Extremophilic hemoglobins: The structure of Shewanella benthica truncated hemoglobin N

Truncated hemoglobins (TrHbs) have an ancient origin and are widely distributed in microorganisms where they often serve roles other than dioxygen transport and storage. In extremophiles, these small heme proteins must have features that secure function under challenging conditions: at minimum, they must be folded, retain the heme group, allow substrates to access the heme cavity, and maintain their quaternary structure if present and essential. The genome of the obligate psychropiezophile Shewanella benthica strain KT99 harbors a gene for a TrHb belonging to a little-studied clade of globins (subgroup 2 of group N). In the present work, we characterized the structure of this protein (SbHbN) with electronic absorption spectroscopy and X-ray crystallography and inspected its structural integrity under hydrostatic pressure with NMR spectroscopy and small-angle X-ray scattering. We found that SbHbN self-associates weakly in solution and contains an extensive network of hydrophobic tunnels connecting the active site to the surface. Amino acid replacements at the dimeric interface formed by helices G and H in the crystal confirmed this region to be the site of intermolecular interactions. High hydrostatic pressure dissociated the assemblies while the porous subunits resisted unfolding and heme loss. Preservation of structural integrity under pressure is also observed in nonpiezophilic TrHbs, which suggests that this ancient property is derived from functional requirements. Added to the inability of SbHbN to combine reversibly with dioxygen and a propensity to form heme d, the study broadens our perception of the TrHb lineage and the resistance of globins to extreme environmental conditions.