Oxygen Transport by the Proteins Myoglobin and Hemoglobin | Chemistry Optional Notes for UPSC PDF Download

Hemoglobin

• 1st. protein whose molecular weight was determined
• 1st protein to be assigned a specific function - dioxygen transport
• has a prosthetic group (non-amino acid) heme group (protoporphyrin IX with a ferrous ion) covalently attached to the protein. The heme group binds dioxygen.
• 1st protein in which a point mutation (single base pair charge) causes a single amino acid change in the protein, marking the start of molecular medicine
• 1st protein with high resolution x-ray structure
• theory for dioxygen binding explain control of enzyme activity
• the binding of dioxygen is regulated by binding of H⁺, CO₂, and bisphosphoglcyerate which bind to sites (allosteric) distant from oxygen binding site.
• crystals of deoxy-Hb shatter on binding dioxygen, indicating significant conformational changes on binding.

Figure 4.1.1: The heme group contains protoporphyrin IX, with four tetrapyrrole rings linked by methene bridges. Attached to the tetrapyrrole structure are four methyl, two vinyl, and two proprionate groups. These can be arranged in 15 ways, only one (IX) occurs in biological systems.

The heme fits into a hydrophobic crevice in the proteins with the proprionate groups exposed to solvent. The Fe²⁺ ion is coordinated to 4 N's on the 4 pyrrole rings, The 5th ligand is a supplied by proximal His (the 8th amino acid on helix F) of the protein. In the absence of dioxygen, the 6 ligand is missing. and the geometry of the complex is square pyramidal with the Fe above the plane of the heme ring. A distal His (E7) is on the opposite side of the heme ring, but too far to coordinate with the Fe. When dioxgen binds, it occupies the 6th coordination site and pulls the Fe into the plane of the ring, leading to octahedral geometry. CO, NO, and H₂S also bind to the 6th site, but with higher affinity than dioxygen, which can lead to CO poisoning. The distal His keeps these ligands (including dioxygen) bound in a bent, non-optimal geometry. This minimizes the chances of CO poisoning.

Myoglobin

• Myoglobin is a relatively small protein that contains 150 amino acids.
• Mb is extremely compact, and consists of 75% alpha helical structure.
• The interior amino acids are almost entirely nonpolar. The only polar amino acids found completely buried are the two His (proximal and distal) found at the active site of dioxygen binding.

Figure 4.1.2: The skeletal structure of the heme prosthetic group found within the structure of myoglobin. The porphyrin ring contains four pyrrole nitrogens bound to a ferrous (Fe(II)) ion center. There are six coordination sites in the Fe(II) ion; four are occupied by the pyrrole nitrogens, one is occupied by a proximal histidine, one site can be occupied by a dioxygen molecule (not shown).

• Difference between Hb and Mb
  

  Hb is a tetramer of two α and two β subunits held together by IMF's (an example of quarternary protein structure), and 4 bound hemes, each of which can bind a dioxygen. In a fetus, two other subunits make up Hb (two zeta - ζand two epsilion - ε subunits -analogous to the two αand two β subunits, respectively). 
• This changes in fetuses to two α and two γ subunits. Fetal Hb has higher affinity for dioxygen than adult Hb. Mb is a single polypeptide chains which has higher affinity for dioxygen than Hb.
• The α and β chains are similar to Mb, which is unexpected since only 24 of 141 residues in the α and β chains of Hb are identical to amino acids in Mb. This suggests that different sequences can fold to similar structures. The globin fold of Mb and each chain of Hb is common to vertebrates and must be nature's design for dioxygen carriers. 
• A comparison of the sequence of Hb from 60 species show much variability of amino acids, with only 9 identical amino founds. These must be important for structure/function. All internal changes are conservative (e.g. changing a nonpolar for a nonpolar amino acid). Not even Pro's are conserved, suggesting there are different ways to break helices. The two active site His are conserved, as is Gly B6 (required for a reverse turn).

Oxygen Transport 

• Many microorganisms and most animals obtain energy by respiration, the oxidation of organic or inorganic molecules by O2. At 25°C, however, the concentration of dissolved oxygen in water in contact with air is only about 0.25 mM. Because of their high surface area-to-volume ratio, aerobic microorganisms can obtain enough oxygen for respiration by passive diffusion of O₂ through the cell membrane. 
• As the size of an organism increases, however, its volume increases much more rapidly than its surface area, and the need for oxygen depends on its volume. Consequently, as a multicellular organism grows larger, its need for O₂ rapidly outstrips the supply available through diffusion. Unless a transport system is available to provide an adequate supply of oxygen for the interior cells, organisms that contain more than a few cells cannot exist. 
• In addition, O₂ is such a powerful oxidant that the oxidation reactions used to obtain metabolic energy must be carefully controlled to avoid releasing so much heat that the water in the cell boils. Consequently, in higher-level organisms, the respiratory apparatus is located in internal compartments called mitochondria, which are the power plants of a cell. Oxygen must therefore be transported not only to a cell but also to the proper compartment within a cell.

Myoglobin and Hemoglobin 

Myoglobin is a relatively small protein that contains 150 amino acids. The functional unit of myoglobin is an iron–porphyrin complex that is embedded in the protein (Figure 4.2.1). In myoglobin, the heme iron is five-coordinate, with only a single histidine imidazole ligand from the protein (called the proximal histidine because it is near the iron) in addition to the four nitrogen atoms of the porphyrin. A second histidine imidazole (the distal histidine because it is more distant from the iron) is located on the other side of the heme group, too far from the iron to be bonded to it. Consequently, the iron atom has a vacant coordination site, which is where O₂ binds.

In the ferrous form (deoxymyoglobin), the iron is five-coordinate and high spin. Because high-spin Fe²⁺ is too large to fit into the “hole” in the center of the porphyrin, it is about 60 pm above the plane of the porphyrin. When O₂ binds to deoxymyoglobin to form oxymyoglobin, the iron is converted from five-coordinate (high spin) to six-coordinate (low spin; Figure 4.2.2). Because low-spin Fe²⁺ and Fe³⁺ are smaller than high-spin Fe²⁺, the iron atom moves into the plane of the porphyrin ring to form an octahedral complex. The O₂ pressure at which half of the molecules in a solution of myoglobin are bound to O₂ (P1/2) is about 1 mm Hg (1.3 × 10⁻³ atm).
Figure 4.2.2: Oxygen Binding to Myoglobin and Hemoglobin. (a) The Fe²⁺ ion in deoxymyoglobin is high spin, which makes it too large to fit into the “hole” in the center of the porphyrin. (b) When O₂ binds to deoxymyoglobin, the iron is converted to low-spin Fe³⁺, which is smaller, allowing the iron to move into the plane of the four nitrogen atoms of the porphyrin to form an octahedral complex.

Hemoglobin consists of two subunits of 141 amino acids and two subunits of 146 amino acids, both similar to myoglobin; it is called a tetramer because of its four subunits. Because hemoglobin has very different O₂-binding properties, however, it is not simply a “super myoglobin” that can carry four O₂ molecules simultaneously (one per heme group). The shape of the O₂-binding curve of myoglobin can be described mathematically by the following equilibrium:

The O₂-binding curve of hemoglobin is S shaped (Figure 4.2.3). As shown in the curves, at low oxygen pressures, the affinity of deoxyhemoglobin for O₂ is substantially lower than that of myoglobin, whereas at high O₂ pressures the two proteins have comparable O₂ affinities. The physiological consequences of unusual S-shaped O₂-binding curve of hemoglobin are enormous. In the lungs, where O₂ pressure is highest, the high oxygen affinity of deoxyhemoglobin allows it to be completely loaded with O₂, giving four O₂ molecules per hemoglobin. In the tissues, however, where the oxygen pressure is much lower, the decreased oxygen affinity of hemoglobin allows it to release O₂, resulting in a net transfer of oxygen to myoglobin.
Figure 4.2.3: The O₂-Binding Curves of Myoglobin and Hemoglobin. Plots of Y (fractional saturation) vs L (pO2) are hyperbolic for Mb, but sigmoidal for Hb, suggesting cooperative binding of oxygen to Hb (binding of the first oxygen facilitates binding of second, etc).

The S-shaped O₂-binding curve of hemoglobin is due to a phenomenon called cooperativity, in which the affinity of one heme for O₂ depends on whether the other hemes are already bound to O₂. Cooperativity in hemoglobin requires an interaction between the four heme groups in the hemoglobin tetramer, even though they are more than 3000 pm apart, and depends on the change in structure of the heme group that occurs with oxygen binding. The structures of deoxyhemoglobin and oxyhemoglobin are slightly different, and as a result, deoxyhemoglobin has a much lower O₂ affinity than myoglobin, whereas the O₂ affinity of oxyhemoglobin is essentially identical to that of oxymyoglobin. Binding of the first two O₂ molecules to deoxyhemoglobin causes the overall structure of the protein to change to that of oxyhemoglobin; consequently, the last two heme groups have a much higher affinity for O₂ than the first two.

The affinity of Hb, but not of Mb, for dioxygen depends on pH. This is called the Bohr effect, after the father of Neils Bohr, who discovered it.

Decreasing pH shifts the oxygen binding curves to the right (to decreased oxygen affinity). Increased [H⁺] will cause protonation of basic side chains. In the pH range for the Bohr effect, the mostly likely side chain to get protonated is His (pKa around 6), which then becomes charged. The mostly likely candidate for protonation is His 146 (on the β chain - CH₃) which can then form a salt bridge with Asp 94 of the β(FG1) chain. This salt bridge stabilizes the positive charge on the His and raises its pKa compared to the oxyHb state. Carbon dioxide binds covalently to the N-terminus to form a negatively charge carbamate which forms a salt bridge with Arg 141 on the alpha chain. BPG, a strongly negatively charged ligand, binds in a pocket lined with Lys 82, His 2, and His 143 (all on the beta chain). It fits into a cavity present between the β subunits of the Hb tetramer in the T state. Notice all these allosteric effectors lead to the formation of more salt bridges which stabilize the T or deoxy state. The central cavity where BPG binds between the β subunits become much smaller on oxygen binding and the shift to the oxy or R state. Hence BPG is extruded from the cavity.

The binding of H⁺ and CO₂ helps shift the equilibrium to deoxyHb which faciliates dumping of oxygen to the tissue. It is in respiring tissues that CO₂ and H⁺ levels are high. CO₂ is produced from the oxidation of glucose through glycolysis and the Krebs cycle. In addition, high levels of CO₂ increase H⁺ levels through the following equilibrium:

In addition, H⁺ increases due to production of weak acids such as pyruvic acid in glycolysis .

Hb, by binding CO₂ and H⁺, in addition to O₂, serves an additional function: it removes excess CO₂ and H⁺ from the tissues where they build up. When deoxyHb with bound H⁺ and CO₂ reaches the lungs, they leave as O₂ builds and deoxyHb is converted to oxyHb.

2,3-BPG

Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG - Figure 4.2.5). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose metabolism. The 2,3-BPG molecule fits into the ‘hole of the donut’ of adult hemoglobin. Such binding of 2,3-BPG favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG, hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen.

Carbon Monoxide

CO is a highly toxic gas without color and odor.  It is commonly produced the partial combustion of carbon-containing compounds. It competes with oxygen for hemoglobin binding. Its binding affinity is ~ 200 fold tighter. 

Summary

• Hemoglobin is a protein found in red blood cells (RBCs) that is comprised of two alpha and two beta subunits that surround an iron-containing heme group. Oxygen readily binds this heme group. The ability of oxygen to bind increases as more oxygen molecules are bound to heme. Disease states and altered conditions in the body can affect the binding ability of oxygen, and increase or decrease its ability to dissociate from hemoglobin.
• Carbon dioxide can be transported through the blood via three methods. It is dissolved directly in the blood, bound to plasma proteins or hemoglobin, or converted into bicarbonate. The majority of carbon dioxide is transported as part of the bicarbonate system. Carbon dioxide diffuses into red blood cells. Inside, carbonic anhydrase converts carbon dioxide into carbonic acid (H₂CO₃), which is subsequently hydrolyzed into bicarbonate (HCO₃⁻) and H⁺. 
• The H+ ion binds to hemoglobin in red blood cells, and bicarbonate is transported out of the red blood cells in exchange for a chloride ion. This is called the chloride shift. Bicarbonate leaves the red blood cells and enters the blood plasma. In the lungs, bicarbonate is transported back into the red blood cells in exchange for chloride. The H⁺ dissociates from hemoglobin and combines with bicarbonate to form carbonic acid with the help of carbonic anhydrase, which further catalyzes the reaction to convert carbonic acid back into carbon dioxide and water. The carbon dioxide is then expelled from the lungs.