The Functional Diversity of Proteins: The Example of Hemoglobin

Two Oxygen-binding Proteins Myoglobin Hemoglobin Skeletal muscle Red blood cells (erythrocytes) Oxygen storage Oxygen transport (lung to tissues) Monomer Four subunits (two each of two different kinds) Prosthetic group*: Prosthetic groups: 1 Heme-Fe+2 4 Heme-Fe+2 (1 per subunit) Binds oxygen Bind oxygen * a nonpolypeptide moiety that forms a functional part of a protein

Myoglobin 153 amino acids

Myoglobin 153 amino acids

Naming Helices and Corners “proximal” His F8

Myoglobin Hemoglobin β-chain Sequences: 26% identical 59% similar

Hemoglobin has: 2 α-chains (α1 and α2) 2 β-chains (β1 and β2) α and β chains are 45% identical

α1 β2 β1 α2

α1 β2 β1 α2

α1 β2 β1 α2

Introduction to Hemoglobinopathies: (Hereditary Disorders of Hemoglobin) Structural variants --altered amino acid sequence >800 known Thalassemias --decreased abundance of one or more of the globin chains Most common single-gene diseases

Sickle Cell Disease Red blood cells Oxygenated Deoxygenated Normal: squeeze through blood capillaries single file Sickle cells: block blood flow (local ischemia) (painful sickling crisis) Sickle cells have weak membranes and break (hemolysis, anemia)

β-chain Glu 6: mutated to Val (Glu6Val) Called hemoglobin S (HbS)

Binding site for β-chain Val6 (normal hydrophobic patch; Ala, Phe, Leu)

Hemoglobin Polymerizes and Precipitates

(heterozygous) “sickle cell trait” 2 bad β-chain genes 1 good β-chain gene (heterozygous) “sickle cell trait” 2 bad β-chain genes (homozygous) “sickle cell disease” Prevalence of sickle cell trait (protects against malaria) African-Americans: 8% trait; 1/600 disease

Heme Porphyrin + Fe+2 Planar. One edge hydrophobic; other hydrophilic (proprionic acid groups, red). Fe2+: ferrous. Two H2O molecules bind to Fe2+ above and below the plane. (Fe3+: ferric. Oxidized, cannot carry O2; “methemoglobin”)

Heme Binding Site (Hydrophobic)

Heme Binding Site (Hydrophobic)

Heme Binding Site (Hydrophobic) Heme carboxyl groups on the surface

Heme Binding Site (Hydrophobic) β-chain Phe42 mutated to Ser Hb Hammersmith: Heme slips out of pocket Hb unstable, precipitates

Oxygen Binding to Heme

Heme Stick Model

Heme from the Edge

Heme from the Edge

Heme from the Edge F8 His “proximal histidine”

Heme from the Edge F8 His “proximal histidine”

Heme from the Edge F8 His “proximal histidine” E7 His “distal histidine”

Heme from the Edge Oxygen F8 His “proximal histidine” E7 His “distal histidine”

Binding of oxygen to hemoglobin results in conformational changes in the protein structure (“allosteric effect”) Deoxyhemoglobin: T-conformation (tissues) (“tense” or “taut”) Oxyhemoglobin: R-conformation (lung) (“relaxed”) Oxygen affinity of R is 150 to 300-fold greater than that of T.

Positive Cooperativity Between Subunits

Mechanism of Cooperativity Red: deoxyhemoglobin (T) Black: oxyhemoglobin (R)

Effect on Adjacent Subunits

β –chain: Asp99Asn (FG corner) Hb Kempsey A hemoglobinopathy caused by mutation of a residue at the α1 – β2 interface β –chain: Asp99Asn (FG corner) Hb Kempsey Locks Hb in high oxygen affinity structure (R-conformation)

R-State (red) T-State (blue)

Definitions: Oxygen saturation: per cent (or fraction) of myoglobin or hemoglobin that has O2 bound. pO2: the concentration of oxygen given as “partial pressure” in units of torr. P50: the concentration of oxygen (pO2) when 50% of myoglobin or hemoglobin has oxygen bound.

Lung capillary pO2 = 90

More on Cooperativity Cooperativity can be measured by the fold-change in O2 needed to go from 10% saturation to 90%. For myoglobin: ≈ 0.11 to 9 torr (81-fold) (no cooperativity) For hemoglobin: ≈ 11 to 52 torr (4.8-fold) (positive cooperativity) (The smaller the range of fold-change, the more cooperativity) Hill coefficient: 1 = no cooperativity >1 = positive cooperativity <1 = negative cooperativity The hemoglobin Hill coefficient is 2.8

Beyond Cooperativity: Role of pH and BPG pH Hb + 4 O2 Hb(O2)4 + nH+ T R [H+] ( pH) tissues lactic acid carbonic acid (CO2)

BPG binds to the β1β2 interface of deoxy Hb (T) but not oxy Hb (R). 2,3-bisphosphoglycerate -- made in RBC’s -- 5 negative charges -- hypoxia (anemia; high altitudes; lung diseases) BPG binds to the β1β2 interface of deoxy Hb (T) but not oxy Hb (R). BPG stabilizes the T conformation; forces equilibrium from R to T.

Enhance delivery of oxygen to tissues Summary [H+] [BPG] Hb Hb (T) (R) Enhance delivery of oxygen to tissues

Different Globin Genes Hb F: high affinity for oxygen; enhances transfer from mother to fetus.

Thalassemias Imbalance of α- and β-globin subunits: Reduced synthesis or stability of one of them. Pathology: the excess subunit precipitates, RBC’s destroyed (anemia). Carrier (heterozygote) is resistant to malaria. α-Thalassemia: mostly α-globin gene deletion. Hemoglobin has 4 β-subunits, which can’t release O2. β-Thalassemia: mostly single base pair mutations, leading to decreased or defective β-chain mRNA.