• Hemoglobin was the first protein to :
  • have its molecular mass accurately determined
  • be characterized by ultracentrifugation
  • be associated with a specific physiological function
  • in which a point mutation was demonstrated to cause a single amino acid change.

1. Hemoglobin Function

• Hemoglobin is a tetramer, a dimer of ab protomers.
• The structural units are related to each other and to myoglobin (Mb).
• Its major physiological role is oxygen transport.

A. Heme

• Mb and each monomer of Hb noncovalently bind a single heme group.
• Protoporphyrin IX holds an FE II.
• In Hb and Mb, the iron atom normally remains in the Fe(II) ferrous oxidation state, whether or not the heme is oxygenated.
• Oxygenation changes the electronic state of the Fe(II)-heme as is indicated by the color change of blood from dark purple venous blood to brilliant scarlet.

B. Oxygen Binding

• Hemoglobin Cooperatively Binds O2.
• Hemoglobin and Myoglobin work together as a pair of oxygen delivery and storage systems.
• Hemoglobin's oxygen dissociation curve is of great significance. It permits the blood to deliver much more O2 to the tissues than it could if Hb had a hyperbolic O2 dissociation curve with the same 26 Torr p50. Such a hyperbolic curve has a YO2 =0.79 and 0.54 at 39 Torr for a difference in YO2 of only 0.25.
• The Hill Equation Phenomenologically Describes Hemoglobin's O2 Binding Curve
• The quantity n, the Hill constant, increases with the degree of cooperatively of a reaction and thereby provides a convenient, although simplistic, characterization of a ligand-binding reaction.
• If n>1 in the Hill equation, the ligand binding is said to be positively cooperative.
• If n<1 in the Hill equation, the ligand binding is said to be negatively cooperative.
• Hill Equation Parameters May Be Graphically Evaluated
• The fourth O2 to bind to Hb does so with 100-fold greater affinity than the first.
• Globin Prevents Oxyheme from Autooxidizing
• Globin no only modulates the O2 binding affinity of heme, but makes reversible O2 binding possible.

C. Carbon Dioxide Transport and the Bohr Effect

• In addition to being an O2 carrier, Hb plays an important role in C02 transport.
• Increasing pH stimulates Hb to bind O2
• The Bohr Effect Facilitates O2 Transport
• In the capillaries, where pO2 is low, the H+ generated by bicarbonate formation is taken up by Hb, which is thereby induced to unload its bound O2. This H+ uptake facilitates CO2 transport by stimulating bicarbonate formation. Conversely, in the lungs, where pO2 is high, O2 binding by Hb releases the Bohr protons, which drive off the CO2.
• These reactions are closely matched and cause little change in blood pH.
• CO2 and Cl- Modulate Hemoglobin's O2 Affinity
• CO2 modulates O2 binding directly and by combining reversibly with the N-terminal amino groups of blood proteins to form carbamates:
• R-NH2 + CO2 <---> R-NH-COO- + H+
• Cl- is a modulator of hemoglobin's O2 affinity

D. Effect of BPH on O2 Binding

• In the absence of BPG, little of this O2 is released since hemoglobin's O2 affinity is increased, thus shifting the O2 dissociation curve significantly towards lower pO2.
• The presence of BPG, CO2, H+ and Cl- accounts for the O2 binding properties of Hb.
• Increased BPG Levels Are Partially Responsible for High-Altitude Adaptation
• Fetal Hemoglobin a Low BPG Affinity

2. Structure and Mechanism

• Life is based on the interactions of complex, structurally well-defined molecules.

A. Structure of Myoglobin

• Mb consists of eight helices labeled A-H that are linked by short polypeptide segments to form an ellipsoidal molecule.
• The structures of oxy and deoxyMb are nearly superimposable.

B. Structure of Hemoglobin

• The tertiary structures of the a and b subunits are remarkably similar, both to each other and to that of Mb.
• The a and b subunits in the tetramer are related by inexact twofold rotations so that the subunits occupy the vertices of a tetrahedron.
• The polypeptide chains of Hb are arranged such that there are extensive interactions between unlike subunits.
• Oxy and Deoxyhemoglobins Have Different Quaternary Structures
• The quaternary structural change preserves hemoglobin's exact twofold symmetry and takes place entirely across its a1-b2 (and a2-b1) interface. The a1-b2 (and a21-b1) contact is unchanged.

C. Mechanism of Oxygen-Binding Cooperativity

• The positive cooperativity of O2 binding to Hb arises from the effect of the ligand-binding state of one heme on the ligand-binding affinity of another.
• They are mechanically transmitted by the protein.
• X-Ray crystal structure analysis has provided snapshots of the R and T states of Hb in various states of ligation but does not indicate how the protein changes states.
• The Movement of FE(II) into the Heme Plane Triggers the T-R Conformational Shift.
• The a1-b2 and a2-b1 Contacts Have Two Stable Positions
• These contacts, which are joined by different but equivalent sets of hydrogen bonds in the two states, act as a binary switch that permits only two stable positions of the subunits relative to each other.
• The side chains therefore act as flexible joints or hinges about which the a1 and b2 subunits pivot during the quaternary change.
• The T state is Stabilized by a Network of Salt Bridges That Must Break to Form the R State.
• Hemoglobin's O2-Binding Cooperativity Derives from the T-R Conformational Shift
• No one subunit or dimer can greatly change its conformation independently of the others. Indeed the two stable positions of the a1C-b2FG contact limit the Hb molecule to only two quaternary forms, R and T.
• The T state has reduced O2 affinity, because its Fe-O2 bond is stretched by steric repulsions between the heme and the O2 and in the b subunits by the need to move ValE22 out of the O2 binding site.
• Unliganded subunits in the R state have an increased O2 affinity because they are already in the O2 binding conformation.
• Hemoglobin's Sigmoidal O2 Binding Curve Is a Composite of its Hyperbolic R and T State Curves

D. Testing the Perutz Mechanism

• C-Terminal Salt Bridges Are Required to Maintain the T State
• Fe-O2 Bond Tension Has Been Spectroscopically Demonstrated

E. Origin of the Bohr Effect

• It arises from pK changes of several groups caused by changes in their local environments that accompany hemoglobin's T-R transition.

F. Structural Basis of BPG Binding

• BPG decreases the oxygen-binding affinity of Hb by preferentially binding to its deoxy state.

G. Role of the Distal Histidine Residue

• Histidine acts as a proton trap.

3. Abnormal Hemoglobins

• Hemoglobin defects give rise to a class of diseases known as thalassemias

A. Molecular Pathology of Hemoglobin

• 1) Changes in surface residues
  Changes of surface residues are usually innocuous because most of these residues have no specific functional role.
• 2) Changes in internally located residues
  Changing an internal residue often destabilizes the Hb molecule.
• 3) Changes Stabilizing Methemoglobin
  Changes at the O2 binding site that stabilize the heme in Fe(III) oxidation state eliminate the binding of O2 to the defective subunits.
• 4) Changes at the a1-b2 contact
  Changes at the a1-b2 contact often interfere with hemoglobin's quaternary structural changes.

B. Molecular Basis of Sickle-Cell Anemia

• 10% of American blacks and 25% of African blacks are heterozygotes for sickle-cell hemoglobin (HbS).
• HbS Fibers are Stabilized by Intermolecular Contacts Involving Val b6 and Other Residues
• The sickling of HbS-containing erythrocytes results from the polymerization of deoxyHbS into rigid fibers that extend throughout the length of the cell.
• The contact involving Val6B is essential for fiber formation.
• The Initiation of HbS Gelation Is Complex
• Upon achieving gelation conditions, there is a reproducible delay that varies according to conditions from milliseconds to days. During this time, no HbS fibers can be detected.
• No other solution process even approaches a 30th power concentration dependence.
• One fiber acts as a nucleus for formation of other fibers.
• The fibers bundle into hex packed groups of 14 with twist along the fiber axis.

4. Allosteric Regulation

• Ligand binding to proteins takes place via other ligands.
• These other ligands are known as effectors or modulators.

A. The Adair Equation

B. The Symmetry Model

• Homotropic Interactions
  For high L values, if a single ligand is to bind, it must force the protein into its less preferred R state. The requirement that all protomers change their conformational states in a concerted manner causes the remaining three ligand binding sites to become available.
• The free energy of ligand-binding stabilizes the R state with respect to the T state.
• Heterotropic Interactions
• The presence of activator therefore increases the protein's substrate-binding affinity. This is a positive heterotropic effect.
• The presence of inhibitor (b>0), which only binds to the T state, reduces the binding affinity for substrate.
• Both homotropic and heterotropic effects can be explained solely by the requirement that the molecular symmetry of the
• These other ligands are known as effectors or modulators.

C. The Sequential Model

• Lock and Key versus Induced Fit
• Induced Fit is a flexible interaction between ligand and protein that induces a conformational change in the protein, which results in its increased ligand binding affinity.
• In the resulting sequential or induced-fit model, ligand binding induces a conformational change in a subunit; cooperative interactions arise through the influences that these conformational changes have on neighboring subunits.
• The strengths of these interactions depend on the degree of mechanical coupling between subunits.
• The essence of the sequential model is that a protein's ligand binding affinity varies with its number of bound ligands, whereas in the symmetry model this affinity depends only on the protein's quaternary state.

D. Hemoglobin Cooperativity

• To a first approximation, Hb largely follows the symmetry model, although it also exhibits some features of the sequential model.
• Such tertiary structural changes are responsibly for the buildup of strain that eventually triggers the T-R transition.