Structures of Myoglobin and Hemoglobin Myoglobin (Mb) - monomeric protein that facilitates the diffusion of oxygen in vertebrates Hemoglobin (Hb) - tetrameric protein that carries oxygen in the blood Heme consists of a tetrapyrrole ring system called protoporphyrin IX complexed with iron Heme of Mb and Hb binds oxygen for transport

Heme Fe(II)-protoporphyrin IX

Protein component of Mb and Hb is globin Myoglobin is composed of 8 a helices Heme prosthetic group binds oxygen His-93 is complexed to the iron atom, and His-64 forms a hydrogen bond with oxygen Interior of Mb almost all hydrophobic amino acids Heme occupies a hydrophobic cleft formed by three a helices and two loops

Sperm whale oxymyoglobin

Hemoglobin (Hb) Hb is an a2b2 tetramer (2 a globin subunits, 2 b globin subunits) Each globin subunit is similar in structure to myoglobin Each subunit has a heme group The a chain has 7 a helices, b chain has 8 a helices

Hemoglobin tetramer (a) Human oxyhemoglobin (b) Tetramer schematic

Oxygen Binding to Mb and Hb Oxymyoglobin - oxygen bearing myoglobin Deoxymyoglobin - oxygen-free myoglobin In oxymyoglobin, six ligands are coordinated to the ferrous ion in octahedral symmetry Oxygen is coordinated between the iron and the imidazole sidechain of His-64

Oxygen-binding site of whale oxymyoglobin Octahedral geometry of coordination complex (six ligands around iron) His-93 (proximal histidine) liganded to Fe His-64 (distal histidine)

Oxygen-binding curves (a) Comparison of O2-binding to Mb and Hb

Oxygen-Binding Curves of Myoglobin and Hemoglobin Curves show reversible binding of O2 to Mb and Hb Fractional saturation (Y) is plotted versus the partial pressure of oxygen, pO2 (oxygen concentration) The shape of the Hb curve shows a positive cooperativity in the binding of 4 O2 molecules (i.e. the O2 affinity of Hb increases as each O2 molecule is bound)

Oxygen-binding curves (a) Comparison of O2-binding to Mb and Hb

O2 binding curves (continued) Mb-O2 binding curve is hyperbolic, indicating a single equilibrium constant for binding O2 Hb-O2 binding curve is sigmoidal, and reflects the binding of 4 molecules of O2, one per each heme group

Oxygen-binding curves (a) Comparison of O2-binding to Mb and Hb

Oxygen-binding curves Binding of the R (high-affinity) and T (low affinity) forms of Hb

Conformational changes in a hemoglobin chain induced by oxygenation Oxygen binding to Fe pulls the His toward ring plane Helix with His shifts position, disrupting some ion pairs between subunits (blue to red position)

Oxygen-binding site of whale oxymyoglobin Octahedral geometry of coordination complex (six ligands around iron) His-93 (proximal histidine) liganded to Fe His-64 (distal histidine)

Hemoglobin is an Allosteric Protein Oxygen binding and release from Hb are regulated by allosteric interactions Allosteric effectors (modulators) bind to a protein at a site separate from the functional binding site (may be activators or inhibitors) The activity of an allosteric protein is regulated by allosteric effectors

Two conformations of hemoglobin: T and R Active (R state) and inactive (T state) forms are in rapid equilibrium in allosteric proteins Binding of substrates and allosteric activators stabilize the R state and shift the equilibrium in the R direction Allosteric inhibitors stabilize the T state and shift the equilibrium in the T direction

Bisphospho-D-glycerate (2,3BPG) 2,3BPG is an allosteric effector of Hb 2,3BPG lowers the affinity of deoxyHb for oxygen (raises the P50 of Hb from ~12 to ~26 torr) Negatively charged 2,3BPG is bound to six (+) charged groups of deoxyhemoglobin

Bisphospho-D-glycerate (2,3BPG)

Binding of 2,3BPG to deoxyhemoglobin (-) Charges on 2,3BPG pair with (+) charges lining the central cavity, stabilizing the DeoxyHb form a-Subunits pink, b-subunits blue, heme groups red

Oxygen-binding curves (a) Comparison of O2-binding to Mb and Hb

Bohr effect Lowering the pH decreases the affinity of Hb for oxygen

Carbamate adduct Carbon dioxide is transported from the tissues to the lungs in two ways: (1) Dissolved bicarbonate ions (2) Carbamate adducts of hemoglobin (N-terminal globin residues react with CO2 to form carbamates)

Review of Relevant Parameters Low P50 indicates high O2 affinity (2) Low pH (through CO2 intake) stabilizes 2,3-BPG and lowers O2 affinity (3) Raising P50 causes unloading of O2

Case Studies HCO3- generates CO2 to the tissues Shock victims are given intravenous HCO3- Why? HCO3- generates CO2 to the tissues and lowers the O2 affinity of Hb, thus releases O2 from HbO2 to the tissues

Case Studies Fetal Hb (HB-F) contains ser in place of the cationic his at position 143 of the b chains of adult Hb (HB-A). Residue 143 faces the central cavity between the b chains Outcomes: his in Hb-A is protonated and thus binds more tightly to negatively charged 2,3-BPG; ser in Hb-F is not protonated and does not bind to 2,3-BPG as strongly; thus Hb-F has a greater fraction of HbO2

Case Studies Fetal Hb (HB-F) contains ser in place of the cationic his at position 143 of the b chains of adult Hb (HB-A). Residue 143 faces the central cavity between the b chains Outcomes: Hb-F has a greater fraction of HbO2 which means greater O2 affinity and lower P50 (18 torr) Since average P50 for Hb-A is 26 torr, oxygen can efficiently be transferred from maternal blood to fetus

Antibodies Bind Specific Antigens Vertebrate immune systems synthesize protein antibodies (immunoglobulins) to eliminate bacteria, viruses, other foreign substances Antibodies specifically recognize and bind antigens Antibodies are synthesized by lymphocytes (white blood cells)

Human antibody structure

Heavy chains (blue) and light chains (red) Disulfide bonds (yellow) Variable domains colored darker

Stereo view of the immunoglobin fold Two antiparallel b sheets linked by nonrepetitive segments

Binding of three different antibodies to an antigen