Crystallographic evidence for a new ensemble of ligand-induced allosteric transitions in hemoglobin:: The T-to-THigh quaternary transitions

A detailed description of hemoglobin cooperativity requires knowledge of the dimer-dimer interactions responsible for the low ligand affinity of the quaternary-T tetramer, the "quaternary-T constraints", along with stereochemical pathways that specify how ligand binding disrupts these quaternary constraints. The recent mutagenic screen of Noble et al. [Noble, R. W., et al. (2001) Biochemistry 40, 12357-12368] has identified the major region of quaternary constraint to be a cluster of residues at the alpha 1 beta 2 interface that is centered at Trp37 beta. In this paper, crystallographic studies are presented for most of the mutant hemoglobins studied by Noble et al. These crystallographic experiments identify structural transitions-referred to as T-to-T-High transitions-between the quaternary-T structure of wild-type deoxyhemoglobin and an ensemble of related T-like quaternary structures that are induced by some mutations in the Trp37 beta cluster and/or by exposing crystals of wild-type or mutant deoxyhemoglobins to oxygen. The T-to-T-High quaternary transitions consist of a rotation of the al l dimer relative to the alpha 2 beta 2 dimer as well as a coupled alpha beta dimer bending component that consists of a small rotation of the alpha 1 subunit relative to the beta 1 subunit (and a symmetry related rotation of the alpha subunit relative to the beta 2 subunit). In addition, differences in subunit tertiary structure associated with the T-to-THigh transitions suggest two stereochemical pathways (one associated with the a subunits and one associated with the subunits) by which ligand binding specifically disrupts quaternary constraints in the Trp37 beta cluster. In the a subunits, ligand binding induces a shift of the heme iron producing tension in a chain of covalent bonds that extends from the Fe-N(is an element of 2)His(F8)alpha 1 bond to the peptide backbone bonds of residues His87(F8)alpha 1 and Ala88(F9)alpha 1. This tension induces an alpha-to-pi transition in the COOH-terminal end of the F-helix that shifts the beta-carbon of Ala88 alpha 1 by similar to 1.5 angstrom directly into the side chain of Tyr140 alpha 1 (a key residue in the Trp37 beta 2 cluster). Collectively these structural changes constitute a relatively short pathway by which ligand binding forces Tyr140 alpha 1 into the alpha 1 beta 2 interface disrupting quaternary constraints associated with the Trp37 beta 2 cluster. In the beta subunits, our analysis suggests a more extended energy transduction pathway in which ligand-induced beta 1-heme movement triggers tertiary changes in the beta 1 subunit that promote alpha 1 beta 1 dimer bending that disrupts quaternary constraints in the Trp37 beta 2 cluster at the alpha 1 beta 2 interface.