Biochem Exam 2 Lecture 9

Myoglobin/Hemoglobin History

• First protein to have mass accurately measured 

• First protein to be studied by ultracentrifugation 

• First protein to be associated w/ a physiological condition 

• First protein to show a point mutation causes pathological conditions 

• First protein to have X-ray structures solved 

• Theories of cooperativity & control explain hemoglobin function & serve as a frame for study of enzymes 

Similarities b/w O2 Binding Sites

both have prosthetic heme groups & bind O2 to heme(s)

Diff b/w O2 Binding Sites

diff binding curves to O2, enabling diff functions/specializations

Myoglobin Function

• Facilitates respiration in rapidly respiring muscle tissue 

• Facilitates O2 diffusion 

  • Rate of diffusion from capillaries to tissue is slow because of oxygen solubility 

Oxygen Storage

myoglobin conc. are 10-fold greater in whales/seals than land mammals 

Hemoglobin Function

• Transports O2 from lungs to tissues 

• O2 diffusion alone is too poor for transport in larger animals because O2 solubility in plasma is low (10^-4M) 

  • Bound to hemoglobin [O2] = 10^-2M, meaning 100x more O2 transport than dissolved O2 alone, allowing enough O2 to reach body tissues 

Hemocyanin (altenerative O2 transporters)

a Cu containing protein (96 O2 binding sites), which is “free floating” in hemolymph of invertebrates 

Hemerythrin (altenerative O2 transporters)

a non-heme containing protein (8 O2 binding sites)

Respiration Overview

• O2 Transportation: O2 transported by Hb bound to the Heme [to Fe(II)]

• CO2 Transportation (route 1): CO2 + H2O → H+ + HCO3- (bicarbonate) 

  • Catalyzed by carbonic anhydrase (main mode of elimination of CO2) 

• CO2 Transportation (route 2): R-NH2 + CO2 → R-NH-COO- (carbamate) + H+ 

  • Formed at N-termini of hemoglobin helping to stabilize T-state (deoxy) 

O2 Binding Chart

• YO2 (y-axis): fractional saturation, aka fraction of myoglobin bound to O2 

• pO2 (torr, x-axis): conc of O2 

• P50 = the partial oxygen pressure for half of the myoglobin molecules to bind to O2 

  • Low P50 = higher affinity 

Myoglobin O2 Binding Chart

• P50 of Myoglobin = 2.8 torr

• Hill Plot ( slope = 1): straight line w/ a slope of one, indicating non-cooperative independent binding of O2 

Hemoglobin O2 Binding Chart

• Sigmoidal Curve: indicates cooperative interaction b/w binding sites (pos/neg)

• S-shaped curve means more O2 can be delivered to tissue

• Hill Plot: slope is 3

Cooperative Interaction

• binding first O2 makes it easier for subsequent O2 to bind  

  • Hemoglobin (Hb) has 4 subunits, each subunit binds to 1 O2 so 1 hemoglobin carries 4 O2 molecules total 

  • Hb subunits compete for who will bind to O2 first 

  • 4th O2 binds to Hb w/ 100 fold greater affinity than first O2 

    • P50(1st O2) = 30 & P50(4th O2): 0.3 

Hill Coefficient

• provides a way to quantify the degree of interaction b/w ligand binding sites 

• higher coefficient = higher cooperativity

• mutations affects hill coefficient

Myoglobin YO2

• Arterial Blood (100 torr): 0.97

• Venous Blood (30 torr): 0.91

• △YO2 = 0.06

Hemoglobin YO2

• Arterial Blood (100 torr): 0.95

• Venous Blood (30 torr): 0.55

• △YO2 = 0.40

Myoglobin Structure

• 153 residues (aka AA) 

• 8 alpha-helices (A-H) 

• Heme (porphyrin) w/ iron atom at the center 

• O2, CO-, NO, & H2S bind to Fe (II) of heme 

• Heme coordinates Fe by 4 nitrogen atoms 

• His F8 is fifth ligand 

• O2 or CO represents the 6th ligand 

Heme Structure

• 4 nitrogen atoms of heme binds to Fe(II) 

• His F8 is fifth ligand (His on helix F, at 8) 

• O2 is sixth ligand 

• CO, NO, & H2S also bind 

• CO binds a factor of 200 more strongly than O2 (CO posioning)

What do the 2 hydrophobic residues in hemoglobin do

• help hold heme in place & help to prevent oxidation of Fe(II) to Fe(III) 

• Moves out the way to allow O2 binding 

T-State (deoxyhemoglobin)

• constrained by ionic bonds & has low O2 affinity 

• Fe is 0.6A out of heme plane 

• TENSE STATE 

R-State (oxyhemoglobin)

• high O2 affinity & is in relaxed state 

• Fe in heme plane 

• Helix containing F8 shifts 

• Change in quaternary structure 

• C-terminal residues (Arge141𝛼, His146𝛽, & Vall𝛼) change interactions and/or ionization state (Bohr Effect) 

α2, β2 dimer (subunits)

2 alpha (α) protein subunits & 2 beta (β) protein subunits of hemoglobin

α2, β2 Dimer (subunits) Interactions

• Extensive interactions between unlike subunits

• α2-B2 or α1-B1 interface has 35 residues

• α1-B2 & α2-B1 have 19 residue contacts

• No contacts b/w α1-α2 or B1-B2 (separated by water channel)

• contacts are mostly hydrophobic w/ some H-bonds & salt bridges

What happens after the first O2 binds in Hb

• shifts the α1-B2 contacts and moves distal His E7 and Val E11 out of the oxygen’s path to the Fe on the other subunits

• inc O2 affinity of heme

What 2 stable positions does α1-B2 have

• T (deoxy) & R (oxy) states

• Binary Switch: one H-bond stabilizes T-state & other one stabilizes R-state & Hb can switch b/w the 2 bonding patterns

Bohr Effect

• Higher pH (more basic): has higher O2 affinity & promotes tigther binding of O2 to Hb

• Lower pH (more acidic): has lower O2 affinity & permits easier release of O2 from Hb

pH & P50 Relationship

• pH inc, P₅₀ dec, O2 binding inc (vice versa)

• At 20 torr, 10% more O2 is released when pH drops from 7.4 to 7.2

Origin of Bohr Effect

• T → R state: pKa dec, so less likely to hold onto proteins (H+)

  • Hb binds to O2, shifts to R state, & H+ ions released

• N-Terminal Amino Group (20-30% of Bohr Effect): binds to H+ in T-state & releases H+ in R-state

• His146B salt bridged w/ Asp 94B (40%): His protonated, forms salt bridge w/ Asp in T state, His loses protons & salt bridge breaks in R-state

H+ & O2 Relationship in Bohr Effect

• Inc H+ = release O2

• Inc O2 = release H+

D-2,3-bisphosphoglycerate (BPG)

• BPG binds to hemoglobin & dec O2 affinity & keeps it in deoxy form

• 1 BPG binds to 1 HB (in T-state) w/ a K=1×10^-5M (strong binding affinity) to deoxy form but weakly to oxy form (BPG cannot fit well due to smaller central cavity)

High Altitude

less O2 binding (arterial) because BPG inc O2 release

Negative Cooperativity

induces R → T transition by allosteric interactions (conformational change after binding of ligand)

CO2 Concentration Effect

induces R → T transition through the Bohr effect and the formation of carbamates

BPG Concentration Effect

induces R → T transition by stabilizing (binding to) the T state