Combined Biochemical, Biophysical, and Cellular Methods to Study Fe–S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET

Cécile Mons (1) , Ioana Ferecatu (1) , Sylvie Riquier (1) , Ewen Lescop (1) , Cécile Bouton (1) , Marie-Pierre Golinelli-Cohen (1)

1 ICSN - Institut de Chimie des Substances Naturelles

MitoNEET is the first identified Fe–S protein anchored to mammalian outer mitochondrial membranes with the vast majority of the protein polypeptide located in the cytosol, including its [2Fe–2S] cluster-binding domain. The coordination of the cluster is unusual and involves three cysteines and one histidine. MitoNEET is capable of transferring its redox-active Fe–S cluster to a bacterial apo-ferredoxin in vitro even under aerobic conditions, unlike other Fe–S transfer proteins such as ISCU. This specificity suggests its possible involvement in Fe–S repair after oxidative and/or nitrosative stress. Recently, we identified cytosolic aconitase/iron regulatory protein 1 (IRP1) as the first physiological protein acceptor of the mitoNEET Fe–S cluster in an Fe–S repair process. This chapter describes methods to study in vitro mitoNEET Fe–S cluster transfer/repair to a bacterial ferredoxin used as a model aporeceptor and in a more comprehensive manner to cytosolic aconitase/IRP1 after a nitrosative stress using in vitro, in cellulo, and in vivo methods.

Mots clés: siRNA; Cluster repair; Cytosolic aconitase; MitoNEET; Cluster transfer; Cluster disassembly; UV–visible absorption spectroscopy; Nuclear magnetic resonance spectroscopy

Domaines: Biochimie, Biologie Moléculaire

Cécile Mons, Ioana Ferecatu, Sylvie Riquier, Ewen Lescop, Cécile Bouton, et al.. Combined Biochemical, Biophysical, and Cellular Methods to Study Fe–S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET. Methods in Enzymology, 2017, 595, pp.83-106. ⟨10.1016/bs.mie.2017.07.010⟩. ⟨hal-01873518⟩