Influence of Heme Post-Translational Modification and Distal Ligation on the Backbone Dynamics of a Monomeric Hemoglobin

The cyanobacterium <i>Synechococcus</i> sp. PCC 7002 uses a hemoglobin of the truncated lineage (GlbN) in the detoxification of reactive species generated in the assimilation of nitrate. In view of a sensing or enzymatic role, several states of GlbN are of interest with respect to its structure–activity relationship. Nuclear magnetic resonance spectroscopy was applied to compare the structure and backbone dynamics of six GlbN forms differing in their oxidation state [Fe­(II) or Fe­(III)], distal ligand to the iron (histidine, carbon monoxide, or cyanide), or heme post-translational modification (<i>b</i> heme or covalently attached heme). Structural properties were assessed with pseudocontact shift calculations. ¹⁵N relaxation data were analyzed by reduced spectral density mapping (picosecond to nanosecond motions) and by inspection of elevated <i>R</i>₂ values (microsecond to millisecond motions). On the picosecond to nanosecond time scale, GlbN exhibited little flexibility and was unresponsive to the differences among the various forms. Regions of slightly higher mobility were the CE turn, the EF loop, and the H–H′ kink. In contrast, fluctuations on the microsecond to millisecond time scale depended on the form. Cyanide binding to the ferric state did not enhance motions, whereas reduction to the ferrous bis-histidine state resulted in elevated <i>R</i>₂ values for several amides. This response was attributed, at least in part, to a weakening of the distal histidine coordination. Carbon monoxide binding quenched some of these fluctuations. The results emphasized the role of the distal ligand in dictating backbone flexibility and illustrated the multiple ways in which motions are controlled by the hemoglobin fold