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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.
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