Evolution of Hemoglobin as Studied by Protein Engineering and X-Ray Crystallography

Abstract

Proteins in the globin family are found in a wide variety of organisms, their physiological function being the storage and transport of oxygen. In vertebrate blood, Hb forms a tetramer consisting of two α and two β chains. Assembly into tetramers was a crucial step in globin evolution, allowing it to acquire its allosteric properties. The tetrameric Hb molecule can form two alternative quaternary structures, the T structure with low oxygen affinity and the R structure with high oxygen affinity (Perutz 1979; Monod et al. 1965). The equilibrium between these two structures allows the four subunits to bind oxygen co-operatively and hence Hb can load and unload oxygen efficiently over a small change in partial oxygen pressure. Various metabolites such as H+, 2,3-diphos- phoglycerate (DPG), HCO3 -, CO2, and ATP, bind to the T structure more strongly and thereby reduce the oxygen affinity (Kilmartin and Rossi- Bernardi 1973). These effectors are called the heterotropic effectors, and help to deliver oxygen preferentially to actively respiring tissues. Many amino acid residues on the subunit surface are involved in intersubunit contacts and the binding of heterotropic ligands; any mutations at these positions are likely to affect the free energy of the R and T states. This imposes additional selective pressure on the Hb molecule.