Reconstitution of the Mia40-Erv1 Oxidative Folding Pathway for the Small Tim Proteins

Abstract

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, 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. 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. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX9C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

“…UnfoldingMia40 substrates contain very stable disulfide bonds that require high concentration of reductants to be broken (16,18,19). We estimated the standard redox potential of Cox19 to be approximately Ϫ0.31 V (data not shown), very close to that reported for Cox17 (Ϫ0.34), Tim9 (Ϫ0.31), and Tim10 (Ϫ0.32), suggesting that Mia40 substrates have similar redox stability.…”

The disease-associated mutation of the mitochondrial thiol oxidase Erv1 impairs cofactor binding during its catalytic reaction

“…Next, the now CPC-reduced Mia40 (rMia40) is re-oxidized by Erv1/ALR to allow for another substrate oxidation cycle. In the case of Erv1, it is the N-terminal Cys 30 -Cys 33 shuttle disulfide that first accepts the electrons from rMia40 and transfers them to the Cys 130 -Cys 133 active-site disulfide, from where they are then passed to Erv1-bound FAD [12,[16][17][18][19]. Finally, the FAD-reduced Erv1 (Erv1-FADH 2 ) is re-oxidized by transferring the electrons to either oxidized cytochrome c (cyt c) or molecular oxygen, thereby regenerating Erv1 activity.…”

Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

“…Excess Mia40 can oxidize substrates in vitro (18,19,26), and recent studies in vivo support that Mia40 is present in a partially reduced state (28,29). Whereas these studies show that Mia40 is reduced, the specific redox state of Mia40 has not been determined.…”

“…The midpoint potentials of the reactive cysteine centers in proteins of the disulfide relay are poised to favor the transfer of electrons from the substrate to the terminal electron acceptors (19). The twin CX 3 C protein Tim13 acquires two disulfide bonds simultaneously when equilibrated in buffers with different redox potentials and has a single midpoint potential of Ϫ310 mV (19).…”

“…The twin CX 3 C protein Tim13 acquires two disulfide bonds simultaneously when equilibrated in buffers with different redox potentials and has a single midpoint potential of Ϫ310 mV (19). Coordinated insertion of two disulfide bonds requires four electrons to be transferred at one time.…”