Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.

Mitochondria contain their own genomes, and unlike nuclear genomes, mitochondrial genomes are inherited maternally. With a high mutation rate and little recombination, special selection mechanisms exist in the female germline to prevent the accumulation of deleterious mutations 1 – 5 . The molecular mechanisms underpinning selection remain poorly understood 6 . Here, using an allele-specific fluorescent in situ -hybridization approach to distinguish wildtype from mutant mtDNA, we have visualized germline selection for the first time. Selection first manifests in the early stages of Drosophila oogenesis, triggered by reduction of the pro-fusion protein Mitofusin. This leads to the physical separation of mitochondrial genomes into different mitochondrial fragments, preventing the mixing of genomes and their products, and thereby reducing complementation. Once fragmentated, mitochondria harboring mutant genomes are less able to make ATP, which marks them for selection through a process requiring the mitophagy proteins Atg1 and BNIP3. Surprisingly, a reduction in Atg1 or BNIP3 decreases the amount of wildtype mtDNA, suggesting a link between mitochondrial turnover and mtDNA replication. Remarkably, fragmentation is not only necessary for selection in germline tissues, but also sufficient to induce selection in somatic tissues where selection is normally absent. Our studies posit a generalizable mechanism to select against deleterious mtDNA mutations that may allow the development of strategies for treatment of mtDNA disorders.

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. However, the Cys residues and the thiol modifications involved are not known. Here we investigate complex I thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. In mitochondria incubated with the thiol oxidant diamide, complex I is only glutathionylated on the 75-kDa subunit. Of the 17 Cys residues on the 75-kDa subunit, 6 are not involved in iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry of complex I from oxidatively stressed bovine heart mitochondria showed that only Cys-531 and Cys-704 were glutathionylated. The other four non-iron-sulfur center Cys residues remained as free thiols. Complex I glutathionylation also occurred in response to relatively mild oxidative stress caused by increased superoxide production from the respiratory chain. Although complex I glutathionylation within oxidatively stressed mitochondria correlated with loss of activity, it did not increase superoxide formation, and reversal of glutathionylation did not restore complex I activity. Comparison with the known structure of the 75-kDa ortholog Nqo3 from Thermus thermophilus complex I suggested that Cys-531 and Cys-704 are on the surface of mammalian complex I, exposed to the mitochondrial glutathione pool. These findings suggest that Cys-531 and Cys-704 may be important in preventing oxidative damage to complex I by reacting with free radicals and other damaging species, with subsequent glutathionylation recycling the thiyl radicals and sulfenic acids formed on the Cys residues back to free thiols.