Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study

Abstract

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co²⁺ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe²⁺, as it is customarily done, Co²⁺ is most often used in experiments because Fe²⁺ is extremely unstable owing to the fast oxidation reaction Fe²⁺ → Fe³⁺. Protein stability is monitored following the conformational changes induced by Co²⁺ binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements. The stability ranking among the wild-type frataxin and its variants obtained in this way is confirmed by a detailed comparative analysis of the XAS spectra of the metal-protein complex at the Co K-edge. In particular, a fit to the EXAFS region of the spectrum allows positively identifying the frataxin acidic ridge as the most likely location of the metal-binding sites. Furthermore, we can explain the surprising feature emerging from a detailed analysis of the XANES region of the spectrum, showing that the longer 81-210 frataxin fragment has a smaller propensity for Co²⁺ binding than the shorter 90-210 one. This fact is explained by the peculiar role of the N-terminal disordered tail in modulating the protein ability to interact with the metal.

Keywords: XAS (XAFS, XANES); frataxin; frataxin mutants; metal ions (Co2+); termal stability.