THE ORIGINS OF SUPEROXIDE AND HYDROGEN PEROXIDE PRODUCTION BY THE MAMMALIAN MITOCHONDRIAL ELECTRON TRANSPORT CHAIN : A COMPUTATIONAL AND EXPERIMENTAL APPROACH

Summary

The mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) as by-products of cellular respiration. Certain pathological conditions including ischemia-reperfusion (IR) injury disrupts mitochondrial ROS homeostasis, causing oxidative stress which can lead to cell death. Despite research efforts in the past few decades, an incomplete understanding of how mitochondria orchestrate ROS production and elimination prevails and hinders the development of efficacious... Show moreThe mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) as by-products of cellular respiration. Certain pathological conditions including ischemia-reperfusion (IR) injury disrupts mitochondrial ROS homeostasis, causing oxidative stress which can lead to cell death. Despite research efforts in the past few decades, an incomplete understanding of how mitochondria orchestrate ROS production and elimination prevails and hinders the development of efficacious therapeutic measures against oxidative stress. A major impediment is the lack of a quantitative framework capable of identifying site-specific sources of mitochondrial ROS production. Many mathematical models of mitochondria have been developed to tackle this challenge. Unfortunately, none is comprehensive and able to quantitatively reproduce a variety of bioenergetic data.The work presented in this dissertation culminates in a mathematical model of ROS production by the ETC complexes coupled with substrate metabolism. First, a variety of bioenergetic data are generated in-house using isolated mitochondria. Generating in-house data permits precise control over experimental conditions, which minimizes hidden variables in model simulations. Second, a biophysically detailed model of the ETC complex II is developed. Finally, the complex II model is integrated with the models developed by Beard and colleagues. These include the bioenergetic model, the kinetic model of complex I and the functional bc1 dimer model. The integrated model is biophysically detailed, mass-and-charge balanced and thermodynamically consistent. The modelling process involves an iterative process of model calibration and validation to ensure the quality of model predictions. Using this model, the flavin mononucleotide of complex I is identified as the primary source of superoxide and hydrogen peroxide in forward electron transport. The model predicts that, under conditions that favor highly reduced quinol and NADH pools in uninhibited mitochondria, both sites IF and IQ produce significant amounts of ROS. The model also reveals that hydrogen peroxide (H2O2) production by site IF underlies the substrate-specific monotonic dependence between net ROS production and oxygen concentrations. When electron flux is perturbed such as in the presence of an inhibitor, the topology of ROS production is altered. Thus, the model can be used to quantify the effects of changes to mitochondrial environment on ROS production, an application that, today, is experimentally limited. The model also highlights the importance of furthering our understanding of the scavenging system under different conditions to establish a complete picture of mitochondrial ROS homeostasis. Show less