Hemoglobin Course Materials

Oxygen-Binding Proteins • Myoglobin, Hemoglobin, Cytochromes bind O2. • Oxygen is transported from lungs to various tissues via blood in association with hemoglobin • In muscle, hemoglobin gives up O2 to myoglobin which has a higher affinity for O2 than hemoglobin. • Oxygen-binding curve for hemoglobin is sigmoidal whereas for myoglobin it is hyperbolic. This facilitates transfer of O2 to myoglobin. • Cytochromes participate is redox reactions and are components of the electron transport chain. Hemoglobin Structure • Hemoglobin is a O2 transport protein found in the RBCs • Hemoglobin is an oligomeric protein made up of 2 αβ dimers, a total of 4 polypeptide chains: α1β1α2β2. • Total Mr of hemoglobin is 64,500. • The α (141 aa) and β (146 aa) subunits have < 50 % identity. • The 3D- structures of α (141 aa) and β (146 aa) subunits of hemoglobin and the single polypeptide of myoglobin are very similar; all three are members of the globin family. • Each Hb subunit consists of 7 (α) or 8 (β) alpha helices and several bends and loops folded into a single globin domain. • Each subunit has a heme-binding pocket. The Prosthetic Heme Group • The heme group is responsible for the O2-binding capacity of hemoglobin. • The heme group consists of the planar aromatic protoporphyrin made up of four pyrrole rings linked by methane bridges. • A Fe atom in its ferrous state (Fe+2) is at the center of protoporphyrin. • Fe+2 has 6 coordination bonds, four bonded to the 4 pyrrole N atoms. The nucleophilic N prevent oxidation of Fe+2. • The two additional binding sites are one on either side of the heme plane. • One of these is occupied by the imidazole group of His. • The second site can be reversibly occupied by O2, which is hydrogen-bonded to another His. Different forms of Hemoglobin • When hemoglobin is bound to O2, it is called oxyhemoglobin. This is the relaxed (R ) state. • The form with a vacant O2 binding site is called deoxyhemoglobin and corresponds to the tense (T) state. • If iron is in the oxidized state as Fe+3, it is unable to bind O2 and this form is called as methemoglobin • CO and NO have higher affinity for heme Fe+2 than O2 and can displace O2 from Hb, accounting for their toxicity. T and R states of Hemoglobin • Hemoglobin exists in two major conformational states: Relaxed (R ) and Tense (T) • R state has a higher affinity for O2. • In the absence of O2, T state is more stable; when O2 binds, R state is more stable, so hemoglobin undergoes a conformational change to the R state. • The structural change involves readjustment of interactions between subunits. Changes Induced by O2 Binding • O2 binding rearranges electrons within Fe+2 making it more compact so that it fits snugly within the plane of porphyrin. • Since Fe is bound to histidine of the globin domain, when Fe moves, the entire subunit undergoes a conformational change. • This causes hemoglobin to transition from the tense (T) state to the relaxed (R) state. • The α1β1 and α2β2 dimers rearrange and rotate approximately 15 degrees with respect to each other • Inter-subunit interactions influence O2 binding to all 4 subunits resulting in cooperativity. O2-binding kinetics • Four subunits, so four O2-binding sites • O2 binding is cooperative meaning that each subsequent O2 binds with a higher affinity than the previous one • Similarly, when one O2 is dissociated, the other three will dissociate at a sequentially faster rate. • Due to positive cooperativity, a single molecule is very rarely partially oxygenated. • There is always a combination of oxygenated and deoxygenated hemoglobin molecules. The percentage of hemoglobin molecules that remain oxygenated is represented by its oxygen saturation. • O2-binding curves show hemoglobin saturation as a function of the partial pressure for O2. Oxygen Saturation Curve • Saturation is maximum at very high O2 pressure in the lungs (pO2 = ~ 100 torr). • As hemoglobin moves to peripheral organs and the O2 pressure drops (pO2 = ~20 torr), saturation also drops allowing O2 to be supplied to the tissues. • Due to co-operative binding of O2 to hemoglobin, its oxygen saturation curve is sigmoid. • Such a curve ensures that at lower pO2, small differences in O2 pressure result in big changes in O2 saturation of hemoglobin. This facilitates dissociation of O2 in peripheral tissues. Effectors of O2 binding • Small molecules that influence the O2-binding capacity of hemoglobin are called effectors (allosteric regulation). • Effectors may be positive or negative; homotropic or heterotropic effectors. • Oxygen is a homotropic positive effector. • Positive effectors shift the O2-binding curve to the left, negative effectors shift the curve to the right. • From a physiological view, negative effectors are beneficial since they increase the supply of oxygen to the tissues. The Bohr Effect • The regulation of O2-binding to hemoglobin by H+ and CO2 is called the Bohr effect • Both H+ and CO2 are negative effectors of O2-binding. • Addition of a proton to His imidazole group at C-terminus of βsubunit facilitates formation of salt bridge between His and Asp and stabilization of the T quaternary structure of deoxyhemoglobin. • CO2 reduces O2 affinity by reacting with terminal –NH2 to form negatively charged carbamate groups • Metabolically active tissues need more O2; they generate more CO2 and H+ which causes hemoglobin to release its O2. 2,3-Bisphosphoglycerate • 2,3-Bisphosphoglycerate is a negative effector. • A single 2,3-BPG binds to a central pocket of deoxyhemoglobin and stabilizes it by interacting with three positively charged aa of each β-chain. • 2,3-BPG is normally present in RBCs and shifts the O2-saturation curve to the right • Thus, 2,3-BPG favors oxygen dissociation and therefore its supply to tissues • In the event of hypoxia, the body adapts by increasing the concentration of 2,3-BPG in the RBC Fetal Hemoglobin • Fetal hemoglobin has 2 α and 2 γ chains • The g chain is 72% identical to the b chain. • A His involved in binding to 2,3-BPG is replaced with Ser. Thus, fetal Hb has two less + charge than adult Hb. • The binding affinity of fetal hemoglobin for 2,3-BPG is significantly lower than that of adult hemoglobin • Thus, the O2 saturation capacity of fetal hemoglobin is greater than that of adult hemoglobin • This allows for the transfer of maternal O2 to the developing fetus