Comprehensive characterization of the major nemoglobin from the giant kidney worm Dioctophyme renale

Globins are widely distributed heme proteins involved in oxygen transport and redox-related processes. Their diversity and functional roles remain poorly understood in nematodes. Dioctophyme renale, the giant kidney worm, is a Clade I nematode that infects mammals, including humans, yet no globin from this group has been experimentally characterized. Here we report the first biochemical and biophysical characterization of a Clade I nemoglobin, P17, an abundant soluble globin isolated from pseudocoelomic fluid of D. renale and responsible for its characteristic red pigmentation. Structural modeling using AlphaFold2 and AlphaFold3 predicts that P17 adopts a canonical globin fold compatible with heme coordination, experimentally supported by circular dichroism spectroscopy. Dynamic light scattering and native mass spectrometry show that P17 is monomeric in solution and binds a single heme group with 1:1 stoichiometry. Comparative analyses of apo- and holoP17 reveal that heme binding does not alter the protein fold but induces near-UV and visible circular dichroism signals consistent with a chiral heme environment. Thermal and chemical denaturation experiments indicate similar global stability for apo and holo forms, while chemical unfolding uncouples protein unfolding from heme dissociation in the holo protein. Functional assays show that apoP17 can be reversibly reconstituted with free heme and can also acquire heme from other hemoproteins. In addition, holoP17 contains a slowly oxidizable ferrous heme and exhibits peroxide-sensitive pseudoperoxidase activity. Together, these results establish P17 as a stable, monomeric heme-binding globin with redox-related properties, providing the first experimental insight into globin function and heme handling in Clade I nematodes.