Mitochondria are responsible for the provision of energy in the form of ATP in mammalian cells. Mitochondrial ATP production begins when electrons are removed from carbon in the Krebs cycle. These electrons are then ferried through the multisubunit respiratory complexes embedded in the mitochondrial inner membrane reducing molecular oxygen to water at the end of the chain. Energy released by the flow of electrons is conserved as an electronchemical gradient of protons (protonmotive force) across the inner membrane, which is then harnessed by complex V to make ATP. Electron flow through the chain is not perfectly coupled to the production of ATP. At various points of the Krebs cycle and respiratory chain, electrons can leak out and prematurely interact with molecular oxygen to form reactive oxygen species (ROS). Although ROS can be dangerous, it is now clear that cells have a bifunctional relationship with these molecules. At high enough levels, ROS can overwhelm antioxidant defenses causing cellular damage, known as oxidative distress. However, at low levels ROS actually serve as secondary messengers, regulating various cellular functions. The ROS-mediated modulation of cell functions is defined as oxidative eustress but is more often called redox signaling. Like any other secondary messenger, the abundance of ROS in the cell is regulated through its rate of production and degradation. Antioxidant defenses, which have been well characterized, are vital for removing ROS and thus serve as an important regulator for redox signaling. However, how ROS production in mitochondria is controlled is not as well characterized.

Research in my laboratory focuses on:
1) The regulation of mitochondrial ROS release by various sites of production through protein S-glutathionylation.
2) Understanding the relative contributions of different sites of production towards overall ROS release in mitochondria.

Protein S-glutathionylation involves the reversible conjugation and removal of the antioxidant glutathione from a cysteine residue. These modifications are highly specific and rapid and occur in response to changes in ROS and NADPH, two factors that influence the redox state of the glutathione pool. Mitochondrial protein S-glutathionylation reactions regulate several physiological functions including heart rate and metabolism, eyesight, blood pressure, and embryogenesis. However, we still have a rudimentary understanding of how these reactions modulate mitochondrial bioenergetics and ROS production, as well as other functions, in different tissues. My group has made considerable progress in understanding the impact of reversible protein S-glutathionylation reactions on the production of ROS from different mitochondrial enzymes in heart, skeletal muscle, and liver tissue (please see publication list). Protein S-glutathionylation research in my lab is currently focused on:

• Fully characterizing the function of S-glutathionylation in regulating ROS release from different sites of production in muscle tissue.
• Identifying sites for S-glutathionylation in the Krebs cycle and respiratory chain in muscle tissue.
• Investigating the effect of S-glutathionylation on solute transport in muscle mitochondria.
• Examining the role of glutaredoxin-2 in regulating mitochondrial fat combustion.
• Characterizing the effect of the partial deletion of glutaredoxin-2 on exercise physiology and the induction of diet-induced obesity and the development of obesity-related disorders.

My second major research focus is characterizing which ROS producing enzymes in mitochondria serve as the major sites of production. Mitochondria can contain up to 12 sites of ROS production which happen to be enzymes associated with carbon catabolism and respiration. These enzymes also happen to be flavoproteins and dehydrogenases that oxidize and reduce the electron carriers, NAD and ubiquinone. Most of the work characterizing the sites of production in mitochondria has been carried out mostly in skeletal muscle and to a lesser extent brain and heart. My group is currently invested in assessing which sites serve as major emitters in the liver and heart tissue. In addition, this work is also devoted to understanding how over production from these sites leads to development of different disorders. Ongoing work includes:

• Examining the impact of partial complex I deficiency and ROS release from this enzyme in cardiac reperfusion injury.
• Assessing which sites serve as major emitters in muscle, heart, and liver mitochondria isolated from glutaredoxin-2 deficient mice.
• Fully characterizing which ROS release sites serve as major ones in liver and heart.
• Understanding the relationship between enzyme activities and mitochondrial bioenergetics with ROS release from individual sites.

Dr. Ryan J. Mailloux received his BSc in Biochemistry and PhD in Biomolecular Sciences from Laurentian University after which he spent 7 years performing postdoctoral work at the University of Ottawa, Carleton University, and Health Canada, Division of Toxicology in the fields of mitochondrial bioenergetics, redox biology, toxicology, and pharmacology