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Biophys J, January 1999, p. 88-97, Vol. 76, No. 1

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

Gregory B. Vásquez,* Michael Karavitis,# Xinhua Ji,* Igor Pechik,* William S. Brinigar,§ Gary L. Gilliland,* and Clara Fronticelli#

 *Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and National Institute of Standards and Technology, Rockville, Maryland 20850,  #Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201, and  §Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122 USA

Three variants of tetrameric human hemoglobin, with changes at the alpha 1beta 2/alpha 2beta 1-interface, at the alpha 1beta 1/alpha 2beta 2-interface, and at both interfaces, have been constructed. At alpha 1beta 2/alpha 2beta 1-interface the beta 93 cysteine was replaced by alanine (beta C93A), and at the alpha 1beta 1/alpha 2beta 2-interface the beta 112 cysteine was replaced by glycine (beta C112G). The alpha 1beta 2 interface variant, beta C93A, and the alpha 1beta 1/alpha 1beta 2 double mutant, beta (C93A+C112G), were crystallized in the T-state, and the structures determined at 2.0 and 1.8 Å resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the beta 112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of beta C112G are essentially those of human hemoglobin A. At the beta 93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of beta 146His, weakening the important interaction of this residue with beta 94Asp. As a result, when Cl- is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the beta 93Cysright-arrowAla substitution, beta C93A and beta (C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between beta 94Asp and Cl- in the salt link with beta 146His in T-state hemoglobin. These results point to an interplay between the beta His146-beta Asp94 salt bridge and the Cl- in solution regulated by the Cys present at position beta 93, indicating yet another role of beta 93 Cys in the regulation of hemoglobin function.

Biophys J, January 1999, p. 88-97, Vol. 76, No. 1
© 1999 by the Biophysical Society   0006-3495/99/01/88/10  $2.00


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[Abstract] [Full Text] [PDF]


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