Part II => PROTEINS and ENZYMES. 2.4 PROTEIN FUNCTION 2.4a Oxygen Transport

Transcription

1 Part II => PROTEINS and ENZYMES 2.4 PROTEIN FUNCTION 2.4a Oxygen Transport

2 Section 2.4a: Oxygen Transport

3 Synopsis 2.4a - Myoglobin (Mb) and hemoglobin (Hb) are two of the best characterized members of the heme-based family of oxygen-binding proteins - While Hb carries oxygen (oxygen carrier) in the bloodstream to make it available to all tissues, Mb largely serves to store oxygen (oxygen reservoir/facilitator) so that it can be quickly made available to the muscle tissue during high oxygen demand (eg strenuous exercise) - Mb exhibits a hyperbolic oxygen-binding curve (a non-cooperative response) such behavior is ideally suited to its role as an oxygen reservoir! - By virtue of its ability to undergo a conformational change, Hb displays a sigmoidal oxygen-binding curve (a cooperative response) such behavior is ideally suited to its role as an oxygen carrier! - The ability of allosteric factors such as ph (Bohr effect) and 2,3- bisphosphoglycerate (BPG) to modulate oxygen binding to Hb is of immense physiological significance

4 Oxygen Transporters: Myoglobin (Mb) and Hemoglobin (Hb) PDBID 1MBO Myoglobin (monomer) myo of muscle heme of blood globin globe(sphere)-like PDBID 1GZX Hemoglobin ( 2 2tetramer) - Oxygen is essential for most living organisms as it is required for the breakdown of food to release energy in a phenomenon referred to as respiration how do organisms obtain oxygen from the air?! - In addition to microbes, more complex organisms such as sponges and jellyfish also rely on direct diffusion of oxygen from the environment without the need for organs such as lungs/heart/blood no brains either!! - However in higher-order organisms such as vertebrates, the direct diffusion of oxygen to specific tissues is not sufficient to maintain life - Vertebrates rely on Hb (the mainstay of red blood cells) to efficiently transport oxygen from lungs to other tissues in the body - On the other hand, Mb (predominantly located in the muscle tissue) binds and relays (facilitates) oxygen from the capillaries to muscle cells additionally, Mb also stores oxygen so that it can be made available in drastic times such as during exercise and diving (particularly in aquatic animals) - Both Mb and Hb harbor a prosthetic group (a type of cofactor) called heme which is buried in a deep hydrophobic cleft within each globin and serves as the oxygen-carrier

5 Mb HbA HbB Mb HbA HbB Evolutionary Relationship Between Mb and Hb: Sequence Alignment MG-LSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALG MV-LSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHF-DLS--H---GSAQVKGHGKKVADALT MVHLTPEEKSAVTALWGKV--NVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFS GILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKE NAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR- DGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH- - The (HbA) and (HbB) chains of Hb are evolutionarily related to each other and to Mb polypeptide chain D - Out of nearly 150 residues, only 28 are IDENTICAL the three polypeptide chains of Mb and Hb only share a meager 18% amino acid identity! - Yet, all three polypeptide chains of Mb and Hb share a remarkably similar 3D structure with each polypeptide comprised of eight helices (designated A-H) wound around each other into a globular fold with an equally remarkable shared function - The only difference being that while Mb adopts a monomeric conformation, Hb is a tetramer ( 2 2 ) C COOH H 2 N

6 Myoglobin and Hemoglobin: Heme Cofactor - In a manner akin to chlorophyll (the light-absorbing pigment critical for photosynthesis in plants), heme is a porphyrin derivative containing four pyrrole groups (designated A-D) linked together via methine bridges - At the heart of heme lies the iron divalent metal ion (Fe 2+ ) hereinafter referred to as Fe(II) 7 (7th residue of helix E) Pyrrole - The porphyrin ring of heme is stabilized via numerous van der Waals contacts with specific sidechain moieties located in the globin - Fe(II) is stabilized via coordination by five ligands: one N atom from each of the four pyrrole rings and an additional N atom courtesy of a highly conserved histidine ( 8) residue located on the proximal face of heme - Oxygen (O 2 ) binds as a sixth ligand to Fe(II) in a reversible manner bound under high concentrations of oxygen (arterial blood) and released in regions with low oxygen (venous blood) - Additionally, an highly conserved histidine ( 7) located on the distal face of heme sandwiches the oxygen molecule via hydrogen bonding this interaction caps the oxygen binding crevice so as to keep larger molecules out Oxygen 8 (8th residue of helix F) Distal Face Proximal Face

7 Myoglobin

8 Myoglobin: Binding of oxygen 7 and 8 histidine residues in human Mb and Hb Globin Length 7 8 Mb 154* H65* H94* Hb 142* H59* H88* Hb 147* H64* H93* *Including the N-terminal MET residue cleaved via post-translational modification (PTM) - 8 histidine serves as one of the five N ligands of heme (note the planar conformation of the porphyrin ring) - 7 histidine hydrogen bonds to oxygen Myoglobin in complex with heme and oxygen O2 Heme 7 - In addition to oxygen (O 2 ), heme also binds to toxic gases such as carbon monoxide (CO), nitric oxide (NO) and hydrogen sulfide (H 2 S) but with much higher affinity thereby accounting for their toxicity 8 PDBID 1A6M - But, there is no obvious route for the entry of O 2 (as well as other toxic gases) to coordinate with heme! So what gives?! Enter allostery.

9 Myoglobin: Allosteric binding of oxygen - Static images do little justice to the remarkable flexibility that the proteins harbor various regions of protein are under constant fluctuations at the very minimum Note the subtle planarization of the porphyrin ring of heme upon oxygen binding - Such protein motions (or dynamics) are central to protein function in a manner akin to human body (flexible) versus a robot (rigid)! 7 - Indeed, many proteins undergo a varying degree of conformational change upon binding to their ligands (small molecules or other protein partners) Heme 8 O2 Fe(II) - Such conformational change (involving changes in either structure and/or dynamics) in response to ligand binding has come to be known as allostery - Allostery plays a key role in the ability of Mb to bind O 2 albeit in a highly subtle manner ie the binding of O 2 induces a conformational change in Mb Animation generated via morphing of deoxymb (PDBID 1A6N) and oxymb (PDBID 1A6M)

10 Myoglobin: Hyperbolic binding of oxygen - The reversible binding of oxygen (O2) to myoglobin (Mb) can be described as: Mb + O2 <==> MbO2 When [Mb] = [MbO2]: => K = [O2] ie K is the which the protein is half-saturated! - The equilibrium dissociation constant (K) is given by: K = [Mb].[O2] / [MbO2] [1] [MbO2] = [Mb].[O2] / K [2] where [Mb] and [MbO2] are respectively the concentrations of free and oxygen-bound equilibrium - Thus, the fractional saturation (Y) of myoglobin with oxygen is: Y = [MbO2] / {[Mb]+[MbO2]} [3] - Combining Eqs [2] and [3] and factoring out the [Mb]/K term yields: Y = {[Mb].[O2]/K} / {[Mb] + {[Mb].[O2]/K}} [4] Y = {{[Mb]/K} [O2]} / {{[Mb]/K} {K+[O2]}} [5] Y = [O2] / {K+[O2]} [6] - Since oxygen is a gas, its concentration can be expressed by its partial pressure to be po2 now, rewriting Eq [6] in terms of the partial pressure of oxygen gives: Y = po2 / (K + po2) [7] - Eq [7] is the equation of a hyperbola a plot of po2 versus Y will almost linearly increase at lower concentrations of oxygen and eventually plateau out at saturating oxygen! - Such hyperbolic behavior is characteristic of many biological phenomena such as drug-receptor interactions and substrate binding to enzymes (Michaelis-Menten kinetics)

11 1 Torr = 1/760 atm = 133 Pa po 2 (21% air) = 160 Torr Myoglobin: Hyperbolic response curve K O2 = 3 Torr = 50*K CO CO binds to Mb by 50-fold stronger than O2! Y T in Torr must be capitalized as a unit named after Torricelli, the inventor of barometer Saturation of Mb in response to oxygen concentration Evangelista Torricelli ( ) po2 / Torr - As shown earlier, the degree of saturation of Mb in response to oxygen concentration is given by: Y = po 2 / (K + po 2 ) - But, K is the concentration of oxygen (po2) at which Mb is half-saturated thus, from the hyperbolic curve shown: K = 3 50% Mb saturation why is CO so toxic?! - The fact that the physiological range of po2 in the blood is Torr is telling lower the K, higher the ligand binding affinity!!!! - This implies that Mb is almost always saturated under physiological conditions, thereby accounting for its ability to not only serve as an efficient oxygen reservoir but also being able to relay oxygen from a region of high (arterial blood) to a region of low concentration (venous blood)

12 Hemoglobin

13 Hemoglobin: Binding of oxygen 7 and 8 histidine residues in human Mb and Hb Globin Length 7 8 Mb 154* H65* H94* Hb 142* H59* H88* Hb 147* H64* H93* *Including the N-terminal MET residue cleaved via post-translational modification (PTM) Hemoglobin in complex with heme and oxygen (Only monomer shown) 7 O2-8 histidine serves as one of the five N ligands of heme (note the planar conformation of the porphyrin ring) Heme 8 Fe(II) - 7 histidine hydrogen bonds to oxygen - As noted earlier for Mb, there is no obvious route for the entry of O2 to coordinate with heme! So what does hemoglobin do? PDBID 1HHO

14 Hemoglobin Allostery: Deoxygenated Conformation (deoxyhb) Valence Electrons Electrons in the outer shells directly involved in bonding between adjacent atoms deoxyhb (Only monomer shown) In the deoxyhb conformation: - Porphyrin ring of heme is non-planar Heme 7 Fe(II) - Fe(II) atom is pulled out of the porphyrin ring toward 8 due to repulsion between the valence electrons in heme and Under such electronic and conformational properties of heme and Fe(II), deoxyhb predominantly absorbs visible light at wavelengths between nm ie venous blood appears dark red! PDBID 2HHB

15 In the oxyhb conformation: - Binding of O2 as a sixth ligand to Fe(II) overcomes the repulsion between the valence electrons in heme and 8 so as to allow Fe(II) to move into the center of the porphyrin ring, thereby resulting in the planarization of the porphyrin ring - The change in the electronic and conformational properties of Fe(II) and heme upon planarization of its porphyrin ring enables oxyhb to predominantly absorb visible light at all wavelengths but above 600nm ie arterial blood appears bright red! Hemoglobin Allostery: Oxygenated Conformation (oxyhb) oxyhb (Only monomer shown) - The planarization of porphyrin ring also draws 8 upwards and closer to heme such movement of 8 subsequently results in the conformational shift of numerous other residues so as to adopt the oxyhb state - The resulting intra-subunit conformational change from deoxyhb to oxyhb state (triggered by O2 binding) ultimately culminates in the likewise conformational re-arrangement of the other three subunits within the 2 2 tetramer to oxyhb (without the intervention of O2!) thereby enabling them to bind O2 much more easily than the first monomer Heme 8 7 O2 Fe(II) - The conformational change in 2 2 tetramer in response to oxygen thus enhances its ability to rapidly load additional molecules of O2 a phenomenon referred to as cooperative binding a form of allosteric regulation PDBID 1HHO

16 Hemoglobin Allostery: Intra-Subunit ( ) Unlike Mb, Hb undergoes a substantial intra-subunit conformational change upon oxygen binding deoxyhb <=> oxyhb (Only monomer shown) 7 Heme O2 Fe(II) Note the planarization of the porphyrin ring of heme upon oxygen binding 8 Animation generated via morphing of deoxyhb (PDBID 2HHB) and oxyhb (PDBID 1HHO)

17 - O2 binding to heme within 2 2 tetramer does not occur simultaneously! - Binding of O2 to one of the four heme groups triggers a conformational transition of its deoxyhb component to oxyhb state - Such intra-subunit conformational transition causes the other three neighboring subunits to quickly adopt the oxyhb conformation, thereby facilitating O2 binding - Thus, the binding of first O2 molecule is slow at first but the subsequent O2 molecules are loaded rapidly by virtue of the ability of 2 2 tetramer to undergo a change in quaternary structure note the narrowing of the central cavity due to the rotation of each protomer by about 15 relative to each other - In thermodynamic terms, the 2 2 subunits of Hb bind O2 in a cooperative manner - How can we thermodynamically rationalize such binding cooperativity? - But, first, the physics of hemoglobin color (spectroscopy)! Hemoglobin Allostery: Inter-Subunit ( 2 2) 2 2 O2 deoxyhb <=> oxyhb ( 2 2 tetramer shown) Central Cavity Fe(II) Heme Animation generated via morphing of deoxyhb (PDBID 2HHB) and oxyhb (PDBID 1HHO) 1 1

18 Hemoglobin Color: Absorption Spectra oxyhb Visible Spectrum oxyhb deoxyhb Bright Red oxyhb: absorb < 600nm reflect > 600nm Red Blue deoxyhb: 500nm < absorb < 600nm reflect < 500nm reflect > 600nm Red Blue + Red = Dark Red/Purple! deoxyhb Dark Red

19 Hemoglobin Color: Why Does Venous Blood Appear Blue?! I 1/ 4 Blue 1/(400) 4 = 4*10-11 Red 1/(700) 4 = 4*10-12 Why is the sky blue? Because of the reflection of light by the sea! True or false? - White light is made up of visible spectrum with wavelength roughly stretching from about nm - Three primary components of white light are blue ( nm), green ( nm) and red ( nm) - Particles in the atmosphere scatter white light from the sun (according to Rayleigh scattering particles much smaller than the wavelength!) on its way to us in a wavelength-dependent manner the intensity (I) of scattered light is inversely proportional to the wavelength ( ) raised to the 4 th (quartic) exponent/power simply put, I 1/ 4 - Thus, blue light (shorter ) is scattered by an order of magnitude greater than red light (longer ) such that the blue light is radiated in all directions making the sky appear blue from the earth s surface - For the same reason, the blue light component of deoxyhb is much more scattered as it travels through the skin than the red component, making venous blood appear BLUE through the skin but, inside the veins, it is DARK RED!

20 O2 Binding to Hemoglobin: Hill Equation - The reversible and cooperative binding of oxygen (O2) to hemoglobin (Hb) was first described by Hill in 1910 via a single step: Hb + no2 <==> Hb(O2)n where n is the number of O2 molecules bound but, more commonly, referred to as the Hill coefficient (or degree of cooperativity) - The above model assumes simultaneous (rather than sequential) binding of n molecules of O2 to Hb thermodynamically valid but not how Hb binds O2 in kinetic terms! - The overall equilibrium dissociation constant (K) is given by: K n = {[Hb].[O2] n } / [Hb(O2)n] [1] [Hb(O2)n] = {[Hb].[O2] n } / K n [2] where [Hb] and [Hb(O2)n] are respectively the concentrations of free and oxygen-bound hemoglobin - Thus, the fractional saturation (Y) of hemoglobin with oxygen is: Y = [Hb(O2)n] / {[Hb]+[Hb(O2)n]} [3] Archibald Hill ( ) - Combining Eqs [2] and [3] and factoring out the [Hb]/K n term yields: Y = {[Hb].[O2] n /K n } / {[Hb] + {[Hb].[O2] n /K n }} [4] Y = {{[Hb]/K n } [O2] n } / {{[Hb]/K n } {K n + [O2] n }} [5] Y = [O2] n / {K n + [O2] n } [6] - Since oxygen is a gas, its concentration can be expressed by its partial pressure to be po2 now, rewriting Eq [6] in terms of the partial pressure of oxygen gives: Y = (po2) n / {K n + (po2) n } [7] - Eq [7] is the Hill Equation describing the binding of oxygen to hemoglobin (n > 1) in a cooperative manner (sigmoidal response curve) a plot of po2 versus Y progresses slowly at the beginning and then rapidly accelerates (due to cooperative binding) before plateauing out at saturating oxygen so as to generate an S-shaped curve!

21 O2 Binding to Hemoglobin: Sigmoidal response curve 1 Torr = 1/760 atm = 133 Pa po 2 (21% air) = 160 Torr Y Hypothetical hyperbolic curve for HB (n=1) K O2 (Hb) = 30 Torr = 200*K CO K O2 (Mb) = 3 Torr = 50*K CO CO binds to Hb by 200-fold stronger than O2! po2 / Torr - As shown earlier, the degree of saturation of Hb in response to oxygen concentration is given by: Y = (po2) n / {K n + (po2) n } - But, K is the concentration of oxygen (po2) at which Hb is half-saturated thus, from the sigmoidal curve shown: K = 30 50% Hb saturation - The partial pressures of oxygen in the VENOUS and ARTERIAL blood are respectively 30 Torr and 100 Torr! - Sigmoidal binding response enables Hb to deliver more O2 to tissues (po2=20 Torr) than would be achieved via hyperbolic binding (indicated by dashed curve) a greater fraction of Hb becomes unloaded below the venous pressure of oxygen (30 Torr) and, conversely, a greater fraction of Hb becomes loaded above this threshold - Such a remarkable virtue of Hb is made possible by its ability to bind oxygen in a cooperative manner in other words, the 2 2 subunits bind oxygen with differential affinities how can we tell?!

22 O2 Binding to Hemoglobin: Linearization of Hill Equation - The Hill equation describing the cooperative binding of oxygen to hemoglobin is as follows: Y = (po2) n / {K n + (po2) n } [1] Hill Plot - Taking the reciprocal of both sides of Eq [1] gives: 1/Y = {K n + (po2) n } / (po2) n => 1/Y = {K n / (po2) n )} + 1 => {(1/Y) 1} = {K n / (po2) n )} => {(1-Y)/Y} = {K n / (po2) n )} [2] - Now, inverting both sides of Eq [2] and taking logs, we have: => {Y/(1-Y)} = {(po2) n / K n } => log{y/(1-y)} = log{(po2) n / K n } => log{y/(1-y)} = log(po2) n log(k n ) => log{y/(1-y)} = nlog(po2) nlogk [3] cf: y = mx + b - Thus, Eq [3] is the equation of a straight line with a slope of n and y-intercept equal to -nlogk! - A plot of log(po2) versus log{y/(1-y)} would yield a straight line (or lines!) from which one can extrapolate the Hill coefficient (n) and the dissociation constant (K) this is the so-called Hill Plot -nlogk log(po2) What does n mean?! n > 1 => Positive cooperativity n = 1 => No cooperativity n < 1 => Negative cooperativity What would you expect n to be for Mb and Hb? n

23 O2 Binding to Hemoglobin: Hill Plot Myoglobin Hill plot is essentially linear indicative of non-cooperative binding as expected (n=1 and K=3 Torr) : -nlogk = log(po2)=0 => logk = 0.5 => K = 10 (0.5) = 3 Torr A B C Hemoglobin Hill plot exhibits three distinct phases (A-C): (A) po2 < 10 Torr => log(po2) < 1 All four subunits compete with each other for binding to O2 in a non-cooperative manner (n=1 and K=100 Torr): -nlogk = log(po2)=0 => logk = 2 => K = 10 2 = 100 Torr (binding affinity of the first subunit!) (C) po2 > 55 Torr => log(po2) > 1.75 Since at least three of the four subunits are already occupied, O2 binds to the fourth subunit independently in a non-cooperative manner (n=1 and K=1 Torr): -nlogk = log(po2)=0 => logk = 0 => K = 10 0 = 1 Torr (binding affinity of the fourth subunit!) log(po2) (B) 10 < po2 < 55 Torr => 1 < log(po2) < 1.75 In between the two extremes (regions A and C), the binding of O2 occurs in a highly cooperative manner as evidenced by an accelerated slope (n=3 and K=30 Torr): -nlogk = log(po2)=0 => logk = (4.5)/3 => logk = 1.5 => K = 10 (1.5) = 30 Torr (the average binding affinity of all four subunits)

24 Hemoglobin as a Model for Allosteric Regulation Allosteric ( other site ) change occurs at a site other than where the ligand binds! allo other stereo solid/site T (-O 2 ) <<<<=> R (-O 2 ) deoxyhb oxyhb - Allosteric regulation is a hallmark of modular and multi-subunit proteins ligand binding induces conformational changes within the protein so as to facilitate subsequent binding - According to the contemporary Equilibrium Shift model, such proteins exist in an equilibrium between two conformations designated T (taut) and R (relaxed) - In the context of Hb, the T and R states would respectively be the deoxyhb and oxyhb conformations In other words T is the unbound (free) state and R is the state that assumes the conformation of oxyhb but in the absence of O2! - In the absence of ligand or substrate (S), the equilibrium lies well over to the left in the direction of T the ligand-free state - Upon the introduction of the ligand S, it only binds to the R state and merely serves to shift the equilibrium in its direction when R becomes usurped upon binding S, more of T will undergo conformational shift to R due to cooperative interactions

25 O2 Binding to Hb Is Coupled to Proton Release - In the T (deoxyhb) state, there exists an intricate network of ion pairs and hydrogen bonding between charged residues such as the sidechain groups of Asp94 and His146 the stabilization of imidazole proton on His146 effectively makes it less likely to be released, thereby increasing its pk to around 8 - Upon O2 binding, the T (deoxyhb) state undergoes equilibrium shift to R (oxyhb) and such conformational switch results in the disruption of the network of ion pairs and hydrogen bonding, including the Asp94-His146 pair this exposes the imidazole proton to solution, thereby decreasing its pk value to around 6 - Consequently, under physiological conditions (ph 7.4), oxygen binding to Hb is coupled to proton release from residues such as His146 one proton released for every two O2 molecules bound! - Thus, a decrease in ph would favor the protonation of Hb (and hence favor deoxyhb over oxyhb), thereby mitigating its affinity for O2 this phenomenon is called the the Bohr effect

26 Bohr Effect: Role of ph on O2 binding to Hb Y Bohr Effect: Lowering the ph lowers the affinity of Hb for oxygen! K O2 (ph7.4) = 30 Torr K O2 (ph7.2) = 35 Tissue po2 (20 Torr): Hb-saturation(pH7.4) = 32% Hb-saturation(pH7.2) = 22% Christian Bohr ( ) po2 / Torr Effect of H + on Hb: - In the tissues, the ph is lower (~7.2) due to metabolic production of CO2 (catalyzed by carbonic anhydrase): CO 2 + H 2 O <==> HCO H + T (deoxyhb) H + H + R (oxyhb) - Thus, the lower ph shifts the equilibrium in favor of the T (deoxyhb) state, thereby facilitating the release of O2 from Hb where it is needed most this is further added by lower po2 in the tissues (20 Torr) neat! - ph thus acts as a negative allosteric effector of Hb and the delivery of O2 to tissues is boosted by about 10% thanks to the Bohr effect

27 Bohr Effect: Role in CO2 Transport to Lungs - In respiring muscles (po2 = 20 Torr), the metabolic production of CO2 generates bicarbonate and Bohr protons which facilitate Hb to unload O2 and bicarbonate is carried in the blood back to the lungs - In the lungs (po2 = 100 Torr), binding of O2 to Hb is favored such that the released Bohr protons combine with bicarbonate to regenerate CO2, which is subsequently exhaled - The bound O2 is transported back to the muscles, where it can either directly diffuse into the cells, or in the case of rapidly respiring muscles, O2 is first taken up by Mb before being made available

28 (-BPG) BPG Effect: Role in O2 Release to Tissues Crystal structure of deoxyhb ( 2 2) bound to BPG (red) Y (+BPG) K O2 (+BPG) = 30 Torr K O2 (-BPG) = 10 Tissue po2 (20 Torr): Hb saturation (+BPG) = 30% po2 / Torr Hb saturation (-BPG) = 80% - Human blood contains high concentrations of 2,3-bisphosphoglycerate (BPG) an indispensable allosteric effector in that Hb would not be able to release O2 to the tissues without it! - In a manner akin to ph, BPG also facilitates the release of O2 from Hb by virtue of its ability to bind to the T (deoxyhb) state but not R (oxyhb) thereby shifting the equilibrium in favor of deoxyhb - BPG specifically docks into the rather wide central cavity of 2 2 tetramer in the T state via an extensive network of ion pairing and hydrogen bonding - Since the central cavity narrows upon T -> R transition (see the animation on Slide 15), the binding of BPG to oxyhb is hindered on steric grounds - By virtue of its ability to act as a negative allosteric effector of Hb, BPG boosts the delivery of O2 to tissues by about 50%!

29 Hemoglobin Variants in Health and Disease Variant Topology Mutation Biochemical and Clinical Consequences HbA 2 2 Wild type Most common variant in human population with > 95% occurrence (K=30Torr). HbC 2 2 -> E7K Prevalent among Africans. May cause hemolytic anemia but also confers resistance to malaria. HbD 2 2 -> E122Q Prevalent among Euroasians. Protects against sickle cell anemia. HbE 2 2 -> E27K Prevalent among Asians. May cause -thalassemia HbF > 2 Fetal hemoglobin (K=20Torr). Binds O2 with greater affinity than HbA due to its diminished interaction with BPG. HbH 4 2 -> 2 Causes -thalassemia (production of chains is impaired). Much lower binding affinity for O2 than HbA. HbO 2 2 -> E122K Prevalent among Arabs and Africans. May cause moderate anemia. HbS 2 2 -> E7V Prevalent among Africans. The E7V mutation induces polymerization of deoxyhb giving erythrocytes the appearance of a sickle the cause of sickle cell anemia. Protects against malaria.

30 Exercise 2.4a - Describe the O 2 -binding behavior of myoglobin in terms of po 2 and K. How is K defined? - Explain the structural basis for cooperative oxygen binding to hemoglobin - Sketch a binding curve (% bound ligand versus ligand concentration) for cooperative and noncooperative binding - Describe how myoglobin and hemoglobin function in delivering O 2 from the lungs to respiring tissues - What is the physiological relevance of the Bohr effect and BPG?