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Updated: Aug 21, 2026

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Peroxynitrite-mediated inactivation of heme oxygenases
Robert Kinobe1, Yanbin Ji1, Kanji Nakatsu1
1Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen's University, Kingston, Ontario, K7L 3N6, CANADA.
Background:
Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases.
Results:
Exposure to ONOO- led to concentration-dependent but reversible decreases in the activity of microsomal rat spleen and brain HO activity. Spleen HO activity was 100-fold more sensitive to ONOO--dependent inactivation compared to that of the brain, with IC50 values of 0.015 +/- 0.005 mM and 1.25 +/- 0.25 mM respectively. Inhibition of both rat spleen and brain microsomal HO activity was also observed with tetra-nitromethane, a tyrosine nitrating agent, as well as two NO donors, S-nitrosoglutathione (GSNO) and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.
Conclusion:
These results indicate that ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.
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