NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein

MitoNEET (mNT) is the founding member of the recently discovered CDGSH family of 2Fe-2S] proteins capable of 2Fe-2S] cluster transfer to apo-acceptor proteins. It is a target of the thiazolidinedione (TZD) class of anti-diabetes drugs whose binding modulate both electron transfer and cluster transfer properties. The 2Fe-2S] cluster in mNT is destabilized upon binding of NADPH, which leads to loss of the 2Fe-2S] cluster to the solution environment. Because mNT is capable of transferring 2Fe-2S] clusters to apo-acceptor proteins, we sought to determine whether NADPH binding also affects cluster transfer. We show that NADPH inhibits transfer of the 2Fe-2S] cluster to an apo-acceptor protein with an inhibition constant (K(i)) of 200 μm, which reflects that of NADPH concentrations expected under physiological conditions. In addition, we determined that the strictly conserved cluster interacting residue Asp-84 in the CDGSH domain is necessary for the NADPH-dependent inhibition of 2Fe-2S] cluster transfer. The most critical cellular function of NADPH is in the maintenance of a pool of reducing equivalents, which is essential to counteract oxidative damage. Taken together, our findings suggest that NADPH can regulate both mNT 2Fe-2S] cluster levels in the cell as well as the ability of the protein to transfer 2Fe-2S] clusters to cytosolic or mitochondrial acceptors.