Hemoglobin: A Paradigm for Cooperativity and Allosteric Regulation

Why do we breathe? http://www.uni.edu/schneidj/webquests/spring04/tvbroadcast/circulatorysystem.html

Cellular Requirement for O2

Diffusion Limited solubility of O2 in Blood and Cell Water Oxygen Carriers Diffusion Limited solubility of O2 in Blood and Cell Water

Myoglobin and Hemoglobin Myoglobin (Mb) Increases O2 solubility in tissues (muscle) Facilitates O2 diffusion Stores O2 in tissues Hemoglobin (Hb) Transports O2 from lungs to peripheral tissues (erythrocytes)

Oxygen Transport HB: Oxygen Transport Mb: Oxygen Diffusion

Myoglobin Small Intracellular Protein in Vertebrate Muscle

Function(s) of Myoglobin Facilitate O2 Diffusion in Muscle O2 Storage (aquatic mammals) Aquatic mammals have 10x more Mb then terrestrial mammals

Structure of Sperm Whale Myoglobin Eight helices (A - H) Heme prosthetic group Connect  helices by loops Figure 7-1

The Heme Prosthetic Group Tightly bound Synthesized separately Porphyrin Four pyrrole groups (A - D) Methene bridges Figure 7-2

Properties of Heme Prosthetic Group in Myoglobin Tightly bound Synthesized separately from myoglobin Fe2+ Coordination Nitrogens of heme (4) His (F8): proximal histidine His (E7): distal histidine Ligands: O2, CO, and NO

Small molecules that bind to proteins by non-covalent interactions Ligands Small molecules that bind to proteins by non-covalent interactions (e.g. O2 to myoglobin)

Ligand Binding usually transient and reversible interaction with others molecule (= ligands) such as metals, hormones often involves “molecular breathing” of the protein, i.e. ability to undergo small conformational changes often induces molecular rearrangements in the protein ligand binding sites are - highly conserved - complementary in size, shape, and charge Transient/reversible: important: ligand has to come off again, evolution did not select the strongest ia possible, but for one that makes sense physiologically; evolution would not select a Dodge RAM for inner city traffic breathing; transient opening of binding sites rearrangements: hormon binds receptor on cell surface -> changes conformation on the inside -> partner proteins sense “new” conformation -> signal transduction

Heme prosthetic (permanent, non-proteinaceous) group of Mb and Hb incorporated into Hb and Mb during folding responsible for reversible O2 binding responsible for red color of blood and muscles prosthetic group: permanent non-protein component of a protein lipoproteins: contain lipids glycoproteins: contain sugars metalloproteins: contain metals hemoproteins: contain heme different from cofactors, which are also non-protein components that are, however, not permanently associated

Heme – Structure central Fe2+ 4 methyl groups 2 vinyl groups (buried in protein) Vinyl group face interior of protein, propionate group face outside 2 propionate groups (exposed)

Heme – Iron Coordination Fe2+ has 6 coordination sites 4 with N of pyrrole rings, 2 perpendicular to ring Mb/Hb: 5th coordination site is occupied with proximal His 6th coordination site: O2 oxyhemoglobin none deoxyhemoglobin CO carboxyhemoglobin

Heme – Binding of CO vs. O2 free heme binds C0 105 times better than O2 kinked binding topology in Mb/Hb favors O2 (100-fold) TOTAL: CO binding ~ 230 fold stronger than O2 binding (Carbon monoxide poisoning) Proximal His F8 – α-helices in Hb are named sequentially with letter, 8 residue in helix F Oxygen molecule is sandwiched between heme iron and his E7 Distal His E7 can only make H-bond to O2/CO when kinked, prevents sterically linear configuration

Function(s) of Myoglobin Facilitate O2 Diffusion in Muscle O2 Storage (aquatic mammals)

Myoglobin (Mb) primarily found in muscle (highly abundant in marine mammals such as whales) single polypeptide (153 aa) with one bound heme very simple oxygen binder: binds oxygen at high pO2, releases it at low pO2 Mb + O2 MbO2 typical globin fold Crystallized Mb from spermwhale, because aquatic mammals have 10x more Mb

MCDB310 – Chapter 5: Protein Function The Globin Fold Right: Heme sandwiched between proximal and distal His Propionate groups face outside Helices labeled A-H, amino acids have 2 numbering schemes, # in primary sequence, but more commonly by location in helix, SO his E7 or His F8 8 helices (A-H) and loops in between MCDB310 – Chapter 5: Protein Function

Myoglobin – Oxygen Binding Curve When we want to interpret this curve, we have to learn more about ligand binding curves in general

Binding/Association Constant Ka Quantitatively describes the affinity of a protein P for its ligand L P + L PL Assumption: every protein molecule can bind max. 1 ligand the higher the binding affinity, the higher Ka

Dissociation Constant Kd P + L PL the higher the binding affinity, the smaller Kd Notice the different units! Ka has some advantages when doing the math (which can get very complex!) Example: Ka = 106 M-1  Kd = 10-6 M

Degree of Saturation,  0    1 Fraction of binding sites that are occupied by ligand at any given ligand concentration Right eqn only valid for proteins with 1 binding site 0    1

Degree of Saturation,  If [L] = Kd   = 0.5 Using  If we divide denominator and numerator by Ka => Kd form If [L] = Kd   = 0.5 Kd is the ligand concentration at which 50% of the binding sites are occupied

Ligand Binding Curve

Some Examples with KD = 1 µM Question: What fraction of the protein has ligand bound when the [L] is 1 µM or 10 µM? [L] = 1 µM: [L] = 10 µM:

Some Examples for Dissociation Constants Avidin/biotin is among the strongest interaction Molecular evolution was not to select for strongest binding, but for appropriate binding (release must be possible) Insulin Rz: insulin conc. In blood is very low

Myoglobin – Oxygen Binding Curve Revisited When ligand is a gas, partial pressures = concentrations Kd is replaced with partial O2 pressure at which 50% of the sites are saturated

Myoglobin – Oxygen Binding Curve Revisited Saturation of Mb depends on the binding constant of Mb for O2 (KD = p50 = 2.8 torr) the concentration of O2 (pO2) Question: What is the fractional saturation of Mb? pO2 = 1 torr: pO2 = 10 torr: Pressure units: 1 atm = 758 torr = 101 kPa Tissue: 20-40 torr = 4 kPa lungs: 95-105 torr = 13 kPa

Myoglobin – An Oxygen Storage! pO2 in lung ~ 13 kPa pO2 in tissue ~ 4 kPa Curve tells us that Mb binds O2 tightly under both conditions 10 kPa = 76 torr

Hemoglobin (22)

Hemoglobin (Hb) present in erythrocytes (makes blood look red, 34% of weight is Hb) Different Hb subtypes: Hb A (adult): two  (141 aa) and two  (146 aa) subunits that are arranged as a pair of identical  subunits (2  subunits) Hb F (fetal): two  and two  chains

Hemoglobin – 3D Structure a2 b1 Individual subunits resemble mb a1 b2

Each subunit has 1 heme, which binds 1 O2 Each subunit contains one prosthetic group called heme, so it is permanently associated with the protein, and tighly bound to it, but not covalently bound, The heme is bound by interactions with nearby amino acid side chains. It has 1 Fe atom, which where the oxygen binds. 1 molecule of oxygen can bind to 1 heme, so 1 hemoglobin can bind 4 oxygen molecules Heme Lehninger, Figure 7-5, 7-6

Hemoglobin Erythrocytes: 1 ml blood: 5 x 109 erythrocytes 1 erythrocyte: 3 x 108 Hb molecules Hb is a good marker for number of red blood cells Homology: 50% of AA are identical between  and  subunits 20% of AA are identical between / and Mb

Function of Hemoglobin O2 binding in lungs O2 release in tissues

Oxygen Transport

Oxygen binds to hemoglobin and myoglobin differently We can measure the binding of oxygen to Hemoglobin as a function of the concentration of oxygen in its environment. The concentration of gases in solution is its partial pressure, or in this case pO2. And binding is measured as the fractional saturation, or the fraction of hemoglobin binding sites occupied by O2. So a theta of 1 means all 4 subunits of every hemoglobin molecule have a bound oxygen. The amount of oxygen in the environment determines how much oxygen is bound to Hb.

Oxygen binding to hemoglobin pO2 = partial pressure of oxygen Θ = fraction of binding sites that are occupied We can measure the binding of oxygen to Hemoglobin as a function of the concentration of oxygen in its environment. The concentration of gases in solution is its partial pressure, or in this case pO2. And binding is measured as the fractional saturation, or the fraction of hemoglobin binding sites occupied by O2. So a theta of 1 means all 4 subunits of every hemoglobin molecule have a bound oxygen. The amount of oxygen in the environment determines how much oxygen is bound to Hb.

p50 is the pO2 where half the binding sites are occupied When talking about ligand binding to a protein (or the specific case of substrates binding to enzymes, which we will look at a lot later) a useful characteristic to talk about is this midpoint of the curve. The point where half protien has ligand bound, since we are talking about Oxygen and pressure this is called the p50. This is generally a fairly steep part of the graph, and physiologically this means that slight changes in pO2 can lead to large changes in oxygen binding.

Hb has evolved to transport O2 In Tissues pO2 In Lungs 38% p50 Hb has evolved so that this point of the curve is poised where it needs to be to function most efficiently. The role of hemoglobin is to bind Oxygen in the lungs and release it in the tissues. Hb has a sigmoidal binding curve, and this shape allows for almost 100% bidning at high PO2, like in the lungs, and significant release at lower pO2 like in the tissues,. Generally about 38% of O2 bound in the lungs is released in the tissues The curve is sigmoidal because of cooperativity bewteen the subunits in Hb, this means binding of 1 oxygen molecule to 1 subunit makes it more likely that another oxygen molecule will bind to another subunit. In Hb the 4th O2 molecule is bound with ~100fold higher affinity than the 1st

Hb gains cooperativity by switching between 2 states T state (Low Affinity) R state (high affinity) For a protein to exhibit cooperativity it must exist in at least 2 states. Hemoglobin has 2 states, a low affinity Tense state, the T state; and a high affinity Relaxed state, or R state. T state is characterized by more stabilizing interactions within the protein, While the R state has fewer stabilizing interactions and is more compact Lehninger Figure 7-10

All or nothing mechanism The Concerted Model All or nothing mechanism T R There are two models prposed to try toexplain cooperativity. The concerted model for cooperativity says that all the subunits in the protein must be in the same state. The Hb tetramers would exist with all subunits in either the T or the R state. The ligand can bind to both states, but with different affinity. As more ligands bind the high affinity state is more likely to form. So while all these states are possible… Lehninger, Figure 7-14

All or nothing mechanism The Concerted Model All or nothing mechanism T R … These (T with less than 2 ligands, R with more than 2 ligands) are more likely to form. Lehninger, Figure 7-14

The Sequential Model The other model is the sequential model. Here each subunit in a protein can be in either of the available states, which immediately allows for many more conformations. Additionally it suggests that the binding of a ligand to one subunit changes the conformation of that subunit and influences the subunits next to it. So that as more ligands are bound more ligands are likely to bind. The concept of cooperativity is that once a ligand is bound more ligands are likely to bind, so both models accuont for that. In the concerted model it is because the high affinity state is more likely to form, and in the sequential model it is due to ligand induced conformational changes. It is difficult to say which model is correct for Hb as they both account for observations that the other does not.

Hb follows a little of both R For example, in hemoglobin there are elements of each in what is known about its cooperativity. All the subunits in Hb are either in the T state or the R state, like in the concerted model. Hemoglobin is in the T state when oxygen is not bound. Oxygen binding to one subunit causes local changes (like in the sequential model). Once at least 2 oxygens have bound to one hemoglobin protein there is enough strain from those changes that all 4 subunits shift to the R state in a concerted manner. Since the T state has such a low affinity 2 Oxygen molecules bound can only happen at relatively high Po2, like in the lungs. And once the empty subunts are in the R state they will quickly bind O2. Lehninger, Figure 7-14

Movements of the Heme and the F Helix During the T —> R Transition Figure 7-8

Local structural changes around the Heme are communicated to the rest of Hb Here we are looking at the side view of the heme group in one subunit of Hb. When oxygen binds to the iron it pulls the it further into the plane of the heme, pulling the protein with it. For this to happen the helices near the heme have to shift These changes pull of the rest of the protein and eventually there is a rearrangement. 2nd movie: Here we are looking at a top view of the entire protein. These changes in the helices we saw in the first movie are communicated through the rest of the individual monomer and then to the rest of the protein, as these subunits shift relative to each other, they make the whole protein more compact, shrinking the central cavity. And that the majority of the shift is along the a1-B2 or a2-B1 interface. By Janet Iwasa, https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html

Changes in the 1–2 Interface during the T —> R Transition in Hemoglobin Lets take a closer look at the local interactions that that are changing Figure 7-9

Changes in the 1–2 Interface during the T —> R Transition in Hemoglobin Figure 7-9 part 1

Changes in the 1–2 Interface during the T —> R Transition in Hemoglobin Figure 7-9 part 2

Networks of Ion Pairs and Hydrogen Bonds in Deoxyhemoglobin Figure 7-10

T vs R State (1) Change at interface between b1a2 and b2a1 (2) R state is more compact, and relaxed (3) T state has additional salt bridges, which makes it more tense (4) In R state individual O2 sites have higher affinity for O2. - better Fe-O2 bond length - fewer steric repulsions associated with oxygen binding.

Without cooperativity Hb could not efficiently transport oxygen Tissues Lungs Hb has cooperativity, but how important is it really?? Let look at what would happen is Hb didn’t switch between the T and the R state All T state too low binding in lungs All R state not enough release in tissues Thus, cooperativity or the shift from the R to the T state allowes for efficient binding of oxygen when its needed and efficient release of oxygen when that’s needed. T state

Allosteric regulation of protein function homotropic, positive (= cooperative binding) Cooperativity is a special case of allosteric regulation of protein function. Hb coop. is an example of positive regulatin because oxygen increases the binding affinity of HB for more oxygen. It is a homotropic regulator because it is the actual ligand that is the regulating binding. Ligand does not fit well in site (low affinity, T-state), binding of ligand in one site induces conf. change in empty site -> high affinity

Allosteric regulation of protein function homotropic, positive (= cooperative binding) There is also heterotropic regulation, where a molecule other than the ligand or substrate binds the the protein to regulate function. and both of regulators, either heterotropic or homotropic, can also be negative regulators. We talked about Hb binding oxygen, and it actually needs a heterotropic negative regulator, or it couldn’t function properly. Ligand fits well into site -> high affinity, modulator induces conf. change that closes pocket -> low affinity heterotropic, negative

The Bohr Effect Hb also binds and transports H+ and CO2 from tissue to lungs and kidneys for secretion H+ and CO2 are negative, heterotropic modulators of Hb metabolizing tissue: H+ and CO2 accumulate  bind to Hb and lower the affinity of Hb for O2  Hb releases O2 lungs: CO2 and H+ dissociate from Hb  increases the affinity of Hb for O2  Hb binds O2 increase the efficiency of Hb as O2 transporter

The Bohr Effect Lungs: pO2 = 100 torr, high pH (7.6), low [CO2]  Hb has high affinity for O2 Tissue: pO2 = 20 torr, low pH (7.2), high [CO2]  Hb has low affinity for O2 CO2 + H2O HCO3- + H+ Lungs: CO2 in blood is in equilibrium with low pCO2 in lungs, bicarbonate -> Co2d -> CO2 (g), reactions consumes protons -> pH increases CO2 + H2O HCO3- + H+

pH Dependence of O2 Binding to Hb Bohr effect

Mechanism of Bohr Effect Protonation of His-146  His-146+ forms salt bridge with nearby Asp-94  stabilizes low affinity T-state  O2 is released as pH drops MCDB310 – Chapter 5: Protein Function

Roles of Hemoglobin and Myoglobin in O2 and CO2 Transport Figure 7-12

Heterotropic Negative Modulator This regulator is is 2,3-bisphosphoglycerate. This is a small molecule that is present in very high concentrations in our red blood cells, all the time. Or: 2,3-Diphosphoglycerate (DPG)

BPG is negatively charged It is a highly negatively charged molecule

BPG binds to the central cavity of Hb And binds to the central cavity of the T state of hemoglobin, stabilizing the low affinity state.

BPG binds to the positively charged central cavity of Hb This is a zoom in of the central cavity with BPG bound Since BPG is such a highly negatively charged molecule the binding site has to be a positively charged environment. This is acheived by positively charged side chains projecting into the cavity to bind BPG. BPG binding stabilizes the T-state, or low affinity state of HB, decreasing oxygen binding. When the oxygen concentration gets high, O2 will still bind shifting Hb to the R state. The protein compacts and the central cavity becomes too small, kicking out BPG By Janet Iwasa, https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html

BPG allows for release of O2 pO2 In Tissues pO2 In Lungs At Sea Level No BPG 5mM BPG The normal binding curve for Hb that we have been looking at is in the presence of 5mM BPG, the physiological conentration of BPG in red blood cells. If there were no BPg the binding curve would look more like this purple curve. Does that look like it could efficiently transport oxygen to tissues?? So the presence of BPG shifts the binding curve and the p50 so that Hb releases O2 in the tissues.

In Class Activity: Lets start applying some of those ideas in our in class activities. You go Skiing in Mammoth this weekend, you get to the top of the ski lift at 10,000 ft and one of your gloves blows away and you have to run after it, what happens to your breathing? A little more difficult to catch your breath?

Ligand Binding can affect Protein Function Cooperativity 1 ligand bound = higher affinity for more ligands Concerted vs Sequential Allosteric regulation 1 regulator binding affects binding of ligand Homotropic vs heterotropic Positive vs Negative Today we have discussed cooperativity, and looked at 2 models of how that could be accomplished, and how actual proteins might do something in the middle. We also looked at allosteric regulation and the general ways that ligand or substrate binding can be modulated by other molecules.

From Protein Structure to Function Hemoglobin and myoglobin: Principles of reversible ligand binding (Antibodies: Principles of specific, high affinity ligand binding) Myosin and actin: Protein activity modulated by ATP Enzymes Today we will cover only 1: introduce the concepts of ligand binding, discuss structural and physical, physiological 2 is not part of the syllabus 3 can be covered in self-study

Hemoglobin Variants Table 7-1

Sickle Cell anemia Glu ——> Val (residue 6 of -chain) Leads to hydrophobic interactions between hemoglobin molecules Hemoglobin fibers Sickling of erythrocytes Increased resistance to malaria

Normal Erythrocytes Figure 7-17a

Sickled Erythrocytes Figure 7-17b

Correspondence between Malaria and Sickle-Cell Gene Figure 7-20