Presentation is loading. Please wait.

Presentation is loading. Please wait.

Protein Function: Oxygen Binding Proteins CH 339K.

Similar presentations


Presentation on theme: "Protein Function: Oxygen Binding Proteins CH 339K."— Presentation transcript:

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". Nature 181 (4610): 662–666.

3 Myoglobin Myoglobin - 17,000 daltons (monomeric) 153 amino acids 8  -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 O 2 - 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. (H 63 in (SWM) The second site can be reversibly occupied by O 2, which is hydrogen- bonded to another His. (His E7, H 94 in SWM)

8

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

10 Absorbance Curves for Mb

11 CO Poisoning Autopsy photo showing characteristic skin discoloration Myoglobin’s affinity for carbon monoxide is ~ 60x its affinity for O2. Hemoglobin’s affinity for carbon monoxide is ~ 230x its affinity for O 2.

12 O 2 Binding Kinetics

13 Reaction : Mb + O 2 ⇌ Mb  O 2 (1) (2 (3) (4) (5)

14 (6) (7) (8) Dr. Ready finally gets to the point!

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

16 So: Converts to:

17 Hyperbolic Binding Kinetics

18 p50 Defines the Curve

19 Oxygen Transport

20 3 o structure overlap: myoglobin,  -globin and  -globin  -Globin (blue)  -Globin (violet) Myoglobin (green)

21 Mb vs. Hb

22

23

24 Hemoglobin O 2 carrying capability Erythrocytes/ml blood: 5 billion ( 5 x 10 9 ) Hemoglobin/red cell: 280 million ( 2.8 x 10 8 ) O 2 molecules/hemoglobin: 4 O 2 ml blood: (5 x 10 9 )(2.8 x 10 8 )(4) = (5.6 x 10 18 ) or (5.6 x 10 20 ) molecules of O 2 /100 ml blood or ~ 0.3 g/l or ~ 9 mM By comparison: Solubility of O 2 in saline: ~ 0.007 g/l or ~ 0.2 mM

25 O2 Binding – Hb vs. Mb

26 O 2 transport capability, a comparison

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

28

29 Cooperative Binding Kinetics Reaction: Hb + nO 2 ⇌ Hb  nO 2 (1) (2) (3)

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

31

32 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

33 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

34 Hemoglobin T and R States Hb is more MWC-like than KNF-like T (Low Affinity)R (High Affinity)

35 Shift from T to R – another view

36

37

38 Structural Basis O 2 Bound conformation does not permit several intersubunit bonds

39 Histidine “Ratchet” locks T and R states Histidine at H 97 of  1 fits into socket between T 41 and P 44 in  2 in the T state In the R state, the valine side chain locks between T 38 and T 41.

40 In the  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 O 2 affinity Shift to the R state by the adjacent  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

41 Bohr Effect The O 2 affinity of hemoglobin decreases with decreasing pH Improves delivery of oxygen to the tissues

42 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.

43 Carbamate Formation Covalent binding at the N-terminus of each subunit CO 2 transport is improved since some CO 2 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 O 2 release.

44 2,3-Bisphosphoglycerate Binding

45

46 Combined Effects CO 2, BPG and pH are all allosteric effectors of hemoglobin.

47 Fetal Hb Fetal hemoglobin has 2 α and 2  chains The  chain is 72% identical to the  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 O 2 saturation capacity of fetal hemoglobin is greater than that of adult hemoglobin This allows for the transfer of maternal O 2 to the developing fetus

48 Fetal Hb Binding Curve is Always to the Left of the Maternal Hb Binding Curve

49 Disease From a Hemoglobin Mutation

50 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 ♀

51 Sickle Cell Complications Above: dactylitis Below: swollen, scarred spleen

52

53 Sickle Cell Cause: Glu  6 changed to Valine by gene mutation Hydrophobic residue binds to pocket on adjacent  chain of deoxygenated form ~5% of American blacks carry gene This is not a neutral mutation

54

55 Geographic Distribution of HbS

56 Malaria Belt

57 Heterozygote Advantage Heterozygous individuals in Nigeria had a 29% higher likelihood of surviving to adulthood than homozygous normals. The gene is maintained in the population by selection against both homozygotes.

58 Other O 2 Binding Proteins (w/o Heme)

59 Other O 2 Binding Proteins Hemocyanins –Molluscs and some arthropods –Copper acts as binding metal –Cu(I) (colorless)  Cu(II) (blue) –75 kDal monomers (arthropods) Each monomer has 2 Cu, binds 1 O 2 –Form dimers or hexamers –Polymers form very large complexes

60 Hemocyanin structures A. 24mer from Eurypelma (a tarantula) B. Single subunit from Limulus (horseshoe crab) C. 20 x 8mer from Haliotis (Abalone) (each individual polypeptide is an 8-fold repeat) d. C-terminal subunit from Octopus.

61 Other O 2 Binding Proteins Hemerythrins –Sipunculids, brachiopods, priapulids, bacteria –Binuclear iron center –Fe(II)  Fe(III) –13-14 kDal monomers Each monomer has 2 Fe, binds 1 O 2 –Form (most often) octamers –Not cooperative Fe Fe-O-O Fe-O-OH \ + O=O \ : \ O-H  O··H  O /  /  / Fe Fe Fe A (deoxy) B C (oxy)

62 SipunculidPriapulidBrachiopod

63 Hemerythrin

64 O 2 Binding Sites

65 Another Heme Protein That Doesn’t Bind O 2

66 The Disease - Chagas

67 Symptoms Acute Phase (weeks to months) Swelling at the infection site Fever Fatigue Rash Body aches Headache Loss of appetite Nausea, diarrhea or vomiting Swollen glands Enlargement of your liver or spleen Chronic Phase (10-20 years post-infection) Irregular heartbeat Inflamed, enlarged heart (cardiomyopathy) Congestive heart failure Sudden cardiac arrest Difficulty swallowing due to enlarged esophagus Abdominal pain or constipation due to enlarged colon

68 Acute Stage – swelling at bite location Chronic Stage – cardiomyopathy, congestive heart failure Chronic Stage - megacolon Chagas Complications

69 The Agent: Trypanosoma cruzi

70 The Vector – Cone Nosed Bugs Triatoma gerstaeckeri (Local) Rhodnius prolixus (Tropical)

71 The Protein - Nitrophorin dimer of 20 kdal monomers Heme contains Fe +3 salivary glands of Triatomid bugs bind nitrous oxide (NO)

72 Nitrophorin Action – slightly dramatized Ravenous insect climbs onto face of peacefully sleeping human victim Inserts hideous proboscis into helpless victim’s flesh Nitrophorin, with NO bound, is injected into the bite wound In the alkaline environment of the ghastly wound, NO is released NO acts as vasodilator, prevents platelet accumulation Empty binding site on nitrophorin binds histamine Antihistamine effect prevents irritation to wake hapless blood donor.

73 Lest you think this is all theoretical… From Emerging Infectious Diseases (2003) 9(1): 103-105 Potential future distribution in Texas


Download ppt "Protein Function: Oxygen Binding Proteins CH 339K."

Similar presentations


Ads by Google