ESTABLISHMENT OF THE PYRUVATE DEHYDROGENASE COMPLEX AS A

Once regarded as “byproducts ” of aerobic metabolism, the production of superoxide/H2O2 is now understood to be a highly specialized and extensively regulated process responsible for exerting control over a vast number of thiol-containing proteins, collectively referred to as the redox-sensitive proteome. Although disruptions within this process, secondary to elevated peroxide exposure, have been linked to disease, delineation of the sources and mechanisms regulating this increased peroxide burden remain poorly defined and as such difficult to target using pharmacotherapy. Herein we demonstrate a role for pyruvate dehydrogenase (PDH) as a key source of H2O2 under physiological constraints in which respiratory chain-dependent electron leak is negligible. PDH is shown to generate H2O2 as a function of glutathione content, matrix metabolic balance, as well as antioxidant reductase activity. With respect to the latter, manipulation of matrix redox buffering reveals a novel mechanism whereby H2O2 producing NADH-linked dehydrogenases, such as PDH, are functionally linked to the ii redox buffering network within skeletal muscle through the activity of nicotinamide nucleotide transhydrogenase (NNT). These findings highlight the importance of NNT and the entire redox buffering system in regulating cytosolic peroxide emission and suggest a novel and pivotal role for PDH as a redox-sensitive reporter of matrix redox buffering integrity and nutrient status. ii