Crystallographic analysis of mutant human haemoglobins made in Escherichia coli.
The expression of beta-globin in Escherichia coli has enabled us to study the functional role of individual amino-acid residues in haemoglobin (Hb) by site-directed mutagenesis. In contrast to mammalian Hbs, some teleost fish haemoglobins show a drastic lowering of oxygen affinity and cooperativity at low pH, a phenomenon known as the Root effect. We have produced the two mutant haemoglobins Hb Nymphéas [Cys(F9)93 beta----Ser] and Hb Daphne [His(H21)143 beta----Arg, Cys(F9)93 beta----Ser] to investigate this allosteric property. Although these substitutions were thought to be responsible for the Root effect, Hb Nymphéas and Hb Daphne show an increased oxygen affinity and a reduced effect of pH on oxygen affinity. Our X-ray crystallographic studies show that the hydroxyl group of Ser 93 beta forms a hydrogen bond with Asp 94 beta which is in equilibrium with the salt bridge between Asp 94 beta and His 146 beta. The oxygen-binding properties of Hbs Nymphéas and Daphne are accounted for by the partial disruption of the salt bridge.
D000596:Amino Acids / Q000032:analysis; D003461:Crystallography; D004926:Escherichia coli / Q000235:genetics; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D006454:Hemoglobins / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
D000596:Amino Acids; D006454:Hemoglobins; D005914:Globins