Haemoglobin & Myoglobin

Haeme definition

A prosthetic group at the site of O₂ binding

Apoprotein

Haeme without a prosthetic group

Holoprotein

Haeme with a prosthetic group

Haeme structure (7)

• Contains protoporphyrin IX with an Fe atom in its center

• The Fe atom is in the ferrous (Fe²⁺) oxidation state in functional myoglobin

• The Fe atom can form 5 or 6 ligand bonds

  • depending on O₂ binding

• Four bonds are to the pyrrole nitrogen atoms of the porphyrin, lying in the plane of the ring

• The 5th and 6th bonds are directed perpendicular to the porphyrin ring

• The 5th coordinate bond is to a nitrogen of histidine imidazole (proximal histidine)

• The 6th coordinate bond is to O₂ or another histidine (distal histidine)

Proximal Histidine

5th coordinate bond is to a nitrogen of histidine imidazole

Distal Histidine

6th coordinate bond is to O₂ or another histidine

Porphyrin Hydrophobic Pocket (5)

• Haeme is positioned within a hydrophobic pocket of each globin subunit

• Non-covalent interactions with approximately 18 residues

  • mainly from apolar side chains + apolar regions of the porphyrin

  • stabilize the haeme

  • 80 interactions

Driving Force for Non-covalent Interactions

The expulsion of H₂O of solvation on association of the hydrophobic haeme with the apolar a.a. side chains in the haeme pocket

Myoglobin characteristics (6)

• 153 amino acids in the polypeptide chain

• 83 invariant residues

  • with 15 identical to mammalian haemoglobin

• A single polypeptide chain with one O₂ - binding site

• Binds and releases O₂ with changes in the O₂ concentration in skeletal muscle cells' sarcoplasm

• Acts as a monomer, preventing other myoglobin molecules from associating

Function of Myoglobin (2)

• Binds and releases O₂ based on changes in the O₂ concentration in skeletal muscle cells

• Essential for oxygen storage in muscle tissue

Myoglobin 2° Structure (2)

Approximately 70% of residues form α-helical structures, generating:

• Seven helical segments in its structure

Haemoglobin Characteristic (3)

• It’s surface residues are designed to form H bonds and nonpolar contacts with other subunits

• Supporting its quaternary (4°) structure

• Tetrameric - interact more extensively with each other

Haemoglobin 2° Structure (3)

Approximately 70% of residues form α-helical structures, generating:

• Seven helical segments in the α chain

• Eight helical segments in the β chain

Helical Regions - M & H (3)

• The interhelical regions are labeled AB, BC, CD, …, GH

• The nonhelical region

  • at the NH₂-terminal end is the NA region

  • at the COOH-terminal end is the HC region

General Properties for M & H (4)

• Changes in amino acid sequences are conservative

  • preserving the physical properties of residues

• The 2° and tertiary (3°) structures of hemoglobin subunits and myoglobin are nearly identical

• Physiological differences arise from small specific changes in structure

• The similarity in tertiary structure shows that the same 3° structure can result from diverse amino acid sequences

Oxygen Binding and Equilibrium in Myoglobin (3)

• Follows a simple equilibrium constant (K_eq)

  • which depends on pH, ionic strength, and temperature

• [K_eq] = mol/L

P₅₀ definition

The equilibrium constant representing the O₂ partial pressure at which 50% of the oxygen-binding sites are occupied

P₅₀ equation (3 + photo!)

• The pressure of O₂ is measured in torr

  • as its partial pressure

• 1 torr = pressure of 1 mmHg at 0°

pO₂ definition

The concentration of O₂ expressed in torr, used to describe the O₂-binding dynamics of proteins like myoglobin

O₂ Saturation Curve (3+photo!!)

• Used to characterize the properties of an O₂-binding protein

• It plots the fraction of O₂-binding sites occupied (Y) on the ordinate (y-axis)

• Versus the partial pressure of O₂ (pO₂) on the abscissa (x-axis)

Y (Fractional Saturation) of Myoglobin (2 + photo!!)

• [MbO2​] is the concentration of oxygen-bound myoglobin

• [Mb] is the concentration of unbound myoglobin

Simplified Equation for Fractional Saturation (Y) (5 + photo!!)

• By dividing through by [Mb]

• Where:

  • Y: fractional saturation

  • P₅₀: equilibrium constant

  • pO₂:​ oxygen concentration in terms of partial pressure

P₅₀​ = pO_2​ when? (3)

• when Y=0.5

  • indicating 50% of available binding sites are occupied

• This defines the equilibrium constant's designation as P₅₀ with the subscript "50" referring to 50% saturation.

Saturation Curve Characteristics (3)

• A plot of Y versus pO₂ generates the oxygen saturation curve for myoglobin

• The curve takes the shape of a rectangular hyperbola

  • Reflects the direct dependence of fractional saturation on oxygen concentration and P₅₀

O₂-binding curves for Myoglobin and Haemoglobin (photo!)

Rearranged O₂-binding Equation (photo!)

A simple algebraic manipulation of the equation used to construct the O₂-binding curve

Hill Equation - Logarithmic Form

Used to assess the cooperativity of oxygen binding to a molecule

Hill Coefficient - Hill Plot (4)

• photo* produces a straight line

• Hill coefficient (n_H):

  • the slope of that straight line

  • reflects the cooperativity of oxygen binding

Cooperativity in Myoglobin (3)

• Myoglobin has a single O₂-binding site per molecule

• It does not exhibit cooperativity

• Hill coefficient is 1

O₂ Saturation Curves - Myoglobin (2)

• Hyperbolic

• Reflecting the simple binding of O₂ without cooperativity

Cooperativity in Haemoglobin (3)

• Hb has 4 monomeric subunits, each with an O₂-binding site (4)

• The binding of O₂ to one subunit enhances the binding of O₂ to the other subunits

• Hill coefficient >1 - reflecting this cooperative behavior

O₂ Saturation Curves - Haemoglobin (4)

• Sigmoidal

  • indicating cooperative binding of O₂

• Steep in the middle

  • demonstrating the enhanced affinity for O₂ as more O₂ molecules bind

T-State (5)

= Quaternary structure of deoxyhaemoglobin

• Lower oxygen affinity

• Tense conformation

• Stabilized by salt bridges

• Hydrogen bonds

Iron Position

In the deoxy state, the Fe is 0.6 Å out of the heme plane (angstrom)

R-State (4)

= Quaternary structure of oxyhaemoglobin

• Higher oxygen affinity

• Relaxed conformation

• Induced by oxygen binding

Oxygenation rotation (2)

• Rotates the α₁β₁ dimer relative to the α₂β₂ dimer by 15°

• Oxygen binding to one subunit facilitates binding to others

Positive Cooperativity of O₂ binding definition

Arises from the effect of one heme's ligand-binding state on the affinity of another

Oxygen Binding

Oxygen pulls the iron back into the heme plane

F Helix Movement

The proximal histidine (His F8) attached to the iron pulls the entire F helix, acting like a lever on a fulcrum

Positive Cooperativity of O₂ binding (photo!)

Oxygen Binding and Affinity Change (5)

• Binding of oxygen to one heme group in hemoglobin is initially more difficult

• However, oxygen binding causes a shift in the α1-β2 contacts

• This shift moves the distal histidine (His E7) and Val E11 out of the oxygen's path

  • facilitating oxygen binding to the Fe²⁺ in the other subunit

• As a result, the affinity of the heme for oxygen increases

α₁-β₂ Contacts and Structural States (3)

• The α₁-β₂ contacts have two stable positions

• These contacts, held together by different but equivalent hydrogen bonds

  • act as a binary switch between the T (tense) and R (relaxed) states of hemoglobin

Transition Between States (8)

• The energy from the formation of the Fe²⁺-O₂ bond drives the T → R transition

• Hemoglobin's O₂-binding cooperativity arises from this transition

• The Fe²⁺ of any subunit cannot move into its heme plane without the reorientation of its proximal histidine (His)

  • prevents this residue from bumping into the porphyrin ring

• The proximal His is tightly packed by surrounding groups

  • making reorientation impossible

  • unless accompanied by the translation of the F helix across the heme plane

• The F helix translation is only possible in concert with the quaternary shift that steps the α1C-β2FG contact one turn along the α1C helix

Methemoglobin (3)

• Produced when Fe(II) to Fe(III) oxidized

• Brown

• Sixth position is coordinated with water instead of oxygen

Brown Color of Methemoglobin (2)

• Visible in dried blood and old meat

• Contributes to less desirable appearance of aged meat

Methemoglobin vs Ascorbic Acid (3)

• Butchers' use Ascorbic Acid

• Reduces methemoglobin back to hemoglobin

• Helps maintain fresher appearance of meat

Enzyme Involvement - Methemoglobin (2)

• Methemoglobin reductase converts methemoglobin to regular hemoglobin

• Restores hemoglobin's ability to bind and carry oxygen

Bohr Effect (2+2)

• Higher pH (lower [H⁺])

  • Promotes tighter binding of oxygen to hemoglobin

• Lower pH (higher [H⁺])

  • Permits easier release of oxygen from hemoglobin

P₅₀ and pH Relationship (2)

• As pH increases, P50 value decreases, indicating increased oxygen binding

• As pH decreases, P50 value increases, indicating decreased oxygen binding

Effect of pH on Oxygen Release

At 20 torr, 10% more oxygen is released when pH drops from 7.4 to 7.2

Carbonic Anhydrase (2)

• Catalyzes the conversion of CO₂ to bicarbonate (HCO₃^-) in red blood cells

• As oxygen is consumed, CO₂ is released