High-Resolution x-ray Study of Deoxyhemoglobin Rothschild 37β Trp→Arg: A Mutation that Creates an Intersubunit Chloride-Binding Site

The mutation site in hemoglobin Rothschild (37β Trp → Arg) is located in the “hinge region” of the αlβ2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under “low-salt” conditions [100 mM Cl^- in 10 mM phosphate buffer at pH 7.0 with polyethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1° rotation of the α subunit about the “switch region” of the αlβ2 interface. The tertiary changes are confined to residues at the αlβ2 interface, with the largest shifts (˜0.4 A) located across the interface from the mutation site at the α subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the α 1β2 interface. Chloride binds at this site as a counterion for Arg 37β. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.