Module 06: Myoglobin & Hemoglobin

What is a ligand?

A molecule that reversibly binds to a protein at a specific binding site

What determines binding site specificity?

Complementarity in size, shape, charge, and hydrophobicity/hydrophilicity

Why are Mb and Hb used as case studies for protein function?

They illustrate reversible ligand (O2) binding, with known 3D structures

What is the role of myoglobin (Mb)?

Stores O2 in muscle and facilitates O2 diffusion to tissues.

What is the role of hemoglobin (Hb)?

Transports O2 from lungs to tissues via blood.

What structural motif do both proteins share?

Globin fold (8 alpha-helices surrounding a heme group)

What is the heme group?

A protoporphyrin ring with a central Fe2+ that binds O2 reversibly

What stabilizes O2 binding to Mb?

HisF8 binds Fe2+, HisE7 H-binds to O2, Val E11 and Phe CD1 stabilize heme

What happened when Fe2+ is oxidized to Fe3+?

O2 can no longer bind—this is methemoglobin (brown color in meat).

What is Kd?

Dissociation constant; [ligand] at which half of the protein is saturated.

What is theta?

Fractional saturation = [PL] / ([P] + [PL]); ranges from 0 to 1.

What is the relationship between Kd and affinity?

Lower Kd = higher affinity

What does p50 represent for O2?

The partial pressure of O2 at which Mb or Hb is 50% saturated

What type of curve does Mb show in O2 binding?

Hyperbolic—non-cooperative, one binding site

What type of curve does Hb show?

Sigmoidal—indicates cooperative binding

Why is Mb better for O2 storage than Hb?

Mb has a lower p50 → higher affinity, holds onto O2 tightly

Why is Hb better for O2 delivery

Hb’s cooperatively allows it to load O2 in lungs and unload in tissues

What is cooperativity?

Binding of one ligand affects binding of additional ligands.

What type of cooperativity does Hb exhibit?

Positive cooperativity—binding O2 at one site increases affinity at others

What equation models cooperative binding?

Hill Equation: YO2 = (pO2^n)/(p50^n + pO2^n)

What does the Hill coefficient (n) represent?

Degree of cooperativity.

n=1

non-cooperative

n > 1

positive cooperativity

n < 1

negative cooperativity

What does a Hill plot graph?

log[Y/(1-Y)] vs. log(pO2); slope = n, x-intercept = log(p50)

What is Hill coefficient for Mb?

n = 1 (non-cooperative, single binding site)

What is the Hill coefficient for Hb?

~3, indicating strong positive cooperativity

What is the O2 pressure in various tissues?

Lungs: ~100 torr, Tissues: ~40 torr, Cytosol: ~10 torr

How does cooperativity benefit O2 transport?

High pO2 (lungs) = strong binding and low pO2 (tissues) = efficient release

Why is CO dangerous?

CO binds heme with much higher affinity than O2, out competing it.

Why is CO binding lower in bound heme than free heme?

Steric hindrance by HisE7 in Mb reduces CO binding

What is the globin fold and why is it important?

A structure of 8 alpha-helices that creates a hydrophobic pocket for heme binding—found in both Mb and Hb.

What is the role of the proximal and distal histidine in heme binding?

Proximal His (F8) binds Fe2+ directly; distal His (E7) stabilizes O2 and blocks CO binding.

What is the function of myoglobin?

Stores and facilitates diffusion of O2 in muscles

What is myoglobin’s structure and MW?

Monomer with 153 residues; ~17.2 kDa; tertiary structure only

Why does Mb have high O2 affinity?

To efficiency store O2 and scavenge it from the bloodstream

What is Kd and what does a low Kd indicate?

Dissociation constant; lower Kd = higher affinity for ligand

What is p50?

Partial pressure of O2, where protein is 50% saturated (p50 = Kd for O2)

What kind of binding curve does Mb show?

Hyperbolic—non-cooperative, always high affinity

What is the quaternary structure of hemoglobin?

a2B2 heterotetramer; describes as a dimer of dimers (a1B1 and a2B2)

What stabilizes dimer interfaces in Hb?

a1B1: hydrophobic interactions (stable) and between dimers: ionic and H-bonds (weaker, breaking during transitions)

What are the 2 major Hb conformations?

T-state (tense): low O2 affinity and R-state (relaxed): high O2 affinity

What is cooperativity in hemoglobin?

O2 binding at one site increases affinity at other sites

What is the Hill coefficient for Hb and Mb?

Hb: ~2.8 (positive); Mb: 1 (no cooperativity)

What structural change initiates the R-state?

Fe2+ pulled into porphyria plane → moves HisF8 → shifts helix F → triggers 15 degrees rotation of dimers

What are the p50 values for each O2 binding event in Hb?

1st O2 (T-state): 30 torr, 2nd/3rd (transition): 12 torr, and 4th O2 (R-state): 0.3 torr

What happens to the central cavity during T → R transition?

It collapses as dimers rotate

What is the Bohr Effect?

Hb’s O2 affinity decreases at low pH and increases at high pH

How does pH affect T and R state stability?

Low pH stabilizes T (more H+ supports salt bridges) and high pH stabilizes R (loss of H+ breaks ionic interactions)

How does the Bohr Effect relate to CO2 transport?

In tissues: CO2 + H2O → H2CO3 → H+ + HCO3- → H+ stabilizes T-state (oxygen delivery). In lungs: H+ + HCO3- → H2CO3 → CO2 + H2O (exhaled)

What is an allosteric effector?

A molecule that binds one site and influences another site on the same protein

How does O2 act as a allosteric effector?

Binds N-terminus of Hb subunits (carbamation) → stabilizes T-state, decreases O2 affinity

How does 2,3-BPG affect Hb?

Binds to central cavity in T-state → stabilizes T → reduces O2 affinity → aids oxygen delivery in tissues

Why is cooperativity important in hemoglobin function?

It allows Hb to load O2 in the lungs and unload it efficiency in tissues, adapting to local pO2

How does environment affect Hb’s oxygen affinity?

Changes in pO2, pH, and CO2 levels promote conformational changes that shift Hb between T and R states

What are typical O2 pressures in the body?

Inspired air: 158 torr, alveolar air: 100 torr, arterial blood: 90 torr, capillary: 40 torr, & cytosol: ~10 torr

What is the T-state of hemoglobin?

“Tense” state with low O2 affinity predominant in deoxygenated blood

What is the R-state of hemoglobin?

“Relaxed” state with high O2 affinity; predominant when O2 is bound

What triggers T→ R transition?

Binding of O2 to Fe2+, which pulls HisF8 and Helix F, altering quaternary structure

What structural change occurs when O2 binds to Fe2+?

Fe2+ is pulled into the heme plane, dragging HisF8 and shifting Helix F

How large is the quaternary rotation between dimers during T → R shift?

~15 degree rotation of a1B1 relative to a2B2

What breaks during the T→ R shift?

Ion pairs at the dimer-dimer interface; hydrophobic aB interactions remain intact

What are the p50 values for O2 binding steps in Hb?

1st O2 (T): 30 torr (no cooperativity), 2nd/3rd O2 (transition): ~12 torr (cooperative), and 4th O2 (R): 0.3 torr (high affinity)

What does the Hill coefficient tell us?

Degrees of cooperativity; Hb’s Hill coefficient is ~2.8-3.0 (Mb =1)

How does O2 binding improve with each successive site?

Initial O2 binding strains the T-state, breaking ion pairs and shifting Hb into R-state, increasing affinity

What is the key mechanisms of hemoglobin’s cooperative behavior?

Conformation strain and quaternary shifts in response to initial ligand binding

What is allostery in hemoglobin?

Binding at one site (O2, H+, CO2, 2,3-BPG) influences affinity at other sites

What concept will follow cooperativity in Hb study?

The Bohr effect—how pH and CO2 levels regulate Hb’s O2 binding

What triggers the T→ R transition in hemoglobin?

O2 binding pulls Fe2+ into porphyrin plane, shifting HisF8 and helix F, causing a quaternary rotation

What types of bonds are broken and maintained during this transition?

Ionic interactions at the dimer-dimer interface are broken; hydrophobic interactions remain

What type of curve does cooperative O2 binding produce?

Sigmoidal curve (s-shaped), indicating variable affinity

What is the Hill equation for O2 binding?

YO2 = (pO2^n)/(p5o^n + pO2^n)

no cooperativity (Mb)

n=1

positive cooperativity (Hb ~2.8-3)

n> 1

negative cooperativity

n< 1

What is an allosteric effector?

A molecule that binds a protein and changes its function at a different site

Name 3 allosteric effectors of Hb and their effect on O2 binding?

H+ (Bohr effect) = decreases O2 affinity, CO2 = decreases O2 affinity, and 2,3-BPG = decreases O2 affinity

What is the Bohr effect?

A decrease in Hb’s O2 affinity at low pH, promoting O2 release in acidic tissues

How does low pH stabilize the T-state?

H+ protonates residues, forming salt bridges that stabilize the T-state

What is the equation for carbonic acid formation?

CO2 + H2O ←> H2CO3 ←> H+ + HCO3-

How does CO2 decrease O2 affinity?

It reacts with the N-termini of Hb subunits to form carbamate groups, stabilizing the T-state

Where does the CO2 bind on hemoglobin?

The N-terminal amino groups, forming negatively charged carbamates

What is 2,3-BPG and where does it bind?

A negatively charged molecule that binds in the central cavity of the T-state of Hb

What effect does 2,3-BPG have on Hb?

Stabilizes T-state → reduces O2 affinity → enhances O2 delivery

How does 2,3-BPG affect fetal vs. adult hemoglobin?

Fetal Hb binds 2,3-BPG less tightly, increasing O2 affinity for better maternal-fetal transfer

What is the concerted (MWC) model?

All subunits switch from T to R together—ligand binding shifts equilibrium toward R

What is the sequential (KNF) model?

Subunits switch individually upon ligand binding—binding at one site induces local change

Which model better explains hybrid states?

The KNF (sequential) model

Which model better explains sigmoidal curve and all-or-nothing transitions?

The MWC (concerted) model

What is the Bohr Effect?

A decrease in pH (increases in [H+]) lower Hb’s O2 affinity; increase in pH raises it.

How does pH shift the O2 binding curve?

Low pH (high H+) → right (↓ affinity, ↑ p50);

High pH (low H⁺) → left shift (↑ affinity, ↓ p50)

What causes this effect at the molecule level?

H+ stabilizes the T-state by forming salt bridges; Hb releases ~0.6 H+ per O2 bound

What system buffers blood pH?

The carbonic acid (H2CO3)/bicarbonate (HCO3-) system

What reaction is catalyzed by carbonic anydrase?

CO₂ + H₂O ⇌ H₂CO₃ ⇌ H⁺ + HCO₃⁻

What happens to H+ and CO2 in lungs vs. tissues?

Tissues: Hb binds H+, forming HCO3- for CO2 transport. Lungs: Hb releases H+, converting HCO3- to CO2 for exhalation

How else does Hb transport CO2?

CO2 binds to N-termini forming carbamates, stabilizing T-state and promoting O2 release

What effect does carbamate formation have on Hb?

Increase Bohr effect via H+ release; promotes T-state

What percent of CO2 transport is Hb responsible for?

~50%

What is BPG’s role in Hb function?

Binds to central cavity T-state → decreases O2 affinity → helps unload O2 in tissues

What happens to BPG during T→ R transition?

Central cavity narrows → BPG is released