Abstract
The enormous success of structural biology challenges the physical scientist. Can biophysical studies provide a truly deeper understanding of how a protein works than can be obtained from static structures and qualitative analysis of biochemical data? We address this question in a case study by presenting the key concepts and experimental results that have led to our current understanding of cooperative oxygen binding by hemoglobin, the paradigm of structure function relations in multisubunit proteins. We conclude that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Edsall, J.T. Hemoglobin and the origins of the concept of allosterism. Fed. Proc. 39, 226–235 ( 1980).
Bohr, C., Hasselbach, K.A. & Krogh, A. Über einen in biologischen Beziehung wichtigen Einfluss, den die Kohlen-sauerspannung des Blutes auf dessen Sauerstoffbindung übt. Skand. Arch. Physiol. 15, 401– 412 (1904).
Adair, G.S. A critical study of the direct method of measuring osmotic pressure of hemoglobin. Proc. R. Soc. London Ser. A, 108A, 627– 637 (1925).
Pauling, L. The oxygen equilibrium of hemoglobin and its structural interpretation. Proc. Natl. Acad. Sci. USA 21, 186– 191 (1935).
Pauling, L., Itano, H.A., Singer, S.J. & Wells, I.C. Sickle cell anemia: a molecular disease. Science 110 , 543–548 (1949).
Perutz, M. F., Bolton, W., Diamond, R., Muirhead, H. & Watson, H. Structure of haemoglobin. An X-ray examination of reduced horse haemoglobin. Nature 203, 687– 690 (1964).
Monod, J., Wyman, J. & Changeux, J.-P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88– 118 (1965).
Koshland, D.E., Nemethy, G. & Filmer, D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5, 365–385 (1966).
Perutz, M.F. Stereochemistry of cooperative effects in haemoglobin. Nature 228, 726–739 (1970).
Perutz, M.F., Wilkinson, A.J., Paoli, M. & Dodson, G.G. The stereochemistry of the cooperative effects in hemoglobin revisited. Ann. Rev. Biophys. Biomol. Struct. 27, 1– 34 (1998).
Rodgers, D., Crepeau, R.H. & Edelstein, S.J. Pairings and polarities of the 14-strands in sickle cell hemoglobin fibers. Proc. Natl. Acad. Sci. USA 84, 6157–6161 (1987).
Eaton, W.A. & Hofrichter, J. Sickle cell hemoglobin polymerization. Adv. Prot. Chem. 40, 63– 279 (1990).
Edelstein, S.J. Extensions of the allosteric model to haemoglobin. Nature 230, 224–227 (1971).
Szabo, A. & Karplus, M. A mathematical model for structure-function relations in hemoglobin. J. Mol. Biol. 72: 163–197 (1972).
Imai, K. The Monod-Wyman-Changeux allosteric model describes haemoglobin oxygenation with only one adjustable parameter. J. Mol. Biol. 167 , 741–749 (1983).
Shulman, R.G., Hopfield, J.J. & Ogawa, S. Allosteric interpretation of haemoglobin properties. Quart. Rev. Biophys. 8, 325– 420 (1975).
Perutz, M.F. Mechanisms of cooperativity and allosteric regulation in proteins. Quart. Rev. Biophys. 22, 139–236 (1989).
Smith, F.R. & Ackers, G.K. Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin. Proc. Natl. Acad. Sci. USA 82, 5347– 5351 (1985).
Sawicki, C.A & Gibson, Q.H. Quaternary conformational changes in human hemoglobin studied by laser photolysis of carboxyhemoglobin. J. Biol. Chem. 251, 1533–1542 (1976).
Rivetti, C., Mozzarelli, A., Rossi, G.L., Henry, E.R. & Eaton, W.A. Oxygen binding by single crystals of hemoglobin. Biochemistry 32, 2888– 2906 (1993).
Liddington, R., Derewenda, Z., Dodson, G.G. & Harris, D. Structure of the liganded T state of hemoglobin identifies the origin of cooperative oxygen binding. Nature 331, 725– 728 (1988).
Sun, D.Z.P., Zou, M., Ho, N.T., & Ho, C. The contribution of surface histidyl residues in the alpha-chain to the Bohr effect of human adult normal hemoglobin: roles of global electrostatic effects. Biochemistry 36, 6663–6673 ( 1997).
Bettati S., Mozzarelli A. & Perutz M.F. Allosteric mechanism of haemoglobin: rupture of salt-bridges raises the oxygen affinity of the T-structure. J. Mol. Biol. 281, 581–585 (1998).
Shibayama, N., & Saigo, S. . Fixation of the quaternary structures of human adult haemoglobin by encapsulation in transparent porous silica gels. J. Mol. Biol. 251, 203 –209 (1995).
Bettati. S. & Mozzarelli, A. T state hemoglobin binds oxygen noncooperatively with allosteric effects of protons, inositol hexaphosphate, and chloride. J. Biol. Chem. 272, 32050 –32055 (1997).
Ackers, G.K. Deciphering the molecular code of hemoglobin allostery. Adv. Prot. Chem. 51, 185–253 ( 1998).
Gill, S.J., Robert, C.H., Coletta, M., Di Cera, E. & Brunori, M. Cooperative free energies for nested allosteric models as applied to human hemoglobin. Biophys. J. 50, 747–752 (1986).
Mozzarelli, A., Rivetti, C., Rossi, G.L., Eaton, W.A. & Henry, E.R. Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals. Protein Sci. 6, 484–489 (1997).
Shibayama, N., Morimoto, H. & Saigo, S. Asymmetric cynanomet valency hybrid hemoglobin: the issue of valency exchange. Biochemistry 37, 6221–6228 (1998).
Gibson, Q.H. The photochemical formation of a quickly reacting form of haemoglobin. Biochem. J. 71, 293–303 (1959).
Antonini, E. & Brunori, M. Hemoglobin and myoglobin in their reactions with ligands (North-Holland Publishing Co., Amsterdam; 1971).
Hopfield, J.J., Shulman, R.G. & Ogawa, S. An allosteric model of hemoglobin: I, kinetics. J. Mol. Biol. 61, 425–443 (1971).
Hofrichter, J., Sommer, J.H., Henry, E.R. & Eaton, W.A. Nanosecond absorption spectroscopy of hemoglobin, elementary processes in kinetic cooperativity. Proc. Natl. Acad. Sci. USA 80 , 2235–2239 (1983).
Jackson, T.A., Lim, M. & Anfinrud, P.A. Complex nonexponential relaxation in myoglobin after photodissociation of MbCO: measurement and analysis from 2 ps to 56 μs. Chem. Phys. 180, 131–140 ( 1994).
Frauenfelder, H., Sligar, S.G. & Wolynes, P.G. The energy landscapes and motions of proteins. Science 254, 1598–1603 ( 1991).
Agmon, N., & Hopfield, J.J. CO binding to heme proteins: a model for barrier height distributions and slow conformational changes. J. Chem. Phys. 79, 2042– 2053 (1983).
Hagen, S.J., Hofrichter, J. & Eaton, W.A. Protein reaction kinetics in a room-temperature glass. Science 269, 959–962 (1996).
Austin, R.H., Beeson, K.W., Eisenstein, L., Frauenfelder, H. & Gunsalus, I.C. Dynamics of ligand binding to myoglobin. Biochemistry 14, 5355– 5373 (1975).
Eaton, W.A., Henry, E.R. & Hofrichter, J. Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin. Proc. Natl. Acad. Sci. USA 88, 4472– 4475 (1991).
Henry E.R., Jones, C.M., Hofrichter, J. & Eaton, W.A. Can a two-state MWC allosteric model explain hemoglobin kinetics? Biochemistry 36, 6511–6528 (1997).
Perrella, M., Colosimo, A., Benazzi, L., Ripamonti, M. & Rossi-Bernardi, L. What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity. Biophys. Chem. 37, 211– 223 (1990).
Dickerson, R.E. & Geis, I. Hemoglobin: structure, function, evolution, and pathology. (Benjamin/Cummings, Menlo Park, California; 1983).
Huang Y.W., Doyle M.L. & Ackers G.K. The oxygen-binding intermediates of human hemoglobin: evaluation of their contributions to cooperativity using zinc-containing hybrids. Biophys. J. 71, 2094–2105 (1996).
Doyle, M.L., Holt, J.M. & Ackers, G.K. Effects of NaCl on the linkages between O2 binding and subunit assembly in human hemoglobin: titration of the quaternary enhancement effect. Biophys. Chem. 64, 271 –287 (1997).
Ackers, G.K. The energetics of ligand-linked subunit assembly in hemoglobin require a third allosteric structure. Biophys. Chem. 37, 371–382 (1990).
Acknowledgements
We thank A. Szabo for numerous helpful discussions on the hemoglobin mechanism and for his comments on the manuscript. We also thank M. Brunori, P. Wolynes, and R. Zwanzig for helpful discussions, and G.L. Rossi for his generous support and collaboration in the single-crystal studies. This work was supported by a NATO Collaborative Research grant. This work was presented by W.A.E. at the Dahlem Workshop on "Simplicity and Complexity in Proteins and Nucleic Acids," Berlin, Germany, May 17–22, 1998 (eds Frauenfelder, H., Deisenhofer, J. & Wolynes, P.G.) Dahlem University Press (in the press).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Eaton, W., Henry, E., Hofrichter, J. et al. Is cooperative oxygen binding by hemoglobin really understood?. Nat Struct Mol Biol 6, 351–358 (1999). https://doi.org/10.1038/7586
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/7586
This article is cited by
-
Mathematical models describing oxygen binding by hemoglobin
Biophysical Reviews (2023)
-
Multivalent cooperativity induced by self-assembly for f-element separation
Communications Chemistry (2021)
-
A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy
Biophysical Reviews (2018)
-
Tertiary and quaternary structural basis of oxygen affinity in human hemoglobin as revealed by multiscale simulations
Scientific Reports (2017)
-
Phthalide Derivatives from Angelica Sinensis Decrease Hemoglobin Oxygen Affinity: A New Allosteric-Modulating Mechanism and Potential Use as 2,3-BPG Functional Substitutes
Scientific Reports (2017)