Chapter 10: Hemoglobin

Myoglobin

-18kDa monomeric oxygen binding protein of skeletal and heart muscle -First protein to have its 3D structure determined, and this surprised people because the model was surprisingly unsymmetrical -Heme group-center of the diagram-oxygen gets bound through coordination to the iron, held in place through interaction with histidine-shown -Very similar in structure to the monomers of hemoglobin -Facilitates oxygen diffusion through the muscle -Was originally assumed to store oxygen, it is now clear that this function is significant only in marine mammals which have Mb concentrations in their muscles 10- to 30-fold greater than those in terrestrial mammals-important for prolonged undersea activities -In terrestrial mammals its major physiological role in terrestrial mammals is to facilitate oxygen transport muscle -Increases the effective solubility of O2 in muscle-rapidly respiring under stress -Functions as a kind of molecular bucket brigade to facilitate O2 diffusion in muscle -Monitoring oxygen binding via absorption spectrum-plotted as a function of oxygen concentration -Looking at oxygen binding properties(how occupied various sites are at varying concentrations of oxygen)-protein is able to bind well at low concentrations, saturates quite quickly, reaches 100% saturation

Positive Cooperativity Diagram

-2 subunit protein-no ligand bound-the whole molecule is flexible -1 ligand, more compact, regular shape-stabilizing the red subunit as ligand binds -One subunit is stabilized, so the other subunit gets more little stabilized-look at colors on the left subunit changing-less room for movement -Because the subunit is closer to what its final conformation will be, it makes the subunit have a high affinity for ligand -As ligand binds, the protein undergoes a conformational change that enhances binding of the additional subunits

Hemoglobin

-65kDa heterotetramer (α2β2-2 alpha, 2 beta, ~16kDa per monomer)-each subunit is structurally similar to Mb -Red blood pigment that transports oxygen from the lungs, gills, or skin of an animal to its capillaries for use in respiration-very small organisms do not require such a protein because their respiratory needs are satisfied by the simple passive diffusion of O2 through their bodies -One of the first complex proteins studied because it is easy to isolate(lots of blood) -Crystalline form was first reported in 1840; first enzyme Crystals(jack bean ureae) were not reported until 1926 -The first protein to have its mass accurately determined, to be characterized by ultracentrifugation, to be associated with a specific physiological function, to have structure solved by x-ray crystallography -Not an enzyme, it acts only as a transporter, but it's oxygen binding properties are highly enzyme-like, so it is a good model system to study properties, structure, and mechanism of protein/enzyme science -Binds oxygen with high Cooperativity-binds all four-affinity for first oxygen is quite low-with every bound oxygen, affinity for the next oxygen is much higher

Myoglobin-Structure

-8 helices 7-26 residues long-126nof 153 residues -Different helix numbering convention peculiar to globins-residues are designated according to their position in a helix or interhelical segment -For example, , Glu EF7(83) of human Mb is the 83rd residue from its N-terminus and the 7th residue in the nonhelical segment connecting its E and F helices -The heme is wedged in a hydrophobic pocket -The structures of oxy-and deoxyMb are nearly super-imposable.

Fetal Hemoglobin

-A fetus obtains its O2 from the mother-BPG is also important in this process -In fetal Hb the 𝛾 subunit replaces the β subunit, which does not bind BPG - This allows fetal Hb to have a higher affinity for oxygen than the maternal Hbr -BPG occurs in the same concentration in adult and fetal erythrocytes but binds more tightly to deoxyHbA(red curve) than to deoxyHbF(black curve

The Sequential Model

-Alternatively the induced-fit or KNF model -Ligand binding induces a conformational change in a subunit-cooperative interactions arise through the influence that these conformational changes have on neighboring subunits -Protein's ligand-binding affinity varies with its number of bound ligands, whereas in the symmetry model this affinity depends only on the protein's quaternary state due to strong coupling -Mechanical coupling between subunits determines the effect of ligand binding on conformation, and thus further ligand binding affinity, and depends on the protein's symmetry -With strong coupling, conformational changes are concerted and preserve oligomeric symmetry(symmetry model) - With looser coupling, conformational changes occur sequentially as more and more ligand is bound(sequential model)-in addition, the subunit bound is the one most effected

The Hill Equation and Hemoglobin

-An attempt to analyze hemoglobin's sigmoidal O2-dissociation curve as an enzyme and substrate model for hemoglobin, as it acts similarly to this mode -Use the same equations as for Mb to describe a multisubunit protein binding a ligand where n = number of subunits -Assume infinite cooperativity (protein has all or none of its sites occupied, so it will give up all 4 oxygen at once)-no observable in- termediates ES1, ES2, etc. -Infinite cooperativity assumption doesn't really happen, yet mathematical representation assumes this -Hill equation is useful but more so for taking data and fitting it to a curve and getting an estimate -Similar to earlier generalized representation of 𝜭-all we are putting into the expression for theta is the number of subunits, n-for myoglobin n=1

fraction bound(𝜭)

-Analyze binding in terms of this - Represents the number of ligand-binding sites occupied at any given ligand concentration. Relationship between this and the dissociation constant is of key importance How we obtain the relationship: -Substituting [PL] with Ka[L][P], we'll eliminate [PL] in the top equation -Eliminating [P] and rearranging gives the result in terms of equilibrium association constant -We write the equation In terms of the more commonly used dissociation constant Why the relation between this and Kd is useful: -typically you know what the ligand concentration is(you are adding it into the protein) -you measure this quantity -From this data you can get the kd to tell you how tight your protein binds ligand ***Note that when [L]=kd, half of the ligand binding sites will be occupied -Gives us an easy relationship to understand the relationship between occupancy and dissociation constant

Globins

-Ancient gene products and are found in bacteria, plants and animals -Generally function in oxygen binding, but not always-some other functions, some unknown function

BPG Binding

-BPG decreases the oxygen-binding affinity of Hb by preferentially binding to its deoxy state - The binding of the physiologically quadruply charged BPG to deoxyHb is weakened by high salt concentrations, which suggests that this association is ionic in character -Studies indicate that BPG binds in the central cavity of deoxyHb on its 2-fold axis -The anionic groups of BPG are within hydrogen bonding and salt bridging distances of Lys, his, and N termini of the β subunits -One of these His residues is substituted in HbF -T→R transition shrinks the central cavity, and widens the distance of the β subunit N-termini, preventing H-bonding with the phosphate groups of BPG and ejecting it -Therefore, the T↔R equilibrium is shifted towards T because BPG stabilizes the T state by crosslinking its β subunits

Ligand Binding

-Binding is a REVERSIBLE process in most biochemical reactions-Reverse direction is of primary importance in biochemistry(such as signal termination)-release and dissociation rates -The ligand is a molecule that binds reversibly to a protein at a binding site-can be a protein, small molecule -The binding site is typically complementary in some way to the ligand (in size, shape, charge, hydrophobic character) -Binding is often highly specific, allowing the protein to discriminate among numerous similar molecules and selectively bind -Ligand binding can be coupled to conformational changes (induced fit)-when lock fits into the key, lock molds around the key(conformational change) -This is why conformational changes in one subunit can affect the others (cooperativity) in hemoglobin-each bound subunit changes the conformation-affinity and structure changes with each step

Methemoglobin-Stabilizing Mutations

-Changes at the O2-binding site that stabilize the heme in the Fe(III) oxidation state eliminate the binding of O2 to the defective subunits -When Fe(II) is converted to Fe(III) we call the protein Methemoglobin (HbM), -Various mutations cause stabilization of the Fe(III) state -Individuals with the mutations have bluish skin from the deoxyHb -All known methemoglobins arise from substitutions that provide the Fe atom with an anionic oxygen atom ligand. -The two here Hb Boston -Substitution of Tyr for His E7-the distal His -the phenolate ion of the mutant Tyr forms of a 5-coordinate Fe(III) complex -This displaces the imidazole ring of His F8 as the apical ligand -Locks the Fe(III) complex in place Hb Milualkee -Glu that replaces Val E11(on the distal side) -Carboxyl group of the Glu forms an ion pair(DOES NOT DISPLACE HIS) with a 5-coordinate Fe(III) complex -This stabilizes the Fe(III) complex

HbS Fibers

-Consequence of the aggregation of deoxyHbS -Causes sickling in erythrocytes -Electron microscopy indicates that these fibers are 220-Å-diameter elliptical rods consisting of 14 hexagonally packed and helically twisting strands of deoxyHbS molecules that associate in parallel pairs -Twisting and packing arrangement similar to amyloid fibrilizarion and aggregation causes the whole cell to change shape

Quantitative Analysis of Ligand Binding

-Consider a process in which a ligand (L) binds reversibly to a site in a protein (P). -This interaction can be described quantitatively by the association rate constant(ka) or the dissociation rate constant (kd) •-After some time, the process will reach the equilibrium where the association and dissociation rates are equal-equations shown -The equilibrium composition is characterized by the equilibrium association constant(Ka) or the equilibrium dissociation constant(Kd) -Typically dissociation constant is of interest in biochemistry

BPG

-D-2,3-bisphosphoglycerate -As well as Carbon dioxide, this independently modulates the binding affinity of Hb to oxygen -When Hb is purified(stripped hemoglobin) it has a much greater affinity for O2 than it does in whole blood -This is because BPG complexes with Hb to reduce its O2 binding affinity in whole blood - Binds deoxyHb but only weakly to oxyHb Curve on the Right: -Removing BPG-Transitions Hb to the R state cause conformational shifts that narrow the binding pocket for BPG -lowers the p50 in stripped Hb -When bound, BPG keeps Hb in deoxy(T) form by stabilizing it, raising the p50 of Hb Curve on the left: -in the absence of BPG, little O2 is released since hemoglobin's overall O2 affinity is increased, thus the O2-dissociation curve is significantly shifted toward lower p50 -The effect of having BPG in solution with blood is essential to the difference in O2 binding with O2 release

T-State

-Deoxy form of Hb -One of the most important changes with the T to R state has to do with the Iron being out of plane with Fe(II) -T state Iron is tethered to the whole protein through the proximal histidine(F8) -The Imidazole ring of the proximal His is oriented such that its direct movement of 0.6 Å toward the heme plane would cause it to collide with the heme, keeping Fe(II) out of the plane

Heme

-Each Hb subunit binds a single one-4 total in hemoglobin -Globin = Hb/Mb without this group -The ring system is a porphyrin derivative and consists of 4 pyrrole rings(A-D) -The central iron atom remains in the Fe(II) oxidation state whether or not oxygen is bound -The Fe atom in deoxygenated Hb and Mb is 5-coordinated by a square pyramid of N atoms: four from the porphyrin and one from a His side chain of the protein -The O2 binds to the Fe(II) on the opposite side of the porphyrin ring from the His ligand -Fe(II) becomes octahedrally coordinated upon oxygen binding-the ligands occupy the six corners of an octahedron centered on the Fe atom -Oxygenation changes the electronic state of the Fe(II)-heme, as is indicated by the color change of blood from the dark purplish hue characteristic of venous blood to the brilliant scarlet color of arterial blood and blood exposed to air(when cut) -Some small molecules (CO, NO, CN-) can coordinate with greater affinity than oxygen, and are therefore toxic -Fe(II) can be oxidized to Fe(III) to form methemoglobin and metmyoglobin -In the Fe(III) state it cannot bind oxygen because it is already octahedrally coordinated -Enzymes can reduce Fe(III) to Fe(II), such as methemoglobin reductase in erythrocytes -Dried blood is oxidized and in the Fe(III) state

HbS gelation.

-Follows an unusual time course in solution and within the cell, follows an unusual time course -A solution of HbS can be brought to conditions under which it will gel: -Lowering the pO2 -Raising the HbS concentration -Raising the temperature -On achieving gelation conditions, there is a reproducible delay that varies according to conditions from milliseconds to days -During this time, no HbS fibers can be detected -Only after the delay do fibers first appear, and gelation is then completed in about half the delay time -At first, HbS molecules sequentially aggregate to form a nucleus after which elongation is rapid -This kinetic hypothesis is why sickle-cell anemia is characterized by episodic crises -If the delay time for sickling is greater than the transit time of the erythrocytes passing through no blood flow blockage occurs -However, small increases in HbS concentration and/or small decreases in HbS solubility can be caused by various conditions: -Dehydration -O2 deprivation -Fever -Once a blockage occurs, the situation can be quickly compounded-symptoms onset quickly -HbS fibers dissolve instantly on oxygenation

Oxygen Binding to Myoglobin

-Fractional saturation(Y): Fraction of oxygen binding sites occupied by oxygen-same as fraction bound(𝜭) -Rearrange for [MbO2] and plug in to get fractional saturation as shown before -Oxygen is a gas, so we can express its concentration by its partial pressure pO2 -Define p50 as the value of pO2 when half of the oxygen binding site sites are occupied(Y=0.5) -If you solve for Kd, you get that Kd=p50 Looking at the graph: -pO2 in lungs is about 13 kPa: it binds oxygen well. -pO2 in tissues is about 4 kPa: Mb will not release it -Shows why myoglobin would not be a good transporter-Has such a high affinity for oxygen(low Kd) it would be way too difficult for oxygen to dissociate, which needs to occur in oxygen transport -Makes it a good storage protein

Alternate oxygen transport and other Globins

-Hemoglobin is not the only way for oxygen transport to occur -The globin superfamily members occur widely in all kingdoms of life -Generally participate in enzymatic and oxygen sensing functions Antarctic icefish: the only adult vertebrates that lack hemoglobin, don't need as much oxygen and blood is cold enough to solublize oxygen Invertebrate Hemoglobins: -Erythrocruorins: invertebrate hemoglobins that are extracellular and up to 3.5 million kDa -Chlorocruorins: found in the blood of certain annelids, have a different porphyrin ring structure making them green when deoxygenated and light red when oxygenated Invertebrates that lack Hb and instead have alternative oxygen transport proteins: -Hemocyanins: found in invertemollusks and arthropods; large extracellular proteins; oxygen binding sites contain Cu ions -Hemerythrins: intracellular proteins found in some marine worms; oxygen binding sites contain non-heme Fe ions Other vertebrate globins: -Neuroglobin: found in brain, retina, and endocrine tissues; protects neurons from hypoxia -Cytoglobin: occurs in most tissues, function unknown

Hemoglobin Cooperativity

-Hemoglobin's fractional saturation curve is closely approx- imated by both the symmetry model and the sequential model - Clearly such curves cannot by themselves be used to differentiate between these two models because either is correct Support of the Symmetry Model: -The functional differences between ⍺ and β subunits can be ignored - The quaternary T→R conformational shift of Hb is concerted as required Supporting the Sequential Model -Ligand binding to the T state does cause small tertiary structural changes as the sequential model predicts -This phenom- enon is evident in the X-ray structure of crystals of human Hb whose ⍺ subunits are fully oxygenated and whose β subunits are unliganded-Hb remains in the T state -Bonds between the porphyrin ring and Fe(II) are 1.5Å closer that in the T state unliganded -Such tertiary structural changes are undoubtedly responsible for the buildup of strain that eventually triggers the T→R transition. -Does the ligand-binding affinity of Hb subunits depend only on Hb's quaternary state (symmetry model) or does this affinity vary with the number of ligands bound to Hb (sequential model)? still unresolved -Evidently, cooperativity arises both from concerted quaternary switching (symmetry model) and from sequential modulation of ligand binding within each quaternary state through ligand-induced alterations in terti- ary structure (sequential model).

Bohr Effect

-In addition to being an O2 carrier, Hb plays an important role in the transport of CO2 by the blood -The pH difference between lungs and metabolic tissues increases efficiency of the O2 transport -This effect explains the mechanism for additional oxygen to be supplied to active muscles -The two reactions involved are matched, so they cause little change in blood pH Most of the CO2 in the blood is carried in the form of bicarbonate, which is generated in the capillaries -The H+ generated is taken up by Hb in the capillaries, resulting in it unloading the bound O2 -THe continuous uptake of H+ in the capillaries moreover pushes the equilibrium towards bicarbonate production and thus facilitates carbon dioxide transport In the Lungs: -Oxygen concentration is high -O2 binding by Hb causes a conformational shift, making the it a slightly better acid -Hb releases H+, which then shifts the equilibrium toward CO2 and it is thus driven off -Highly active muscles generate acid so fast that they lower the pH of the blood from 7.4 to 7.2 -At pH 7.2, Hb releases ~10% more oxygen than at pH 7.4-more cooperative

T→R Conformational Shift

-In the T state, the Fe(II) is situated 0.6 Å out of the heme plane on the side of the proximal His -This is because because the Fe-N porphyrin bonds are too long to allow the Fe to lie in the porphyrin plane -The change in the heme's electronic state upon binding O2 causes the Fe-N porphyrin bonds to contract by ~0.1 Å -The Fe's movement drags the proximal His along with it, which tilts the attached F helix and translates it 1 Å across the heme plane, reorienting the heme and bringing the Fe(II) into the heme plane -The Fe-O2 bond is thereby strengthened because of the relaxation of the steric interference between the O2 and the heme -In addition, in the T-state of the β subunits(not ⍺) Val E11 partially occludes the O2-binding pocket so that it must be moved aside before O2 binding can occur

Carbon Dioxide

-Independently modulate Hb's oxygen binding affinity with BPG -Dtripped Hb can be made to have the same oxygen-dissociation curve as the Hb in whole blood by adding this and BPG in the concentrations found in erythrocytes

Changes at the ⍺-β Interface of Hb

-Likely to interfere with quaternary structure -In Hb this results in an increased O2 affinity that impairs release of O2 to the tissues -Compensation with an increase in erythrocyte concentration causes patients to have a reddish complexion -Various mutations can change the relative stability of the R and T forms thus altering O2 affinity and cooperativity

Oxygen dissociation curve

-Myoglobin's O2-dissociation curve closely follows the hyperbolic curve described by the Y=pO2/(pO2+p50) equation -Oxygen is bound over a wide range and so it would not be a good transporter -Hemoglobin has a sigmoidal curve, which is NOT described by the Y equation -Hemoglobin's sigmoidal O2-dissociation curve is of great physiological importance; it permits the blood to deliver much more O2 to the tissues than it could if Hb had a hyperbolic O2-dissociation curve with the same p50(shown by dotted line) -looking at the dotted line- Hb wont be fully loaded in the lung-only be 70% bound, then released in the tissues-not as big a difference as the sigmoidal curve -much more oxygen can get transported with sigmoidal curve -Binding is cooperative-that is, binding of one molecule affects the binding of the others-in this case, increasing the affinity of Hb for binding additional oxygen -Higher fractional occupancy in the lung and Lower fractional occupancy in the tissue capillaries means that Hb is a good transporter -Thus, the two proteins form a sophisticated O2 transport system that delivers O2 from lung to muscle

Molecular pathology of Hemoglobin

-Over 1000 variant or abnormal hemoglobins which typically have a single amino acid substitution have been analyzed -Location can sometimes explain the physiologic effect of an amino acid substitution Changes in surface residues -Generally these residues are not as important and mutations(as we would expect from their roles in packing) can be tolerated (sickle cell HbS is the major exception) -HbE (GluB8(26)β→Lys) is the most common Hb mutant after HbS and has no known manifestations, even though there is a change from a negative to positive charge -Around half of Hb mutations are of the like Changes in internal residues -Internal mutations often destabilize proteins, having more severe pathology -The structure of Hb is so delicately balanced that small structural changes may render it nonfunctional -Through the weakening of the heme hydrophobic association with the hydrophobic globin pocket -Or a consequence of other conformational changes -Destabilized Hb is degraded and can result in granular precipitates that adhere to erythrocyte cell membranes, making it more prone to lysis -Many other mutations have been identified that destabilize Hb

Oxygen Transport

-Oxygen is picked up in the lungs, binds to hemoglobin -Hemoglobin acts as an oxygen transporter in red blood cells -Oxygen is delivered to the tissues where it is unloaded -Stored by a protein called myoglobin(from the globin gene tree) -Oxygen is then used in respiration -CO2 has the opposite pattern -O2 is poorly soluble in aqueous solutions-needs to be some innovation to allow it to get to tissues -Diffusion is ineffective beyond a few millimeters-some small animals do not need hemoglobin because they use diffusion, in general most animals need hemoglobin -Evolution of large multicellular animals required proteins for O2 transport-required the development of circulatory systems that actively transport O2 and nutrients to the tissues-The blood of such organisms must contain an oxygen transporter such as Hb because it is not soluble enough -No amino acid side chains are well suited for O2 binding -Metals like iron can bind well to O2 -In solution, metal binding to O2 would generate free radicals (which can cause DNA damage) -Organometallic compounds like heme are more suitable, but Fe2+ could be oxidized to Fe3+, which is very reactive Biological solution-evolved: -Capture the O2 molecule with heme(nice oxygen binding) that is protein bound(protects iron from oxidation) -Myoglobin is used for storage and hemoglobin for transport-Both bind O2 via a protein-bound heme

Hill Plots for Hemoglobin(visual)

-Plot the hill equation from hemoglocin -Mb is non-cooperative-has a slope of 1-this no change to the affinity as more ligand binds -For normal human Hb, the Hill constant is between 2.8 and 3.0; that is, hemoglobin oxygen binding is highly, but not infinitely(if n=1), cooperative At Y valuesnear 0,when few Hb molecules have bound even one O2 molecule, the Hill plot of Hb assumes a slope of 1 because the Hb subunits independently compete for O2 as do molecules of Mb -At Y values near 1, when at least three of each of hemoglobin's four O2-binding sites are occupied, the Hill plot also assumes a slope of 1 because the few remaining unoccupied sites are on different molecules and therefore bind O2 independently -Its maximum slope, which occurs near pO2=p50 [(YO2=0.5; YO2/(1 -YO2)=1], is normally taken to be the Hill constant -Middle region is when you can see a change in affinity based on the fraction bound-cooperativity -Extrapolating the lower asymptote to the horizontal axis according to the equation on the right side of the graph, p50 =30 torr (binding the first O2 to Hb) -Extrapolating the upper asymptote yields p50=0.3(binding hemoglobin's fourth O2) -Thus the fourth O2 to bind to Hb does so with 100-fold greater affinity than the first

Erythrocytes

-Red Blood Cells -Hemoglobin is contained inside these -All vertebrates have these except ice fishes-don't need RBC or hemoglobin

Molecular Basis of Sickle-Cell Anemia

-Sickle-cell hemoglobin (HbS) arises from Glu A3(6)β→Val mutation on the surface of the protein -Leads to hemolytic anemia and blood flow blockages due to the irregularly shaped and inflexible erythrocytes -Sickling results from aggregation of deoxyHbS into rigid fibers that extend throughout the cell(fribrilization stretches out the cell) Glu 6β residues are shown as yellow dots - - notice their positioning at the interface The new Val residue fits into this hydrophobic pocket. The WT glutamic acid is negative and would not fit as well in this pocket -Looking at the mutations -Valine only interferes at the β2 subunit -Hydrophibic interaction will cause aggregation The structure of the fibers: -Only one of the two Val 6's per Hb molecule contacts a neighboring mol- ecule. In this contact, the mutant Val side chain occupies a hydrophobic surface pocket on the subunit of an adja- -Note that the only intermolecular association in which the mutant residue Val 6 participates involves subunit β2, not β1 -The mutant Val 6β2 fits neatly into a hydrophobic pocket formed mainly by Phe 85 and Leu 88 of an adjacent β1 subunit -This pocket is absent in oxyHb and is too hydrophobic to contain the WT Glu 6β2 side chain

Binding Affinity

-Smaller dissociation constant correlates with higher binding affinity-this means that a small amount of ligand will occupy the antibody at much lower concentrations because the dissociation constant is so small -Biotin-avitin is an irreversible binding interation-because it is so tight-wouldn't want to think as a typical on/off relationship as having this high of an affinity-not going to dissociate -enzyme-substrate constants are generally a bit weaker -antibodies have higher affinity -Generally, at the 10-6 to 10-8 range-above this is generally a weakly interacting protein

Homotropic Interactions

-Symmetry model of allosterism can be described by the shown equation(do not worry about how to use, just the plot) -Plot the equation for a tetramer (n=4) as a function of α for different values of L and C Recall: -The equilibrium constant is L - if L=1, the T and R states are in equilibrium -⍺ is the normalized ligand concentration -c increases with the ligand-binding affinity of the T state relative to that of the R state- the smaller this value is the tighter the R state binds 3 points can be drawn: 1. The degree of upward curvature exhibited by the initial sections of these sigmoid curves is indicative of their level of cooperativity 2. LEFT GRAPH -c=0, meaning only the R state binds ligand -Cooperativity increases as L increases(T is preferred more-think of like deoxyHb) -For high L values, if a single ligand is to bind, it "forces" the protein into the R state, opening three ligand-binding sites, promoting the binding of subsequent ligands - a positive homotropic effect **Note that cooperativity and ligand binding affinity are different quantities; for c=0, curves indicative of high ligand-binding affinity (low L-more R states) exhibit low cooperativity -EQUILIBRIUM FAVORS THE T STATE IF NO O2 IS BOUND 3. RIGHT GRAPH -L is large, meaning the T state is highly preferred -Cooperativity increases with decreasing c (as the R state's affinity for ligand grows larger relative to the T state) -At low ligand concentrations (⍺) the amount of ligand bound(y axis) increases with the ligand-binding affinity of the T state (increasing c) since the protein is largely in the T state -As ⍺ increases, however, the amount of ligand bound to the R state surpasses that of the T state, thereby resulting in a cooperative effect. -This is because the free energy of ligand binding stabilizes the R state with respect to the T state -R STATE WILL BIND MORE TIGHTLY THAN THE T STATE

Nitric Oxide

-Synthesized in many tissues and functions in signaling, inducing vasodilation (dilation of blood vessels which decreases blood pressure) -Must deliver its message and then be eliminated to terminate the signal because it is highly reactive and toxic -Mb can detoxifies it by converting to nitrate -Mb Fe gets oxidized to Fe(III)-metMb -metMB must then be reduced back to Mb by metmyoglobin reductase

Hemoglobin-Structure

-Tetramer of two αβ(2 alpha, 2 beta) protomers that are symmetrically related -The polypeptide chains of Hb are arranged such that there are extensive interactions between unlike subunits -⍺-β subunit interactions are largely hydrophobic in character, although numerous hydrogen bonds and several ion pairs are also involved -In contrast, contacts between like subunits, ⍺1-⍺2 and β1-β2, are few and largely polar in character -Oxygenation causes extensive structural changes -If deoxy crystals are exposed to oxygen, the crystals shatter -oxygenation rotates the ⍺1-β1 dimer 15° with respect to the ⍺2-β2 dimer -Some atoms at the ⍺1-β2 interface shift by as much as 6 Å relative to each other -The quaternary structural change preserves hemoglobin's exact 2-fold symmetry and takes place entirely across its ⍺-β interfaces -T state (tense-blue): Deoxy-Hb-notice central cavity is larger-where BPG is -R state (relaxed-red): Oxy-Hb, and bound to any other small molecule

Limitations of the Hill Equation and the Adair Equation

-The Hill Equation is based on all-or-none O2 binding-requires that once one binds, all four bind simultaneousy in a concerted manner -Not the actual case because Hb molecules can be partially oxygenated -Dissociation do not have to all be equal for each site -Although this is the most general relationship describing ligand binding to a protein and is widely used to do so, it provides NO PHYSICAL INSIGHT as to why various microscopic dissociation constants differ from each other For a protein such as Hb with 4 Ligand-binding sites, the reaction sequence is as shown: -Ki are the macroscopic or apparent dissociation constants for binding the ith ligand to the protein -Each binding event can be represented by a microscopic or intrinsic dissociation constant, ki -The numerator of the statistical factors in front of the Ki values represent the concentration or number of ligand binding sites not bound- 4 for step 1 -The numerator of the statistical factors in front of the Ki values represents the concentration or number of sites bound in the products of that step- 1 for step 1 -How to Derive the statistical factor is the bottom equation-where n is the number of sites, i is the number ligand being bound

Hill Plot equation

-The Hill constant, n, and the dissociation constant, K, that best describe a saturation curve can be graphically determined by rearranging the hill equation(1.) -Then taking the log of both sides to yield a linear equation(2.) -The linear plot of log[YS/(1-YS)] versus log[S](eq 2.) is the the hill plot equation-has a slope of n and an intercept on the log[S] axis of (log K)/n -Sustitute pO2 for [S] in the hill equation(3.) -K becomes (p50)^n(4.) -Substitute 4. into 5. -Hill plot for Hemoglobin(6.)-has a slope of n and an intercept on the log pO2 axis of log p50

T-State Stability

-The R state is stabilized by ligand binding -In the absence of ligand, the T state must be more stable -In the electron density maps of R-state Hb, the C- terminal residues of each subunit (Arg 141 and His 146) appear as a blur -Suggests that these residues are free to wave about in solution -Maps of the T form, however, show these residues firmly anchored in place via several intersubunit and intrasubunit salt bridges that evidently help stabilize the T state -The structural changes accompanying the T→R transition break the salt bridges in a process driven by the Fe-O2 bonds' energy of formation

Graphical Analysis of Ligand Binding

-The fraction of bound sites(Y) depends on the free ligand concentration and Kd -Experimentally: Ligand concentration is known, and Kd can be determined graphically or via least-squares regression -Graph of ligand concentration(known) versus fraction occupied(Y-measured through some sort of signal) -Visualize the dependency of ligand bound on Kd-at half bound, ligand concentration is Kd

T and R conformations

-The main difference between the T and R conformation occurs in the ⍺1-β2 subunit interface -The quaternary change results in a 6Å relative shift at the ⍺1C-2FG interface -In both conformations, the "knobs" on one subunit mesh nicely with the "grooves" on the other -Any potential intermediates are likely too strained -Hence these contacts, which are joined by different but equivalent sets of hydrogen bonds in the two states act as a binary switch that permits only two stable positions of the subunits relative to each other

Experimentally Testing the Hb Mechanism

-The mechanism is a description of dynamic behavior that is based on STATIC structures of the R and T states -Some aspects have been backed up experimentally The proposed function of the C-terminal salt bridges in stabilizing the T state: -Removal of the C-terminal Arg 141 with carboxypeptidase B followed by reconstitution drastically reduces the cooperativity of O2 binding (Hill constant n=1.7 reduced from n=2.8) -Cooperativity is decreased further by removal of the other C-terminal residue, His 146 (n=1.0) -Human deoxy-Hb with its C-terminal residues removed crystallizes in a form very similar to that of normal human oxyHb(R) -Thus, in the absence of its C-terminal salt bridges, the T form of Hb is unstable Spectroscopic evidence supports Fe-O2 bond tension-measurements through crystallography Detaching the proximal His from the F helix largely eliminates cooperativity -His to Gly mutation cuts off ligand induced conformational changes upon O2 binding -This cuts the covalent bond that links the ligand induced movement of the Fe into the heme plane to the accompanying movement of F helix according to the mechanism -These changes increase affinity for O2 and decrease cooperativity

Hb Cooperativity Mechanism

-The positive cooperativity of O2 binding to Hb arises from the effect of the ligand-binding state of one heme on the ligand-binding affinity of another. -The heme's are 25-37 Å apart-long range interaction-must be a dynamic conformational shift -The protein is transmitting this information through large scale conformational shifts Binding of O2 triggers a series of coordinated movements(small scale to large scale): 1: The Fe(II) cannot move into the plane without reorientation of the proximal His 2: The proximal His is tightly packed and cannot reorient unless the movement is accompanied by movement of the entire F helix 3: The F helix can only move in concert with the quaternary shift that snaps the helices between the T and R state across its ⍺-β interfaces 4: This shift must occur simultaneously between the two ⍺-β interfaces ***No one portion of the protein can change its conformation independently, limiting the mechanism to two quaternary forms, R and T -Interfaces packing of grooves and knobs are both stabilized-either one is allowed, but no in between ***Rationalization of Cooperativity: -Any deoxyHb subunit binding O2 is constrained to remain in the T conformation -T state has reduced O2 affinity because its Fe-O2 bond is stretched beyond its normal length by the steric repulsions between the heme and the O2(and by the need to move Val E11 out of the O2-binding site in the β subunits) -As more O2 is bound to the Hb tetramer, this strain, which derives from the Fe-O2 bond energy, accumulates in the liganded subunits until it is of sufficient strength to snap the molecule into the R conformation -ALL the subunits are thereby converted to the R state whether or not they are liganded -Unliganded subunits in the R state have an increased O2 affinity because they are already in the O2-binding conformation -This is why nearly saturated Hb still has a high affinity for O2

Distal and Proximal His

-The proximal His coordinated with the heme Fe(II), and connects the movements of Fe(II) to the movement of the F helix and the entire Hb structure -The distal His has a pK of 5.5 and is therefore neutral at neutral pH- its unprotonated N atom faces the heme pocket, acting as a proton trap,protecting Fe(II) from oxidation

Free Energies of T and R state

-The relative stabilities of the T and R states vary with fractional saturation -In the absence of ligand, the T state is more stable than the R state, and vice versa when all ligand- binding sites are occupied -The formation of Fe-O2 bonds causes the free energy of both the T and the R states to decrease (become more stable) with oxygenation-although the rate of this decrease is smaller for the T state as a result of the strain that liganding imposes on T-state subunits -The R↔️T transformation is, of course, an equilibrium process, so that Hb molecules, at intermediate levels of fractional saturation (1, 2, or 3 bound O2 molecules), continually interconvert between the R and the T states**** -The O2-binding curve of Hb can be understood as a composite of those of its R and T states -For pure states, such as R or T, these curves are hyperbolic because ligand binding at one protomer is unaffected by the state of other protomers in the absence of a quaternary structural change -At low pO2's, Hb follows the low-affinity T-state curve -At high pO2's, it follows the high-affinity R-state curve -At intermediate pO2's, Hb exhibits an O2 affinity that changes from T-like to R-like as pO2 increases -The switchover results in the sigmoidal shape of hemoglobin's O2-binding curve

Heterotropic Interactions

-The symmetry model of allosterism is also capable of accounting for heterotropic effects -This comes about by assuming that each protomer has specific and independent binding sites for the three types of ligands: 1. A substrate, S, that we assume binds only to the R state (c=0) 2. An activator, A, that only to the R state 3. An inhibitor, I, that binds only to the T state -Note that the equation for this interaction differs from the homotropic Equation for c=0 only in that the second term in the denominator is modulated by terms related to the amounts of activator and inhibitor bound to the oligomer Consequences of effector binding to a tetramer: 1. Activator binding (𝛾 > 0) INCREASES the concentration of the substrate-binding R state (the second term in the denominator decreases) because it is the only state capable of binding activator -INCREASES the protein's substrate-binding affinity (a positive heterotropic effect) -DECREASES the protein's degree of substrate-binding cooperativity (compare Curves 1 and 2 in Fig. 10-31). 2. The presence of an inhibitor (β>0), ,INCREASES the concentration of the T state (the second term in the denominator increases)because it is the only state capable of binding inhibitor -DECREASES the protein's substrate-binding affinity(a negative heterotropic effect) -INCREASES increases the cooperativity of substrate binding as well as that for activator binding -this is because the substrate must "work harder" to convert the oligomer to the substrate-binding R state

Allosteric Interactions

-These cooperative interactions occur when the binding of one ligand at a specific site is influenced by the binding of another ligand, known as an effector or modulator, at a different (allosteric) site on the protein -Homotropic effect: when the ligands are identical -Heterotropic effect: when the ligands are different (allows for activators and inhibitors) -These interactions can be positive or negative, depending on whether the effector increases or decreases the protein's ligand-binding affinity -Allosteric effects generally result from interactions among subunits of oligomeric proteins-can also be one large protein, but typically apply to only multi-subunit proteins -Using these terms, The effect of O2 on hemoglobin is said to be positively homotropic -Carbon dioxide, cyanide, and BPG are negatively heterotropic -Even though hemoglobin catalyzes no chemical reaction, it binds ligands in the same manner as do enzymes -Since an enzyme cannot catalyze a reaction until after it has bound its substrate(s), the enzyme's catalytic rate varies with its substrate binding affinity -Consequently, the cooperative binding of O2 to Hb is taken as a model for the allosteric regulation of enzyme activity

High Altitude Adaptation

-This is a complex physiological process that involves an increase in the amount of hemoglobin per erythrocyte and in the number of erythrocytes -Normally takes several weeks to complete, but even a 1-day stay in high altitude results in a noticeable degree of adaptation Curve on the right: -This results from an Increased BPG levels inside erythrocytes, decreasing overall O2 binding affinity(p50) and thus an increased amount of O2 that Hb unloads into the capillaries Curve on the left: -Between the sea level arterial and venous pO2 values, Hb normally unloads 38% of the O2 it can maximally carry -However, when the arterial pO2 drops to 55 torr, as it does at an altitude of 4500 m, this difference is reduced to 30% in nonadapted blood -High-altitude adaptation increases the BPG concentration in erythrocytes, which shifts the O2-dissociation curve of Hb to the right, dropping the -The amount of O2 that Hb delivers to the tissues is thereby restored to 37% of its maximum load.

Picket Fence Fe(II) Porphyrin

-This is one of the reasons why cooperatively happens at a molecular scale -Globin not only modulates the O2-binding affinity of heme, but makes reversible O2 binding possible -Fe(II)-heme by itself is incapable of binding O2 reversibly -In the presence of O2 it autooxidizes irreversibly to the Fe(III) form through the intermediate formation of a complex consisting of an O2 bridging the Fe atoms of two hemes(top left) -To prove that the globin is needed for reversibility, these molecules were synthesized -The oxidation reaction can be inhibited by derivatizing the heme with bulky groups that sterically prevent the close approach of two hemes and hemes with other molecules(such as CO) -These can bind O2 reversibly -Globins are thought to function similarly- they surround the heme preventing exposure to solvent, allowing reversibility

Cooperitivity and the Hill Equation

-When n=number of subunits, the assumption of the hill equation is that a protein has all or none of its sites occupied, so it will give up all 4 oxygen at once -This is is a physical impossibility -Nevertheless, n may be taken to be a noninteger parameter related to the degree of cooperativity among interacting ligand-binding sites rather than the number of subunits per protein -The Hill equation then becomes a useful empirical curve-fitting relationship rather than an indicator of a particular model of ligand binding -The quantity n, the Hill constant, increases with the degree of cooperativity of a reaction and thereby provides a simple characterization of a ligand-binding reaction -If n=1, the hill equation describes a hyperbola, as for Mb, and the ligand-binding reaction is said to be noncooperative -A reaction with n>1 is described as positively coop- erative: Ligand binding increases the affinity of E for further ligand binding (cooperativity is infinite in the limit that n is equal to the number of ligand-binding sites in E) -Conversely, if n<1, the reaction is termed negatively cooperative: Ligand binding reduces the affinity of E for sub- sequent ligand binding. -Sigmoidal for n=4-positive cooperativity

Antarctic Icefish

-the only adult vertebrates lacking hemoglobin because they live in the cold, making their blood colorless -At such low temperatures they have a lower need for oxygen, which is more soluble at low temperatures -It is very energy expensive to make RBCs and hemoglobin, so through evolution the need to produce them was lost

Theraputics for Sickle-Cell Anemia

1. Gene therapy -Could be highly promising but not yet a viable therapy 2. Compounds that bind and disrupt intermolecular interactions leading to fiber formation-small molecule interference -The body contains a large amount of Hb (~400g) so doses would need to be quite high and toxicity becomes a problem at high doses 3. Increase affinity of Hb for O2 -Cyanate can carbamoylate the N-terminal amino groups eliminating some salt bridges that stabilize the T state, increasing affinity of Hb for O2 -Works in vitro but cyanate is toxic-can be done in the lab, but not a great therapeautic -Idea is good though-maybe there is something that works a similar way that is less toxic 4. Lowering HbS concentration in erythrocytes-most viable strategy -Alter erythrocyte membrane permeability to promote influx of water -Looked at the most out of these

Pitfalls of the Symmetry Model

1. The symmetry model does not explain all situations-oligomeric symmetry is not always preserved (hybrid states of some T and R states are possible) 2. Negative homotropic effects are known(Binding of ATP to the cis ring of GroEL prevents ATP from binding to the trans ring), but the symmetry model only allows for positive homotropic effects 3. The symmetry model implicitly assumes the "lock-and-key" model of ligand binding in which ligand-binding sites of proteins are rigid, pre-formed and complementary in shape to their ligand -A more sophisticated extension of the lock-and-key model, known as the induced-fit hypothesis, postulates that a flexible interaction between ligand and protein induces a conformational change in the protein, which results in its increased ligand- binding affinity -In the resulting sequential model (alternatively, the induced-fit or KNF model), ligand binding induces a confor- mational change in a subunit; cooperative interactions arise through the influence that these conformational changes have on neighboring subunits

DoubleNucleation Process for HbS

2 steps in the process 1. Initial homogeneous nucleation process -(Taking place in solution) accounts for very high concentration dependence -In this phase, aggregation of HbS molecules (spheres) occurs very slowly because this process is thermodynamically unfavorable Hence the intermediates tend to decompose rather than grow -Once an aggregate reaches a certain critical nucleus, further growth becomes thermodynamically favorable, leading to rapid fiber formation 2. Secondary heterogeneous nucleation process -(Taking place on a surface of a fiber) is responsible for the rapid onset of gelation -Each fiber can nucleate the growth of other fibers, leading to the explosive appearance of polymer

The Symmetry Model

The best model for describing cooperative ligand binding to a protein, aka MWC model 4 rules: 1. An allosteric protein is an oligomer of protomers that are symmetrically related (for hemoglobin, assume all 4 subunits are functionally identical) 2. Each protomer can exist in (at least) two conformational states(T and for Hb); these states are in equilibrium whether or not ligand is bound to the oligomer 3. The ligand can bind to a protomer in either conformation. Only the conformational change alters the affinity of a protomer for the ligand 4.The molecular symmetry of the protein is conserved during conformational change. Protomers must therefore change conformation in a concerted manner-the conformation of each protomer is constrained by its association with the other protomers-there are no oligomers that simultaneously contain R- and T-state protomers Equations can be used to describe the model: -The species and reactions permitted under this model of allosterism are shown - -The equilibrium constant L for the conformational interconversion of the oligomeric protein in the absence of ligand -⍺ may be considered a normalized ligand concentration -c is the ratio of the ligand-binding dissociation constants; c increases with the ligand-binding affinity of the T state relative to that of the R state.