Slide 1Slide 2HemeHeme (cont.)Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Oxygen TransportBeth A. BouchardBIOC 212: Biochemistry of Human DiseaseSpring 2006PROPERTIES OF O2- Limited solubility in aqueous solutions: arterial blood contains 0.13mmol/L dissolved O2- Transported in blood in complex with hemoglobin, which results in an ~60-fold increase in the O2 content of blood (8.6 mmol/L)-Stored in skeletal and striated muscle in complex with myoglobin (in the cytoplasm) -Delivered as needed to the mitochondria for electron transportHeme- Incorporated into proteins during synthesis - Stabilized by hydrophobic residues found in interior of the protein: protective environment that prevents oxidation of Fe2+ to Fe3+ or “rusting”. In this state it can not react with O2. - Iron is normally chelated by 6 atoms: 4 N atoms in the porphyrin ring; and two histidines in the heme binding pocket* Proximal histidine has an imidazole nitrogen that is close enough to bond directly to the Fe2+ atom* Distal histidine is important for allowing binding of O2 to the Fe2+ atomHeme (cont.)154362In deoxygenated globins, the 6th position is vacantPorphyrin nitrogen atomCHARACTERISTICS OF GLOBIN PROTEINS~150 aa: 75% associated with α-helicesHighly soluble: polar (charged) aa on surfaceHemoglobin-Synthesized in RBC precursor cells: reticulocytes and erythroblasts- Tetramer of 2 -globin and 2  -globin chains- Best described as a dimer of the heterodimer ()Hemoglobin synthesis is tightly controlled by [heme]cellHCI = heme controlledinhibitorReduced initiation of translationINTERACTIONS WITH O2* Can bind up to 4 O2 molecules* Binding of O2 is cooperative: the binding of 1 O2 influences the binding of anotherInteractions between the heterodimers is stronger in the “T”-stateDEOXYGENATED VS. OXYGENATEDHEMOGLOBIN (CONT.)- The transition of hemoglobin from the T- to the R-state is not well-defined- Best explained as a combination of a sequential and a concerted model - It is unknown whether the  and  subunits differ in O2 affinity and which subunit binds to (or releases) O2 first.INTERACTIONS WITH ALLLOSTERIC EFFECTORS- Allosteric proteins are typically multisubunit proteins- Small molecules know as allosteric effectors bind to the protein at sites that are spatially distinct from the ligand binding site and exert either a positive or negative effect on ligand binding- These effects are accompanied by changes in tertiary and/or quaternary structure- Hemoglobin is modified negatively (i.e. decreased affinity for O2) by a number of allosteric effectors including H+ (Bohr Effect), CO2 and 2,3-bisphosphoglycerate (2,3-BPG) - It is unknown whether the  and  subunits differ in O2 affinity and which subunit binds to (or releases) O2 first.INTERACTIONS WITH ALLLOSTERIC EFFECTORS (CONT.)- As the curve shifts from A to B (to the right) the affinity for O2 decreases- The effects of these molecules appears to be additive- Increasing temperature will also shift the curve to the rightThe Bohr Effect- Describes the rightwards shift in the O2 saturation curve (i.e. decreased O2 affinity) with increasing H+ concentration (decreasing pH)- N-terminal amino group of the  -chain and side chains of His122 and His146 are the residues most involved-These residues are more extensively protonated in the T-state. When hemoglobin binds O2, protons dissociate. In acidic media, protonation inhibits O2 binding. Lungs (high pO2)- Promotes O2 saturation- Forces protons from the molecule tostabilize the R-statePeripheral tissues/Capillaries (lower pH)O2-saturated hemoglobin will acquire some “excess” protons, shift towards the T-state and release O2 for tissue uptakeEffect of CO2: increased pCO2 in venous capillaries decreases the affinity for O21. CO2 reacts reversibly with the N-terminal amino groups of the globin polypeptides to form carbamino-hemoglobin -Shifts the equilibrium towards the T-state thereby promoting the dissociation of O22. In peripheral tissues, hydration (H2CO3) followed by dissociation (H+ + HCO3-) generates additional protons available to participate in the Bohr Effect and facilitate CO2-O2 exchange (more O2 can be released)Transport and Removal of CO2- Blood transports two forms of CO2 to the lungs: carbamino-hemoglobin and H2CO3/HCO3- (carbonic acid-conjugate base pair)1. Carbamino-hemoglobin: exposure to low pCO2 results in the reversal of the carbamination reaction through mass action and O2 binding is again favored. CO2 is expelled by the lungs.2. H2CO3/HCO3-: in the pulmonary capillaries RBC carbonic anhydrase converts H2CO3 into CO2 and H20, which are expelled in their gaseous forms into the atmosphereWorking Muscles…Produce H+ and CO2 via aerobic metabolism and liberate heatAs the binding of O2 isexothermic (produces heat), affinity of O2 decreases as temp-erature increases More efficient release of O2 to the surround-ing tissueDeoxygenated HbStabilizes the “T”-state:Marked increase in P50 (without it the curve would look like Mb) pO2BPGElectrostatic interactions[rbcs] = 4.1 mMCARBON MONOXIDE (CO) POISONING- Affinity of globin bound heme for CO is 104 times more then that for O2- Like O2, it binds to the 6th position of the heme iron- Bound CO allosterically activates hemoglobin (shifts O2 saturation curve to the left) - Hemoglobin becomes trapped in the R-state- Any O2 already bound cannot be released so its transport to tissues becomes seriously compromised- Prolonged exposure would be virtually irreversible (t1/2 = 4-5 hr) and leads to highly toxic levels of carboxyhemoglobin- Hyperbaric O2 therapy (administration of 100% O2 at 200-300 kPa) is used to treat CO poisoning - This results in arterial and tissue pO2 of 2000 and 4000 mmHg, respectively, displacing the bound CO, and immediately resulting in a reduction in the t1/2 to less then 20 minHEMOGLOBIN VARIANTSType Structure CommentsHbA(95%)22HbA2(4%)22Functionally, this variant is indistinguishable from HbAMutations in -globin are without effectHbF(1% in adults – predominate form in the fetus during the 2nd and 3rd trimesters of pregnancy)22His143 ()  Ser ()Interaction with 2,3-BPG is weaker resulting in an increased affinity for O2 and a greater stabilization of the R state. This allows for a more efficient transfer of O2 from maternal to fetal hemoglobinHEMOGLOBINOPATHIES> 600