Allosteric intermediates indicate R2 is the liganded hemoglobin end state.

Hemoglobin has been a long-standing paradigm for understanding protein allostery. Here, the x-ray structures of two chemically crosslinked, fully liganded hemoglobins, α2β82CA82β and α2β82ND82β, are described at 2.3 Å and 2.6 Å resolution, respectively. Strikingly, these crosslinked hemoglobins assume intermediate conformations that lie between those of R and the controversial liganded hemoglobin state R2 rather than between R and T. Thus, these structures support only a T ↔ R ↔ R2 allosteric pathway and underscore the physiological importance of the R2 conformation.