High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.
The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 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 poly(ethylene 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 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. 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.
Kavanaugh
J S
JS
Department of Biochemistry, University of Iowa, Iowa City 52242.
Rogers
P H
PH
Case
D A
DA
Arnone
A
A
D000838:Anions; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides / Q000378:metabolism*; D003460:Crystallization; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006455:Hemoglobins, Abnormal / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D015394:Molecular Structure; D009154:Mutation; D011487:Protein Conformation; D014961:X-Ray Diffraction
D000838:Anions; D002712:Chlorides; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D046911:Macromolecular Substances; C024993:hemoglobin Rothschild; C100003:deoxyhemoglobin