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Cysteines beta 93 and beta 112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces



G B. Vasquez, M Karavitis, X Ji, I Pechik, W S. Brinigar, G L. Gilliland, C Fronticelli


Human hemoglobin is a tetrameric protein containing two types of subunits, [alpha] and [Beta]. They assemble forming two different interfaces. The [alpha1[Beta}1 (and [alpha]2[Beta]2) interface stabilize the two dimers and is not modified by the state of ligation of the protein. The two [alpha][Beta]-dimers assemble forming the [alpha]1[Beta]2/[alpha]2[Beta]1-interfaces that mediate the quaternary changes associated to ligand binding (Ackers et al., 1992). At the [alpha]1[Beta]2/[alpha]2[Beta]1-interface a cysteine is present at position [Beta]93, this residue is highly conserved in mammalian hemoglobins. In R-state hemoglobin [Beta]93Cys is exposed to the solvent, but in the deoxy T-state it is concealed behind the salt bridge formed by [Beta]146His and [Beta]94Asp. The cysteine at [Beta]112 forms part of the [alpha]1[Beta]1/[alpha]2[Beta]2-interface and is not highly conserved in mammalian hemoglobins. Since these are the only cysteines present in the [Beta]-chains, their replacement by other residues might improve the yield in the refolding of recombinant hemoglobin in vitro and could be used to probe conformational and functional events occurring at the two different interfaces upon ligand binding. Thus, we have constructed three recombinant hemoglobins carrying either a replacement of one of the two cysteines or of both cysteines. At [Beta]112 the cysteine was replaced by glycine so to avoid effects produced by the characteristics of the side chain of the substituting amino acid. At [Beta]93, cysteine was replaced by alanine, the small hydrophobic residue was selected to prevent H-bonding to [Beta]94Asp, which was observed when [Beta]93Cys was replaced with serine. In [Beta](C93A+C112G) the two mutations are combined. [Beta]C112G and [Beta](C93A+C112G) were crystallized in the deoxy state and the structures determined at 2.0 and 1.8 A resolution, respectively. The crystallographic analysis indicated that neither the single or double amino acid replacement alters the quaternary assembly or tertiary folding of the protein. At the [Beta]112 site, the void left by the cysteine side chain is filled by a water molecule, and the functional characteristics of [Beta]C112G are essentially those of human hemoglobin A (HbA). At the [Beta]93 site the void left by the mutation does not appear to be filled with a water molecule and the cysteine to alanine substitutions increases the conformational freedom of [Beta]146His, weakening the important interaction of this residue with [Beta]94Asp. As a result, the Bohr effects of the two mutants carrying the [Beta]93 cysteine to alanine substitution, [Beta]C93A and [Beta](C93A+C112G) are dramatically modified with C1 is present in the solution. These data suggest that [Beta]93Cys plays an important role in the interaction between [Beta}146His, [Beta]94Asp and C1 in modulating the Bohr effect.
Biophysical Journal


B-globin cysteines, Bohr effect, conformational probe, hemoglobin, oxygen equilibrium, protein crystallography


Vasquez, G. , Karavitis, M. , Ji, X. , Pechik, I. , Brinigar, W. , Gilliland, G. and Fronticelli, C. (1999), Cysteines beta 93 and beta 112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces, Biophysical Journal (Accessed July 20, 2024)


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Created January 1, 1999, Updated February 19, 2017