Globular proteins

• Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form in a biologically functional way.

Globular proteins

• Globular proteins, or spheroproteins comprising "globe"-like proteins that are more or less soluble in aqueous solutions.

• This main characteristic helps distinguishing them from fibrous proteins (the other class), which are practically insoluble.

Globular proteins

• Unlike fibrous proteins which only play a structural function, globular proteins can act as:

1. Enzymes.

2. Messengers, by transmitting messages to regulate biological processes. This function is done by hormones (i.e. insulin etc).

3. Transporters of other molecules through membranes.

Globular proteins

Globular hemeprotein

• Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group.

Prosthetic group is a tightly bound, specific non-polypeptide unit required for the biological function of some proteins.

The prosthetic group may be organic (such as a vitamin, sugar, or lipid) or inorganic (such as a metal ion), but is not composed of amino acids.

• The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein.

• For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced.

• In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide .

• In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen.

Globular hemeprotein

Structure of heme

• Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+).

• The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.

• The heme (Fe2+) can form two additional bonds, one on each side of the planar porphyrin ring.

Myoglobin

• For example, in myoglobin and hemoglobin, one of these

positions is coordinated to the side chain of a histidine

residue of the globin molecule, whereas the other position is

available to bind oxygen.

Structure and function of myoglobin

• Myoglobin hemeprotein present in heart and skeletal muscle, functions both as a reservoir for oxygen, and as an oxygen carrier that increases the rate of transport of oxygen within the muscle cell.

• Myoglobin consists of a single polypeptide chain that is structurally similar to the individual subunit polypeptide chains of the hemoglobin molecule.

1. α-Helical content

• Myoglobin is a compact molecule, with approximately 80% of its polypeptide chain folded into eight stretches of α-helix.

• These α-helical regions are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix ,or by β-bends and loops stabilized by hydrogen bonds and ionic bonds.

2. Location of polar and nonpolar amino acid residues

• The interior of the myoglobin molecule is composed almost entirely of nonpolar amino acids.

• They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues .

• In contrast, charged amino acids are located almost exclusively on the surface of the molecule, where they can form hydrogen bonds, both with each other and with water.

3. Binding of the heme group

• The heme group of myoglobin sits in a crevice in the molecule, which is lined with nonpolar amino acids.

• Notable exceptions are two histidine residues .

• One, the proximal histidine, binds directly to the iron of heme.

• The second, or distal histidine, does not directly interact with the heme group, but helps stabilize the binding of oxygen to the ferrous iron.

Structure and function of hemoglobin

• Hemoglobin is found exclusively in red blood cells, where its main function is to transport oxygen from the lungs to the capillaries of the tissues.

• Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains ; two alpha (α) chains and two beta (β) chains held together by noncovalent interactions .

• Each subunit has stretches of α-helical structure, and a heme binding pocket similar to that described for myoglobin.

• However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin.

• For example, hemoglobin can transport H+ and CO2 from the tissues to the lungs, and carry four molecules of O2 from the lungs to the cells of the body.

• Further, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors .

Structure and function of hemoglobin

Quaternary structure of hemoglobin

• The hemoglobin tetramer can be envisioned as being composed of two identical dimers (αβ )1, and (αβ)2, in which the numbers refer to dimers one and two.

• The two polypeptide chains within each dimer are held tightly together, primarily by hydrophobic interactions.

• Hydrophobic amino acid residues are localized not only in the interior of the molecule, but also in a region on the surface of each subunit.

• Interchain hydrophobic interactions form strong associations between α-subunits and β-subunits in the dimers.

• Ionic and hydrogen bonds also occur between the members of the dimer.

• In contrast, the two dimers are able to move with respect to each other, being held together primarily by polar bonds.

• The weaker interactions between these mobile dimers result in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin:

• T form

• R form

Quaternary structure of hemoglobin

• The deoxy form of hemoglobin is called the "T" or taut (tense) form.

• In the T form, the two α β dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains.

• The T form is the low oxygen-affinity form of hemoglobin.

T form

R form

• The binding of oxygen to hemoglobin causes the rupture of some of the ionic bonds and hydrogen bonds between the α β dimers.

• This leads to a structure called the "R," or relaxed form, in which the polypeptide chains have more freedom of movement.

• The R form is the high oxygen-affinity form of hemoglobin.

Binding of oxygen to myoglobin and hemoglobin

• Myoglobin can bind only one molecule of oxygen because it contains only one heme group.

• In contrast, hemoglobin can bind four oxygen molecules—one at each of its four heme groups.

• The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100 percent (all sites are full).

Oxygen dissociation curve

• A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen dissociation curve.

• The curves for myoglobin and hemoglobin show important differences .

• Myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin.

• The partial pressure of oxygen needed to achieve half-saturation of the binding sites (P50) is approximately 1 mm Hg for myoglobin and 26 mm Hg for hemoglobin.

• [Note: The higher the oxygen affinity (that the more tightly oxygen binds), the lower the P50.

• The oxygen dissociation curve for myoglobin has a hyperbolic shape. This reflects the fact that myoglobin reversibly binds a single molecule of oxygen.

• Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:

• The equilibrium is shifted to the right or to the left as oxygen is added to or removed from the system.

• [Myoglobin is designed to bind oxygen released by hemoglobin at the low PO2 found in muscle. Myoglobin, in turn, releases oxygen within the muscle cell in response to oxygen demand.]

a. Myoglobin:

• The oxygen dissociation curve for hemoglobin is sigmoidal in shape, indicating that the subunits cooperate in binding oxygen.

• Cooperative binding of oxygen by the four subunits of hemoglobin means that the binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule .

• This effect is referred to as heme-heme interaction .

• Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen occurs with high affinity, as shown by the steep upward curve in the region near 20 to 30 mm Hg.

b. Hemoglobin

Allosteric effects

• The ability of hemoglobin to reversibly bind oxygen is affected by:

• The pO2 (through heme-heme interactions)

• The pH of the environment.

• The partial pressure of CO2 (pCO2).

• The availability of 2,3- bisphosphoglycerate.

• These are collectively called allosteric ("other site") effectors, because their interaction at one site on the hemoglobin molecule affects the binding of oxygen to heme groups at other locations on the molecule.

• [Note: The binding of oxygen to myoglobin is not influenced by the allosteric effectors of hemoglobin.]

1. Heme-heme interactions:

• The sigmoidal oxygen-dissociation curve reflects specific

structural changes that are initiated at one heme group and transmitted to other heme groups in the hemoglobin tetramer.

• The net effect is that the affinity of hemoglobin for the last oxygen bound is approximately 300 times greater than its

affinity for the first oxygen bound.

• The cooperative binding of oxygen allows hemoglobin to deliver more oxygen to the tissues in response to relatively small changes in the partial pressure of oxygen.

• For example, in the lung, the concentration of oxygen is high and hemoglobin becomes virtually saturated (or "loaded") with oxygen.

• In contrast, in the peripheral tissues, oxyhemoglobin releases (or "unloads") much of its oxygen for use in the oxidative metabolism of the tissues .

a. Loading and unloading oxygen:

1. Heme-heme interactions:

a. Loading and unloading oxygen:

2. Bohr effect

• The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased partial pressure of CO2 (pCO2).

• Both result in a decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen dissociation curve and both, then, stabilize the T state.

• This change in oxygen binding is called the Bohr effect.

• Conversely, raising the pH or lowering the concentration of CO2 results in a greater affinity for oxygen, and a shift to the left in the oxygen dissociation curve and stabilization of the R state.

a. Source of the protons that lower the pH:

• The concentration of both CO2 and H+ in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air.

• In the tissues, CO2 is converted by carbonic anhydrase to carbonic acid:

• which spontaneously loses a proton, becoming bicarbonate (the major blood buffer):

2. Bohr effect

a. Source of the protons that lower the pH:

• The proton produced by this pair of reactions contributes to the lowering of pH.

• This differential pH gradient (lungs having a higher pH , tissues a lower pH) favors the unloading of oxygen in the peripheral tissues, and the loading of oxygen in the lung.

• Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of oxygen.

2. Bohr effect

b. Mechanism of the Bohr effect

• The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin .

• This effect is caused by ionizable groups, such as specific histidine side chains that have higher pKas in deoxy hemoglobin than in oxyhemoglobin.

• Therefore, an increase in the concentration of protons (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (also called salt bridges).

• These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity.

2. Bohr effect

• The Bohr effect can be represented schematically as:

• where an increase in protons (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in protons) shifts the equilibrium to the left.

b. Mechanism of the Bohr effect

2. Bohr effect

3. Effect of 2,3- Bisphosphoglycerate on oxygen affinity

• 2,3-Bisphosphoglycerate (2,3-BPG) is an important regulator of the binding of oxygen to hemoglobin.

• It is the most abundant organic phosphate in the red blood cell, where its concentration is approximately that of hemoglobin.

• 2,3-BPG is synthesized from an intermediate of the glycolytic pathway.

a. Binding of 2,3-BPG to deoxyhemoglobin

• 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin.

• This preferential binding stabilizes the taut conformation of deoxyhemoglobin.

3. Effect of 2,3- Bisphosphoglycerate on oxygen affinity

b. Binding site of 2,3-BPG:

• One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer.

• This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG.

• [Note: A mutation of one of these residues can result in hemoglobin variants with abnormally high oxygen affinity.]

• 2,3-BPG is expelled on oxygenation of the hemoglobin.

3. Effect of 2,3- Bisphosphoglycerate on oxygen affinity

c. Shift of the oxygen-dissociation curve

• Hemoglobin from which 2,3-BPG has been removed has a high affinity for oxygen.

• However, as seen in the red blood cell, the presence of 2,3- BPG significantly reduces the affinity of hemoglobin for oxygen, shifting the oxygen-dissociation curve to the right.

• This reduced affinity enables hemoglobin to release oxygen efficiently at the partial pressures found in the tissues.

3. Effect of 2,3- Bisphosphoglycerate on oxygen affinity

4. Binding of CO2

• CO2 produced in metabolism is hydrated and transported as bicarbonate ion.

• However, some CO2 is carried as carbamate bound to the uncharged α-amino groups of hemoglobin (carbamino-hemo-globin which can be represented schematically as follows:

• The binding of CO2 stabilizes the T (taut) or deoxy form of hemoglobin, resulting in a decrease in its affinity for oxygen , In the lungs CO2, disassociates from the hemoglobin, and is released in the breath.

• Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carbon monoxyhemoglobin (HbCO) or carboxy hemoglobin.

• When carbon monoxide(CO) binds to one or more of the four heme sites, hemoglobin shifts to the relaxed (R) conformation, causing the remaining heme sites to bind oxygen with high affinity.

• This changes the normal sigmoidal shape toward a hyperbola.

• As a result, the affected hemoglobin is unable to release oxygen to the tissues

5. Binding of CO

• [Note: The affinity of hemoglobin for CO is 220 times greater than for oxygen.

• Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carbon monoxy hemoglobin in the blood.

• For example increased levels of CO are found in the blood of tobacco smokers.

• CO toxicity appears to result from a combination of tissue hypoxia and direct carbon monoxide-mediated damage at the cellular level.

• CO poisoning is treated with 100% oxygen at high pressure which facilitates the dissociation of CO from the hemoglobin

5. Binding of CO

Summary

• Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two αchains and two βchains, α2β2) held together by noncovalent interactions.

• The subunits occupy different relative positions in deoxyhemoglobin compared with oxyhemoglobin.

• The deoxy form of hemoglobin is called the “T,” or taut (tense) form.

• It has a constrained structure that limits the movement of the polypeptide chains.

• The T form is the low-oxygen-affinity form of hemoglobin.

• The binding of oxygen to hemoglobin causes rupture of some of the ionic and hydrogen bonds.

• This leads to a structure called the “R,” or relaxed form, in which the polypeptide chains have more freedom of movement.

• The R form is the high-oxygen-affinity form of hemoglobin.

• The oxygen dissociation curve for hemoglobin is sigmoidal in shape (in contrast to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding oxygen.

• Cooperative binding of oxygen by the four subunits of hemoglobin means that the binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule.

Summary

• Hemoglobin’s ability to bind oxygen reversibly is affected by the pO2(through heme-heme interactions), the pH of the environment, the pCO2, and the availability of 2,3-bisphosphoglycerate (2,3-BPG).

• For example, the release of O2 from Hb is enhanced when the pH is lowered or the pCO2 is increased (the Bohr effect), such as in exercising muscle, and the oxygen dissociation curve of Hb is shifted to the right.

• To cope long-term with the effects of chronic hypoxia or anemia, the concentration of 2,3-BPGin RBCs increases.

• 2,3-BPG binds to the Hb and decreases its oxygen affinity, and it, therefore, also shifts the oxygen-dissociation curve to the right.

• Carbon monoxide(CO)binds tightly (but reversibly) to the hemoglobin iron, forming carbon monoxy hemoglobin (HbCO)

Summary