Biochemical and structural characterization of human aconitase, a key mitochondrial oxidant target

Mitochondrial aconitase (m-aconitase), an essential enzyme that catalyzes the reversible isomerization of citrate to isocitrate in the TCA cycle, is very sensitive to reactive oxygen and nitrogen species due to its particularly labile [4Fe-4S] prosthetic group which yields an inactive [3Fe-4S] cluster upon oxidation. Several cell and animal studies revealed m-aconitase as a main oxidant target during ageing and in pathologies in which mitochondrial dysfunction is implied. Other post-translational modifications have been reported such as nitration, succinylation, acetylation, phosphorylation and glutathionylation. In order to characterize how post-translational modifications of human (h) m-aconitase would impact in mitochondrial structure and function, we designed and expressed a recombinant hm-aconitase with a His-tag on its N-terminus. We obtained a high yield of pure (≥99%) enzyme of 83.002 ± 40 Da in agreement with the theorical molecular weight. The predicted structure of the hm-aconitase was build based on the Sus scrofa heart aconitase from the PDB database as both proteins share a 97% of identity. Hm-aconitase exhibits a specific activity of 22 ± 2 U/mg after being activated with 50 µM Fe 2+ and 10 mM DTT during 90 minutes under argon saturated atmosphere. Kinetic characterization of hm-aconitase yields K M of 950 ± 60, 80 ± 10 and 8.0 ± 0.7 µM for citrate, isocitrate and cis-aconitate respectively. Peroxynitrite and H 2 O 2 reacted with hm-aconitase with second rate order constants of 10 5 M -1 s -1 and 10 2 M -1 s -1 , respectively as previously reported for other mammalian aconitases, yielding a [3Fe-4S] reactivable enzyme. Thermal denaturation assessed by Trp fluorescence of hm-aconitase resulted in melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 oC for [4Fe-4S] and [3Fe-4S] forms of hm-aconitase, sustaining lower enzyme stability upon cluster oxidation while addition of substrate or inhibitors modified the unfolding mechanism of the active enzyme.