blood proteins 1

Functional Comparison of Myoglobin and Haemoglobin

Myoglobin ($Mb$) and Haemoglobin ($Hb$) are both oxygen-binding proteins, but they are distinguished by their distinct physiological roles and locations within the body. Myoglobin functions primarily in the muscle tissue, where it serves as an oxygen storage reservoir or "sponge." In contrast, Haemoglobin is located in the blood and acts as an oxygen carrier, transporting $O_2$ from the lungs to the tissues.

Structure and Coordination of Myoglobin

Myoglobin is a monomeric protein with a molecular weight of approximately $18\,kDa$. It consists of a single globin polypeptide chain and a haem prosthetic group.

Globin Chain Composition

• The globin chain consists of $153$ amino acids.

• The secondary structure is dominated by $8$ $\alpha$-helices, designated by the letters $A$ through $H$.

The Haem Prosthetic Group

• The haem group is composed of a Protoporphyrin IX ring with a central iron atom in the ferrous state ($Fe^{2+}$).

• In the Protoporphyrin IX structure, the $Fe^{2+}$ ion is coordinated by $4$ nitrogen atoms within the plane of the porphyrin ring.

• There are two additional coordination positions, identified as positions $5$ and $6$, perpendicular to the ring plane.

• Position 5: This site is occupied by a nitrogen atom from the imidazole side chain of Histidine 93 ($His93$), which is known as the Proximal His.

• Position 6: This is the binding site for the oxygen molecule ($O_2$).

Oxygen Stabilization and the Formation of Metmyoglobin

The binding of oxygen to Myoglobin involves critical stabilization by the protein environment to prevent the release of reactive species.

The Distal Histidine

• Histidine 64 ($His64$), referred to as the Distal His, plays a vital role in stabilizing the bound $O_2$ molecule via hydrogen bonding ($H$-bonding).

• This stabilization helps prevent the accidental release of the superoxide radical ($\text{O}_2^-$).

Electronic States and Metmyoglobin

• The binding can be viewed as an electronic equilibrium: $Fe^{2+} \cdots O=O \rightleftharpoons Fe^{3+} \cdots O=O^-$.

• If the iron is oxidized to the ferric state ($Fe^{3+}$), the protein is converted into Metmyoglobin.

• Metmyoglobin is physiologically inactive as it is unable to bind $O_2$.

Myoglobin Binding Kinetics and Saturation

The saturation curve for Myoglobin is hyperbolic, reflecting its high affinity for oxygen.

• $P_{50}$: The partial pressure of oxygen ($pO_2$) at which half of the Myoglobin molecules are oxygenated ($MbO_2$).

• Physiological Behavior: Most Myoglobin molecules remain bound to oxygen at normal tissue $pO_2$ levels. Oxygen is only released during periods of intense exercise or specific physiological states, such as in whales during diving.

Haemoglobin Structure and Properties

Haemoglobin is a much larger and more complex protein than Myoglobin, with a molecular weight of approximately $65\,kDa$.

Quaternary Structure

• Haemoglobin is a tetramer consisting of two types of globin chains ($2 \alpha$ and $2 \beta$ subunits), forming a pair of $\alpha\beta$ dimers ($\alpha_1\beta_1$ and $\alpha_2\beta_2$).

• The $\alpha$ subunit contains $141$ amino acids.

• The $\beta$ subunit contains $146$ amino acids.

• Each monomer shares approximately $44\%$ sequence identity with Myoglobin, and the characteristic "globin fold" ($8$ $\alpha$-helices) is strictly maintained across all subunits despite sequence drift.

Physiological Concentrations and Solubility

• There are approximately $300 \times 10^6$ Hb molecules per red blood cell ($RBC$).

• The concentration of Hb is roughly $150\,g\,dm^{-3}$ of blood.

• Solubility Enhancement: The solubility of $O_2$ in plasma alone is only $10^{-4}\,mol\,dm^{-3}$; the presence of Hb increases this solubility to $10^{-2}\,mol\,dm^{-3}$.

Optical Properties

• OxyHb: Bright red in color.

• DeoxyHb: Dark red in color.

• These forms can be distinguished using spectroscopy at a wavelength of $660\,nm$.

Conformational Changes and the Mechanism of Oxygen Binding

The transition between the deoxygenated and oxygenated states of Haemoglobin involves both small-scale and large-scale structural shifts.

The Atomic Mechanism

1. When $O_2$ binds, electronic rearrangements cause the radius of the $Fe^{2+}$ ion to shrink.

2. This shrinkage allows the iron atom to move further into the plane of the protoporphyrin ring by approximately $0.04\,nm$ ($0.4\,\text{Angstroms}$).

3. As the central iron moves, it pulls the Proximal His ($F8$) along with it.

4. The movement of the Proximal His exerts a pull on the F helix.

Large-Scale Shifts

• The movement of the F helix alters bonds at the dimer-dimer interface.

• This triggers a "Quaternary shift," characterized by a $15^{\circ}$ rotation of the $\alpha\beta$ dimers relative to each other. Departure of $O_2$ reverses this entire process.

Cooperativity and the Tense-Relaxed Transition

Haemoglobin exhibits cooperativity, meaning the binding of oxygen to one subunit increases the affinity of the remaining subunits for oxygen. This results in a sigmoidal (S-shaped) saturation curve.

Tense (T) State

• This is the deoxygenated form (Deoxy Hb) with low oxygen affinity.

• It is stabilized by inter-subunit salt bridges that resist movement: $\alpha_1 \rightarrow \beta_2$, $\alpha_2 \rightarrow \beta_1$, and $\alpha_1 \rightarrow \alpha_2$.

Relaxed (R) State

• This is the oxygenated form (Oxy Hb) with high oxygen affinity.

• The salt bridges are broken, and the quaternary structure is rotated into the Relaxed state.

Mathematical Analysis: The Hill Plot

The Hill plot is a log/log graph used to display the binding characteristics of a protein. It plots $\log\left(\frac{\%S}{100 - \%S}\right)$ against $\log(pO_2)$.

• Slope ($n$):

  • If $n = 1$: The binding is non-cooperative (e.g., Myoglobin).

  • If n > 1: The binding is cooperative.

• Haemoglobin Hill Coefficients:

  • At very low $pO_2$ (T state predominates) and very high $pO_2$ (R state predominates), the slope is $1$.

  • In the transition region, the slope is approximately $3$, indicating strong cooperativity.

Theoretical Models of Cooperativity

Two primary models describe the transition between the T and R states in Haemoglobin.

Model 1: Concerted (MWC Model)

• This model suggests an "all-or-nothing" transition.

• All subunits within the tetramer must be in the same state (either all-T or all-R).

• Every oxygen molecule that binds to the T-form increases the statistical probability of the entire tetramer flipping to the R-form.

Model 2: Sequential (KNF Model)

• This model suggests that subunits can change state individually.

• The binding of $O_2$ to one subunit flips only that subunit from T to R.

• This change induces conformational strain in neighboring subunits (creating a T' state), easing their transition upon further $O_2$ binding.

Empirical Reconciliation of the Models

Experimental data suggests that neither model is perfectly accurate on its own, and the reality of Haemoglobin binding involves aspects of both.

• Subunit Heterogeneity: The $\alpha$ and $\beta$ subunits are not identical. The $\alpha$ subunit can bind $O_2$ in both the T and R states, whereas the $\beta$ subunit can only bind $O_2$ when it is in the R state.

• Affinity Gradation: Hb with only one $O_2$ bound is almost entirely in the T-form, yet it possesses approximately $\times 3$ the oxygen affinity of completely empty Hb.

• The Quaternary Shift: Hb with three $O_2$ bound is almost always in the all-R form. A minimum of $2$ bound $O_2$ molecules are generally required to trigger the quaternary shift to the all-R configuration.