Journal of Biological Chemistry
Volume 286, Issue 49, 9 December 2011, Pages 42679-42689
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Cell Biology
14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide*

https://doi.org/10.1074/jbc.M111.271973Get rights and content
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Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1–5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

Heme
Hypoxia
Nitric Oxide
Phosphorylation
Protein-Protein Interactions
Neuroglobin

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*

This work was supported, in whole or in part, by National Institutes of Health Grants R01 HL098032, R01 HL096973, and P01 HL103455 (to M. T. G.) and R01 HL096376, R01 HL097376, and R01 HL098174 (to R. K. M.). This work was also supported by the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania (to M. T. G.). M. Tiso and M. T. Gladwin are listed as co-inventors on a patent application entitled “Neuroglobin as a Six-to-Five Coordinate-regulated Nitrite Reductase.”

2

Recipient of a Merit Review from the Department of Veteran Affairs.