Nitric oxide sensitivity of the aconitases

Area of Science:

• Biochemistry
• Cellular Biology
• Toxicology

Background:

• Aconitases are crucial enzymes in cellular metabolism.
• Nitric oxide (NO) is known to cause cellular damage, with aconitases being identified as potential targets.
• The exact mechanisms of NO-induced aconitase inactivation are not fully understood.

Purpose of the Study:

• To investigate the inactivation of aconitase by nitric oxide (NO) in both bacterial (Escherichia coli) and mammalian (porcine mitochondrial) systems.
• To elucidate the specific NO-derived species responsible for aconitase inactivation.
• To understand the conditions influencing NO-mediated aconitase inactivation.

Main Methods:

• Exposure of E. coli and porcine mitochondrial extracts to controlled atmospheres of NO.
• Assay of aconitase activity under various conditions (pH, presence of substrates, aerobic/anaerobic).
• Investigation using superoxide dismutase and glutathione-deficient strains to probe reactive species.

Main Results:

• Aconitase activity in E. coli decreased by approximately 70% after 2 hours of exposure to 120 ppm NO.
• Inactivated aconitase showed poor reactivation in E. coli.
• NO-mediated inactivation was enhanced at acidic pH and in the presence of substrates, particularly cis-aconitate.
• Porcine mitochondrial aconitase was sensitive to NO at pH 6.5 but not pH 7.5, with inactivation occurring during catalysis.
• Superoxide dismutase and glutathione levels did not significantly alter inactivation, suggesting peroxynitrite and S-nitrosoglutathione are not primary mediators.

Conclusions:

• Nitric oxide (NO) directly inactivates aconitases, with NO-derived species playing a significant role.
• The inactivation is dependent on pH and substrate availability, explaining previous in vitro resistance.
• A proposed mechanism involves S-nitrosation of the aconitase [4Fe-4S] center.
• This NO-mediated inactivation contributes to cellular NO toxicity.