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. 2004 Sep 8;24(36):7771-8.
doi: 10.1523/JNEUROSCI.1842-04.2004.

Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase

Laszlo Tretter  1 , Vera Adam-Vizi
Affiliations

Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase

Laszlo Tretter et al. J Neurosci. .

Abstract

Alpha-ketoglutarate dehydrogenase (alpha-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 alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-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 alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) 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(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-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 alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

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Figures

Figure 1.
Figure 1.
H2O2 release from synaptosomes as measured by a direct assay with Amplex Red (a, b) or by the activity of endogenous aconitase (c). a, Synaptosomes (0.5 mg of protein/ml) were incubated for 1 hr in glucose-free medium, and then Amplex Red and HRP were added. The rate of H2O2 release was measured after addition of glucose (10 mm) or α-KG (5 mm). The results are expressed as percentage of the rate of H2O2 release from synaptosomes observed in the absence of added substrates (12.7 ± 0.17 pmol/min/mg synaptosomal protein; ±SEM; n = 4). b, Synaptosomes were incubated for 1 hr in glucose-free medium (control) or in the presence of glucose (10 mm) or α-KG (5 mm), and then the rate of H2O2 release was measured. Bars indicate the rate of H2O2 release as percentage of control (12.1 ± 0.77 pmol/min/mg synaptosomal protein; ±SEM; n = 4). c, Synaptosomes were incubated in the standard medium for 1 hr in the absence (control) or presence of α-KG (5 mm), and then the activity of aconitase was measured as described in Materials and Methods. Aconitase activity is expressed as percentage of control (6.3 ± 0.19 nmol/min/mg synaptosomal protein; ±SEM; n = 5). Asterisk indicates significant difference from the corresponding controls.
Figure 2.
Figure 2.
H2O2 production by isolated α-KGDH. H2O2 formation was measured with Amplex Red as detailed in Materials and Methods. For a-c, α-KGDH, HS-CoA (0.12 mm), and α-KG (1 mm) were added as indicated. For a, catalase was given before α-KGDH. NAD+ (b) or NADP+ (c) (5 μm) was given as shown. Traces have been offset for clarity.
Figure 3.
Figure 3.
Inhibition of H2O2 production (a, b) and stimulation of NADH formation (c) by isolated α-KGDH in response to NAD+. α-KGDH, HS-CoA (0.12 mm),α-KG (1 mm), and NAD+ in different concentrations (50 nm to 1 mm) were applied as indicated. H2O2 and NADH formations were measured simultaneously in the same samples, as described in Materials and Methods. Traces have been offset for clarity. b, Inhibition of H2O2 generation is shown as a function of NAD+ concentrations. Points represent mean values from three experiments ±SEM. Rectangular hyperbola was fitted to the experimental points. H2O2 generation in the absence of NAD+ was 1.07 ± 0.01 pmol/sec (n = 6).
Figure 4.
Figure 4.
Stimulation of α-KGDH-mediated H2O2 production by NADH. α-KGDH, HS-CoA (0.12 mm), α-KG (1 mm), and NADH (1 μm) were applied as indicated. H2O2 production was measured as for Figures 2 and 3.
Figure 5.
Figure 5.
H2O2 formation by isolated α-KGDH as a function of α-KG. The experimental protocol was as for Figure 2, except that α-KG was added in different concentrations. The rate of the initial H2O2 formation was measured after addition of α-KG without (•) or with (○) subsequent application of NAD+ in 1 mm concentration. H2O2 formation at low concentrations of α-KG (1-10 μm) in the absence of NAD+ is shown in the inset. Points represent mean ± SEM from three experiments. SEM is within the size of symbols unless otherwise indicated.
Figure 6.
Figure 6.
The effect of NADH/NADH plus NAD+ ratio on the α-KGDH-mediated H2O2 and NADH formation. Experiments were performed as described for Figure 3; however, subsequent to α-KG, NADH plus NAD+ was added in different ratios shown in the abscissa at final concentrations of 100, 250, or 500 μm (a) and 100 or 500 μm (b) as indicated. The rate of H2O2 (a) and NADH formation (b) were measured after addition of NADH plus NAD+. Addition of NADH in this concentration range resulted in a sharp increase in the Amplex signal because of the presence of some H2O2 contamination always present in NADH solutions. Thereafter, the signal became linear, and the slope was taken as a measure of H2O2 generation under the indicated conditions. Catalytic activity of α-KGDH was followed by the reduction of NAD+, measuring absorbance changes at 340 nm with a GBC Scientific Equipment (Dandenong, Australia) UV-visible 920 spectrophotometer, using the extinction coefficient E340 = 6.22 mm-1*cm-1. Points represent mean values from three experiments (±SEM). SEM is within the size of symbols unless otherwise indicated.
Figure 7.
Figure 7.
a, b, The effect of free [Ca2+] on the H2O2 (a) and NADH formation (b) measured with isolated α-KGDH. Isolated α-KGDH was incubated in a buffer containing different concentrations of ionized Ca2+ (0-100 μm). Ca2+ concentrations were controlled as described in Materials and Methods. The experimental protocol was as described for Figure 3, but ADP and Mg2+ were not present in the medium. H2O2 formation was measured in the absence (•) or presence (○) of NAD+ (1 mm). Values represent mean ± SEM from three experiments. Asterisk indicates points significantly different from the control (measured in the absence of Ca2+).
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