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 • Protein binding prevents oxidation • If oxidized, becomes metmyoglobin o This is responsible for browning of cooked meat of old meat • 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 α 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 • P50=2.8 torr  Hyperbolic curve indicates that individual myoglobin binds O2 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 α subnits  2 β 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 α and β 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 α,β 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)/P50n + P(O2)n  n=number of O2 bound in a single step (aka: Hill coefficient) 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 β chain contracts Thr 38 one turn back along C-helix  T-state: His 97 β chain contracts Thr 41 in α chain  In both conformations, “knobs” on one subunit mesh with “grooves” on another Α and β pairs move 15° relative to each other in TR transition  In T-state, C terminal residues of each subunit make salt bridges network  T R transition breaks salt bridges and strength of salt bridge is 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- +H+ • Leads to more O2 delivery in actively respiring