Myoglobin and Hemoglobin

1. Myoglobin and Hemoglobin BL4010 10.12.06

2. Myoglobin & Hemoglobin Objectives • Identify biological functions. • Identify parts of Mb & their roles in O2 transport. • Identify how Hb differs from Mb.

3. Myoglobin • O2 transport, storage in cells. • Two parts: protein and heme prosthetic group. • Protein: • 155 amino acids, ~ 17 kDa. • Compact, globin fold. • 75% helix.

4. Heme • Heme is composed of porphyrin and Fe2+. • Porphyrin is non-polar, except two proprionate groups. • Porphyrin binds O2, CO. • CO 10,000x tighter than O2.

5. Heme Ligands • Fe is 6-coordinate. • Four N of heme group • One N from proximal His. • One from H2O or O2. • Can also bind CO.

6. carboxyl group of proprionate. Heme – Protein Interaction • Heme stabilizes protein fold. • Binds through hf interactions • Prooprionate groups on surface.

7. Myoglobin • Protein completely surrounds heme. • Function of protein: • ↑ heme solubility. • ↓ heme oxidation (Fe2+ → Fe3+) • metmyoglobin inactive. • ↓ CO binding.

8. O O O O C C Fe Fe Fe O2 vs. CO binding. • CO binds tightly; linear. • O2 binds less tightly, bent structure. • Distal His forces bent binding of both, weakens CO binding. Distal His Proximal His

9. Hemoglobin • Tetrameric protein • Dimer of dimers, (ab)2 • a,b chains resemble Mb. • Each chain contains, heme, prox. histidine. • Each binds 1 eq. O2. • ab dimer interface different from aa, bb interface. • Marked by salt bridges that stabilize the deoxy structure.

10. Oxygen Binding Curves Objectives. • Understand O2 binding curve for Mb. • Understand O2 binding curve for Hb. • Identify mechanism of cooperative binding.

11. Quantify O2 binding. • Measure deoxy- vs. oxy by visible absorption (Soret band). • Reaction is Mb + O2 MbO2 • Equil. Const. given by: Ka = [MbO2]/[Mb][O2] • Plot fraction bound: Y = [MbO2] / ([Mb] + [MbO2]) • Recast in terms of measureable quantities: Y = pO2 / (pO2 + pO2,50)

12. Direct Plots • Plot fractional saturation versus partial pressure of O2. • Most relevant part of plot in range of cell pO2 (~ 15-25 mm Hg).

13. Direct Plot • Binding characterized by pO2,50. • Partial pressure of O2 where Mb is half saturated. • If lower oxygen affinity, curve shifts right; if higher, curve shifts left. pO2,50 = 3 mm Hg.

14. 4° Structure of Hb alters O2 binding. • Interactions between dimers alters oxygen binding. • Direct plot shows Hb has lower affinity than Mb. • Sets up delivery system. • O2 bound by Hb in lungs; released in tissues. Mb Hb

15. Cooperative regulation Hb oxygen binding: • Start binding with given affinity in deoxy state, subsequent binding enhances affinity. • Defines positive cooperative regulation. • Only seen in multi-domain proteins. • Hill coefficient ~ number of interacting subunits. Advantage: binding is more sensitive to small changes in [ligand].

16. R T Molecular Model • In deoxy state, Fe out of heme plane; domed. • Bind O2, moves Fe. • This moves proximal His and its helix. • Moving helix alters a/b interface. • Deoxy = Tense (T) • Oxy = Relaxed (R)

17. T → R Structural Changes. R form: • All are broken. T form: • H143 - D94 in b1. • K40 - H146 CO2-. • R141 - D126 a2/a1. oxy deoxy

18. Small changes translate to large movements. Deoxy State

19. Small changes translate to large movements. Oxy state

20. Modifying O2 Binding in Hb Objectives. • Identify allosteric effectors • Describe molecular basis of each.

21. log Q D94 H146 log pO2 Bohr Effect • Oxygen affinity sensitive to pH. • ↓ pH; ↑ pO2,50 (lowers sensitivity). • D94 ↔ H146 salt bridge in T state only. • Excess H+ forms salt bridge, favors deoxy state.

22. log Q R-NH2 + CO2 R-NH-CO2- + H+ In lungs, Low CO2 log pO2 In tissues, High CO2 CO2 • Produced during aerobic metabolism. • Reacts with N terminal amino; carbamylation reaction. • Negative charge forms salt bridge with aR141, stabilizes deoxy state.

23. CO2 is Coupled to Bohr Effect In Tissue • CO2 is bound by Hb or converted to bicarbonate by carbonic anhydrase. • Buffers blood pH. • Hb binds 2H+ / 4 O2 released, also buffers (Bohr effect). In Lungs • Low pCO2; reaction reverses; • CO2 and H+ released from Hb, • pO2,50 decreased (increased oxygen affinity). From Lange’s Biochemistry

24. Chloride Anion Binding • Also favors T state • Forms salt bridge with R141, V1 in T state. • Released in R state.

25. 2,3-BPG • Side product of glycolysis. • indicates active respiration, need O2. • Binds cationic region in T-form. • Favors deoxy, releases O2 to tissues. • [2,3-BPG] is high, responsible for observed pO2,50 of 27 mm Hg. • stripped Hb has pO2, 50 ~ 8 mm Hg.

26. Another look at 2,3-BPG • BPG acts as a “wedge” and drives the R state to the T state. • Forces release of bound O2 in active tissue. • BPG increases at high altitude.

27. NO potent vasodilator Produced by Nitric Oxide Synthase (NOS). Arginine → Citrulline + NO Activates soluble guanylyl cyclase, signal transduction cascade. Reacts with Hb, which inactivates the NO (can’t react with sGC). Interactions with Hb Binds to HbO2 to form nitrates (NO3-) Binds to deoxy-Hb to form iron-nitrosyl (Fe2+-NO). Rapid reaction in vitro, but slow in vivo due to nature of blood flow along endothelium and diffusional barriers. NO and Hb

28. NO also binds to Cys93 on b-chains. Forms S-NO bond (SNO-Hb). Transfers NO to glutathione, which functions as a storage form for NO (won’t react with HbO2). Transport of NO by Hb

29. Transfer reaction depends on T/R state. R-state promotes binding to Cys93 SNO-Hb formed in lungs. NO released in T-state. Effectively delivers NO to vasculature. Vasodilation then enhances O2 delivery. But wait a minute … [SNO-Hb] 10,000 x lower than HbO2. NO transfer from SNO-Hb is slow. Amount released can’t compete with NO produced by NOS in erythrocytes. NO transport by Hb

30. Clinical relevance • If NOS impaired, SNO-Hb is effective transport, O2 delivery system. • Therapeutic value in treatment of sickle cell? • Use in blood substitutes?