Identification of the Intermediate Allosteric Species in Human Hemoglobin Reveals a Molecular Code for Cooperative Switching

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-02, Vol.88 (4), p.1110-1114
• Main Authors: Daugherty, Margaret A., Shea, Madeline A., Johnson, Jeremy A., LiCata, Vince J., Turner, George J., Ackers, Gary K.
• Format: Article
• Language: English
• Subjects: allosteric properties; Allosteric Site; Amino Acid Sequence; Biochemistry; Biological and medical sciences; Code switching; Dimers; Free energy; Fundamental and applied biological sciences. Psychology; haemoglobin; Hemoglobin A - metabolism; Hemoglobins; Humans; Hybridity; Interactions. Associations; Intermolecular phenomena; Kinetics; Ligands; Ligation; man; Methemoglobin - analogs & derivatives; Methemoglobin - metabolism; Molecular biophysics; Molecular Sequence Data; mutation; Protons; structure; temperature; Thermodynamics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.4.1110

The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unligated (T) and fully ligated (R) species. To determine the nature of the intermediate allosteric state, we have studied the effects of pH, temperature, and single-site mutations on its free energy of quaternary assembly, in parallel with corresponding data on the deoxy (T) and fully ligated (R) species. Results indicate that the intermediate allosteric tetramer has the deoxy (T) quaternary structure. This finding, together with the resolved energetic distribution of the 10 microstates reveals a symmetry rule for quaternary switching-i.e., switching from T to R occurs whenever a binding step creates a tetramer with one or more ligated subunits on each side of the α1β2intersubunit contact. These studies also reveal significant cooperativity within each α1β1dimer of the T-state tetramer. The ligand-induced tertiary free energy alters binding affinity within the T structure by 170-fold prior to quaternary switching.