1. Oxygen Transport (II) 1

2. Special features of myoglobin 1.Isolated haem can bind oxygen but in doing so risks having its Fe oxidised from Fe(II) to Fe(III), a form of iron that no longer binds oxygen. This reaction requires the formation of a haem-oxygen-haem sandwich. Mb encloses haem in a deep cleft and thus sterically hinders the formation of this sandwich intermediate and prevents oxidation to Fe(III). “Picket-fence” Fe porphyrin 2

3. Proximal Histidine Distal Histidine 2. The binding environment of the haem reduces its affinity for carbon monoxide (CO) from 25,000 to 200 times greater than its affinity for oxygen. Under normal conditions about 1% of Mb is occupied by CO rather than O 2. Special features of myoglobin 3

4. Oxygen dissociation curve for Mb By definition, Substituting from the expression for K d : 4 Mb + O 2 Mb-O 2 Y Oxygen partial pressure (atm) 1.0 0.5 At [O 2 ] = K d, Y = 0.5. Thus K d represents the concentration at which Mb is half-occupied. Thus, since [O 2 ]  pO 2, we can write: where p 50 is the partial pressure of oxygen at which Mb is half-occupied.

5. Oxygen dissociation curve for Mb For Mb, p 50 = 0.0013 atm (1 torr). In humans, pO 2 (lungs) = 0.13 atm and pO 2 (tissue) = 0.026 atm Mb would be an inefficient O 2 transporter (less than 4% of the oxygen picked up in the lungs is released in the tissue) because it binds O 2 too tightly. It stores a reservoir of oxygen in cells that are likely to need rapid access to it under certain conditions; thus Mb will release O 2 under conditions of strenuous exercise when the tissue concentration drops to very low levels. Y lungs = 0.990 Y tissue = 0.952  Y = 0.038 Y Oxygen partial pressure (atm) 1.0 0.5 0.00130.0260.13 lungstissue 5

6. Haemoglobin (Hb) - oxygen transporter Haemoglobin is similar in some ways to myoglobin but has a number of important differences. Hb is an  2  2 tetramer (4 polypeptide chains): 2  chains (141 amino acids) 2  chains (146 amino acids) Max Perutz (1914-2002) 6

7. Haemoglobin (Hb) - oxygen transporter  and  have about 20% sequence identity with myoglobin and are structurally homologous to it. Both chains possess a haem group and bind oxygen in the same way. Haemoglobin can bind up to 4 molecules of oxygen (one per monomer). Myoglobin Haemoglobin 7

8. Haemoglobin (Hb) - oxygen transporter Hb binds a co-factor, 2,3-diphosphoglycerate (DPG) at the  interface. DPG 8

9. Haemoglobin (Hb) - oxygen transporter The oxygen binding curves for Hb and Mb are significantly different. In contrast to Mb, Hb (i)binds O 2 less tightly (ii)displays co-operativity (i.e. binding of one molecule of O 2 increases the affinity for subsequent O 2 binding). How do we account for this behaviour? Y Oxygen partial pressure (atm) 1.0 0.5 0.00130.0260.13 Lungs Tissue Hb Mb 9

10. Oxygen dissociation curve for Hb Monod-Wyman-Changeux model The model makes a number of simple assumptions: The protein is an oligomer (>1 polypeptide chain) The protein can exist in 2 states: Tense (T) and Relaxed (R) T-state has low affinity for oxygen (K T large) R-state has high affinity for oxygen (K R small) All the subunits of any one molecule are either in the T-state or the R-state (concerted model) 10

11. Oxygen dissociation curve for Hb 11

12. Y is defined as the fraction of occupied sites, so: Oxygen dissociation curve for Hb 12

13. To simplify this expression, we can substitute using our expressions for K T and K R and make the following definitions: Oxygen dissociation curve for Hb 13

14. This gives us: Plotting Y against ,we get: Y  1.0 Oxygen dissociation curve for Hb 14

15. Oxygen dissociation curve for Hb Limiting Cases Note that for Hb, L ≈ 3 x 10 5 and c ≈ 0.01. 1. Low oxygen concentration (  ≈ 1, i.e. [S] ≈ K R ): This is a hyperbolic equation describing low affinity binding. Y  15

16. Oxygen dissociation curve for Hb Limiting Cases 2. High oxygen concentration (  >> 1, i.e. [S] >> K R ): This is a hyperbolic equation describing high affinity binding. Y  16

17. Oxygen dissociation curve for Hb The observed oxygen-binding curve for Hb shows that over the range of oxygen partial pressures at which the molecule has to operate (between 0.13 atm in the lungs and 0.026 atm in the tissues), there is a large  Y. Thus, Hb is well “tuned” to its transport function. Y pO 2 (atm) 1.0 0.5 0.0260.0340.13 Hb Mb  Y ≈ 0.66 LungsTissue 17

18. Molecular mechanism of Hb co-operativity The tense state is stabilised by the binding of the negatively charged co-factor, DPG. DPG 18

19. Molecular mechanism of Hb co-operativity DPG plays a major role in T-state stabilisation; in the absence of DPG, Hb switches to the R-state (high affinity) and is not capable of displaying co-operativity. Y  Hb without DPG Hb 19