Globular Hemeproteins and Hemoglobin

• Globular proteins are generally characterized by a spherical shape and good water solubility.
• They often play a catalytic/regulatory/transport role
• Globular heme proteins contain heme as a prosthetic group.
• Functions of globular hemeproteins include:
  • Electron carriers
  • Part of the active enzyme site
  • Transport of O2 and CO
  • Storage of O2

Hemoglobin

• Is a hemoprotein found in the cytoplasm of erythrocytes.
• The normal concentration of hemoglobin in the blood for adult males is 13.5 – 16.5 g/dL and for adult females 12 – 15 g/dL.
• Actual blood carries very little oxygen in solution.
• Hemoglobin is required to carry oxygen in the blood.

Structure of Heme

• Heme contains Protoporphyrin IX and ferrous iron (Fe2+).
• Heme consists of a conjugated system of double bonds, which gives it a red color.
• It contains 4 nitrogen atoms.
• It has 1 iron cation (Fe2+) bound in the middle of tetrapyrrole structure by coordination covalent bonds.
• Iron can form six bonds: four with porphyrin nitrogens, plus two additional bonds.
• The additional bonds are one above and one below the planar porphyrin ring.

Properties of Iron in Heme

• Iron can form six bonds: 4x pyrrole ring (A,B,C,D), 1x link to a protein and 1x link to an oxygen.

Heme Group Location

• Heme groups can be found in hemoproteins, hemoglobin (Hb), myoglobin (Mb), cytochromes, catalases, and peroxidases.

Structure and Function of Myoglobin

• Myoglobin is a heme protein present in heart and skeletal muscle.
• Myoglobin works as both an oxygen reservoir and an oxygen carrier.
• Consists of a single polypeptide chain that is similar the polypeptide chains of hemoglobin
• Approximately 80% of the peptide is in 8 stretches of α-helix which are labeled A to H.
• The helical stretches are terminated by Pro or ẞ-bends and loops which are stabilized by H bonds and ionic bonds.
• The interior is made up of hydrophobic amino acids stabilized by hydrophobic interactions.
• The surface has charged amino acids, which form H bonds with water.
• Heme is in crevice lined with non-polar amino acids, except 2 Histidine (His) residues.
• Proximal histidine binds directly to Fe2+ of heme
• Distal histidine stabilizes binding of O2 to Fe2+

Proximal and Distal Histidines

• Proximal His F8 is found above heme
• Distal His E7 is found below heme

Structure and Function of Hemoglobin

• Hemoglobin is found exclusively in red blood cells (RBCs) and functions in the transportation of O2.
• Hemoglobin A (HbA) is the predominant form in adults, consisting of 4 polypeptide chains α2β2.
• Each subunit is similar to myoglobin.
• Hemoglobin can transport oxygen (O2) and carbon dioxide (CO2).
• O2 binding properties are affected by allosteric effectors, different from myoglobin.

Process of Oxygen Binding to Hemoglobin

• Hemoglobin can exist in 2 different forms: T-form and R-form.
• T-form (T = tense) has a lower oxygen affinity than the R-form.
• The subunits of Hb are held together by electrostatic interactions.
• The binding of the first O2 molecule to subunit of the T-form leads to a local conformational change that weakens the association between the subunits → R-form („relaxed“) of Hb.
• Increased oxygen partial pressure results in the conversion of T-form to R-form.
• Hb + ↑pO2 ↔ HbO2
• Binding of O2 is cooperative.
• Hemoglobin binds O2 weakly at low oxygen pressure and tightly at high pressure.
• Oxygen binding at the four heme sites in hemoglobin does not happen simultaneously.
• Once the first heme binds oxygen, this introduces small changes in the protein structure which nudge the neighboring chains into a different shape, making them bind oxygen more easily.
• When blood is in the lungs, oxygen easily binds to the first subunit and then quickly fills up the remaining ones.
• Then, as blood circulates through the body, the oxygen level drops while the carbon dioxide level increases.
• As soon as the first oxygen molecule drops off, hemoglobin releases its bound oxygen, changing its shape and prompting the remaining three oxygens to be quickly released.
• This prompts the release of the remaining three oxygens.
• This enables hemoglobin to pick up the largest possible load of oxygen.

Oxygen Dissociation Curve

• Myoglobin → one heme binds one O2
• Hemoglobin → 4 heme’s binds 4 O2
• Hemoglobin binding indicates degree of saturation (Y) from 0 to 100%
• For myoglobin, P50 is 1 mm Hg.
• For hemoglobin P50 is 26 mm Hg
• The oxygen-dissociation curve for Hb is steepest at the oxygen concentrations that occur in the tissues.
• This permits oxygen delivery to respond to small changes in pO2.

Myoglobin Oxygen Dissociation Curve

• The O2 dissociation curve is hyperbolic
• This reflects that myoglobin binds single O2
• Mb + O2 ↔ MbO2 they exist in equilibrium
• There is an exchange between Hb and Mb, Mb and muscle cells depending on equilibrium
• Myoglobin binds O2 released from Hb, releases when O2 drops
• Myoglobin then releases the O2 into the muscle cell if there is an O2 demand.

Hemoglobin Oxygen Dissociation Curve

• O2 dissociation curve is sigmoidal
• Cooperative bind of O2 (increased affinity for Hb with more binding)
• Heme-heme interaction where the binding of O2 at one heme increases affinity for O2 at others

Allosteric Effects and Binding of Oxygen

• The ability of Hb to bind O2 depends on allosteric("other site”) effectors, including PO2, pH of environment, PCO2, and 2,3-disphosphoglycerate availability.
• Allosteric factors do not affect myoglobin.

Oxygen Unloading in Tissues

• An increase in PCO2 will cause increased unloading of O2.

Heme-Heme Interactions and Oxygen Affinity

• Structural changes in one heme group are transmitted to others
• The affinity for the last O2 is ~300X the affinity for the first O2
• More O2 can be delivered to tissues with small changes in PO2
• A sigmoidal curve gives increasing affinity of O2 for Hb with increasing partial pressure while a hyperbolic curve is a straight line in that range

Binding of Carbon Dioxide Regulation

• Most of the carbon dioxide (CO2) in the blood is transported as bicarbonate.
• CO2 + H2O ↔ H2CO3
• H2CO3 ↔ HCO3 + H+
• Some CO2 binds to the terminal –NH2 of the α and β chains forming carbaminoHb.
• The binding of CO2 stabilizes the “taut” form of Hb (deoxyHb).

Binding of Carbon Monoxide (CO)

• Carbon monoxide (CO) binds reversibly to the Fe2+ the same way that O2 does.
• CO + Hb ↔ HbCO (carbon monoxy Hb)
• Hemoglobin Affinity for carbon monoxide is 220X the affinity for oxygen
• Binding of CO to Hb increases affinity of remaining sites for O2
• The O2 dissociation curve shifts to left and becomes hyperbolic
• If there is greater than 60% carbon monoxy Hb, the condition is fatal
• This condition is treated with O2 therapy

Bohr Effect on Hemoglobin and Oxygen

• The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased pCO2
• In both cases, there are decreased oxygen affinity of hemoglobin.

Source of Lower pH Protons

• There are two general sources of protons: lactic acid produced by anaerobic metabolism in muscles and increased production of CO2 by cell utilization of O2 through respiration:
• CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3
• In the lungs, the equilibrium of this reaction is towards the left because CO2 is lost through exhaling
• A decreased affinity of Hb for O2 under the Bohr effect conditions results is a greater off loading (release) of O2 in the tissues.

Regulatory Effects of Bisphosphoglycerate (BPG)

• 2,3-bisphosphoglycerate (BPG) is an important regulator of Hb binding O2.
• BPG is the most abundant organic phosphate in red blood cells as much as the concentration of hemoglobin.
• BPG is synthesized from intermediate of glycolysis.
• BPG binds to deoxyhemoglobin stabilizing it.
• BPG decreases affinity of Hb for O2.
• Blood stripped of 2,3-BPG has a high affinity for O2.
• 2,3-BPG shifts the O2-dissociation curve to the right allowing decreased affinity of Hb for and effective unloading of O2 in tissues.
• The effect is to similar to the Bohr effect but no difference exists between lungs and tissues

BPG Response to Hypoxia

• 2,3-BPG levels increase during response to chronic hypoxia or anemia.
• Chronic hypoxia can be caused by pulmonary emphysema, high altitudes, or chronic anemia.
• Increased 2,3-BPG shifts O2 dissociation further to right allowing greater unloading of O2.

Hemoglobin Derivatives

• Oxyhemoglobin (oxyHb) results from hemoglobin (Hb) with O2.
• Deoxyhemoglobin (deoxyHb) result from hemoglobin without O2.
• Carbonylhemoglobin (HbCO) – CO binds to Fe2+ in heme in case of CO poisoning or smoking; CO has 200x higher affinity to Fe2+ than O2.
• Carbaminohemoglobin (HbCO2)- CO2 is non-covalently bound to globin chain of Hb. HbCO2 transports CO2 in blood (about 23%).
• Glycohemoglobin (HbA1c) is formed spontaneously by nonenzymatic reaction with Glucose. People with Diabetes have more HbA1c than normal (> 7%). Measurement of blood HbA1c is useful to get info about long-term control of glycemia.

Minor Hemoglobin Components for Normal Adults

• HbA which has the chain composition α2β2 make up 90%
• HbF which has the chain composition α22 make up <2%
• HbA2 which has the chain composition α2δ2 make up 3-9%
• HbA1c which has the chain composition is α2β2 make up 2%-5%

Hemoglobinopathies

• Hemoglobinopathies are a group of genetic disorders caused by the production of a structurally abnormal hemoglobin molecule.
• Insufficient quantities of normal hemoglobin or very rarely, both of the case.
• Examples include Sickle cell anemia (HbS), Hemoglobin C disease (HbC) (HbC), hemoglobin SC disease (HbS + HbC) and the thalassemias which have clinical consequences.

Sickle Cell Anemia

• Sickel Cell Anemia(HbS) causes a chain mutation of glu 6 ->val 6.
• Because glutamate(glu)is negatively charged while valine (val) is nonpolar, valine residues aggregate together by hydrophobic interactions leading to precipitation of Hb within RBCs
• RBCs assume sickle-shaped leading to fragility of their walls and a high rate of hemolysis. -Symptoms are hemolytic anemia, painful crises, poor circulation, frequent infections in affected individuals.
• The lifetime of erythrocyte in sickle cell is less than 20 days, compared to 120 days for normal RBCs.

Hemoglobin C Disease

• Like HbS, Hb C is a mutant Hb in which glutamic acid in 6th position of ẞ-chain is replaced by lysine.
• The RBCs will be large oblong and hexagonal.
• The heterozygous form (HbAC) is asymptomatic.
• The homozygous form (Hb CC) causes anemia, tissue anoxia and severe pain.

Thalassemia

• A group of genetic diseases has a defect in the rate of synthesis of one or more of Hb chains, but the chains are structurally normal.
• There is a defect or absence of one or more of genes responsible for synthesis of a or ẞ chains which leads to premature death of RBCs.
• In β-thalassemia the synthesis of ẞ chains is decreased or absent in individuals with β globin gene defects.
• -β -thalassemia minor (β –thalassemia trait) occurs when the synthesis of only one β -globin gene is defective or absent
• These individuals will make some ẞ chains and usually do not need specific treatment.
• -β -thalassemia major ( Cooley anemia) occurs if both genes are defective.
• Babies will be severely anemic during the first or second year of life and so require regular blood transfusion, but bone marrow replacement has proven safer.

α-Thalassemia

• α-thalassemia is when is which the synthesis of a globin chain is defective or absent.
• There are four copies of gene responsible for synthesis of a globin chains so patients may have various conditions when these genes are altered:
  • Silent carrier of α-thalassemia with no symptoms: if one gene is defective
• α-thalassemia trait: if two genes are defective, the presentation will be minor anemia.
• Hb H disease - if three genes are defective, moderate anemia will be present.
• Hydrops fetalis - Lacks all 4 genes; the fetus may survive till birth then will die.

Methemoglobin (HbM)

• Methemoglobin contains Fe3+ instead of Fe2+ in heme groups.
• Oxidation of Fe converts hemoglobin and myoglobin to metHb and metmyoglobin.
• Methemoglobin (HbM) can not bind to oxygen
• Oxidation can be caused by drugs like nitrates, H2O2 or free radicals, or has mutations ina-or ẞ-chain of globin
• A deficiency of NADH-cytochrome b5 reductase (responsible for the conversion of methemoglobin to hemoglobin) leads to accumulation of HbM
• RBCs of newborns halve the capacity of this enzyme, therefore they are more susceptible to oxidation

Hemoglobin questions

• In deoxy hemoglobin (Hb), the Fe (II) is coordinated to four nitrogens in heme and at the proximal Histadine Histadine of Hemoglobin.
• If iron in hemoglobin is oxidized to 3+, Methemoglobinemia may occur.
• Hemoglobin will have the highest affinity for oxygen when 2,3-bisphosphoglycerate levels are low.