The H2O2-Resistant Fe–S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe–S Protein

International audience; Human mitoNEET (mNT) is the first identified Fe−S protein of the mammalian outer mitochondrial membrane. Recently, we demonstrated the involvement of mNT in a specific cytosolic pathway dedicated to the reactivation of oxidatively damaged cytosolic aconitase by cluster transfer. In vitro studies using apo-ferredoxin (FDX) reveal that mNT uses an Fe-based redox switch mechanism to regulate the transfer of its cluster. Using the "gold standard" cluster recipient protein, FDX, we show that this transfer is direct and that only one of the two mNT clusters is transferred when the second one is decomposed. Combining complementary biophysical and biochemical approaches, we show that pH affects both the sensitivity of the cluster to O 2 and dimer stability. Around physiological cytosolic pH, the ability of mNT to transfer its cluster is tightly regulated by the pH. Finally, mNT is extremely resistant to H 2 O 2 compared to ISCU and SufB, two other Fe−S cluster transfer proteins, which is consistent with its involvement in a repair pathway of stress-damaged Fe−S proteins. Taken together, our results suggest that the ability of mNT to transfer its cluster to recipient proteins is not only controlled by the redox state of its cluster but also tightly modulated by the pH of the cytosol. We propose that when pathophysiological conditions such as cancer and neurodegenerative diseases dysregulate cellular pH homeostasis, this pH-dependent regulation of mNT is lost, as is the regulation of cellular pathways under the control of mNT. I ron−sulfur (Fe−S) clusters are evolutionarily ancient and highly conserved prosthetic cofactors. Composed of only iron and sulfur, they are involved in many essential biological processes. 1,2 MitoNEET (mNT), also known as CISD1, is the first identified Fe−S protein of the mammalian outer mitochondrial membrane (OMM). 3,4 This is a small homodimeric protein (13 kDa for each monomer) anchored to the OMM by its 32-amino acid N-terminus with the major part of the protein, including the C-terminal Fe−S binding domain, located in the cytosol. 4 Each monomer accommodates one [2Fe-2S] cluster coordinated by three cysteines (C72, C74, and C83) and one histidine (H87) in a CDGSH domain 5−8 as other members of the NEET protein family, 9 which also includes Miner1 (or CISD2) and Miner2 (or CISD3) in mammals. 10 Although the biological activity of mNT is still debated, 11 studies have shown that it is involved in the regulation of iron/reactive oxygen species homeo-stasis, 12−14 in the regulation of lipid and glucose metabolism , 13,15 and in cell proliferation in breast cancer. 16 In vitro studies revealed that holo-mNT (the form of the protein with the cluster) is able to transfer its Fe−S cluster to very diverse apoprotein (an Fe−S protein, which has lost its cluster) recipients assembling either a [2Fe-2S] cluster as ferredoxin from various organisms, 17,18 human anamorsin 19 and CISD2, 20 or a [4Fe-4S] cluster as mammalian iron regulatory protein-1 (IRP-1)/cytosolic aconitase (c-aconi-tase). 14 On the basis of in cellulo experiments, we showed that mNT is able to repair the oxidatively damaged Fe−S cluster of human IRP-1/c-aconitase by transferring its cluster to the damaged protein. 14 Recently, we started to investigate in depth the in vitro cluster transfer reaction, focusing on the transfer from holo-mNT to [2Fe-2S] recipient protein. We unambiguously demonstrated that oxidized mNT ([2Fe-2S] 2+) triggers cluster transfer, whereas reduction of its cluster abrogates this transfer. Moreover, while O 2 significantly affects the lability of the oxidized mNT cluster, it does not interfere with the cluster