Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Abstract

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ϳ2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

“…GbX has a nitrite reduction bimolecular rate constant of 26.7 ± 2.0 M −1 ·s −1 at 37°C (Fig. 3C); this rate is 5-to 10-fold faster than the value for Mb or R-state Hb, 25-to 50-fold faster than Ngb or Cygb, and 200-fold larger than T-state Hb (5,21,22). To assess the production of bioavailable NO from the reduction of nitrite, we used chemiluminescence detection techniques.…”

Neuroglobins, Pivotal Proteins Associated with Emerging Neural Systems and Precursors of Metazoan Globin Diversity

“…The in vivo function of Ngb remains undefined despite a major effort over the last decade. Suggested functions include the oxygen (O 2 ) supply in hypoxia and ischemia (6), scavenging of reactive oxygen free radicals (7), protection from apoptosis (8), redox-regulated nitrite reductase activity (9), and involvement in respiratory chain function (10). In murine models of human neuropathology, Ngb is also expressed in reactive astrocytes, a subtype of glia cells in the nervous system (11).…”

Electrochemical Evidence for Neuroglobin Activity on NO at Physiological Concentrations

“…The constant position of the peak potential at different levels of O 2 suggests that Ϫ1 for NGB with reduced and oxidized states of the internal disulfide bridge, respectively (34,35). The P 50 value was reported in the range of 1-8 torr depending on pH, temperature, and the state of the internal disulfide bound (21 (40). The role of NGB in blocking apoptosis through binding other proteins has been also suggested in the literature: g, NGB can bind to the GDP-bound form of the ␣-subunit of heterotrimeric G-protein (G␣ i ) with K d Ϸ 6 ϫ 10 2 nM (41).…”

“…1). The thiol/disulfide couple in NGB has a reduction potential of Ϫ0.194 V measured through equilibrium redox titration with glutathione (40). This value suggests that in a cell at normal conditions the disulfide bond is rather reduced (40), whereas the cysteine residues in the isolated protein are oxidized in air forming the disulfide bridge (21,34).…”