Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

Abstract

Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (E(GSH)) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with E(GSH)-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of E(GSH) in the IMS, thus explaining a steady-state E(GSH) in the IMS which is similar to the cytosol. Moreover, we show that the local E(GSH) contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells.

Inaccurately Assembled CytochromecOxidase Can Lead to Oxidative Stress-Induced Growth Arrest

“…Nevertheless, it is also possible that the reduced levels of cytochrome c oxidase complexes lead to an accumulation of electrons at the level of complex III, which is known to have the potential to produce large amounts of superoxide (13). In vivo, ROS levels are presumably controlled by a number of mitochondrial reducing enzymes and by GSH, which diffuses across the outer membrane through porins (28,34).…”

Retro-translocation of mitochondrial intermembrane space proteins

“…Under these conditions, mitochondrial biogenesis slows because oxidative phosphorylation is not needed. The redox environment in the cytosol has been reported to be more reducing during fermentation than during respiration (30), likely affecting the environment of the IMS (31). The in vivo analysis of protein steady-state levels revealed a decrease in MIA substrate proteins (i.e., Cox12, Cox17, Mix17, and Pet191) upon both inhibition of protein synthesis with cycloheximide (CHX) under respiratory conditions or transfer to glucose (Fig.…”

Divergent Molecular Evolution of the Mitochondrial Sulfhydryl:Cytochrome c Oxidoreductase Erv in Opisthokonts and Parasitic Protists

“…LTERV was cloned previously (9). Constructs encoding N-terminally MRGS(H) 6 GS-tagged PfErv and LtErv were subcloned by PCR using the BamHI and HindIII restriction sites of the Escherichia coli expression vector pQE30 (Qiagen). Cysteine point mutations of LTERV C17S , LTERV…”

“…A construct encoding C-terminally LE(H) 6 -tagged PfErv I2V was PCRcloned via the restriction sites NcoI (causing the point mutation) and XhoI of pET28 (Novagen). The same restriction sites were used to clone untagged PFERV I2V into the yeast expression vectors pYX143 and pYX232.…”

“…Erv1/ALR serves as a redox switch, accepting one electron pair and transferring two single electrons separately to two molecules of cytochrome c. Accordingly, mitochondrial protein import is linked to the respiratory chain. Alternatively, Erv1/ALR was suggested to directly transfer electrons to molecular oxygen (1)(2)(3)(4)(5)(6). Even though the Mia40/ Erv1 system is essential in opisthokonts (a eukaryotic lineage including yeast and mammals), inactivation of Mia40 was reported to have no detectable deleterious phenotype in Arabidopsis (7).…”