Redox characterisation of Erv1, a key component for protein import and folding in yeast mitochondria.

The mitochondrial import and assembly (MIA) pathway plays a vitally important role in import and oxidative folding of mitochondrial proteins. Erv1, a member of the FAD-dependent Erv1/ALR disulphide bond generating enzyme family, is a key player of the MIA pathway. Although considerable progress has been made, the molecular mechanism of electron transfer within Erv1 is still not fully understood. The reduction potentials of the three redox centres were previously determined to be -320 mV for the shuttle disulphide, -150 mV for the active-site disulphide and -215 mV for FAD cofactor. However, it is unknown why FAD of Erv1 has such a low potential compared with other sulfhydryl oxidases, and why the shuttle disulphide has a potential as low as many of the stable structural disulphides of the substrates of MIA pathway. In this study, the three reduction potentials of Erv1 were reassessed using the wild-type and inactive mutants of Erv1 under anaerobic conditions. Our results show that the standard potentials for the shuttle and active-site disulphides are approximately -250 mV and -215 ~ -260 mV, respectively, and the potential for FAD cofactor is -148 mV. Our results support a model that both disulphide bonds are redox-active, and electron flow in Erv1 is thermodynamically favourable. Furthermore, the redox behaviour of Erv1 was confirmed, for the first time using Mia40, the physiological electron donor of Erv1. Together with previous studies on proteins of MIA pathway, we conclude that electron flow in the MIA pathway is a thermodynamically favourable, smoothly downhill process for all steps. DATABASE: Erv1: EC 1.8.3.2.