Characteristics of α‐glycerophosphate‐evoked H₂O₂ generation in brain mitochondria

Abstract

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on aglycerophosphate (a-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DY m ) and released H 2 O 2 in parallel with an increase in NAD(P)H fluorescence in the presence of a-GP (5-40 mM). H 2 O 2 formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H 2 O 2 formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by a-GP, but not by succinate. ROS under these conditions are most likely to be derived from a-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in a-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q o ) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of a-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to a-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by a-GP at complex III is evident only when this complex is inhibited by antimycin. Keywords: brain mitochondria, a-glycerophosphate, oxidative stress, reactive oxygen species, succinate. The main site of mitochondrial ROS production has been a topic of scientific debate, and mainly complex I and complex III of the respiratory chain have been the focus of investigations using isolated mitochondria respiring on either NADH-linked substrates or succinate (Boveris and Chance 1973;Cadenas et al. 1977;Turrens and Boveris 1980). Recently, a growing body of evidence has suggested that complex I is a more important source of ROS under normal and pathological conditions (Hansford et al. 1997;Barja 1999;Herrero and Barja 1999;Votyakova and Reynolds 2001;St Pierre et al. 2002), but depending upon the substrates and experimental conditions, different mechanisms might be responsible for ROS production in isolated mitochondria. Very recently a new ROS-forming site, the a-ketoglutarate dehydrogenase enzyme (a-KGDH) has been described (Bunik and Sievers 2002;Starkov et al. 2004b; Received 21 July, 2006; revised manuscript received September 4, 2006; accepted September 5, 2006. Address correspondence and reprint reqeusts to Prof. Vera Adam-Vizi MD, PhD, Department of Medical Biochemistry, Semmelweis University, Budapest, H-1444, P.O. Box 262, Hungary. E-mail: av@puskin.sote.huAbbreviations used: BCECF, 2¢,7¢-bis(carboxyethyl)-5,6-carboxyfluorescein; BSA, bovine serum albumin; a-GP, a-glycerophosphate; a-GPDH, a-glycerophosphate dehydrogenase; DY m , membrane potential; FCCP, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon; pH m , pH in the mitochondrial matrix; Q o , quinol-oxidizing center; RCR, respiratory control ratio; RET, rever...

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A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

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