Display omitted •HGbI, a hemeprotein from extremophile, exhibits very low electrochemical potential.•Electrochemistry data indicated a pKa of 6.9 associated with an unknown sixth ligand.•Cyanide and imidazole bind unusually well to ferrous HGbI.•HGbI might be involved in redox-related catalytical processes. Methylacidiphilum infernorum is a methanotroph bacterium that survives under extreme temperature and pH conditions in which five globins were identified. Among them, Hell’s Gate Globin I (HGbI) is a hemeprotein that presents peculiar features if compared to other globins, like stability at pH 2.8, very high oxygen affinity, and pH-dependent hexacoordination. All these features are likely related to the FeIII/II ion of the heme group whose redox behavior needs to be understood. Herein, we present the spectroelectrochemical determination of the redox potential of the HGbI protein in the absence and presence of relevant heme-binding molecules (cyanide, imidazole, carbon monoxide, nitric oxide and oxygen) in solution and also at different pH values (3.5 to 9.5). The spectroelectrochemical studies were performed giving midpoint redox potential (Em) values of −305, −338, and −442 mV vs normal hydrogen electrode (NHE) for the HGbI unliganded protein and bonded to imidazole and cyanide, respectively. The impressive negative value of the unliganded HGbI indicates it is very improbable to keep it reduced for binding and/or store O2. Interestingly, it was noticed an unusual high affinity of ferrous HGbI to imidazole and cyanide. In addition, the Em values showed pH-dependence, exhibiting a sigmoidal response whose pKa value was calculated at 6.9 likely associated to a sixth heme ligand. In presence of CO and NO, the spectroelectrochemical curves showed hysteresis due to the complete dissociation of the ligands in the oxidized state. Oxidation potentials of 385 and 130 mV vs NHE were determined for CO-HGbI(FeII) and NO-HGbI(FeII) species, respectively. Altogether, these results suggest HGbI protein is likely not involved in the storage nor in the transport of oxygen in the Methylacidiphilum infernorum microorganism. Nevertheless, it is implied the physiological function of HGbI might be related to redox-based processes, which remains to be further elucidated.