5.1 Oxygen-Binding Proteins · Biochemistry 2027

Oxygen binds to the haem prosthetic group

Haem = porphyrin ring plus a central Fe; four nitrogens coordinate the iron
Six coordination positions: four porphyrin N, the fifth from the proximal histidine (F8), the sixth is the O2 site
Distal histidine (E7) fixes the orientation of bound oxygen and stabilises it
Normal oxygen binding is reversible and requires Fe(II) haem
Fe(III) methaemoglobin cannot transport oxygen; oxygenation is not the same as oxidation of the iron

Figure 5.1-2Coordination structure of haem with the roles of the proximal and distal histidines (simplified).

Globins form a family of oxygen-binding proteins

The globin family shares an α-helical globin fold
Myoglobin serves muscle; haemoglobin transports oxygen in erythrocytes
Fetal HbF (α₂γ₂) differs in subunit composition from adult HbA (α₂β₂), so its binding properties differ
Function is set by haem binding plus protein location, local conditions and inter-chain contacts

Table 5.1-1Globins compared: composition, location and cooperativity

Figure 5.1-3Symbolic comparison of the globin fold; actual protein coordinates are not reproduced.

Myoglobin has a single oxygen-binding site

One polypeptide with one haem gives one site, so no cooperativity is possible
Occupancy rises hyperbolically with oxygen partial pressure
Typical physiological example: P50 about 2 to 3 mmHg, so occupancy is high at tissue pO2
The protein therefore behaves as a storage and diffusion-facilitating protein

Figure 5.1-4Calculated comparison of the hyperbolic Mb curve with the sigmoid Hb curve (Mb P50 = 2.8, HbA P50 = 26.6 mmHg).

Protein–ligand binding can be described quantitatively

A ligand is a molecule or ion that binds a protein selectively: Mb + O2 ⇌ Mb·O2
Kd = [Mb][O2]/[Mb·O2]; a smaller Kd means higher occupancy at the same free concentration
Occupancy Y = [Mb·O2]/([Mb] + [Mb·O2])
Using partial pressure, Y = pO2/(P50 + pO2), where P50 is the half-saturation pressure
A smaller P50 means higher affinity; Kd and P50 use different units and are not interchangeable
Example: P50 2.8 mmHg at pO2 12 mmHg gives Y = 12/(2.8 + 12) ≈ 0.81

Figure 5.1-5Oxygen binding and dissociation equilibrium at a single site, with its quantitative expression.

Protein structure determines how a ligand binds

The haem iron alone does not explain the binding properties of a globin
A hydrophobic pocket adjusts the water environment around haem and ligand
Proximal and distal histidines set the coordination geometry and the direction of binding
Access routes, steric hindrance and hydrogen bonding together determine selectivity and affinity
Identical haem in different protein environments gives different relative reactivity toward O2 and CO

Figure 5.1-6Roles of the proximal and distal histidines in the haem pocket; a concept diagram of relative positions.

Haemoglobin transports oxygen in the blood

HbA is an α₂β₂ tetramer with one haem per subunit, so up to four O2 per molecule
Molecular mass about 64.5 kDa; most oxygen travels bound reversibly to Hb in erythrocytes
Saturation is the fraction of haem sites filled; it differs from the total oxygen content of blood
Dissolved oxygen in plasma is small; haemoglobin is the main carrier

Figure 5.1-7Simplified α₂β₂ tetramer of HbA; each subunit carries one haem.

Haemoglobin subunits resemble myoglobin structurally

The α and β chains differ in sequence from Mb but share the globin fold
Each chain carries one haem-binding pocket
The difference is the quaternary structure formed by subunit contacts
Those contact surfaces are the route by which binding at one haem affects the others

The structure of haemoglobin changes when oxygen binds

O2 binding moves the iron and rearranges the proximal histidine and the F helix
The subunit interface then changes and transmits the change to neighbouring chains
T (tense) state has low affinity; R (relaxed) state has high affinity
Real haemoglobin also occupies partially ligated states and several conformations, so T/R is a simplification

Figure 5.1-8Concept diagram of the T↔R conformational change; oxygen binding shifts the equilibrium between the two states.

Haemoglobin binds oxygen cooperatively

Positive cooperativity means binding at one site makes binding at the others more favourable
The curve rises slowly at low pO2, steeply in the middle range and then saturates
The sigmoid shape allows efficient loading in the lung and substantial unloading in tissue
Without cooperativity, transport efficiency would fall substantially

Figure 5.1-9Occupancy states from Hb(O₂)₀ to Hb(O₂)₄; colour marks the number of bound O₂.

Cooperative binding can also be described quantitatively

Hill equation: Y = pO2^n/(P50^n + pO2^n), where n is the Hill coefficient
For HbA in the mid-saturation range n ≈ 2.7, an index of positive cooperativity
n is not the number of binding sites (there are four) and the equation is an empirical approximation
Linearised form: log[Y/(1−Y)] = n·log pO2 − n·log P50, so the slope gives n
The slope can vary between pO2 ranges, so the fitted interval must be stated
Example: P50 26.6 mmHg, n 2.7, pO2 40 mmHg gives Y ≈ 0.750

Figure 5.1-10Mathematical model of the Hill linearisation; n = 2.7 reflects positive cooperativity, not the number of sites.

Two classical models explain the mechanism of cooperativity

MWC (concerted): subunits switch together between T and R, and ligand shifts the equilibrium
KNF (sequential): each binding event changes structure and passes the change to neighbours
Both are classical simplifications and neither matches the full structural dynamics
The models differ in how far a conformational change is shared across subunits

Figure 5.1-11Summary comparison of the assumptions of the MWC and KNF models of cooperativity.

[Separate note 5-1] Carbon monoxide: the quiet killer

CO is a colourless, odourless toxic gas that binds haem iron far more strongly than O2 (often cited as 200-fold or more)
Two toxic routes: it occupies oxygen sites and it raises the affinity of the remaining sites, hindering release in tissue
It is also toxic to cellular respiration
A standard two-wavelength finger pulse oximeter cannot distinguish CO-Hb from O2-Hb and may miss the defect
Move the casualty to safety and seek emergency care; diagnosis uses a CO-Hb measuring device
Generating or inhaling CO in school experiments is never permitted

Figure 5.1-12Two routes by which CO interferes with oxygen binding; based on literature and models, not exposure experiments.

Haemoglobin also contributes to H⁺ and CO₂ transport

Bohr effect: falling pH and rising CO2 lower oxygen affinity and shift the curve to the right
This promotes release in tissue; in the lung, CO2 removal and higher pH favour binding
Rising temperature also favours oxygen release
CO2 travels in three forms: bicarbonate, dissolved CO2 and carbamino compounds on haemoglobin
Carbonic anhydrase accelerates CO2 + H2O ⇌ HCO3− + H+
CO2 does not bind the haem oxygen site; it reacts mainly with the N-terminal amino group of globin

Figure 5.1-13Conceptual shift of the binding curve with pH and CO₂; P50 values are teaching examples.

Figure 5.1-14Red-cell-mediated CO₂ transport and its relation to the carbonate–bicarbonate buffer system.

2,3-BPG modulates oxygen binding by haemoglobin

2,3-BPG is a glycolytic metabolite of erythrocytes and carries negative charge
It binds the central cavity of deoxyhaemoglobin, stabilising the T state and shifting the curve right
The result is more oxygen release in tissue; it does not compete with O2 at the haem site
HbF interacts only weakly with 2,3-BPG, so its affinity is higher (example P50 19 versus 27 mmHg)
This favours transfer from mother to fetus; actual P50 varies with pH, temperature, CO2 and 2,3-BPG

Figure 5.1-15Binding of 2,3-BPG in the central cavity of deoxyhaemoglobin, emphasising the principle.

Figure 5.1-16Model curves comparing the P50 of adult HbA and fetal HbF.

Sickle cell disease is a molecular disease of haemoglobin

A β-globin gene variant produces HbS; the classic change is β6 Glu to Val (written Glu7Val in some numbering)
The hydrophobic Val lets deoxy HbS contact other HbS molecules, nucleating polymers and long fibres
Consequences include loss of red-cell deformability, sickling, vaso-occlusion and haemolysis
The key point is a surface change from one substitution, not the loss of the haem site
Low oxygen promotes polymerisation through a molecular and cellular cascade

Figure 5.1-17Simplified schematic of the β-globin substitution in HbS and its polymerisation at low oxygen.

Oxygen delivery and release can be read numerically

With the teaching Hill approximation (P50 26.6 mmHg, n 2.7), lung pO2 100 gives about 97.3% saturation
Resting tissue pO2 40 gives about 75.0%, and active tissue gives more release at lower pO2
Rising temperature and CO2, and changes in pH and 2,3-BPG, can be modelled as a higher P50
Actual delivery also depends on Hb concentration, blood flow and how fully capillary equilibration occurs
A saturation difference alone does not explain the whole physiology of oxygen transport

Figure 5.1-18Calculated oxygen partial pressures and fractional saturation in the lung, resting tissue and active tissue.

Figure 5.1-19Calculated comparison of the reference curve with a model curve of increased P50.

References

**Figure 5.1-2**Coordination structure of haem with the roles of the proximal and distal histidines (simplified).

**Table 5.1-1**Globins compared: composition, location and cooperativity
Protein	Typical composition	Main location and function	Cooperativity
Myoglobin (Mb)	One chain, one haem	Muscle: oxygen storage, facilitated diffusion	None
Adult HbA	α₂β₂, four haem groups	Erythrocytes: oxygen transport	Positive
Fetal HbF	α₂γ₂, four haem groups	Favours oxygen supply to the fetus	Positive

**Figure 5.1-3**Symbolic comparison of the globin fold; actual protein coordinates are not reproduced.

**Figure 5.1-4**Calculated comparison of the hyperbolic Mb curve with the sigmoid Hb curve (Mb P50 = 2.8, HbA P50 = 26.6 mmHg).

**Figure 5.1-5**Oxygen binding and dissociation equilibrium at a single site, with its quantitative expression.

**Figure 5.1-6**Roles of the proximal and distal histidines in the haem pocket; a concept diagram of relative positions.

**Figure 5.1-7**Simplified α₂β₂ tetramer of HbA; each subunit carries one haem.

**Figure 5.1-8**Concept diagram of the T↔R conformational change; oxygen binding shifts the equilibrium between the two states.

**Figure 5.1-9**Occupancy states from Hb(O₂)₀ to Hb(O₂)₄; colour marks the number of bound O₂.

**Figure 5.1-10**Mathematical model of the Hill linearisation; n = 2.7 reflects positive cooperativity, not the number of sites.

**Figure 5.1-11**Summary comparison of the assumptions of the MWC and KNF models of cooperativity.

**Figure 5.1-12**Two routes by which CO interferes with oxygen binding; based on literature and models, not exposure experiments.

**Figure 5.1-13**Conceptual shift of the binding curve with pH and CO₂; P50 values are teaching examples.

**Figure 5.1-14**Red-cell-mediated CO₂ transport and its relation to the carbonate–bicarbonate buffer system.

**Figure 5.1-15**Binding of 2,3-BPG in the central cavity of deoxyhaemoglobin, emphasising the principle.

**Figure 5.1-16**Model curves comparing the P50 of adult HbA and fetal HbF.

**Figure 5.1-17**Simplified schematic of the β-globin substitution in HbS and its polymerisation at low oxygen.

**Figure 5.1-18**Calculated oxygen partial pressures and fractional saturation in the lung, resting tissue and active tissue.

**Figure 5.1-19**Calculated comparison of the reference curve with a model curve of increased P50.