Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Jesús Tejero,Swati Basu,Sheila Frizzell,Virgil Simplaceanu,Lisa Geary,Thottala Jayaraman,Ivan Azarov,Daniel B Kim-Shapiro,Mark T Gladwin,Sruti Shiva,Xunde Wang,Chien Ho,Calli Shapiro,Mauro Tiso

doi:10.1074/jbc.m110.159541

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 ∼2000 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-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Highlights

0.12 Ϯ 0.02 0.062 Ϯ 0.005 0.060 Ϯ 0.008 0.058 Ϯ 0.006 259 Ϯ 8 267 Ϯ 16 a Values are from Ref. 18. b The reaction of Mb H64L is significantly slower than wild type or H64A sperm whale Mb and apparently independent ofNO2Ϫin the concentration range studied (1–5 mM)
Our experiments reveal the following: 1) neuroglobin can function as a nitrite reductase, producing NO from nitrite; 2) the redox state of the surface thiols of Cys-46 and Cys-55 modulates the heme coordination, nitrite affinity, and NO generation and signaling; 3) replacement of the His-64 side chain locks the heme in a five-coordinate geometry with increased reactivity toward nitrite; and 4) under hypoxic con
A mechanistic proposal is presented in Scheme 1 where the first step of the reaction is the dissociation of His-64 from the heme iron

Summary

The abbreviations used are

We have recently discovered that the mitochondrial protein cytochrome c can reduce nitrite to NO more rapidly than either hemoglobin or myoglobin, but only when it assumes the five-coordinate conformation [21] This conformation only occurs during the interaction with anionic phospholipids or upon oxidation or nitration of protein residues, suggesting a post-translational tertiary structure regulation of nitrite reduction and NO generation. Disulfide bond formation is accompanied by a decrease in the distal histidine binding affinity to heme iron (KHis has been shown to decrease from ϳ3000 to 280, and values are calculated as kon/koff and are dimensionless) [23] This in turn increases the subpopulation of five-coordinate neuroglobin and increases the affinity for endogenous ligands such as oxygen (P50 shift from about 9 to 1 mm Hg) [22]. Such functionality may underlie hypoxic neuroprotective signaling and the control of hypoxic cellular respiration

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION