Characterization of the Physiologic Functions of the Proteolytic and Diaphorase Activities of Dihydrolipoamide Dehydrogenase

Reactive oxygen species (ROS) production has been implicated in numerous human diseases, including neurodegenerative disorders, ischemia-reperfusion injury, aging and cancer. Thus, identification of the sources of ROS and their role in physiology and pathophysiology is of clear importance. A major site of ROS production is the mitochondrion. The canonical view has been that, within mitochondria, the major site of ROS production is the electron transport chain, principally complex I and complex III. Several recent studies, however, have called this view into question and identified the alpha-ketoglutarate dehydrogenase complex and its dihydrolipoamide dehydrogenase (DLD) component as an important source of ROS.;DLD is a highly conserved, ∼50 kDa mitochondrial protein. As a homodimer, DLD is a common component of four multienzyme complexes in humans: the pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acid dehydrogenase complexes as well as the glycine cleavage system. In this context, DLD catalyzes the oxidative regeneration of a lipoic acid cofactor covalently bound to E2 with production of NADH. Thus, DLD is essential to central carbohydrate metabolism as well as the metabolism of branched-chain amino acids and glycine.;However, DLD also possesses two alternative or "moonlighting" functions which are likely responsible for its ROS production. DLD has long been known to possess diaphorase activity, which is the ability to catalyze the oxidation of NADH to NAD+ using different electron acceptors. Characterization of diaphorase activity has largely been limited to in vitro studies, and the role of this activity in vivo remains poorly defined. We hypothesized that DLD might primarily play a pro-oxidant role in vivo by reducing O2 to superoxide radical and hydrogen peroxide, or by reducing Fe3+ to Fe2+, which would in turn result in the production of highly toxic hydroxyl radical. DLD also possesses a second moonlighting function, a serine protease activity catalyzed by a highly conserved catalytic dyad (S456-E431) buried in the DLD dimer interface. DLD's proteolytic activity was identified in vitro using human frataxin, a mitochondrial protein essential for mitochondrial and cellular iron homeostasis. We hypothesized that, in vivo, proteolytic activity might also contribute to ROS production by cleaving frataxin and altering the balance between protein bound and free iron resulting in iron-catalyzed oxidative damage.;The mechanism by which DLD is able to modulate its multiple activities was not entirely understood, however, several studies suggested that destabilization of the DLD dimer might alter its function. The dehydrogenase active site requires a DLD homodimer. However, diaphorase and proteolytic activity were predicted to be most active in monomeric DLD. To this end, we utilized a series of human mutations in the homodimer interface domain of DLD which were predicted to destabilize the DLD dimer. We hypothesized that these mutations would increase diaphorase and proteolytic activity and provide both an in vitro and in vivo system for the characterization of the alternative activities of DLD. DLD mutations in human patients result in a great deal of clinical heterogeneity and clinical phenotypes do not completely correlate with the loss of dihydrolipoamide dehydrogenase activity, suggesting alternative disease mechanisms. In this work, we investigated how human mutations alter the alternative functions of DLD both in vitro and in vivo and the role which these activities might play in the pathology of DLD deficiency.