Studies of the effect of cysteine and heme pocket mutations of the nitric oxide dioxygenase and nitrite reductase activities of human Cytoglobin and Androglobin heme domain

Oxygen delivery to hypoxic tissues along with the interplay between hemoglobins and hypoxic tissues have been an area of interest, the nitrite reductase activity (NiR) and the nitric oxide dioxygenase activity (NOD) working in tandem is a proposed regulatory response which enhances oxygen levels in hypoxic tissues, serving to protect tissues against hypoxia. In hypoxic conditions, NO is generated via the NiR activity, while in normoxic condition NO is consumed via the NOD activity. With NO being a versatile signalling molecule and a potent relaxant of the vasculature, NO homeostasis by the NiR and NOD activity regulates vascular tone in response to oxygen demand. In traditional oxygen carriers like hemoglobin (Hb) and myoglobin (Mb), NO homeostasis via the NOD and NiR activities is seen as an additional function and is well studied and characterised in these globins. Scientific inquiry on hemoglobin superfamily and advances in genomics have led to the discovery of new hemoglobins, including an expanded human hemoglobin family with the discovery of additional members like Neuroglobin (Ngb) Cytoglobin (Cygb) and Androglobin (Adgb). These fresh additions are hexacoordinate, unlike Mb and Hb which have their sixth coordination site vacant in the absence of external ligands, Cygb and Adgb have their sixth coordination site occupied, by a histidine and glutamine respectively. Cygb is a low abundance vertebrate globin of ubiquitous expression, distantly related to Mb and upregulated during cellular hypoxia. Crystallographic data places Cygb as a monomer with free sulfhydryl and dimer with an intermolecular bond between two monomeric subunits. Biochemical studies have observed Cygb also existing as a monomer with an intramolecular disulfide bond, this form of the protein lacks crystallographic data. Adgb is a chimeric globin with a rearranged globin domain, Adgb lacks biochemical characterisation with little information obtained about its biochemical properties. However, Adgb highly is expressed in the testes and gene expression upregulated during spermatogenesis. Despite efforts to probe the biochemical activities of these additional members, their physiological roles are still not clearly defined. In this study, we report that the formation of an intramolecular disulfide bond between cysteines C38 and C83 enhances the ii nitrite reductase activity by 50-fold over that of the monomer with free sulfhydryl or 140-fold over that of the dimer with intermolecular disulfide bonds. The NO dioxygenase reactivity of cytoglobin is also very rapid with or without disulfide bond. We have targeted amino acid residues (H81A, L46F, L46W, C38,83S) in Cygb by mapping mutations targeting specific properties of hemoglobin (Hb) and myoglobin (Mb), including O2 binding and NO binding/scavenging as well as the NiR activity of Ngb, in addition to other rationalised mutations (C38,83S). Mutations of amino acid residues at the distal pocket successfully enhanced or attenuated the NiR and NOD of Cygb, by understanding how particular mutations can affect specific reactivities, these mutations may be used to target the NiR or NOD (e.g. by CRISPR/Cas9) in cell or animal models to help understand the precise role (or roles) of Cygb under physiological and pathophysiological conditions. Ultrafast and laser flash photolysis aided probing of the roles of the cysteines on ligand migration pathway, data suggests the cysteines play a role in ligand entry and exit from Cygb. We have re-evaluated the structural sequence assigned to the globin domain of Adgb (Adgb-GD), and we propose that part of the original structural sequence alignment was in error. Upon correction, we have expressed the Adgb-GD as a stable protein at neutral or alkali pH. Additionally, with a stable form of the protein expressed, we have identified a unique feature of the Adgb-GD. Adgb-GD binds nitric oxide in 5-coordinate heme configuration, unlike any other human globin, but similar to that observed with guanylate cyclase, cytochrome c’. Adgb-GD displays a high nitrite reductase activity, which is influenced by the redox state of the disulfide bond. Spectra characterisation depicts Adgb-GD as a hexacoordinate globin, comparative spectra analysis with Cytochrome C’ suggest Adgb-GD may possess similar ligand binding properties.