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N-terminal contributions of the -subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts

¹ Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA
² Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, Computing Services, Rockefeller University, New York, New York 10021, USA

Reprint requests to: J.M. Manning, Department of Biology, Mugar Life Sciences Building, Room 414, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA; e-mail: jmanning{at}lynx.neu.edu; fax: (617) 373-4496.

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the -subunit of fetal hemoglobin, which differs from the ß-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the -subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the ß-subunit by the corresponding residues in the -subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 () of HbF with its high pK_a of 8.1 compared with a 7.1 value for Val 1 (ß) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the -subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (ß) by Glu 5 () promotes tetramer strengthening. The Glu Asp replacement at position 7 enhances this effect because of the lower pK_a of Asp but the Val Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.

Keywords: Hemoglobin; long-range interaction; site-directed mutagenesis

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