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Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair
Cécile Mons1, Ioana Ferecatu2, Sylvie Riquier1
1Institut de Chimie des Substances Naturelles (ICSN), CNRS UPR 2301, Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
Abstract:
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.