| A new pathway for protein import into the mitochondrial intermembrane space (IMS) was recently identified, which involves a disulfide relay between Mia40 and Erv1 that results in the oxidation of the imported substrates. To understand this process better I analyzed the redox environment of the IMS, measured the midpoint potential of the components of the pathway, and reconstituted the disulfide relay in vitro. To analyze the redox environment of the IMS I targeted a redox sensitive GFP (iGFP) to the compartment using the sorting sequence from Yme1. After in vivo thiol trapping of the iGFP, as well as a roGFP in the matrix and the cytosol, I quantitated the fraction of the roGFP that was reduced. From these measurements I calculated the redox potential of each compartment. The IMS has a redox potential of -215 mV, which is considerably more oxidizing than either the matrix at -330 mV, or the cytosol at -290 mV. Measurements of the midpoint potentials of Tim13 and Mia40 were conducted using circular dichroism, monobromobimane addition, and monitoring internal tryptophan fluorescence. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of -310 mV via Mia40 with an intermediate midpoint potential of --290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of --150 mV. I reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate, monitoring the redox state of Tim13 by AMS addition. Mia40 served as a direct oxidant of Tim13 and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Thus, Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation. These studies showed that the IMS is conducive to disulfide bond formation, and demonstrates how Mia40 and Erv1 cooperate within this compartment to correctly oxidize their substrates. |
