TY - JOUR
T1 - Characterization of the mechanism and magnitude of cytoglobin-mediated nitrite reduction and nitric oxide generation under anaerobic conditions
AU - Li, Haitao
AU - Hemann, Craig
AU - Abdelghany, Tamer M.
AU - El-Mahdy, Mohamed A.
AU - Zweier, Jay L.
PY - 2012/10/19
Y1 - 2012/10/19
N2 - Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for ∼ 40% of the activation in control cells and ∼ 60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.
AB - Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for ∼ 40% of the activation in control cells and ∼ 60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.
UR - http://www.scopus.com/inward/record.url?scp=84867795031&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84867795031&partnerID=8YFLogxK
U2 - 10.1074/jbc.M112.342378
DO - 10.1074/jbc.M112.342378
M3 - Article
C2 - 22896706
AN - SCOPUS:84867795031
SN - 0021-9258
VL - 287
SP - 36623
EP - 36633
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -