Workshop: Oxygen-Binding Proteins Structure & Function (PDF)

Insight into the Structure & Function of Oxygen-Binding Proteins Dr. Oladayo Folasire Faculty of Health Sciences & Medicine Oxygen binds to Fe2+ atom of haeme...

Insight into the Structure & Function of Oxygen-Binding Proteins Dr. Oladayo Folasire Faculty of Health Sciences & Medicine Oxygen binds to Fe2+ atom of haeme group within each Level 3, room 3_16 subunit of adult haemoglobin (HbA) [email protected] Learning Objectives Understand the key structural features of proteins, myoglobin (Mb) and haemoglobin (Hb), that enable O2 to bind. Explain the structural differences between Mb and Hb that enable their roles in storage and transport respectively. Describe the term, co-operative binding, as it relates to the transition from deoxyHb to oxyHb. Describe the term, allosteric regulation, and the role of H+(Bohr effect)., CO2, CO, 2,3-BPG in effecting changes in the affinity of Hb to O2 Interpret O2-dissociation curves (ODCs) and predict the direction of shift in the curve in response to altered environmental conditions. Understand how genetic differences between normal adult (HbA), foetal (HbF) and sickle cell (HbS) proteins impact on their ability to bind O2. Explain the clinical consequences of carbon monoxide poisoning and sickle cell anaemia (HbS). Explain how Hb can help regulate blood pressure.  The Basics of Understanding Oxygen Protein Structure Dissociation Curves Structure Sickle Cell ∝ Function Oxygen-Binding Anaemia Proteins Hb vs Mb How foetal Hb structure works Small molecules affect O2 uptake/offload Introduction … A Requirement for O2 Clinicians are concerned with optimizing the delivery of blood (& O2) to tissues as a means of maintaining homeostasis & promoting healing. O2 is poorly soluble in blood thus, to meet demand, mammals have evolved O2-binding proteins with a special prosthetic group (haeme) to bind iron (Fe2+)* which binds O2  Myoglobin (Mb) is found with muscle and stores O2  Haemoglobin (Hb) is found within erythrocytes & transports O2 By binding to Hb, we achieve a ~60-fold increase in O2-saturation (compared to dissolved O2) Note: Free ferrous ion (Fe2+) is toxic to cells yet it’s this form of iron that is able to bind O2 whereas Fe3+ cannot bind O2  Measuring O2 Content in Blood The arterial oxygen saturation (S ) is expressed An aO2 oximeter as a % (represents the overall % of Hb binding sites occupied by O2 ) In healthy individuals at sea level, SaO2 is ~96%- 98%. The maximum amount of O2 the blood can carry Measuring the amount when fully saturated is of O2 is performed termed in 2 ways: the O2 carrying capacity,  which, (less Pulse oximeter withaccurate) a normalMeasures haemoglobin the pO2 in arterial blood concentration, is ~20 of & gives an indication mL oxygen how much per O2 is100 mL in arterial blood. present blood. Nb. Unable to differentiate dyshaemoglobins (COHb/MetHb).  CO Oximeter – (more accurate) Measures of the SaO2 in arterial blood - able to differentiate oxyHb and dyshaemoglobins (COHb/MetHb). Best for critical care.  home Protein Structure (a hierarchy) Proteins are composed of a sequence of amino acids linked by peptide bonds (primary structure). Short sequences of amino acids form into stable elements such as 1o a-helices, b-sheets, b-turns etc (secondary structure) A single polypeptide chain of a globular proteins (eg myoglobin) folds up into a 2o 3D shape. (tertiary structure) For multi-subunit proteins (eg haemoglobin), each subunit associates with its 3o neighbours through mainly non-covalent forces to form a stable complex (quaternary structure) 4o A protein’s surface reflects its environment. Aqueous (eg blood) – protein surface consists of polar amino acids Lipid (non-polar) environment (e.g. membrane proteins)  Myoglobin - O2 storage Found primarily in muscle tissue. Single polypeptide chain (153 amino acids) Globular protein with 8 a-helical segments, joined by bends. Contains one haeme prosthetic group, binds one O2 molecule Myoglobin has a high affinity for O2 which makes it a good storage molecule. Mb has a hyperbolic-shaped O2 dissociation  What is an Oxygen Dissociation Curve? A graph showing the relationship between the amount of O (saturation) 2 bound to Mb or Hb versus pO2. Below is the ODC for Myoglobin - Mb is almost completely saturated at high pO2 which stays bound unless the pO2 gets very low. This is great for a STORAGE protein! lungs  13 kPa Notice tissues  the hyperbolic shape O2 sat.  Mb has high affinity for O2 Hyperbolic ODC of Mb Question: Refer to the %saturation for Mb at pO2 of tissues and lungs, can you determine why Mb would be a poor transporter?  home Mb has High Affinity for O2 & a low KD Reversible binding of O2 to Mb is described by the simple K equilibrium: Protein + ligand K A Protein-ligand complex D [ 𝑝𝑟𝑜𝑡𝑒𝑖𝑛− 𝑙𝑖𝑔𝑎𝑛𝑑 𝑐𝑜𝑚𝑝𝑙𝑒𝑥] 𝐾 𝐴= and [ 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 ] [ 𝑙𝑖𝑔𝑎𝑛𝑑 ] KD (dissociation constant) is a measure of affinity of a protein to its ligand. Note the inverse relationship (ie a small KD reflects a high affinity) tissues lungs  13 kPa Mb K has high affinity for O2 so has a low KD. D also has a practical meaning: - KD = pO2 at which 50% the binding sites are θ= 𝑝 𝑂2 filled with O2 (P50) 𝑝 𝑂2 + 𝐾 𝐷 KD* 0.26 kPa 𝑇h𝑒𝑡𝑎=𝑂 2 𝑠𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 Haemoglobin (Hb) – O2 Transport Hb has 4 subunits (tetramer), each of which is very similar in 3D to b- subunit of Mb Hb has 2 identical a-subunits and 2 identical b-subunits (a2b2) a-subunit is 141 amino acid long, b-subunit is 146 amino acid long 1 haeme and 1 Fe2+/subunit Hb binds up to 4 O2 molecules. O2 One subunit Hb is better at transporting O2 and is designed to be sensitive to small changes in the pO2  Structure of Haeme Haeme is composed of a porphyrin ring covalently bound in a deep pocket in each Hb subunit (protect the Fe2+!) Fe2+ _ ion in centre. Fe2+ makes 6 covalent bonds. 4 to the planar haeme ring, one to a Histidine amino acid of Hb and one to O2. O2 Haeme has a concave shape in deoxy (T) state Whereas it has a flat, planar shape in oxy (R) state  Colour of Oxygenated & Deoxygenated Haemoglobin in Blood When O2 binds the electronic configuration in haeme complex changes turning from darker red (deoxyHb) to scarlet red (oxyHb). This underpins how the pulse oximeter works But also reflects molecular changes in Hb that affect O2 binding. Visible absorption spectra of Oxygenated & Deoxygenated Haemoglobin Fe2+- O2 Fe2+- empty site Curious about blue blood ? In fair skin people, blood in veins * MetHb is brown appears blue because shorter l blue light doesn’t penetrate as far (colour of old meat!) through skin so is reflected back. Fe - OH2 3+  home Key Principles of Protein Structure/Function Reversible, non-covalent binding of small molecules (ligands) to proteins occurs at sites on the protein called binding sites. The binding site is complementary to the ligand in size, shape, charge & polarity (specificity) Proteins (& ligands) are mobile (not rigid) & so can wiggle around for the best fit. Ligand binding is accompanied by a conformational change in the protein (&/or ligand) which can facilitate and impact on its function. E.g.  Binding of H+ to haemoglobin causes a conformational change leading to off- loading of O2 at tissues. Proteins with high affinity for a ligand bind it more tightly & don’t release it as easily.  Mb has high affinity for O2.  DeoxyHb has a low affinity for O2 whereas OxyHb has a high affinity for O. Cooperative Binding in Hb changes its Affinity towards O2 Hb responds to its environment such as changes in partial pressure of O2. In the lungs, where pO2 is high, the binding of O2 to one subunit of deoxyHb can increase the affinity of neighbouring subunits to O2, effectively making it easier to for O2 bind. This enhances the transition to oxyHb. This is called cooperative binding Take home message:  Hb can change its affinity for O through cooperative binding, 2 from high affinity (in the lungs) to low affinity (at the tissues) which means it can easily pick up or release O2 respectively in the appropriate environment.  Whereas Mb, being only a single subunit protein, only has one affinity for O2 – that is, high affinity.  Transition of DeoxyHb to OxyHb DeoxyHb is in the T (tense) form with a low affinity for O2. The subunits are stabilized by ion pairs. Once 2 subunits, in the T state, bind O2 a T  R transition occurs causing a change to the R (relaxed) state. This makes it easier for the remaining subunits to bind O2. The main difference is in the alignment of the a and b subunits which slide past one another. As they do, a number of ionic bonds are broken. This is called Cooperative binding This change in affinity at different pO2 can be observed in a sigmoidal-shaped O2 dissociation curve.  T  R Transition Animation: Haemoglobin: “Studying the T to R Transition” by Janet Iwasa https://biochem.web.utah.edu/iwasa/projects/hemoglobin.html  Interpreting Hb’s Oxygen Dissociation Curve Unlike Mb, Hb can change its affinity for O2 from high to low affinity (this is characterised by a sigmoidal shaped curve. This is achieved by responding to changes in the environment. Eg. pH, CO2, Temp., 2,3-BPG Hb can remain highly saturated even with large decreases in pO2. O2 rapidly combines with Hb as the pO2 increases (& rapidly dissociates as pO2 decreases. High affinity in the lungs (oxyHb/R state) to load up O2 Low affinity in the tissues (deoxyHb/T state) to offload O2 **S-shaped curves can only be produced by proteins with  home Binding of Small Molecules to Hb changes its affinity towards O2 As we’ve seen partial pressure O2 changes the affinity of Hb towards O2. A number of other factors influence the affinity of Hb for O2 Haemoglobin can bind other small molecules at sites away from the O2-binding site. These molecules are called allosteric effectors. They include:-  H+ (increased acid load/dec. pH)  CO (reflecting an increased acid load, volatile acid) forming 2 carbaminoHb  2,3-bisphosphoglycerate (2,3-BPG) Their binding causes a conformational change in Hb structure which leads to a changed affinity for O2 and altered functionality. This is called allosteric regulation. The binding of H+, CO2 and 2,3-BPG stabilise the T-state  The BOHR Effect At the Periphery At the Lungs ↑ H+ (lower pH) ↓ H+ (inc. pH) Hb binds H+ (acts TR transition as buffer) causes Hb to When H+ binds release H+ Hb, T state is R state is more more stable, thus stable thus able off-loading O2 to to pick up O2 periphery. Causes a shift to Causes a shift to the left of the the right of the ODC. ODC. (Curve B) (Nb. For a given pO2 less O2 saturation) HbH+ + O2  HbO2 + H+ deoxyHb oxyHb Bicarbonate buffer system: H+ + HCO3-  H2CO3  H2O + CO2 (CO2 is exhaled)  O2-off-loading is Regulated by 2,3- Binding of 2,3-BPG dramatically decreases Hb affinity forBPG O Normal RBCs have ~ 5mM 2,3-BPG 2 2,3-BPG stabilizes T state, enabling off-loading O2 Without 2,3-BPG, Hb would not off-load much O 2 at all. 2,3-BPG levels increase at high altitude (during ACCLIMITISATION). This increases delivery of O2 to the tissues under conditions where O 2 levels are lower. 2,3 BPG binds in a crevice between b-subunits of T-state Only 1 *A small molecule, very molecule of negatively charged – binds 2,3-BPG binds to a positively charged per tetrameric binding site on Hb. Hb 2,3-BPG is produced via a shunt off glycolytic pathway within RBCs (a major use of glucose metabolism in RBCs)  Effect of Increased 2,3-BPG on Peripheral O2 Off-loading Under normal conditions there is ~38% off-loading capacity between lungs & tissues (ie between 13  4 kPa respectively). At high altitude this difference is reduced to ~30% causing shortness of breath and fatigue (& other symptoms of altitude sickness) During acclimatization levels of 2,3-BPG within erythrocytes are increased which stabilizes the T state of Hb providing more O2 to tissues. The effect is reflected in a shift to the right of the ODC curve & regaining ~37% off-loading Binding of 2,3-BPG stabilizes capacity the low Foetal Haemoglobin (HbF) During foetal development, O2/CO2 transfer across the placental wall is necessary since the blood of mother & foetus never mix. Foetal Hb is the main O2 carrier for the foetus during Hb F (newborn): development and persists 50-80%, Hb F (6baby in the newborn mths): 8%, upHbuntil F (> 6 mths): ~ 61% to 2% months as it is gradually replaced by HbA. Question: How does O2 preferentially transfer over to the foetus? Compare the structures of adult Hb (HbA) and foetal Hb (HbF) (next slide). They’re look very similar.  Key difference – HbF contains 2 g subunits instead of b subunits. (a2g2). A genetic mutation in HbF within the 2,3-BPG binding site results in 2,3-BPG not binding as well. This leads a higher affinity of HbF towards O2 compared to HbA. This facilitates transfer of O2 across the placental wall. Hierarchy of Hb Affinity to O2: alpha > gamma > beta  Superimposition of Adult & Foetal Hb 2,3-BPG Protein backbones haeme Green ribbons – HbA alpha subunits Beige ribbons – HbA beta subunits Blue ribbon - HbF The blue atoms In HbF, mutation in the 2,3-BPG site here provide +ve mean less positive charge to stabilize 2,3- charge to facilitate –ve 2,3BPG in BPG into site. This means HbF remains in binding site. a higher affinity state compared to HbA in the periphery.  Clinical Significance & Take Home Message forpOODC For people with normal Shiftsor 10-13kPa) shifts in (80-100mmHg 2 the curve are not significant (ie no clinically significant change in Hb’s ability to pick up & transport O2 from lungs and drop off in tissues. But for those with abnormally low pO2 even small shifts in their ODC can significantly effect Hb’s ability to pick up & release O 2. HbH+ + O2  HbO2 + H+ Can use the same equilibria for CO2 & 2,3-BPG. time  Carbon Monoxide Poisoning Carbon monoxide (CO) is a odourless, tasteless gas (a silent killer) Fe2+ in haeme binds to CO with a much higher binding affinity than O2 (~200X). When CO is bound it prevents O2 from binding thus effectively lowering the total [O2] being delivered. In the periphery it also causes O2 to bind with higher affinity in the tissues, soaking up the O2 that is there, effectively asphyxiating cells. CO  Sickle Cell Haemoglobin (HbS) Sickle cell anaemia is a genetic disorder characterised by fragile, sickle shaped red blood cells (RBCs) & caused by a single point mutation in the b chain of Hb. Mutation of glutamate (a charged, polar amino acid) for a non-polar valine residue in position 6 of beta chain of HbA.  In the tissues, when HbS is in the T state, valine is exposed on the SURFACE of Hb - a highly unfavourable situation in an aqueous environment (HbS is insoluble). (Confirming conformation change occurs!) ‘Sticky’ spots are created on Hb surface cause other similarly affected HbS to clump together leading to insoluble fibres in the RBC, ultimately changing the shape & elasticity of the cell. Sickle RBCs are more fragile & tend to rupture as they pass through the narrow capillaries leading to occlusive events & causing severe pain. HbS has a much lower O2 carrying capacity causing an array of complications.  Hb helps regulate blood pressure Hb binds nitric oxide (NO)  NO binds to specific cysteine residues in Hb (Cysβ93) and also to the Fe2+ ions in haeme.  NO is produced from Arginine by NO synthase present in many tissues.  Its released in small amounts, acts locally & very briefly before being oxidized (NO can be converted to other forms involved in other pathways) NO & its derivates have numerous roles.  E.g. NO-induced relaxation of cardiac muscle (via cGMP-signalling pathway) Same response produced by nitroglycerin & other nitro- vasodilators to relieve angina. NO affects the walls of blood vessels, causing them to relax. This in turn reduces the blood pressure. Since Hb binds NO, it inhibits its bioavailability & thus aids in the regulation of blood pressure. Loss of NO bioavailability however can contribute to disease (inc. haemolytic anaemias eg sickle cell disease, Resources Baynes & Dominiczak Medical Biochemistry (Elsevier) 2014 Collins J-A, Rudenski A, Gibson J, et al. Relating oxygen partial pressure, saturation and content: the haemoglobin– oxygen dissociation curve. Breathe 2015; 11: 194–201 http://doi.org/10.1183/20734735.001415 Nelson, Lehninger & Cox (2013) Lehninger Principles of Biochemistry 6e New York (W.H. Freeman) Haemoglobin Animations  http://biochem.web.utah.edu/iwasa/projects/hemoglobin.html Appendix 1 - Amino Acid Chart Non-polar amino acid side chains contain mainly carbon and hydrogen. Polar amino acid pKa = 6.0 side chains contain polar functional groups pKa = 10.5 pKa = 12.5 like hydroxyl, carbonyl, thiol, carboxylate, amine, amide groups etc pKa = 3.65 pKa = 4.25  Appendix 2: Bicarbonate Buffer System *Le Chatelier’s principle for equilibrium tells us: (1)An increase in product forces equilibrium to the left (curve to the right) & vv.