1. Oxygen Transport Beth A. Bouchard BIOC 212: Biochemistry of Human Disease Spring 2006

2. PROPERTIES OF O 2  Limited solubility in aqueous solutions: arterial blood contains 0.13mmol/L dissolved O 2  Transported in blood in complex with hemoglobin, which results in an ~60-fold increase in the O 2 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 transport

3. Heme  Incorporated into proteins during synthesis  Stabilized by hydrophobic residues found in interior of the protein: protective environment that prevents oxidation of Fe 2+ to Fe 3+ or “rusting”. In this state it can not react with O 2.  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 Fe 2+ atom * Distal histidine is important for allowing binding of O 2 to the Fe 2+ atom

4. Heme (cont.) 1 5 4 3 6 2 In deoxygenated globins, the 6 th position is vacant Porphyrin nitrogen atom

5. CHARACTERISTICS OF GLOBIN PROTEINS ~150 aa: 75% associated with α-helices Highly soluble: polar (charged) aa on surface

6. Hemoglobin  Synthesized in RBC precursor cells: reticulocytes and erythroblasts  Tetramer of 2  -globin and 2  -globin chains  Best described as a dimer of the heterodimer (  )

7. Hemoglobin synthesis is tightly controlled by [heme] cell

8. HCI = heme controlled inhibitor Reduced initiation of translation

9. INTERACTIONS WITH O 2 * Can bind up to 4 O 2 molecules * Binding of O 2 is cooperative: the binding of 1 O 2 influences the binding of another

10. Interactions between the heterodimers is stronger in the “T”-state

11. DEOXYGENATED VS. OXYGENATED HEMOGLOBIN (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 O 2 affinity and which subunit binds to (or releases) O 2 first.

12. 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 O 2 ) by a number of allosteric effectors including H + (Bohr Effect), CO 2 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.

13. INTERACTIONS WITH ALLLOSTERIC EFFECTORS (CONT.)  As the curve shifts from A to B (to the right) the affinity for O 2 decreases  The effects of these molecules appears to be additive  Increasing temperature will also shift the curve to the right

14. The Bohr Effect  Describes the rightwards shift in the O 2 saturation curve (i.e. decreased O 2 affinity) with increasing H + concentration (decreasing pH)  N-terminal amino group of the  -chain and side chains of His 122  and His 146  are the residues most involved  These residues are more extensively protonated in the T-state. When hemoglobin binds O 2, protons dissociate. In acidic media, protonation inhibits O 2 binding. Lungs (high pO 2 )  Promotes O 2 saturation  Forces protons from the molecule to stabilize the R-state Peripheral tissues/Capillaries (lower pH) O 2 -saturated hemoglobin will acquire some “excess” protons, shift towards the T-state and release O 2 for tissue uptake

15. Effect of CO 2 : increased pCO 2 in venous capillaries decreases the affinity for O 2 1.CO 2 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 O 2 2. In peripheral tissues, hydration (H 2 CO 3 ) followed by dissociation (H + + HCO 3 - ) generates additional protons available to participate in the Bohr Effect and facilitate CO 2 -O 2 exchange (more O 2 can be released)

16. Transport and Removal of CO 2  Blood transports two forms of CO 2 to the lungs: carbamino-hemoglobin and H 2 CO 3 /HCO 3 - (carbonic acid- conjugate base pair) 1. Carbamino-hemoglobin: exposure to low pCO 2 results in the reversal of the carbamination reaction through mass action and O 2 binding is again favored. CO 2 is expelled by the lungs. 2. H 2 CO 3 /HCO 3 - : in the pulmonary capillaries RBC carbonic anhydrase converts H 2 CO 3 into CO 2 and H 2 0, which are expelled in their gaseous forms into the atmosphere

17. Working Muscles… Produce H + and CO 2 via aerobic metabolism and liberate heat As the binding of O 2 is exothermic (produces heat), affinity of O 2 decreases as temp- erature increases More efficient release of O 2 to the surround- ing tissue

18. Deoxygenated Hb Stabilizes the “T”-state: Marked increase in P50 (without it the curve would look like Mb) pO 2 BPG Electrostatic interactions [rbcs] = 4.1 mM

19. CARBON MONOXIDE (CO) POISONING  Affinity of globin bound heme for CO is 10 4 times more then that for O 2  Like O 2, it binds to the 6 th position of the heme iron  Bound CO allosterically activates hemoglobin (shifts O 2 saturation curve to the left)  Hemoglobin becomes trapped in the R-state  Any O 2 already bound cannot be released so its transport to tissues becomes seriously compromised  Prolonged exposure would be virtually irreversible (t 1/2 = 4-5 hr) and leads to highly toxic levels of carboxyhemoglobin  Hyperbaric O 2 therapy (administration of 100% O 2 at 200-300 kPa) is used to treat CO poisoning  This results in arterial and tissue pO 2 of 2000 and 4000 mmHg, respectively, displacing the bound CO, and immediately resulting in a reduction in the t 1/2 to less then 20 min

20. HEMOGLOBIN VARIANTS TypeStructureComments HbA (95%) 2222 HbA 2 (4%) 2222 Functionally, this variant is indistinguishable from HbA Mutations in  -globin are without effect HbF (1% in adults – predominate form in the fetus during the 2 nd and 3 rd trimesters of pregnancy)  2  2 His 143 (  )  Ser (  ) Interaction with 2,3-BPG is weaker resulting in an increased affinity for O 2 and a greater stabilization of the R state. This allows for a more efficient transfer of O 2 from maternal to fetal hemoglobin

21. HEMOGLOBINOPATHIES > 600 mutations: several hundred of these result in a pathological phenotype HbC (Glu6  to Lys) cellular crystallation of oxygenated protein; increased fragility of rbcs mild anemia and splenomegaly