Lecture 3

the main structural difference between myoglobin and hemoglobin is the

number of subunits with heme groups

• both have the conserved globin fold with 8 alpha helices connected by short loops

• the functional difference between the two proteins is the release of oxygen (cooperativity in hemoglobin’s quaternary structure, whereas no cooperativity for myoglobin’s single subunit)

myoglobin

• “myo” = muscle

• myoglobin is a globular (spherical) protein that exists in muscle cells

• water-soluble protein

• ligand = O2

• function: binds and releases O2 to muscle cells

• structure: 1 polypeptide chain

• has 1 heme group buried in the pocket with ferrous Fe2+ ion (does not oxidize to ferric Fe3+ which cannot bind to O2), therefore each myoglobin can bind 1 O2 molecule reversibly

hemoglobin

• “hemo” = blood

• hemoglobin is a globular (spherical) protein found in red blood cells

• water-soluble protein

• ligand = O2

• function: takes O2 from the lungs to the tissues (including muscle)

• structure: 4 polypeptide subunits (two alpha and two beta)

• each polypeptide subunit has a heme group buried in the pocket with ferrous Fe2+ ion (does not oxidize to ferric Fe3+ which cannot bind to O2)

• each polypeptide subunit can bind 1 O2, therefore, each hemoglobin can bind 4 O2 molecules reversibly

hemoglobin is an

oxygen transport protein

• oxygen from lungs enters the red blood cell since it is poorly soluble in water and binds to the hemoglobin inside of the red blood cell, and then the oxygen is released to the tissue cells

• proteins load O2 where it is plentiful (high partial pressure pO2 in the lungs) and release O2 where it is scarce (low partial pressure pO2 in the tissue)

  • driven only by the partial pressure differences

the relative oxygen affinities of hemoglobin and myoglobin reflect their

physiological roles

• venous blood carries the partly deoxygenated red blood cells back to the lungs, hemoglobin reloads to full capacity, and it delivers back to the tissue

• hemoglobin only unloads the fraction of O2 that the local partial pressure pO2 calls for, so venous blood returns to the lungs still carrying a reserve of O2

  • this allows for tissues to draw more O2 from hemoglobin when demand rises

myoglobin has a hyperbolic curve since it has a

single polypeptide subunit, thus there is no cooperativity or switching between a low or high affinity state for loading or unloading of O2

hemoglobin has a sigmoidal S-shaped curve where it

switches from a low to high affinity state (multiple subunits allow for salt bridges to be broken, hydrogen bonding interactions to be changed, and subunits to be rotated so that the affinity of the heme groups to O2 can change)

• high affinity for loading of O2 for storage, low affinity for unloading of O2 for transport

• unloading depends on the amount of O2 that the tissue needs (can change the affinity by cooperativity)

could myoglobin do the job of hemoglobin?

• pO2 in lungs: 13 kPa

• pO2 in tissues at rest: 4kPa

  • myoglobin has a high affinity for O2, thus there is tight binding and it fairly fully saturated at both pO2 values, and does not give up O2 readily

would lowering the affinity of myoglobin to oxygen help?

more O2 can be released at the pO2 levels in the tissue from the lungs, but there is not as high of an amount of O2 bound to the myoglobin and loaded in the lungs, thus it delivers O2 poorly since the myoglobin cannot be filled

• if the affinity is reduced, O2 can be unloaded from myoglobin from the lungs to the tissue, but cannot be picked up and bound in the lungs due to its low affinity

• a single subunit has no way to change its affinity, while a cooperative tetramer does, which is why hemoglobin is an efficient transporter

myoglobin in tissue, especially in muscle tissue, accepts

oxygen from hemoglobin and stores it in the muscle tissue

• both myoglobin and hemoglobin can approach oxygen saturation in the lungs at 100 mmHg

• when oxygen pressures are very low, myoglobin reserves supply oxygen by unloading it

• hemoglobin can readily supply oxygen to cells at normal oxygen levels and for storage in myoglobin when it is well-oxygenated, but it can be exhausted in intense muscle activity by unloading at low pO2 levels

oxygen binding affinities for myoglobin and hemoglobin

• there is a high oxygen affinity even at a low partial pressure for myoglobin ((hyperbolic curve, extracts oxygen efficiently from the blood in the tissue)

  • myoglobin stays nearly saturated across the whole physiological range of partial pressures of pO2 due to its high binding affinity, and gives up only 20% of its O2 between the lungs and resting muscle

• there is a low initial oxygen affinity for hemoglobin, but the affinity for oxygen increases due to cooperative binding

hemoglobin excels at

O2 transport because it can switch affinities (due to its tetrameric structure which allows for cooperativity and transitioning from a low to high affinity state which creates the sigmoidal curve as a transition state from the high and low affinity curves)

• hemoglobin releases about 60% of its O2 between the lungs to the tissue

• in the lungs, pO2 is high thus hemoglobin is in the R state: high affinity - binds O2 in the lungs

• in the tissue, pO2 is low thus hemoglobin is in the T state: low affinity - unloads O2 in the tissue

hemoglobin adopts a

low affinity tense (T) state and a high affinity relaxed (R) state

T (tense) state

oxygen is unbound (deoxyhemoglobin)

• tense due to salt bridging and ionic binding in the core which keeps it fixed

• regulatory effectors can bind in the central cavity and stabilize the T state

• low affinity due to steric hindrance in the binding site and the tilt of the proximal F8 histidine

R (relaxed) state

bound oxygen (oxyhemoglobin)

• oxygen binding to the cavity breaks the ionic bonds and salt bridges and rearranges the hydrogen bonds between the subunits, which transitions the tetramer from the T to R state

  • this process releases energy

oxygen binding to the heme group reduces the

radius of the Fe2+, forming a planar heme

• Fe2+ is slightly too large to fit and lies below the plane of the porphyrin ring in deoxyhemoglobin (high spin state which gives Fe2+ the larger radius, thus the form is puckered)

• O2 binds to the 6th coordination and pulls electron density away from Fe2+, and iron can now fit into the tetrapyrrole ring which stabilizes it in oxyhemoglobin (low spin state which gives Fe2+ the smaller radius)

hemoglobin structure changes in the

presence or absence of oxygen

• in oxyhemoglobin, His F8 and the F helix move together with Fe2+ towards the heme as the ferrous Fe2+ binds to O2 (stabilizes the structure)

  • this is the first mechanical step of the conformational change to change the affinity of the tetramer since the F8 proximal His residue on the F helix moves with the Fe2+ as it binds to O2

  • movement occurs in both alpha and beta subunits

oxygen binding induces a

rotational movement of heterodimers relative to each other

• the shift in the F helix changes the contacts at the interfaces between the alpha and beta subunits, which changes the binding affinity of hemoglobin

• transition from T to R state; one pair of a-b subunits rotates by 15°

  • one hydrogen bond is broken from the alpha and beta interface and then moved to a different position between different amino acids on the alpha and beta interfaces gives the change in conformation

  • changes from deoxyhemoglobin (does not bind O2) to oxyhemoglobin (binds O2)

cooperativity enhances

oxygen delivery

• the sigmoidal (S-shaped) curve is best described as the transition from the T-state to the R-state curve and describes positive cooperativity

  • the first ligand makes it easier for subsequent ligands to bind (transition from T to R state)

  • a high ligand concentration drives ligand binding even when the affinity is low (the first O2 does not bind with a high affinity, but as the next oxygens bind, they bind easier and with a higher affinity)

the high affinity R state lets hemoglobin fill completely in the

lungs, and as the red blood cells move hemoglobin from the lungs to the tissue, the low affinity T state unloads O2

• negative cooperativity decreases affinity and makes it harder for subsequent ligands to bind (release of O2)

cooperativity allows oxygen to

shift between high and low affinity states to the hemoglobin subunits based on the partial pressure of O2 in the lungs vs. the tissue

• each bound O2 raises the affinity of the empty sites from the T to R state (sensitive to small changes in pO2)

• each released O2 lowers the affinity of the full sites from the R to T state (speeds up the release of O2 at low pO2)

allostery is a

remodeling of a protein’s structure and activity by the binding of a molecule at a site other than the active one

• positive allostery: binding at one site raises the affinity for binding at other sites

• negative allostery: binding at one site lowers the affinity for binding at other sites

• homotropic allostery: the ligand and regulatory effector are the same molecule

• heterotropic allostery: the ligand and regulatory effector are different molecules

  • effector binds to a different site and changes how the ligand binds at the first site

oxygen binding to hemoglobin is an example of

positive homotropic allostery

• homotropic, positive = cooperative binding

  • homotropic due to the ligand and effector both being O2, and positive due to the binding of O2 increasing the affinity for more O2 molecules to bind

heterotropic, negative allostery

• effector is not the ligand if it is heterotropic (effector binds to a regulatory site, not the binding site)

• if it is negative, the binding of the effector makes it more difficult for the ligand to bind

  • protons, CO2, Cl, 2,3-bisphosphoglycerate

cooperative ligand binding can be described

quantitatively

• for a protein with n binding sites, the equilibrium becomes: P + nL ⇌ PLn

• the expression for θ becomes: θ = [L]^n / [L]^n + Kd

• rearranging, then taking the log of both sides yields: θ / 1 - θ = [L]^n / Kd

  • log (θ / 1 - θ) = n log [L] - log Kd

  • this is the Hill equation (a plot of log [ θ / (1 - θ) ] versus log [L] is called a Hill plot (slope measures cooperativity)

    • coefficient n between 1 and the number of sites determines that the binding is cooperative but not all-or-none

temperature affects the

affinity of hemoglobin for oxygen

• the curve shifts to the right due to the increase in temperature, the binding is loosened and O2 can be unloaded easier to the tissue (lower binding affinity with increased temperature)

• O2 binding to the heme releases heat, thus adding heat pushes it back to the unbound deoxyhemoglobin form

decreasing the pH

decreases the affinity of hemoglobin for oxygen (shifts the curve to the right), allowing the body to titrate binding in different tissues (unloads O2 to the tissue)

• less O2 is released to the resting tissue if there is an increase in the pH (left-shifted curve, higher affinity for O2, less acidic)

H+ is an

antagonist of oxygen binding by Hb and the saturation curve of Hb for O2 is displaced to the right as acidity increases (pH decreases, more H+)

• R state binds O2 well and T state binds H+ well, so raising the H+ concentration and raising the O2 concentration pulls the tetramer in opposite directions

• negative heterotropic effector which decreases the O2 binding to hemoglobin and more unloading of O2 to the tissue (shifts the curve to the right)

  • moves hemoglobin from the R state to the T state

active metabolic tissues generate

protons that accumulate on amino acid residues in hemoglobin

• H+ and CO2 both enhance O2 release, which makes them negative allosteric effectors of hemoglobin, and this is described by the Bohr effect

Bohr effect

• protons (low pH) promotes the formation of salt bridges that stabilize the T state (N terminal amino groups on the alpha subunits and C terminal histidines of the beta subunits become protonated, which can interact with negatively charged aspartate residues)

  • lower the O2 binding affinity of hemoglobin and favor the release of O2

• carbon dioxide (CO2) is the product of oxidative metabolism and also decreases O2 affinity of hemoglobin; it binds to N-terminal groups of the alpha and beta chains in the interface resulting in carbamino-hemoglobin

pH and CO2 ensure that oxygen is released

preferentially in actively metabolizing tissue where it is most needed

• only 14% of CO2 is transported by hemoglobin

• CO2 is also transported in the blood as bicarbonate (HCO3-) hydrated by carbonic anhydrase

as hemoglobin reaches tissue with high CO2 concentration, its affinity for O2

decreases

• the pH difference between the lungs and tissues increases the efficiency of O2 transport, since the tissues are more acidic than the lungs, so hemoglobin arriving to the tissue meets both a low pO2 and low pH (high H+ concentration), thus it is pushed into the T state and O2 is released

• in the lungs, there is a higher pH (low H+ concentration) and high pO2, thus hemoglobin is pushed into the R state and O2 is loaded

  • Bohr effect

• negative, heterotropic allosteric effectors: hydrogen ions and CO2 promote the release of oxygen

CO2 also promotes the

dissociation of O2 from hemoglobin

• hemoglobin stripped of its 2,3-bisphosphoglycerate binds O2 too tightly to work as a transporter (high binding affinity even at low pressures of O2)

  • the 2,3-bisphosphoglycerate effector lowers the affinity of hemoglobin for O2 and shifts the curve to the right, which matches the body’s needs (negative, heterotropic effector)

• the curve shifts to the right from Hb with CO2, Hb with BPG, and Hb with CO2 and BPG

• oxygen transport → oxygen uptake in the lungs → binding with hemoglobin → transport to tissues → oxygen release

carbon dioxide must be

removed from tissues, and some is removed by hemoglobin

• CO2 leaves the tissues in three forms

  • dissolved directly in the plasma, converted to bicarbonate, or bound to hemoglobin itself

CO2 acts on O2 affinity in two ways

one through the pH it creates and one through direct binding

• binds to the amino group on the N terminus to form carbamate, which releases H+ and turns the end of the chain from neutral to negative (forms a salt bridge with a positive residue and contributes to the Bohr effect which reduces O2 binding)

• high CO2 in the tissues lowers O2 affinity since hemoglobin gives up O2 and picks up CO2, low CO2 in the lungs increases O2 affinity since hemoglobin binds O2 and releases CO2 for exhalation

the majority of dissolved carbon dioxide is

transported as bicarbonate

• carbonic anhydrase hydrates carbon dioxide to carbonic acid

• CO2 + H2O ⇌ H2CO3 ⇌ H+ + CO3-

  • HCO3- leaves the red blood cell in exchange for a Cl- ion entering, which keeps the charges balanced and carries bicarbonate back into the lungs in plasma

  • the hydration reaction is also the source of protons for the Bohr effect, since it raises the H+ concentration and lowers the pH (CO2 is thus a negative heterotropic effector)

2,3-bisphosphoglycerate (2,3-BPG) binds to hemoglobin

allosterically

• binds in the central cavity (positively charged) with its negative O atoms and thus stabilizes the T state (deoxyhemoglobin)

  • T state has a larger central cavity than the R state and the positively charged groups repel each other, thus 2,3-BPG and Cl- shield the charges from destabilizing the T state

2,3-BPG binding can assist with

oxygen release in tissues (binds to deoxyhemoglobin rather than oxyhemoglobin to stabilize it as O2 is released)

• holds the tetramer in the low affinity T state, thus O2 binds less readily

• the oxygenated R state closes the cavity, so 2,3-BPG cannot bind the R state (pushes equilibrium towards release rather than binding)

left shift (higher affinity of hemoglobin to O2) is caused by

• decreased H+ (increased pH)

• decreased 2,3-BPG

• decreased CO2 and Cl-

• decreased temperature

right shift (lower affinity of hemoglobin to O2) is caused by

• increased H+ (decreased pH)

• increased 2,3-BPG

• increased CO2 and Cl-

• increased temperature

the T-state prevails at

low pO2 values and has a relatively high affinity for protons, organic phosphates, and chloride

• R state and T state exist in an equilibrium, and every effector shifts the equilibrium from one direction to another

  • the R state binds O2 well

  • the T state binds H+, CO2, Cl-, and 2,3-BPG well (raise the P50 O2 amount needed to change the T state to the R state)

• high pO2 values switch the low-affinity T state to the high-affinity R state (the growing steepness is the source of cooperativity)

the Bohr effect is largely caused by

proton binding to the imidazole side chain of the terminal histidines (His) in the two beta chains

• the protonated histidine forms a salt bridge with an aspartate in the same chain, which stabilizes the low affinity T state

several factors regulate the

oxygen affinity of hemoglobin

• temperature, pH, concentration of CO2, and concentration of 2,3-BPG

  • a rise in any of these factors favors the T state and shifts the curve to the right

  • a fall in any of these factors favors the R state and shifts the curve to the left

• this allows for a single protein to read the conditions of a tissue and set its affinity to match

2,3-BPG allows for

changes in altitude

• the air at altitude is thinner, so the total pressure and partial pressure pO2 are lower, so hemoglobin cannot fully load in the lungs compared to at sea level

  • the body answers by unloading a larger fraction of what it does carry by making more 2,3-BPG to lower the affinity of hemoglobin for O2 and shift the curve to the right

• when 2,3-BPG levels are normal, the difference in fractional saturation in the lungs at 4,500 m and tissues is about 33% (0.90 - 0.57 = 0.33)

• when 2,3-BPG levels are elevated, there is a bigger difference in fractional saturation in the lungs at 4,500 m and tissues (0.85 - 0.45 = 0.40)

fetal hemoglobin (HbF) binds oxygen

more tightly, allowing the fetus to out-compete the parent (draws O2 from the parent’s blood more tightly with a higher affinity)

• the fetal hemoglobin replace the two beta chains with two gamma chains

  • the gamma subunits bind 2,3-BPG more weakly than the beta subunits, and since 2,3-BPG lowers the binding affinity between hemoglobin and O2, less binding of 2,3-BPG means a higher affinity for O2

• the beta gene takes over the gamma gene after birth, so the newborn shifts from fetal hemoglobin to adult hemoglobin with the beta subunits

a histidine substitution in fetal hemoglobin causes

2,3-BPG to not bind well to the central cavity, causing a higher affinity for O2 (curve is shifted towards the left)

• the His residue at position 143 of the chain is replaced by a neutral Ser residue in the fetal gamma chains

• because 2,3-BPG binds to the central cavity by pairing its negatively charged O groups to the positively charged His side chains, by replacing the positively charged His residue with a neutral Ser residue, this prevents the interaction from occurring in the cavity

in sickle cell anemia,

sickle hemoglobin (HbS) displays an altered affinity for oxygen

• amino acid substitution from glutamate to valine at position 6, which causes the hemoglobin to sickle in the tissue but not in the lung (T state)

  • glutamate is negatively charged and keeps the surface of the subunit soluble, whereas valine creates the hydrophobic surface which sticks to a complementary hydrophobic pocket in a neighboring hemoglobin in its T state (deoxygenated)

    • deoxyhemoglobin S polymerizes into long fibers whereas oxygenated HbS does not (sickling this appears under the low oxygen conditions of the tissues and not in the lungs)

  • distorted cells are rigid and fragile, and clog vessels and break apart easily which cuts off O2 supply and causes pain

• HbSS shifts to the right: increased tissue unloading (higher P50)

• anemia itself can also cause a rightward shift due to the increased production of 2,3-DPG

carbon monoxide has a higher

binding affinity with hemoglobin than oxygen

• CO has a higher binding affinity with the Fe2+ atom than O2, and thus blocks O2 from binding (heterotropic positive allostery) but pushes the tetramer towards the R state, which raises the affinity of the remaining hemes for O2 and it is held too tightly to reach the tissue

  • CO has a smaller size that O2 and thus is not limited by steric hindrance, so in order to prevent poisoning, the E7 distal His residue creates steric hindrance against CO and forces it to become bent, which creates strain in its binding despite its high binding affinity and this is a protective feature

nitric oxide has a

high affinity ligand for hemoglobin

• NO is a signaling molecule that relaxes blood vessels, and binds the heme Fe2+ atom far more tightly than O2

• why isn’t NO instantaneously bound by Hb?

  • the presence of O2 causes the bound NO to be oxidized to nitrate and the iron is left as ferric methemoglobin

  • the oxygenated R state causes disulfide bridges to shift into SH groups, which keeps the NO off of the Fe2+ atom and shields it from oxidation (promotes O2 delivery)