Oxygen Storage in Muscle Tissue Myoglobin (Mb) Originally isolated from sperm whales 10X abundance greater in aquatic- than terrestrial-mammals Mb knockout mice exhibit normal exercise capacity Detoxification of reactive nitric oxide a signaling molecule

Myoglobin Oxygen Binding Curve Quantification of Myoglobin’s O 2 -Binding Behavior Mb dissociation: MbO 2 ↔ Mb + O 2 Dissociation constant: K = [Mb][O 2 ]/[MbO 2 ] Myoglobin-O 2 term: [MbO 2 ] = [Mb][O 2 ]/K Fractional saturation: Y = [MbO 2 ]/([Mb] + [MbO 2 ]) Substitute for MbO 2 Y = [Mb][O 2 ]/K/([Mb] + [Mb][O 2 ]/K) Simplify: Y = [O 2 ]/(K + [O 2 ]) O 2 partial pressure: Y = pO 2 /(K + pO 2 )

Myoglobin Protein Structure Single polypeptide chain with 8 alpha helices (A-H) Prosthetic heme group; only Fe (II) Central Fe (II) atom bound to 4 porphyrin N atoms N of His F8 and O 2 binds below and above the Fe (II)

Globin Evolution Homologous proteins with a common ancestor Genetic mutations with invariant residues Embryonic hemoglobin Adult hemoglobin

Hemoglobin: Oxygen Transport in Mammals Hemoglobin features: Located in red blood cells; 4 subunits Cooperative oxygen binding (allosteric protein) Deoxygenated T-form and Oxygenated R-form

Hemoglobin Cooperativity Enhances O 2 Delivery Proximal His binds Fe (II) Distal His disfavors CO binding O 2 → Fe (II) shift from out of plane to porphyrin plane Limitations with Hemoglobin as an oxygen transporter?

Conformational Change in Hemoglobin Which is the T and R configuration? Is this enzyme conversion concerted or sequential?

2,3-Bisphosphoglycerate Stabilizes Hemoglobin T Configuration Central cavity size difference T versus R? Amino acids that comprise the central cavity?

How does 2,3-BPG affect T versus R configuration? Hemoglobin Central Cavity with 2,3-Bisphosphoglycerate

Bohr Effect: Protons and Carbon Dioxide Promote the T State Stabilizing the T state increases hemoglobin O 2 release T - more sigmodial R - more hyperbolic T ↔ R + H +

T-State Stabilization by Salt Bridge Formation How is CO 2 linked with H + concentration?

Carbon Dioxide Acidifies the Hemoglobin Environment

Tissue-Specific Hemoglobin Affinity for Oxygen What is the favorable hemoglobin state (T/R) in capillaries near muscle tissue and lungs?

How must fetal hemoglobin oxygen binding vary from the mother? Adjusted Hemoglobin-Oxygen Affinity with Environmental Changes

Fetal hemoglobin (α 2 γ 2 ) has a Ser substitution for His Two less positive charges in central cavity means what? Adjusted Hemoglobin-Oxygen Affinity with Environmental Changes

Hemoglobin Mutation: Sickle-Cell Anemia Sickle-Cell Features Lower hemoglobin (Hb) solubility for Hb S than Hb A (normal) Amino acid substitution Glu → Val Reduced T-state solubility O 2 affinity and allosteric properties unaffected Heterozygotes asymptomatic

Do changes in the oxygen-binding prosthetic group preclude cooperative O 2 binding? Copper Oxygen Carrier Present in Lobster Hemoglobin

Site-Directed Mutagenesis Experimentation Convert proximal His → Gly Exogenous imidazole binds with porphyrin iron Would modified hemoglobin (i)exhibit cooperative binding? (ii) generate Fe (III)?

Substitution for 2,3- Bisphosphoglycerate in Bird Hemoglobin Which compound(s) are well suited to bind in the central cavity of bird hemoglobin?

Physiological [CO 2 ] and [2,3-BPG] at pH = 7 Curve matching: Increase in [CO 2 ] Increase in 2,3-BPG Increase in pH Loss of 4° structure Hemoglobin Fractional Saturation Curve Shift