Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria.
Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.
Vrbacký
Marek
M
Department of Bioenergetics, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ 142 20 Prague, Czech Republic.
Drahota
Zdenek
Z
Mrácek
Tomás
T
Vojtísková
Alena
A
Jesina
Pavel
P
Stopka
Pavel
P
Houstek
Josef
J
D002001:Adipose Tissue, Brown / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D019069:Cell Respiration; D006224:Cricetinae; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives / Q000737:chemistry; D005292:Ferricyanides / Q000494:pharmacology; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D005994:Glycerophosphates / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010101:Oxygen Consumption; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism
D005292:Ferricyanides; D005994:Glycerophosphates; D017382:Reactive Oxygen Species; C067883:dihydroethidium; C032456:antimycin; D014451:Ubiquinone; C007931:hexacyanoferrate III; D000968:Antimycin A; D005993:Glycerolphosphate Dehydrogenase; D014450:Electron Transport Complex III; D004996:Ethidium