Volumetric properties underlying ligand binding in a monomeric hemoglobin: a high-pressure NMR study.

The 2/2 hemoglobin of the cyanobacterium Synechococcus sp. PCC 7002, GlbN, coordinates the heme iron with two histidines and exists either with a b heme or with a covalently attached heme. The binding of exogenous ligands displaces the distal histidine and induces a conformational rearrangement involving the reorganization of internal void volumes. The formation of passageways within the resulting conformation is thought to facilitate ligand exchange and play a functional role. Here we monitored the perturbation induced by pressure on the ferric bis-histidine and cyanide-bound states of GlbN using (1)H-(15)N HSQC NMR spectroscopy. We inspected the outcome with a statistical analysis of 170 homologous 2/2 hemoglobin sequences. We found that the compression landscape of GlbN, as represented by the variation of an average chemical shift parameter, was highly sensitive to ligand swapping and heme covalent attachment. Stabilization of rare conformers was observed at high pressures and consistent with cavity redistribution upon ligand binding. In all states, the EF loop was found to be exceptionally labile to pressure, suggesting a functional role as a semi-flexible hinge between the adjacent helices. Finally, coevolved clusters presented a common pattern of compensating pressure responses. The high-pressure dissection combined with protein sequence analysis established locations with volumetric signatures relevant to residual communication of 2/2 hemoglobins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.