Analytical Chemistry | Biochemical Phenomena, Metabolism, and Nutrition | Biochemistry | Biology | Systems Biology
Mitochondria are essential organelles in most eukaryotic cells because of their role in metabolism and the production of ATP by the oxidative phosphorylation (OXPHOS) pathway, as well as other key cellular processes. Metal cofactors, such as copper (Cu) and iron (Fe), are incorporated into OXPHOS protein complexes of yeast located within the inner membrane of the mitochondria. Misincorporation or modulation of these available metals in mitochondrial enzymes leads to the production of reactive oxygen species (ROS). ROS are reactive molecules containing oxygen such as peroxides, superoxide, and hydroxyl radicals. Yeast are a good model for studying aging and the effect of ROS on lifespan because they are easy to grow, and many of the genes and proteins involved in determining yeast lifespan are conserved in humans and other mammals.
Mitochondrial OXPHOS protein complexes are the primary site of superoxide formation. A primary defense of free radical damage is the neutralization of superoxide by the enzyme superoxide dismutase 1 (Sod1), which requires copper and zinc (Zn) as cofactors. Copper is additionally utilized in the OXPHOS complex cytochrome c oxidase as an electron transferring group. Our first aim was to investigate the role of copper in the production of mitochondrial ROS as a part of normal aerobic respiration utilizing a yeast model. Specifically, we worked to quantify the effect of exogenous copper treatment on the relative protein expression of copper-dependent cytochrome c oxidase (COX) subunits and overall COX complex assembly. Our previous work has indicated a protective behavior of copper treatment on yeast lifespan, and we propose this is due to copper inducing more robust electron movement through a more functional electron transport chain (ETC) complexes. Improved efficacy of the ETC is thought to minimize premature electron leakage to oxygen and lessen mitochondrial ROS levels. We showed that addition of 0.25 mM copper to yeast media increases lifespan of wild type (WT) and lys7Δ cells, but the addition of 0.25 mM copper to the media only effects the growth of sod1Δ yeast cells in the short term. During this granting period we performed protein analysis of mitochondrial proteins showing an increase in subunits 1, 2, and 4 of Complex IV (cytochrome c oxidase, CcO) of the ETC. Our second aim was to determine how copper levels, ROS levels, and enzyme activity are related during yeast chronological aging. We are able to use biochemical staining assays utilizing fluorescent molecules to detect and quantify ROS levels. Specifically, we can track superoxide generation via dihydroethidium (DHE) with some of our results presented in Figure 1. We are also able to use live cell imaging via fluorescent microscopy to assess superoxide levels again using DHE, and an additional mitochondrial specific stain MitoSox (Molecular Probes).
Future directions are to increase the yeast lifespan experimental length and to determine effects of copper on protein activity via in-gel analysis, and at the level of transcription level for respiratory proteins. This work hopes to contribute to our understanding of the copper-utilizing components of this mitochondrial pathway, and this metal’s impact on local ROS production. Ultimately, we will also use both spectroscopy and live cell imaging via fluorescent microscopy to assess superoxide levels in multiple yeast strains as well as in the presence and absence of copper. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan.
Bestwick, Megan, "Yeast Copper Proteins and Reactive Oxygen Species in Effecting Lifespan" (2020). Post-Grant Reports. Report. Submission 198.