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

Heme Plane Shift upon O2 Binding

Max Perutz &John Kendrew First protein crystal structure in 1950s.1MBD.pdb

by rearrangement of Fe electron

Ferrous Oxymyoglobin [Fe2+/O2]

Ferric Oxymyoglobin [Fe3+/O2-]

Ferrous Deoxymyoglobin [Fe2+] + O2

Metmyoglobin [Fe3+] + O2-X

toxicityO

(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.

Maintenance of Heme Functionality

Tetrameric Structure of Hemoglobin

• Hemoglobin Chain vs. Myoglobin : 25% Identity

• Hemoglobin Chain vs. Myoglobin : 24% Identity

• Hemoglobin subunits and myoglobins share an evolutionarily conserved

structural pattern called “globin fold”.

Max Perutz, horse heart 1A3N.pdb

Oxygen Binding Properties of Myoglobin and Hemoglobin

• 2,3-bisphosphoglycerate in red blood cell significantly lowers oxygen

binding affinity of hemoglobin.

• 4 independent oxygen bindings ?

• Cooperative bindings ?

Mb : Hyperbolic

P50 = 2 torr Hb : Sigmoidal

P50 = 26 torr

• 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)

Loading & Unloading of Oxygen by Hemoglobin

X10 than myoglobin, X1.7 than noncooperative protein

• 15 degree rotation of 11 dimer and 22 dimer upon oxygen binding

• Overall structure of dimer themselves is relatively unchanged.

• The interface between 11 dimer and 22 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

O2 Binding Changes the Quaternary Structure of Hemoglobin

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 dimers. Cooperative oxygen binding by hemoglobin

Specific Binding of 2,3-BPG to T-State Hemoglobin

• 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.

• [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 []2 vs. Fetal Hemoglobin []2

• In chain, serine residue has replaced 143 Histidine residue of 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.

Cargo Transport Efficiency of Hemoglobin vs. 2,3-BPG

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 2, His146 of 1 and Asp94 of 1 stabilize the T form

of hemoglobin.

• Protonation on His146 of 1 enhances salt bridge formation with Asp94 of 1.

• 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.

The Bohr Effect (2) : Carbon Dioxide (1)

CO2 decrease the affinity of hemoglobin for oxygen beyond the effect due to decrease in pH

Constant 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 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

• In Sickle-Cell Anemia, Glu6 of chain is mutated to Val6 (HbS substitution).

• The deoxy HbS chains can establish aberrant hydrophobic interactions involving

Val6 of one chain and Phe85 and Val88 of another 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.

Sickle-Shaped RBC Hemoglobin FibersDeoxyhemoglobin S