Rucete ✏ Lehninger Principles of Biochemistry In a Nutshell
5.1 Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins
This chapter introduces the principles of reversible ligand binding to proteins, using myoglobin and hemoglobin as classic examples to illustrate protein-ligand interactions, oxygen transport, allosteric effects, cooperative binding, and molecular basis of disease.
Oxygen Binding and Heme Prosthetic Group
• Oxygen is poorly soluble in water and cannot be efficiently transported or stored by simple diffusion; multicellular organisms evolved proteins to bind and carry oxygen.
• None of the standard amino acid side chains can reversibly bind oxygen. This function is accomplished using transition metals, especially iron, embedded in a prosthetic group called heme.
• Heme consists of a protoporphyrin ring with a central ferrous iron (Fe2+) atom that can form six coordination bonds—four in the plane of the ring, and two perpendicular (one binds to protein, one to O2).
• Heme is sequestered deep within globin proteins, restricting access to its reactive iron and preventing unwanted oxidation and reactivity.
• Binding of O2 to heme changes the electronic properties and color of the protein. Carbon monoxide (CO) and nitric oxide (NO) can also bind heme, often with higher affinity than O2, making CO highly toxic.
Globin Family and Oxygen Storage/Transport
• Globins are a widespread protein family with a highly conserved tertiary structure known as the "globin fold" (eight α-helices connected by loops).
• Myoglobin (Mb): Monomeric, abundant in muscle, facilitates O2 diffusion and storage, especially in diving mammals.
• Hemoglobin (Hb): Tetrameric, found in erythrocytes (red blood cells), responsible for O2 transport in blood.
• Other globins include neuroglobin (protects brain under hypoxia) and cytoglobin (regulates nitric oxide in blood vessel walls).
Protein-Ligand Binding: Quantitative Description
• Protein-ligand binding is reversible and can be described by equilibrium constants (association constant Ka, dissociation constant Kd).
• The fraction of occupied binding sites (Y) shows a hyperbolic dependence on ligand concentration for single-site proteins (e.g., myoglobin).
• Kd is the ligand concentration at which half the binding sites are occupied; lower Kd indicates higher affinity.
• Binding of oxygen to myoglobin follows this hyperbolic relationship, but hemoglobin (multisubunit) displays sigmoidal (cooperative) binding.
Protein Structure and Ligand Specificity
• The protein environment around heme in myoglobin and hemoglobin modulates ligand binding, enhancing selectivity for O2 over CO.
• Specific histidine residues in the globin pocket stabilize O2 binding via hydrogen bonding and steric effects, and control access to the heme pocket.
• Protein "breathing" (small conformational fluctuations) allows O2 to access or leave the buried heme pocket.
Hemoglobin: Structure, Function, and Cooperative Binding
• Hemoglobin is composed of two α and two β subunits, each structurally similar to myoglobin and containing its own heme.
• Strong subunit interactions (hydrophobic, hydrogen bonds, ion pairs) stabilize the quaternary structure. Oxygen binding causes conformational changes (T state ↔ R state) affecting subunit contacts.
• Hemoglobin exhibits cooperative oxygen binding: binding of one O2 increases the affinity of remaining subunits—a phenomenon described by a sigmoidal binding curve and Hill coefficient.
• Allosteric proteins change shape and function upon ligand binding (homotropic or heterotropic interactions).
• Cooperative binding allows efficient O2 loading in lungs and unloading in tissues.
Regulation of Oxygen Binding
• Bohr effect: Hemoglobin’s affinity for O2 decreases at low pH and high CO2, favoring O2 release in tissues; H+ and CO2 stabilize the T state via additional salt bridges and carbamate formation.
• 2,3-Bisphosphoglycerate (BPG) binds in the central cavity of deoxyhemoglobin, stabilizes the T state, and reduces O2 affinity, aiding O2 delivery especially at high altitude or in hypoxic conditions.
• Fetal hemoglobin (α2γ2) binds BPG less tightly, increasing O2 affinity to extract O2 from maternal blood.
Molecular Disease: Sickle Cell Anemia
• Sickle cell anemia is caused by a single amino acid substitution (Glu → Val) in the β chain of hemoglobin (HbS), creating a hydrophobic patch.
• Deoxy-HbS molecules aggregate into fibers, deforming red blood cells into a sickle shape, causing anemia, pain, and organ damage.
• Heterozygous carriers have resistance to malaria—a case of balanced polymorphism maintained by natural selection.
In a Nutshell
• Protein-ligand binding is fundamental to protein function and can be quantitatively analyzed using equilibrium and kinetic constants.
• Myoglobin and hemoglobin exemplify how structure determines reversible oxygen binding, storage, and transport, and how cooperativity and allosteric regulation allow fine control of function.
• Small changes in protein structure or environment have profound effects on ligand binding, as seen in regulation by pH, CO2, BPG, and in genetic diseases like sickle cell anemia.
• Understanding protein-ligand interactions is key to physiology, disease, and biomedical applications.