Efficient intracellular electron transfer is essential to organisms that use oxygen. An exquisite balance between oxidized and reduced enzymes and cofactors, termed redox homeostasis, must be carefully regulated to maintain cellular function. Loss of redox balance underlies molecular changes associated with aging and age related diseases. Consequences of acute or chronic disruption in redox homeostasis include neurodegenerative diseases, cancer, diabetes mellitus, atherosclerosis, and rheumatoid arthritis. To investigate the specific molecular and chemical events that govern redox balance, structural and biochemical studies of enzymes that preserve cellular redox potential will be examined. In particular, the proposed research will consider the enzymatic pathways responsible for the maintenance of reduced thioredoxin and glutathione pools by addressing the following specific aims: (i) Characterize the electron transfer cascade catalyzed by mitochondrial thioredoxin reductase at the molecular level. The goal of this aim is to use structural analysis and biochemical characterizations to examine the mechanistic details of the thioredoxin system as it pertains to mitochondrial redox homeostasis. We will determine the crystal structures of mitochondrial thioredoxin and glutaredoxin alone and in complex with mitochondrial thioredoxin reductase. (ii) investigate the structural and mechanistic features of essential glutathione homeostasis enzymes. The goal of this aim is to gain insight into the regulation of glutathione levels. We will examine the molecular details of allosteric regulation of glutamate cysteine ligase, which catalyzes the committed step of glutathione bisoynthesis. We will also examine the auto-activation mechanism of g-glutamyl transpeptidase, an ectoenzyme required for glutathione salvage, and the effects of self-processing on catalytic activity. Structural characterizations of these key thioredoxin and glutathione systems will provide new insights into mechanisms of cellular redox homeostasis. Understanding the crucial details may translate to new therapeutic targets in the array of difficult health problems caused by oxidative damage.
