Chapter 7
Hemoglobin:
Portrait of a Protein in Action
Heme Allows Myoglobin and Hemoglobin to Bind Oxygen
• Heme = Fe2+ (ferrous) + Protoporphyrin
• Protoporphyrin = 4 pyrrole rings + 4 methine bridges +
4 methyl groups + 2 vinyl groups +
2 propionate side chains
• 5th coordination site occupied by the imidazole ring of
proximal histidine of myoglobin (or hemoglobin)
• 6th coordination site occupied by oxygen
Max Perutz &John Kendrew First protein crystal structure in 1950s.1MBD.pdb
Heme Plane Shift upon O2 Binding
by rearrangement of Fe electron
Maintenance of Heme Functionality
Ferrous Oxymyoglobin [Fe2+/O2]
O
Ferrous Deoxymyoglobin [Fe2+] + O2
toxicity
Ferric Oxymyoglobin [Fe3+/O2-]
X
Metmyoglobin [Fe3+] + O2(unable to bind O2)
• Oxygen must leave as dioxygen rather than superoxide. Why?
• Reversible oxygen binding and storage
• The hydrogen bonding between the distal histidine and oxygen stabilizes the
ferric (Fe3+) form of oxymyoglobin and does not allow superoxide release.
• Oxygen can be released only from the ferrous (Fe2+) form of oxymyoglobin.
Tetrameric Structure of Hemoglobin
Max Perutz, horse heart 1A3N.pdb
• Hemoglobin a Chain vs. Myoglobin : 25% Identity
• Hemoglobin b Chain vs. Myoglobin : 24% Identity
• Hemoglobin subunits and myoglobins share an evolutionarily conserved
structural pattern called “globin fold”.
Oxygen Binding Properties of Myoglobin and Hemoglobin
Mb : Hyperbolic
P50 = 2 torr
Hb : Sigmoidal
P50 = 26 torr
• 2,3-bisphosphoglycerate in red blood cell significantly lowers oxygen
binding affinity of hemoglobin.
• 4 independent oxygen bindings ?
• Cooperative bindings ?
Loading & Unloading of Oxygen by Hemoglobin
X10 than myoglobin, X1.7 than noncooperative protein
• O2 Uptake by Hemoglobin in Lung (pO2 ~100 torr; 98% occupancy)
• O2 Release in Typical Peripheral Tissues (pO2 ~20 torr; 32% occupancy; 66% Release)
• O2 Unloading in Resting Muscle (pO2 ~40 torr; 77% occupancy; 21% Release)
• O2 Unloading in Exercising Muscle (pO2 ~20 torr; 32% occupancy; 67% Release)
O2 Binding Changes the Quaternary Structure of Hemoglobin
• 15 degree rotation of a1b1 dimer and a2b2 dimer upon oxygen binding
• Overall structure of dimer themselves is relatively unchanged.
• The interface between a1b1 dimer and a2b2 dimer is significantly changed.
• Deoxyhemoglobin : T (for Tense) State; Stronger Inter-Subunit Interactions
• Oxyhemoglobin : R (for Relaxed) State; Weaker Inter-Subunit Interactions
• In R state, oxygen binding sites are free of strain and show higher affinity
Hemoglobin Cooperativity (1)
Concerted Model (MWC Model)
• Oxygen Binding Affinity : T state < R state
• Oxygen binding shifts the quaternary structure of hemoglobin from T to R.
• Oxyhemoglobin favors R state, whereas deoxyhemoglobin is in T state.
• This model postulates that all 4 subunits are in the same conformation.
Hemoglobin Cooperativity (2)
Sequential Model
• Oxygen Binding Affinity : square state (
) < quarter circle state (
)
• Oxygen binding modulates the tertiary structure of the neighboring subunits.
• This model postulates that the individual subunits can have different conformations.
Hemoglobin Cooperativity (3)
The Truth ?
• Neither the concerted model nor the sequential model is fully accurate.
• Oxygen Binding Affinity  Hemoglobin-[O2]0 (T) : Hemoglobin-[O2]3 (R) = 1 : 20
(Concerted Model Works !!!)
• Oxygen Binding Affinity  Hemoglobin-[O2]0 (T) : Hemoglobin-[O2]1 (T) = 1 : 3
(Sequential Model Works !!!)
Oxygen Binding to Heme Changes Interface Structures
• Oxygen binding causes T to R quaternary structure changes. HOW ???
• Oxygen binding induces heme plane movement.
 Heme plane movement prompts conformational changes of hemoglobin subunit
via proximal histidine residue, which is the 5th coordination site of the heme iron.
 The C-terminal end of the individual subunit, which is located in the inter-subunit
space, is relocated upon oxygen binding.
 The structural transition at the iron in one subunit is directly transmitted to the
other subunits changing the interface between the ab dimers.
 Cooperative oxygen binding by hemoglobin
Specific Binding of 2,3-BPG to T-State Hemoglobin
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The T state of hemoglobin is highly unstable. (themodynamically not favored…)
R  T transition is essential for efficient oxygen unloading.
Thus, an additional mechanism is necessary to promote R  T transition !!!
In red blood cell, [Hb] = [2,3,-BPG] = ~2 mM.
2,3-BPG can bind to the central pocket located in the center of the tetramer.
2,3-BPG binding pocket can be generated only in the T-state.
Thus, 2,3-BPG can stabilize the T form population.
Cargo Transport Efficiency of Hemoglobin vs. 2,3-BPG
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[2,3-BPG]LOW  R > T and [2,3-BPG]HIGH  R < T (Allosteric Regulation)
O2 unloading efficacy  2,3-BPG(-) : 2,3-BPG(+) = 8% : 66%
Adult Hemoglobin [ab]2 vs. Fetal Hemoglobin [ag]2
In g chain, serine residue has replaced 143 Histidine residue of b chain, which
provides a positive charge critical for optimum 2,3-BPG binding.
Thus, 2,3-BPG binding is much less efficient for fetal hemoglobin.
Oxygen affinity : fetal hemoglobin > maternal hemoglobin.
The Bohr Effect (1) : Hydrogen Ion (pH)
• Rapidly metabolizing tissues such as contracting muscle generate large amounts of
hydrogen ions.
• Decrease of pH from 7.4 to 7.2 increases oxygen unloading efficiency by 11%.
• Salt bridges involving Lys40 of a2, His146 of b1 and Asp94 of b1 stabilize the T form
of hemoglobin.
• Protonation on His146 of b1 enhances salt bridge formation with Asp94 of b1.
The Bohr Effect (2) : Carbon Dioxide (1)
Constant pH!
• Actively metabolizing tissues release large amounts of CO2 resulting in pH decrease.
• Carbonic Anhydrase : Carbon Dioxide  Carbonic Acid (pKa 3.5)  Bicarbonate + H+
• 40 torr CO2 increases oxygen unloading efficiency by 11% at pH 7.2.
CO2 decrease the affinity of hemoglobin for oxygen beyond the effect due to decrease in pH
The Bohr Effect (3) : Carbon Dioxide (2)
• CO2 can react with the N-terminal amino group of the hemoglobin subunits and
yield carbamate groups, which produce additional negative charges.
• The amino termini reside in the interface of ab dimers.
• These CO2-induced negative charges in the N-termini of hemoglobin subunits
consequently promote salt bridges formation stabilizing the T form.
Carbon Dioxide Deportation
• Carbamate yielding reactions contribute to process 14% of the total CO2 released
from the peripheries.
• The majority of the CO2 released from the peripheries is uptaken by the red blood
cells and processed to HCO3-, and eventually exhaled in lung as CO2 via reverse
conversion reactions (i.e. HCO3-  CO2).
Sickle Cell Anemia
Sickle-Shaped RBC
Deoxyhemoglobin S
Hemoglobin Fibers
• In Sickle-Cell Anemia, Glu6 of b chain is mutated to Val6 (HbS substitution).
• The deoxy HbS b chains can establish aberrant hydrophobic interactions involving
Val6 of one b chain and Phe85 and Val88 of another b chain (i.e. between different
hemoglobin tetramers).  Deoxyhemoglobin polymerization  Formation of
hemoglobin fibers (i.e. insoluble aggregates of hemoglobin)
• The HbS substitution does not alter the properties of oxyhemoglobin because the
hydrophobic interaction between Val6 and Phe85/Val88 is not allowed in R state.