Oxygen Storage in Muscle TissueMyoglobin (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 O2-Binding Behavior

Mb dissociation: MbO2 ↔ Mb + O2

Dissociation constant: K = [Mb][O2]/[MbO2]

Myoglobin-O2 term: [MbO2] = [Mb][O2]/K

Fractional saturation: Y = [MbO2]/([Mb] + [MbO2])

Substitute for MbO2 Y = [Mb][O2]/K/([Mb] + [Mb][O2]/K)

Simplify: Y = [O2]/(K + [O2])

O2 partial pressure: Y = pO2/(K + pO2)

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 O2 binds below and above the Fe (II)

Globin EvolutionHomologous proteins with a common ancestor

Genetic mutations with invariant residues

Embryonic hemoglobin

Adult hemoglobin

Hemoglobin: Oxygen Transport in MammalsHemoglobin features:

• Located in red blood cells; 4 subunits• Cooperative oxygen binding (allosteric protein)• Deoxygenated T-form and Oxygenated R-form

Hemoglobin Cooperativity Enhances O2 Delivery

• Proximal His binds Fe (II) • Distal His disfavors CO binding• O2 → 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 O2 release

• T - more sigmodial

• R - more hyperbolic

• T ↔ R + H+

T-State Stabilization by Salt Bridge Formation

How is CO2 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 (α2γ2) 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

• O2 affinity and allosteric properties unaffected

• Heterozygotes asymptomatic

Do changes in the oxygen-binding prosthetic group preclude cooperative O2 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 [CO2] and [2,3-BPG] at pH = 7

Curve matching:

Increase in [CO2]

Increase in 2,3-BPG

Increase in pH

Loss of 4° structure

Hemoglobin Fractional Saturation Curve Shift