Figure 6.

Potential changes in the electron flow to resazurin in normal (A) and perturbed (B) metabolism, based on the enzyme activity data after 24 h incubation of cells with oxythiamin (Figure 2 and Figure 3, 24 h). Due to transamination of the 2-oxo acids which accumulate upon inhibition of the 2-oxo acid dehydrogenases [17,22], amino acids become substrates for oxidation, generating NAD(P)H, FADH₂, ubiquinone QH₂ [23]. These compounds are in redox equilibrium with other acceptors of the electron transport chain, such as redox sites of Complexes I and II (C I and C II). Driven by inhibition of the 2-oxo acid dehydrogenases, the pathophysiological increase in the amino acid oxidation (e.g., through the GABA shunt and proline oxidation) may increase side reactions, including the resazurin reduction, due to unphysiological electron flux. The substrate-dependent NAD(P)H production is depicted by brown arrows. Other electron transfers are shown by blue arrows. The enzymes assayed in Figure 2 are presented within the light-blue rectangles. Well-characterized enzymatic complexes of the TCA cycle enzymes are shown by merged rectangles. Electron-transport chain and its Complexes I (NADH:ubiquinone oxidoreductase) and II (succinate dehydrogenase) are shown in blue. For the sake of readability, the schemes omit cellular compartmentation of the processes, assuming exchange of the intermediates through the substrate/electron shuttles, and show only the relevant intermediates, reactions, and protein partners. In particular, (i) the electron flow is schematically shown as floating electrons instead of separate redox active sites of the electron transport chain, as the distribution of electrons between the sites would depend on specific conditions and metabolism; (ii) to manifest the consequences of the PDH and OGDH inhibition, the enzymes are omitted from (B). The decreased activity of malic enzyme concomitant to elevated NAD(P)H:resazurin oxidoreductase (Figure 3K) is shown by dashed line in (B). Dashed lines of the reactions, catalyzed by the mitochondrial MDH and Complex II refer to inhibition of the indicated transformations upon accumulation of oxaloacetate and malate. Oxaloacetate increases due to intensified transamination of 2-oxoglutarate as a consequence of the OGDH inhibition. Malate increases due to decrease in malic enzyme and/or inhibition of malate oxidation by the product oxaloacetate. Additional inhibition of the succinate oxidation to fumarate by Complex II may be due to alternative generation of fumarate from the 2-oxoglutarate-stimulated oxidation of tyrosine [17]. Dashed line from NAD(P)H to Complex I refers to decreased NADH from the physiological NADH suppliers (PDH and the TCA cycle enzymes) of Complex I. See text for further discussion.