Truncated hemoglobins (trHbs) constitute a family of small oxygen binding heme proteins occurring in bacteria, protozoa and plants, forming a distinct group within the hemoglobin superfamily. They are nearly ubiquitous in the plant kingdom, occur in many aggressively pathogenic bacteria, and are held to be of very ancient origin. None have been detected in the genomes of archaea or metazoa. Characteristically, trHbs occur at nano- to micromolar intracellular concentration, hinting at a possible role as catalytic proteins. Many trHbs display amino acid sequences 20-40 residues shorter than (non-) vertebrate hemoglobins to which they are scarcely related by sequence similarity.
Herbaspirillum seropedicae is a b-proteobacteria and an obligate endophytic diazotroph found in association with grasses, rice, and sugarcane. Our group cloned and expressed a truncated hemoglobin from H. seropedicae named Hs-trHb 1. Mass spectrometry data confirmed the identity of the protein and also the coincidence between theoretical and predicted molecular weight with that of the purified recombinant protein. In this work, we describe a structural characterization of Hs-trHb 1 by NMR and CD spectroscopy. CD spectroscopic analysis with the apo and holo forms, suggests a proper folding and similar a-helical content. The ¹⁵N isotopic labeled protein was analyzed by NMR using HSQC experiments showing good behavior in solution and chemical shift dispersion compatible with a globin fold. The Soret band was used to verify the binding properties of the heme group and the protein binds heme in a molar ratio of 1:1. Cianide and CO binding will also be investigated in order to stabilize the reduced form. Oxidized and reduced states will be analyzed by NMR for further 3D structural determination of H. seropedicae truncated hemoglobin Hs-trHb 1. We intend to use the paramagnetic effect of metal ion to identify the amino acids involved on the heme group coordination.
Financial Support: CNPq, CAPES, MCT, FINEP, FAPESC, FAPERJ, PRONEX, ICGEB, TWAS