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

Abstract

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 li-gands 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 afunctional role. Herewemonitored the perturbation induced bypressureon the ferric bis-histidine and cyanide-bound states of GlbN using 1H- 15N 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, asrepresentedbythe variationofanaverage chemical shift parameter, was highly sensitive to ligand swapping and heme covalent attachment. Stabilization of rare conformers was observedathigh pressures and consistent with cavity redistribution upon ligand binding. Inall states, the EF loop was foundtobeexceptionally 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. © 2013 Elsevier B.V. All rights reserved.