Bio-chemical study of Nitric oxide and its effect on TET enzymes activity

Nitric oxide (•NO) is a ubiquitous, free radical signaling molecule that has a plethora of roles in biological systems that are often ambivalent in action. It is produced by a group of oxygen dependent enzymes called nitric oxide synthase (NOS). •NO can react with O2 or O2- to form the stable end products; nitrate and nitrite. Intermediates generated during these reactions result in oxidative and nitrosative stress. The steady-state •NO concentration [NO]ss depends on a balance between the rates of •NO production and •NO metabolism. Since •NO production and metabolism both are oxygen dependent process; oxygen plays an important role in determining the [NO]ss. We measured the [NO]ss in media from activated murine macrophages in cultured monolayer under hypoxic and normoxic conditions. We observed no considerable difference between [NO]ss under normoxic and hypoxic conditions. Dinitrosyliron complexes (DNIC), which are known to be the most abundant intracellular NO-adducts, were studied under the same conditions. We found that their concentrations were equivalent under both hypoxic and normoxic conditions. Possibly because free •NO is responsible for DNIC formation. We also observed DNIC were predominantly formed in the cytoplasm. In another set of experiments, we tested the effect of •NO on the activity of an iron-dependent enzyme; Ten-Eleven translocation (TET) enzymes. This enzyme catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and is hence responsible for epigenetic modification that can lead to gene regulation. The reaction of •NO with iron to form DNIC renders this iron unavailable for its catalytic action. We studied changes in 5hmC levels as marker of TET activity and observed a decrease in 5hmC levels upon exposure to •NO. In conclusion, the study of [NO]ss and its effect on DNIC formation under hypoxic and normoxic conditions will help us understand their role in various physiological and pathophysiological conditions such as enzyme function and gene regulation.