UC San Diego

MitoNEET (mNT) is an outer mitochondrial membrane iron- sulfur (FeS) protein and a target of the thiazolidinedione (TZD) class of anti-diabetes drugs. The [2Fe-2S] cluster in mNT is ligated by a rare 3-Cys-1-His coordination. As FeS proteins usually function in electron transfer (redox) or in transfer of their clusters to apo-acceptor proteins, we investigated whether the TZDs affect the protein's redox potential (EM,7). We found that TZD binding negatively shifts EM, 7 by ̃100 mV, whereas in the cluster -coordinating His87 to Cys mutant (H87C) this effect is not observed. This suggests that His87 is critical to TZD communication with the [2Fe-2S] cluster of mNT. We further investigated the importance of residues near the cluster and discovered that mNT could tolerate an array of mutations that modified EM, 7 over a range of ∼700 mV. This is the largest range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV). Therefore, mNT is potentially useful for both physiological redox studies and industrial applications as a stable, water-soluble, redox agent. We investigated whether mNT functions as a cluster transfer protein and if TZDs affect transfer. We observed facile [2Fe-2S] cluster transfer between oxidized mNT and apo-ferredoxin (a-Fd) both in vitro and with a mitochondrial iron detection assay in cells. The H87C mutant inhibits transfer of the [2Fe-2S] clusters both in vitro and in cells. Importantly, TZDs inhibit iron transfer from mNT to mitochondria. This finding is interesting in light of the role of iron overload in diabetes. Finally, we show that NADPH shifts EM, 7 negatively and inhibits cluster transfer in a manner similar to the TZDs, 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 a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's anti- diabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster and protect cells from iron-overload