Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production

Abstract

The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC₅₀ for aconitase inhibition ranging from 7 to 24 μM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ˜60 mU/mg in liver to 321 mU/mg in kidney at 21% O₂. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O₂ reflecting aconitase inactivation by oxygen-derived oxidants at 21% O₂. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O₂^●− and H₂O₂ formation by the respiratory chain.

Keywords::

• aconitase 2
• free radicals
• hydrogen peroxide
• metabolic control
• mitochondria
• superoxide radical

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Acknowledgments

We thank Dr. Celia Quijano, Universidad de la República, Uruguay, to her helpful comments, and Dr. Frederick E. Domann, University of Iowa, USA, for careful reading and English editing.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by grants from the Agencia Nacional de Innovación e Investigación (ANII, FCE_ 398 to LC and FCE_2486 to RR) and Comisión Sectorial de Investigación Científica, Universidad de la República.