation.

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Allosteric Proteins: Myoglobin and Hemoglobin The transition from anaerobic to aerobic life was a major step in evolution because it uncovered a rich reservoir of energy. Fifteen times as much energy is extracted from glucose in the presence of oxygen. Vertebrates have evolved two principal mechanisms for supplying their cells with a continuous and adequate flow of oxygen.

Circulatory system actively delivers oxygen to cells.
Oxygen carrying molecules to overcome the limitation imposed by the low solubility of oxygen in water. a. hemoglobin serves as the oxygen carrier in blood and also plays an important role in the transport of CO 2 and H+ b. myoglobin, located in muscle, provides oxygen storage in diving mammals and facilitates diffusion of O 2 from hemoglobin to the tissues Hemoglobin and myoglobin are the best researched globular proteins. They illustrate many important principles of protein conformation, dynamics, and function. Their 3-dimensional structures, known in atomic detail, reveal much about how proteins fold, bind other molecules, and integrate information. Hemoglobin is the best understood allosteric protein. The discovery of mutant hemoglobins first revealed that disease can result from a change in a single amino acid in a chain. Hemoglobin has been a rich source of insight into the molecular basis of evolution. Both contain the heme prosthetic group. The heme consists of an organic part, Protoporpyhrin IX, and an iron atom. The properties of the heme group are dependent on the Fe(II) that is bound to the heme group. Fe(II) has 6 coordination sites, four are bound to the N’s on the heme group, one is bound to a proximal histidine, and the 6th^ coordination site is for reversible binding to O2. Myoglobin Found in high concentration in muscle tissue Storage in diving mammals and off-loading in others Is a single polypeptide chain that contains 8 sections of -helix Extremely compact Heme group is located in a hydrophobic crevice in the myoglobin molecule. The highly polar propionate side chains of the heme are on the surface of the molecule. The rest of the heme is inside the molecule where it is surrounded by nonpolar residues except for two histidines. One is the proximal histidine which is bonded to the Fe, the other is the distal histidine which is near but not bonded. The protein provides the protective environment for Fe to bind O2. Three distinct forms possible Form Oxidation state 5 th^ Position 6 th^ position Deoxymyoglobin Fe+2^ His F8 empty Oxymyoglobin Fe+2^ His F8 O 2 Ferrimyoglobin Fe+3^ His F8 H 2 O If the Fe is not protected, it is oxidized to the +3 state which cannot bind oxygen. Even though the oxygen-binding site comprises only a small fraction of the volume of the molecule, the large protein part is necessary to protect the Fe from oxidation.

Hemoglobin Oxygen transporter in erythrocytes Four polypeptide chains- 2  (141 residues) chains and 2 chains (146 residues) in adults Embryos and fetuses have distinctive hemoglobins to facilitate diffusion of O 2 from the maternal blood Sub-unit interactions are crucial to the ability of hemoglobin to transport O 2 , CO 2 , and H+ in a physiologically responsive way. Comparison of hemoglobins from different species shows 9 invariant residues; F8 His Proximal heme-linked E7 His Distal CD1 Phe Heme contact F4 Leu Heme contact B6 Gly Allows close approach of B and E helices C2 Pro Helix termination CH2 Tyr Cross-links H and F helices H10 Lys Uncertain C4 Thr Uncertain Invariant residues stabilize oxygen binding site or stabilize helical segments Amino acid residues in the interior vary considerably, but are highly conservative. The non-polar interior is maintained. Exterior residues are highly variable and are not conserved. Hemoglobin has more complex structure and more complicated binding properties. Hemoglobin is an allosteric protein, whereas myoglobin is not. This difference is expressed in three ways:

The binding of O 2 to hemoglobin at one site enhances the binding of additional O 2. O 2 binds Cooperatively to hemoglobin.
The affinity of hemoglobin for oxygen depends on pH and CO2.
The oxygen affinity is further regulated by organic phosphates, BPG. The result is that hemoglobin has a lower oxygen affinity than does myoglobin. Cooperative Binding: Oxygen binding curves of myoglobin ( hyperbolic) vs hemoglobin (sigmoidal) Hb has a high oxygen affinity in the lungs where pO 2 is high and has a low affinity in the tissues where the pO 2 is low. Heme groups communicate with each other; binding enhances further binding, unloading enhances further unloading. Role of myoglobin: High affinity at both high and low partial pressures of oxygen. Releases O 2 only when concentrations are very low and assists movement of O 2 from blood to muscle. The major physiological role of myoglobin is to facilitate oxygen transport in rapidly respiring tissue. The rate that O 2 can diffuse from capillaries to tissue and thus the level of respiration is limited by O 2 ’s low solubility in aqueous solution. Mb increases the solubility of O 2 in muscle, the most rapidly respiring tissue under conditions of high exertion. Storage function of Mb is significant only in seals and whales. The dissociation curve is well to the left of Hb. It releases O 2 only when the pO 2 is very low and has a higher affinity for O 2 than Hb, hence the O 2 moves easily from blood to muscle. Conformational changes on O 2 binding: In deoxyhemoglobin, the iron atom is about 0.4 A out of the porphyrin plane toward the proximal histidine so that the heme group is domed. On oxygenation, the Fe moves into the plane to form a strong bond with O 2 and the heme becomes more planar.

Hb(O 2 )nHx + O 2 Hb(O

2 )n+ 1 +^ xH

Capillaries: Low pH; low pO2; protons bind to Hb, cause unloading of O2; causes more CO 2 to be converted to bicarbonate to be transported in the bloodstream.

CO 2 (g) CO 2 +^ H 2 O H 2 CO 3 H

(^) +

HCO 3

(aq)

Lungs: High pH; high pO 2 ; protons released from Hb; enhances O 2 binding ; release of H+ drives release of CO 2 to be exhaled from lungs. Side chains of histidines 146 and 122 and the  amino group of the  chain account for much of the Bohr effect. Most of the CO 2 is transported as bicarbonate formed by carbonic anhydrase. The remainder binds to the N-terminal groups as carbamates. The carbamates form salt bridges that stabilize the T form. Hence, the binding of CO 2 lowers oxygen affinity of hemoglobin.

RNH 2 +^ CO 2 R N

H

C O-

O

+ H+

carbamate

BPG -only one molecule is bound to deoxyhemoglobin and stabilizes the structure; creates additional salt links that must be broken for O 2 to bind. High altitudes: after about two weeks, body adjusts by making more erythrocytes; until then, the BPG conc increase to ensure maximum release of O2. BPG is also a glycolytic intermediate; it cannot diffuse out of the cells because of its charge, but it may be utilized for other purposes; fresh blood vs week old blood. Fetal Hemoglobin-higher O 2 affinity at low pO 2 facilitates transfer from maternal Hemoglobin can bind 4 O2, 4H+, 4 CO2, and 1 BPG

Allosteric Proteins: Myoglobin and Hemoglobin - Oxygen Transport in Living Organisms - Pro, Study notes of Biochemistry

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