Proton‐Dependence of the MitoNEET [2Fe‐2S] Cluster : An Electrochemical and Structural Investigation

Abstract

MitoNEET is a novel 3‐Cys, 1‐His ligated redox active [2Fe‐2S] cluster binding protein, whose biological function remains unclear. MitoNEET is of interest due to its interaction with the anti‐diabetes drug pioglitazone, a member of the TZD drug family, and its unique cluster ligation. Here, a characterization of the clusters unique ligand environment is presented. Using protein film voltammetry, the potential of the mitoNEET cluster is found to be 0 mV at pH 7, where upon binding of pioglitazone reduces the potential to −100 mV. This is one of the first reports of PFV being used to monitor drug binding. Additionally, the mitoNEET cluster demonstrates unusual proton coupled‐electron transfer as well as marked proton‐dependent cluster instability. Through a series of site‐directed mutations of conserved residues around the [2Fe‐2S] cluster, both of these phenomena have been examined, demonstrating the presence of multiple sites of protonation that affect both cluster midpoint potential and cluster stabilization. The high conservation of these residues, which reduce cluster stability, suggest a physiological role for cluster loss and lead to a putative model, in which mitoNEET may sense cellular oxidation state, proton concentration, or iron levels. Changes in MitoNEET cluster binding could potentially mediate either cluster transfer or additional signaling events within the cell.NIH‐NIGMS R01‐GM072663