SUMMARY Mitochondrial fission mediated by the GTPase dynamin-related protein-1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial Complex I-dependent O2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g. 50 μM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing Complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (Ki>1.2 mM). Overall, results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit Complex I and modify mitochondrial ROS production may contribute to effects observed in disease models.

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␣-Ketoglutarate dehydrogenase (␣-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that ␣-KGDH is able to generate H 2 O 2 and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated ␣-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H 2 O 2 after addition of ␣-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD ϩ ). NAD ϩ , which proved to be a powerful inhibitor of ␣-KGDH-mediated H 2 O 2 formation, switched the H 2 O 2 forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H 2 O 2 formation by ␣-KGDH, and for this, neither ␣-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD ϩ ratio determined the rate of H 2 O 2 production. The higher the NADH/NAD ϩ ratio the higher the rate of H 2 O 2 production. H 2 O 2 production as well as the catalytic function of the enzyme was activated by Ca 2ϩ . In synaptosomes, using ␣-ketoglutarate as respiratory substrate, the rate of H 2 O 2 production increased by 2.5-fold, and aconitase activity decreased, indicating that ␣-KGDH can generate H 2 O 2 in in situ mitochondria. Given the NADH/NAD ϩ ratio as a key regulator of H 2 O 2 production by ␣-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus ␣-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

In this study we addressed the function of the Krebs cycle to determine which enzyme(s) limits the availability of reduced nicotinamide adenine dinucleotide (NADH) for the respiratory chain under H 2 O 2 -induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H 2 O 2 proved to be aconitase, being completely blocked at 50 M H 2 O 2 . ␣-Ketoglutarate dehydrogenase (␣-KGDH) was also inhibited but only at higher H 2 O 2 concentrations (Ն100 M), and only partial inactivation was achieved. The rotenone-induced increase in reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] fluorescence reflecting the amount of NADH available for the respiratory chain was also diminished by H 2 O 2 , and the effect exerted at small concentrations (Յ50 M) of the oxidant was completely prevented by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNUinsensitive decline by H 2 O 2 in the rotenone-induced NAD(P)H fluorescence correlated with inhibition of ␣-ketoglutarate dehydrogenase. Decrease in the glutamate content of nerve terminals was induced by H 2 O 2 at concentrations inhibiting aconitase. It is concluded that (1) aconitase is the most sensitive enzyme in the Krebs cycle to inhibition by H 2 O 2 , (2) at small H 2 O 2 concentrations (Յ50 M) when aconitase is inactivated, glutamate fuels the Krebs cycle and NADH generation is unaltered, (3) at higher H 2 O 2 concentrations (Ն100 M) inhibition of ␣-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H 2 O 2 -induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of ␣-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.