The Enigma of the Liganded Hemoglobin End State:  A Novel Quaternary Structure of Human Carbonmonoxy Hemoglobin

The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the “classical” liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between β2His97 and α1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T → R3 and T → R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the α-heme, reduced steric contact between the β-heme ligand and the distal residues, smaller α- and β-clefts, and reduced α1−α2 and β1−β2 iron−iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure−function relationships that are now based on T → R, T → R → R2, or T → R2 → R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.