Estudos espectroscópicos e cromatográficos da globina recombinante Hell’s Gate I (HGbI) do Methylacidiphilum infernorum

Abstract

HGbI protein from Methylacidiphilum infernorum belongs to the globin Family, exhibiting exciting properties such as resistance to autoxidation of the metallic center, stability of heme in acidic medium and pH-dependent hexacoordination of Fe2+. Although this protein has been discovered for a while, many aspects about it are still unknown. Here, it was investigated conformational aspects of HGbI at different conditions, especially varying pH and Fe heme redox state/coordination. In addition, exploratory studies of typical catalytic activities of hemeproteins were carried out. For these purposes, it was employed techniques such as UV-Vis electronic spectroscopy, circular dichroism, fluorescence, and also analytical gel filtration chromatography. CD assays indicate that approximately 90% of secondary structure is α-helix. Iron redox state influences tertiary structure of the protein according to CD and fluorescence studies. Thermal stability was measured, where it was higher in the ferric form (Tm = 81ºC, pH 7.0). Additionally, expressive tolerance to organic solvents is highlighted, using up to 30% of DMSO or 50% of methanol. Chromatographic studies indicates the existence of a quaternary structure in the ferrous form, suggesting a monomer-dimer equillibrium, that was not shown in the ferric form. HGbI interaction to cyanide and imidazole showed a binding profile analogous to neuroglobin. Regarding to the potential catalytic activity, HGbI was shown to be active catalyzing peroxidases and dealoperoxidase reactions, although it has been lesser than myoglobin. Thus, taking into account the robust characteristics of HGbI and preliminary catalytic results, it emerges great perspectives for HGbI optimizations with mutations and/or insertion of unnatural prosthetic groups, in order to improve its enzymatic properties, as has been done to certain proteins.