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1、Biochemistry Lecture (Oct. 9, 2012)Providing paradigms for understanding protein structure, function andaction mechanism (Hb has been dubbed as an “honorable enzyme”.) Tanford, C. and Reynolds, J. Natures Robots : A History of Proteins, 2001, Oxford University Press, USA. Hemoglobin transports O2, N
2、O, and CO2 in vertebrates.Myoglobin transports and stores O2 inmuscle tissuesOther O2-binding proteins,some not heme-containing,have been found ininvertebrates, bacteriaand plants!97% of the red blood cells dry content lIn invertebrates, fungi, plants, and bacteria.Hemocyanins(molluscs) Hemerythrin(
3、marine invertebrates)Leghemoglobin(soybeans)A hypothesized model for trHbO to deliver O2 in M. tuberculosis.The dimer to monomerchange may decreasethe O2 affinity of trHbOLiu, C. He., Y. and Chang Z. (2004) Truncated hemoglobin of Mycobacterium tuberculosis: The oligomeric state change and the inter
4、action with membrane components” Biochem Biophys Res Commun. 316:1163-1172. Truncated hemoglobin in mycobacterialFirst successful crystallization by Otto Funke (1851).lOptical absorption spectrum and reversible binding of O2 observed by Felix Hoppe-Seyler (1866). lThe structure determined by Max Per
5、utz (1960).Otto Funke(1828-1879)Felix Hoppe-Seyler(1825-1895) Max Perutz(1914-2002)Hoppe-Seyler (1862) initially described the two characteristic absorption bands of the red blood pigment (who coined the term “hemoglobin” and “prosthetic group”) Stokes (1864): when red blood is treated with a reduci
6、ng agent it becomes purplish and its two strong absorption bands are replaced by a single and more diffuse band; when this reduced solution is shaken with air, the colour and the absorption spectrum return to their original states; He correlated the red and purple blood with arterial and venous bloo
7、d; reduced the haematin in the presence of ammonia and obtained a pigment with two very strong absorption bands of reduced haematin. lHoppe, F. (1862) lVirchows Arch. 23, 446 - 449 Stokes GG (1864) On the Reduction and Oxidation of the Colouring Matter of the Blood. Proc. Royal Soc 13: 355-364 Red P
8、urpleChristian Bohr (1855-1911)(father of Niels Bohr)lObserved with blood, not pure HemoglobinlWith PaO2 above 80 mmHg, the hemoglobin is almost completely saturated; with PaO2 below 50-60 mmHg it releases O2 quite easily.lHigher PaCO2 and lower pH decreases the O2 affinity. Bohr C et al “Concerning
9、 a Biologically Important Relationship - The Influence of the Carbon Dioxide Content of Blood on its Oxygen Binding Skand. Arch. Physiol. 16, 401-412 (1904) “ CO2 causes O2 binding of hemoglobin to decrease significantly at low partial pressure of O2. At a partial O2 pressure of 150mm, almost no eff
10、ect on O2 binding can be observed. Because all experiments so far that investigated the O2 binding of hemoglobin have been carried out at a high partial O2 pressure, this may account for the fact that the influence of CO2 has been overlooked.” O2 binding by whole dog blood (solid lines) as a functio
11、n of O2 partial pressure at different partial presusres of CO2.(dashed line: rectangular hyperbola curves obtained by Hufners group,using hemoglobin solution)Conclusion: CO2 promotes O2 release but does not directly compete in binding to heme!lHill R (1933) Oxygen affinity of muscle haemoglobin. Nat
12、ure 132:897-898.lHill AV. The Combinations of Haemoglobin with Oxygen and with Carbon Monoxide. I. Biochem J. 1913 7:471-480. Archibald Hill(1886-1977)Oligomeric Hb assumed to be formedPartially saturated Hb assumed very unstableThere is no Hb2O2 but only Hb2(O2)2lHill Equation: lA plot of log/(1- )
13、 versus log pO2 is called a Hill Plot.lThe slope of Hill plot, nH (the Hill coefficient) reflects the degree of cooperativity (i.e., cooperation of the binding sites):When nH = 1, the binding is not cooperative;When nH 1, the binding is positively cooperative;When nH 1, the binding is negatively coo
14、perative.1- = n log pO2 log P50log - fraction of occupied sites; L - free ligand concentration; Kd- Apparent dissociation constant KA- ligand concentration producing half occupation. The macromolecule is assumed to bind to n ligands simultaneously (where n is to be determined)Hill plots of oxygen bi
15、nding for myoglobin and hemoglobin. lWholly deoxygenated hemoglobin molecules are in the deoxy conformation, so as O2 is added, the binding initially occurs along the line corresponding to the weak-binding state. lPartial oxygenation favors transition to the strong-binding oxy state. As O2 is bound,
16、 more and more of the remaining available sites are in hemoglobin molecules that have this conformation. The binding curve then passes over to that for the strong-binding state. lAs the last few sites are filled, all the molecules have adopted the strong-binding form nHlElemental analysis revealed t
17、he minimal formula (Zinoffsky, 1886): C712 H1130 N214 S2 Fe O245; Minimal MW being 16, 000.lThe osmotic pressure method (Adair, 1925): MW is 67,000.l Sedimentation equilibrium ultracentrifugation (Svedberg, 1926): four subunits of 16,700, i.e. 66,800.Adair, G. S. (1925) A Critical Study of the Direc
18、t Method of Measuring the Osmotic Pressure of Hemoglobin, Proc. Royal Soc. London, 108A:627-637.Svedberg, T & Fhraeus R. (1926) A New Direct Method for the Determination of the Molecular Weight of the Proteins, J. Am. Chem. Soc. 48:430-438. (ultracentrifugation)lTreatment of hemoglobin with acid gav
19、e a colorless albuminoid constituent (globin) and a red iron-containing material (by 1870).lThe structure of heme was elucidated to be a tetrapyrrol (porphyrin) derivative by chemical synthesis (Hans Fischer, 1929).The heme group = protoporphyrin IX + Fe2+The tetrapyrrol ringSix coordinationbonds wi
20、ll beFormed for the Ferrous iron (Fe2+)O2 would not bind if the iron is initially in the ferric (Fe3+) state!pyrrolpropionatevinylThe red color of blood is due to the porphyrin moiety of hemoglobin, not the iron itself !Hemoproteins have diverse biological functions: transportation of diatomic gases
21、, catalysis, diatomic gas detection, and electron transfer. The Nobel Prize in Chemistry 1930for his researches into the constitution of haemin and chlorophyll and especially for his synthesis of haemin Hans Fischer GermanyTechnische Hochschule (Institute of Technology) Munich, Germany b. 1881d. 194
22、5Kendrew et al. (1958) A Three-Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis. Nature 181: 6626. Perutz et al (1960) Structure of Hmoglobin: A Three-Dimensional Fourier Synthesis at 5.5-. Resolution, Obtained by X-Ray Analysis Nature 185:416-422. The distal His(His E7, or His
23、64)The proximal His(His F8, or His93)CO (colorless, odorless and tasteless) can also bind to heme, with about 250 X greater affinity than O2, accounting for its toxicity.Heme is noncovalently bound! !His E7His F8Carboxyhemoglobin A ferrous (Fe2+)to ferric (Fe3+)seems to occurafter O2 is bound.The qu
24、aternary structure of the a a2 2b b2 2 Hb tetramer (Max Perutz, 1960) The greatest surprise for Perutz: the four hemes are far from each other!Heme 3Heme 4Heme 1Heme 2The hemes are0.24-0.4 nmaway from each other.The Nobel Prize in Chemistry 1962for their studies of the structures of globular protein
25、s Max Ferdinand Perutz John Cowdery Kendrew 1/2 of the prize 1/2 of the prize United Kingdom United Kingdom MRC Laboratory of Molecular Biology Cambridge, United Kingdom MRC Laboratory of Molecular Biology Cambridge, United Kingdom b. 1914(in Vienna, Austria)d. 2002b. 1917d. 1997Mb(1947- 1959)Kendre
26、w et al. (1960)Nature 185:422Hb(1937-1960)Perutz et al. (1960)Nature 185:416Kendrewin 1997The father ofprotein X-raycrystallography! The Nobel Prize in Physiology or Medicine 1962for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer i
27、n living material Francis Harry Compton Crick(Ph.D. student of Perutz)James Dewey Watson(Postdoc of Kendrew) Maurice Hugh Frederick Wilkins Mb, Hba a, and Hbb b share a similar tertiary structure although their sequence identity is only about 18%.Deoxy-HbOxy-HbConformational differences between the
28、T- (Taut) and R (Relaxed)-states: Tertiary Structures changed little, only the two pairs of abab dimers slide past each and rotate.lThe O2 affinity of Hb in the whole blood was found to be much lower than the purified Hb (1921).lThe regulatory substance was found to be 2,3-bisphosphoglycerate (1967)
29、.lMore BPG was found to be produced in erythrocyte when one moves to high altitudes.lThe fetal Hb (a a2 2g g2 2) has a much lower affinity for BPG than the adult Hb, thus a higher affinity for O2, allowing O2 to be effectively transferred from maternal to fetal red cells.Benesch R. and Benesch, R. E
30、. (1967) The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin, BBRC, 26:162-167.lBohr, Hasselbalch, and Krogh (1904): observed cooperative homotropic binding of oxygen by hemoglobin and the heterotropic control exerted by CO2 in diminishing the oxyge
31、n affinity. lV. Hill n(1910): explained cooperative phenomena by postulating reversible aggregation of these monomer units. lAdair and Svedberg (1925, 1926): independently showed that hemoglobin contains four subunits.lHaurowitz (1938): demonstration that crystal structure (conformation) changed on
32、oxygenationlPerutz (1960): Revealed in molecular detail the nature of the change associated with ligand binding. lRreciprocal interactions between oxygen binding and the uptake of the heterotropic ligands, proton, CO2, diphosphoglycerate. lReversible conformational transitions are the basis for both
33、 homotropic and heterotropic interactions in allosteric proteins.lMonod, J., Changeux, JP, and Jacob, F. (1963) Allosteric proteins and cellular control systems. J Mol Biol. 6:306-29. lMonod, J., Wyman, J. and Changeux (1965) On the nature of allosteric transitions: A plausible model, J. Mol. Biol.
34、12:88-118.lKoshland DE Jr, Nmethy G, Filmer D. (1966) Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365-85. A current understanding on the cooperative oxygen-binding process: the strain-transmitting model O2 binds at the left of the ir
35、on atom shown in the upper left of diagram. This causes the iron atom to move backward into the heme which holds it, tugging the histidine residue (the red pentagon on the right of the iron) closer, This, in turn, pulls on the protein chain holding the histidine. Fetal hemoglobin (HbF) binds oxygen
36、with higher affinity than adult hemoglobin (HbA). So the fetus can acquire oxygen from the maternal circulation. lHerrick JB (1910). Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia“ Archives of Internal Medicine 6: 51721. Electron micrographof deoxy-Hb S fiberssp
37、illing out of a ruptured erythrocyte.The sickled cells are fragile. Their breakdown leads to an anemia that leaves the victim susceptible to infections and diseases. lPauling, L. Itano, HA, Singer SJ, and Wells IC (1949) Sickle cell anemia a molecular disease. Science. 110:543-8. lIngram, V. M. (195
38、6) A Specific Chemical Difference Between the Globins of Normal Human and Sickle-Cell Anmia Hmoglobin Nature 178:792-794. lIngram, V. M. (1957) Gene Mutations in Human Hmoglobin: the Chemical Difference Between Normal and Sickle Cell Hmoglobin, Nature 180, 326 - 328 Sickle cell anemia is caused by a
39、 single amino acid substitution, which isresulted from a single base change in the encoding Hb S gene. b b subunit of Hb Ab b subunit of Hb SlWishner BC, Ward KB, Lattman EE, Love WE. (1975) Crystal structure of sickle-cell deoxyhemoglobin at 5 A resolution. J Mol Biol. 98:179-94. lHarrington DJ, Ad
40、achi K, Royer WE Jr. (1997) The high resolution crystal structure of deoxyhemoglobin S. J Mol Biol. 272:398-407. lJia L, Bonaventura C, Bonaventura J, Stamler JS. (1996) S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature. 380:221-6. lBiagioli et al. (2009) Unexpected expression of a a and b b-globin in mesencephalic dopaminergic neurons and glial cells. PNAS, 106:15454-15459.Even for a protein as extensively studied as hemoglobin, there is still a lot to learn about its structure, function, regulation and mechanism (especially in living cells)!
生物化學(xué)：Chapter 5-2 Protein Function,Modulation and evolution
