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Protein Function: Oxygen Binding Proteins

Protein Function: Oxygen Binding Proteins. CH 339K. Myoglobin. Sperm Whale Myoglobin was the first protein to have its 3-dimensional structure determined John Kendrew(1958) Shared the 1962 Nobel in chemistry Solving the structure wasn’t hard, but getting the samples was a real achievement….

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Protein Function: Oxygen Binding Proteins

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  1. Protein Function:Oxygen Binding Proteins CH 339K

  2. Myoglobin • Sperm Whale Myoglobin was the first protein to have its 3-dimensional structure determined • John Kendrew(1958) • Shared the 1962 Nobel in chemistry • Solving the structure wasn’t hard, but getting the samples was a real achievement… • Kendrew, JC; Bodo, G; Dintzis, HM; Parrish, RG; Wyckoff, H; Phillips, DC (1958). "A three-dimensional model of the myoglobin molecule obtained by x-ray analysis". Nature181 (4610): 662–666.

  3. Myoglobin • Myoglobin - 17,000 daltons (monomeric) 153 amino acids • 8 a-helices, designated A - H • Conjugated protein - A conjugated protein has a non-protein part in addition to a polypeptide component.

  4. Myoglobin – naming of helices

  5. Heme

  6. Heme Function • The heme group is responsible for the O2 -binding capacity of hemoglobin. • The heme group consists of the planar aromatic protoporphyrin made up of four pyrrole rings linked by methane bridges. • A Fe atom in its ferrous state (Fe+2) is at the center of protoporphyrin.

  7. Heme Binding • Fe+2 has 6 coordination bonds, four bonded to the 4 pyrrole N atoms. The nucleophilic N prevent oxidation of Fe+2. • The two additional binding sites are one on either side of the heme plane. • One of these is occupied by the imidazole group of His F8. (H63 in (SWM) • The second site can be reversibly occupied by O2, which is hydrogen-bonded to another His. (His E7, H94 in SWM)

  8. Oxygenation state can be measured spectrophotometrically Reflectance spectra of myoglobin (1),  metmyoglobin (2) and oxymyoglobin (3).

  9. Absorbance Curves for Mb

  10. CO Poisoning Myoglobin’s affinity for carbon monoxide is ~ 60x its affinity for O2. Hemoglobin’s affinity for carbon monoxide is ~ 230x its affinity for O2. Autopsy photo showing characteristic skin discoloration

  11. O2 Binding Kinetics

  12. Reaction: Mb + O2⇌ MbO2 (1) (2 (3) (4) (5)

  13. (5) (6) (7) (8) Dr. Ready finally gets to the point!

  14. Remember Dalton’s Law – the concentration of a gas in a liquid … … is proportional to the partial pressure of that gas over the liquid

  15. So: Converts to: Big blue arrows are usually a clue to the student

  16. Hyperbolic Binding Kinetics

  17. p50 Defines the Curve

  18. Oxygen Transport

  19. 3o structure overlap: myoglobin, a-globin and b-globin a-Globin (blue) b-Globin (violet) Myoglobin (green)

  20. Mb vs. Hb

  21. Hemoglobin O2 carrying capability • Erythrocytes/ml blood: 5 billion ( 5 x 109 ) • Hemoglobin/red cell: 280 million ( 2.8 x 108 ) • O2 molecules/hemoglobin: 4 • O2 ml blood: (5 x 109)(2.8 x 108)(4) = (5.6 x 1018) • or (5.6 x 1020) molecules of O2/100 ml blood • or ~ 0.3 g/l • or ~ 9 mM • By comparison: • Solubility of O2 in saline: ~ 0.007 g/l • or ~ 0.2 mM

  22. O2 Binding – Hb vs. Mb

  23. O2 transport capability, a comparison

  24. Cooperativity Substrate affinity changes with substrate concentration or (rephrased) Substrate affinity changes with substrate binding Characteristic of (many) proteins with multiple binding sites.

  25. Cooperative Binding Kinetics Reaction: Hb + nO2 ⇌ HbnO2 (1) (2) (3)

  26. (5) (6) (7) (8) (9) (10) (11) Hill Equation Myoglobin Hemoglobin

  27. Cooperativity Models: Concerted Monod, Wyman, and Changeaux (MWC) (1965) Only T and R conformations exist The two states are in equilibrium T R transition involves shift in equilibrium constant

  28. Cooperativity Models: Sequential Koshland, Nemethy, and Filmer (KNF) (1966) There are intermediate conformations between T and R Intermediate conformations have intermediate binding affinities Change involves gradual conformational shift from more T-like states to more R-like states

  29. Hemoglobin T and R StatesHb is more MWC-like than KNF-like T (Low Affinity) R (High Affinity)

  30. Shift from T to R – another view

  31. Structural Basis • O2 Bound conformation does not permit several intersubunit bonds

  32. Histidine “Ratchet” locks T and R states • Histidine at H97 of b1 fits into socket between T41 and P44 in a2 in the T state • In the R state, the valine side chain locks between T38 and T41.

  33. In the b chains, the C teminal His makes a salt link with Asp FG1 This holds the F helix in a position that keeps the Fe+2 out of the plain of the heme ring That in turn lowers the O2 affinity Shift to the R state by the adjacent a chain breaks salt link to C-terminal His, which moves it out of position to bind Asp FG1 Relaxation of F helix allows heme Fe+2 to assume high-affinity position

  34. Bohr Effect • The O2 affinity of hemoglobin decreases with decreasing pH • Improves delivery of oxygen to the tissues

  35. Bohr Effect • C-terminal Histidine of the  subunits is protonated at low pH • His 146 can then form a salt link with Asp94 in the deoxy (T) conformation • This stabilizes the T state of the protein.

  36. Carbamate Formation Covalent binding at the N-terminus of each subunit • CO2 transport is improved since some CO2 is now being carried back to the lungs directly by hemoglobin • The release of H+ decreases pH and increases the Bohr effect • Negatively charged carbamylated N-termini form salt link to the positive charge on Arginine 141. This salt link stabilizes the deoxy (T) form of the molecule and favors O2 release.

  37. 2,3-Bisphosphoglycerate Binding

  38. Combined Effects CO2 , BPG and pH are all allosteric effectors of hemoglobin.

  39. Fetal Hb • Fetal hemoglobin has 2 α and 2 g chains • The g chain is 72% identical to the b chain. • A His involved in binding to 2,3-BPG is replaced with • Ser. Thus, fetal Hb has two less + charges than adult Hb. • The binding affinity of fetal hemoglobin for 2,3-BPG is • significantly lower than that of adult hemoglobin • Thus, the O2 saturation capacity of fetal hemoglobin is greater than that of adult hemoglobin • This allows for the transfer of maternal O2 to the developing fetus

  40. Fetal Hb Binding Curve is Always to the Left of the Maternal Hb Binding Curve

  41. Disease From a Hemoglobin Mutation

  42. Sickle Cell • Misshapen cells cause vascular occlusion • Chronic anemia • Periodic episodes of pain • Autosplenectomy after infarct • Complications • Infection • Stroke • Renal Failure • Retinopathy • Life expectancy much improved since 60’s, but still shortened: 42 ♂ 48 ♀

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