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9 25 Key Concepts Structures and function of myoglobin Equation describing O2 binding Definition of disassociation constant Kd Importance of Kd for myoglobin function Hemoglobin structure and function Hill equation Ch 7 Protein structure and function Hemoglobin and myoglobin o Hemoglobin transport O2 in blood o Myoglobin transport O2 form blood to tissue Structure o Both bind to heme which carries O2 Heme binds Fe2 which coordinates O2 called a prosthetic group Type of porphrin N form rings coordinates Fe2 at four positions N form His of myoglobin on F helix occupies 5th position When O2 binds its occupies 6th position below plane of heme o Fe must be in 2 oxidative state or else it doesn t bind to O2 meat Protein binding prevents oxidation If oxidized becomes metmyoglobin o This is responsible for browning of cooked meat of old CO NO H2S can bind with heme with higher affinity than O2 o This causes toxicity o CO has 200 s higher affinity than O2 Myoglobin 153 residues 8 helices A H binds heme between helices E F Monomer o Eauations describing O2 binding Mb Mb O2 Kd dissacociation constant Mb O2 MbO2 Units M Smaller the Kd tighter the binding O2 concentrations measured by partial pressure or YO2 P O2 Kd P O2 O2 binding by myoglobin is HYPERBOLIC Kd is equal to the concentration at half the saturation or when Kd P O2 P O2 P50 P O2 0 5 Aka P50 Kd Steepness of plot indicates higher affinity Hyperbolic curve indicates that individual myoglobin binds O2 P50 2 8 torr independantly P50 for Mb 2 8 torr PO2 in atrial blood 100 torr PO2 in venous blood 30 torr Mb 91 saturated at veinous O2 PO2 in muscles cells drops drastically during exercise Stored O2 Mb helps provide necessary O2 Hemoglobin Structure and Function o Hb is highly homologous to myoglobin o Forms a tetramer 2 subnits 2 subunits Together they make a diner of dimers o C2 symmetry Hb bind 4 O2 Structure determined by Max Perutz in 1960 Mb determined by John Kendrew in 1959 Shared Nobel prize in 1962 o Folds of and H bonds are very similar despite only about 18 identity o Main difference is that Hb undergoes large conformational change upon O2 binding o 2 conformations T state deoxyhemoglobin T taut R state oxyhemoglobin R relaxed Oxygenation rotates one dimer 15 with respect to the other dimer Conformation changes relate to cooperativity of O2 binding Unlike Mb O2 binding hyperbolic Hb is sigmodel o Sigmodel 4 binding sites on Hb Hb nO2 Hb O2 n o Hill Equation YO2 P O2 P50 n number of O2 bound in a single step aka Hill coefficient n P O2 n o Does the binding of 1 O2 influence binding of more O2 C coopertivity n is degree of cooperativity n 1 no coopertivity n 1 coopertivity n 1 coopertivity being at one subunit decreases the affinity of the other subunits o Hill plot helps us determine n Log YO2 1 YO2 nlog PO2 nlog P50 For Hb If n 4 then plot would be linear and all subunits would bind simultainiously At low PO2 n 1 At Med PO2 n 2 8 3 At high PO2 n 1 9 27 Key Concepts Mechanisms of cooperativity in hemoglobin Albstery o MWC mode o Sequential model Hemoglobin effectors sickle cell anemia Antibiotics Coopeirtivity of Hb is important for its function o Hb must bind O2 in lungs and release it in capillaries o Hb is nearly saturated with O2 in lungs o PO2 100 torr in atrial blood o PO2 30 torr in capillaries half saturated 0 4 difference in saturation By comparison if Hb was hyperbolic difference would only be 25 o Coopertivity increases effcetivness of O2 delivery Structural changes in Hb o Conformation change intitated by heme Deoxy hb Fe2 lies 0 55 above plane of heme T conformation Oxy Hb Fe2 moves into plane Displaced only 0 2 Angstroms above plane o Fe2 pulls His on helix F along with it o Motion of F helix coupled to large scale changes in subunit information R state His 97 chain contracts Thr 38 one turn back along C helix T state His 97 chain contracts Thr 41 in chain In both conformations knobs on one subunit mesh with grooves on another and pairs move 15 relative to each other in T R transition T R transition breaks salt bridges and strength of salt bridge is In T state C terminal residues of each subunit make salt bridges network determined by pH pH id thus important in T R equilibrium Transmission of O2 binding state from one subunit to the other is basis for coopertivity Cooperitivty Allostery Albseterism o 2 common models for mechanism of allosteric regulation o MWC symmetry model and Sequential model o MWC model After Monad Wyman and Changeux 2 conformational state of the protein complex T and R R high affinity T low affinity Substrate S binds cooperatively because it shifts equilibrium to R state thus trapping other monomers in R state o Sequential model Aka Koshland Nemethy Filmer KNF Protein is Oligomeric Ligand induced conformational changes in adjacent subunit Makes them high affinity Difference with MWC doesn t require all subunits to be in the same state Attractive feature of sequential model is that is allows for negative coopertivity as well as positive o Mechanism of Hb allostery is mixure of KNF and MWC models Binding O2 favors sequential conformational changes But only 2 conformationas R T Effectors Modulate Allostery o Hb effectors modulate O2 affinity H CO2 BPG Biophosphoglycerate o H promotes disassociation of O2 from Hb called Bohr effect after Christian Bohr o Deoxy Hb has higher affinity for H than any oxy Hb Decreased pH leads to increased O2 dissociation pH lower in active muscle lactic acid formation promotes O2 release CO2 also promotes O2 dissociation CO2 produced in tissue leads to proton production CO2 H2O HCO3 Leads to more O2 delivery in actively respiring tissue but reverse H occurs in lungs o BPG negative allosteric effector of Hb BPG binds to T state Only weakly binds to R state Binding inhibits conformational change and stabilizes T state Binding of O2 displaces BPG Hb only has sigmoid binding affinity in presence of BPG o Fetal H has higher affinity of O2 because of lower affinity foe Hb Fetus dependent on mother for O2 but circulatory system id entirely independent Gas exchange take place across placenta Fetal Hb has Gamma chains instead of chains Diseases associated with Hemoglobin 2 and 2 o Some mutations are harmless but some cause changes in O2 affinity coopertivity or protein stability o Often lead to hemolytic anemia Loss of hemoglobin due to degradation o Sickle Cell Anemia Caused by single amino acid substitution in Hb Mutant protein called HbS Cause Hb to form filament Glu

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FSU BCH 4053 - Study Guide

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