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

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.

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.

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.

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.

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.

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.

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.

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⁺).