Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

Abstract

Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H 2 O 2 production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone, ␣-ketoglutarate supported the highest rate of H 2 O 2 production. In the absence of ADP or in the presence of rotenone, H 2 O 2 production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of ␣-ketoglutarate. Isolated mitochondrial ␣-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H 2 O 2 . NAD ϩ inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H 2 O 2 production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H 2 O 2 than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

Targeting the Prodromal Stage of Alzheimer's Disease: Bioenergetic and Mitochondrial Opportunities

“…Key enzymes involved in mitochondrial function, such as PDH and α-ketoglutarate dehydrogenase, are often targeted by ROS, leading to deceased enzyme activity and decreased efficiency of mitochondrial electron transport (Fig. 2) [106]. In AD, elevated oxidative stress is detectable in the form of lipid peroxides, 8-oxoguanine, and other oxidized proteins [107][108][109][110].…”

Targeting Mitochondrial Function for the Treatment of Acute Spinal Cord Injury

“…As a consequence, these oxyradicals accumulate and contribute to the formation of lipid peroxidation, protein oxidation, and peroxynitrite byproducts that interact with and inactivate critical mitochondrial enzyme complexes. One approach to combat the increase in free radical accumulation is to develop modified antioxidants that target the mitochondrial matrix, where these oxyradicals are produced [18,[58][59][60]. A separate review dedicated to antioxidant treatment in acute SCI is included in this issue and the reader is referred the accompanying review by Hall et al for details.…”

Bioenergetic dysfunction in Huntington's disease human cybrids

“…Pyruvate, alpha-ketoglutarate and malate dehydrogenases activities were measured as described previously (Nulton-Persson and Szweda, 2001;Starkov et al, 2004;Zhou et al, 2008). Total cell extracts were obtained using 0.05 mg/ml in 25.0 mM KH 2 PO 4 and 0.5 mM EDTA, pH 7.25, containing 0.01% Triton X-100 and protein quantified using BioRad protein assay.…”

“…For pyruvate dehydrogenase (PDH), 15 μg of cell sample were mixed with 2.5 μM rotenone, 0.5 mM of NAD + , 0.2 mM thiamine pyrophosphate (TPP + ), 0.04 mM CoASH, and 4 mM pyruvate (Zhou et al, 2008). For alpha-ketoglutarate dehydrogenase (KGDH) activity, 15 μg of cell sample were mixed with 0.3 mM TPP + , 10 mM CaCl 2 , 0.2 mM MgCl 2 , 5 mM alpha-ketoglutarate (KG), 1 μM rotenone, 0.2 mM NAD + , and 0.14 mM CoASH (Starkov et al, 2004). For malate dehydrogenase activity, 15 μg of protein were used and mixed with 40 μM rotenone, 5 mM MgCl2, 25 mM malate, 1 unit/ml of citrate synthase, 0.3 mM acetyl-CoA, and 10 mM NAD + (Nulton-Persson and Szweda, 2001).…”