P20 - Disruption of the iron-sulfur cluster of aconitase by myeloperoxidase-derived oxidants

Abstract

Oxidative damage catalysed by the heme enzyme myeloperoxidase (MPO) has been linked with multiple inflammatory pathologies. The major oxidant species generated by MPO are hypochlorous (HOCl) and hypothiocyanous acids (HOSCN). The damage induced by HOCl is well characterized and has been linked to multiple diseases, however the role of HOSCN is less well understood. It is known that HOSCN can cause selective damage, as this oxidant selectively targets thiol (e.g. Cys) residues and selenium-containing species. The aim of the current study was to assess whether HOCl and HOSCN can disrupt the [4Fe-4S] cluster of aconitase causing iron release and loss of activity.It is shown that HOSCN induces rapid and efficient release of iron from aconitase, with 80% removed at an oxidant concentration of 3 micromoles/mg protein; this is markedly more efficient than HOCl. In contrast the extent of loss of enzymatic activity was comparable between the two oxidants at the same concentration. Blocking the [4Fe-4S] cluster inhibited HOSCN-mediated inactivation, but did not have dramatic effects on HOCl-mediated damage, consistent with HOSCN, but not HOCl, interacting with the cluster. This data is supported by peptide mass mapping studies that indicate that HOSCN oxidises Cys385 of the [4Fe-4S] cluster. In contrast HOCl damaged multiple sites.Exposure of human coronary artery endothelial cells (HCAEC) to 0-50 micromolar HOCl or 0-150 micromolar HOSCN resulted in an increase in intracellular iron, loss of aconitase activity and a loss of mitochondrial aconitase protein. In contrast cytosolic aconitase was not affected.These data indicate that aconitase - and particularly the mitochondrial form - is a target for MPO-mediated damage with HOSCN showing a selectivity for the [4Fe-4S] cluster and inducing greater iron release. This damage, and the release of iron, may exacerbate oxidative stress in cells at sites of inflammation where active MPO is present.