Iron Binding and Oxidation Kinetics in Frataxin CyaY of Escherichia coli

Abstract

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (Kd∼4 μM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O2. However, oxidation occurs rapidly and completely with H2O2 through a non-enzymatic process with a stoichiometry of two Fe(II)/H2O2, indicating complete reduction of H2O2 to H2O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H2O2 binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV–visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25–26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.