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Native rates of superoxide production from multiple sites in isolated mitochondria measured using endogenous reporters.

TitleNative rates of superoxide production from multiple sites in isolated mitochondria measured using endogenous reporters.
Publication TypeJournal Article
Year of Publication2012
AuthorsQuinlan CL, Treberg JR, Perevoshchikova IV, Orr AL, Brand MD
JournalFree Radic Biol Med
Volume53
Issue9
Pagination1807-17
Date Published2012 Nov 01
ISSN1873-4596
KeywordsAnimals, Calibration, Cytochromes b, Electron Transport, Female, Glutamic Acid, Hydrogen Peroxide, Kinetics, Malates, Mitochondria, Muscle, NADP, Oxidation-Reduction, Rats, Rats, Wistar, Superoxides
Abstract

Individual sites of superoxide production in the mitochondrial respiratory chain have previously been defined and partially characterized using specific inhibitors, but the native contribution of each site to total superoxide production in the absence of inhibitors is unknown. We estimated rates of superoxide production (measured as H(2)O(2)) at various sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site I(F)) was calibrated to the reduction state of endogenous NAD(P)H. Similarly, the rate of superoxide production by the complex III site of quinol oxidation (site III(Qo)) was calibrated to the reduction state of endogenous cytochrome b(566). We then measured the endogenous reporters in mitochondria oxidizing NADH-generating substrates, without added respiratory inhibitors, with and without ATP synthesis. We used the calibrated reporters to calculate the rates of superoxide production from sites I(F) and III(Qo). The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site I(F) was the dominant superoxide producer. Under nonphosphorylating conditions, overall rates were higher, and sites I(F) and III(Qo) and unidentified sites (perhaps the complex I site of quinone reduction, site I(Q)) all made substantial contributions to measured H(2)O(2) production.

DOI10.1016/j.freeradbiomed.2012.08.015
Alternate JournalFree Radic. Biol. Med.
PubMed ID22940066
PubMed Central IDPMC3472107
Grant ListR01 AG033542 / AG / NIA NIH HHS / United States
P01AG025901 / AG / NIA NIH HHS / United States
R01AG033542 / AG / NIA NIH HHS / United States
PL1AG032118 / AG / NIA NIH HHS / United States