Sites of reactive oxygen species generation by mitochondria oxidizing different substrates

Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H(2)O(2) production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site I(Q)), with small contributions from the flavin site in complex I (site I(F)) and the quinol oxidation site in complex III (site III(Qo)). However, with glutamate plus malate as substrate, site I(Q) made little or no contribution, and production was shared between site I(F), site III(Qo) and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site II(F)) was a major contributor (together with sites I(F) and III(Qo)), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo