Electron Transport Chain

By interrupting the electron transport chain (ETC) with ferricyanide, the efficiency with which various substrates of the mitochondrial metabolic reactions were used by isolated mitochondria was measured. As shown in Fig. 1, succinate and, especially, fumarate were the most effective substrate for the ETC based on the rate of ETC. The other substrates were clearly insignificantly used in the process comparing to these two substrates, with glutamate and ß-hydroksybutirate as the least used substrates, then lactate, pyruvate, followed by malate and aspartate.

Figure 1. The effect of different substrates on the rate of electron transport process in isolated mitochondria.

Being the most effective substrate for ETC, fumarate, together with

1.), glutamate, ß-hydroxybutyrate, lactate, pyruvate, malate and aspartate had a negligible effect on the rate of ETC. All these substrates are either intermediates in the Krebs cycle or their precursors. It was expected that adding them to isolated mitochondria would activate suitable enzymes which would subsequently catalyse the oxidation of these substrates by reducing NAD+ to NADH. Consequently, either Complex I or II of ETC or even both would be expected to be activated [1, 3]. Nevertheless, the rate of ETC remained practically unaffected and Complex I and II remained inactive. Adding glutamate to isolated mitochondria led to oxidation of α-ketoglutarate which was then oxidised to succinate, a common Complex II substrate. However, Complex II was not activated. The rate of subsequent reactions is lower than the rate of preceding, thus, the Krebs cycle could not be sustained and succinate production was unsuccessful [4]. ß-hydroxybutyrate and pyruvate were metabolised, by ß-hydroxybutyrate dehydrogenase and pyruvate dehydrogenase, respectively, to acetyl Coenzyme A, but oxaloacetate for the Krebs cycle was absent in the isolated mitochondria [5]. Likewise, lactate was metabolised to pyruvate, which, as mentioned, was unsuccessful in ETC activation [6]. Malate or aspartate alone cannot support respiration of isolated mitochondria since their successor oxaloacetate cannot be metabolised in the absence of a source of

1.) were succinate and fumarate. It was expected that adding succinate, a common substrate in ETC, to isolated mitochondria will increase the rate of ETC. Complex II or succinate dehydrogenase catalysed the oxidation of succinate to fumarate. The electrons derived from this reaction via FAD were transferred to the next acceptor and ETC was established [1, 4]. As mentioned, fumarate is the product of succinate oxidation, and, also, a part of the Krebs cycle. Alike substrates that did not affect ETC rate because of deficiency of components of the Krebs cycle due to mitochondria isolation, it could be expected that fumarate would not affect ETC rate. Nonetheless, fumarate was the most effective substrate used in ETC of isolated mitochondria (Fig. 1.). This condition can be explained by investigating anaerobic metabolism. Studies on anaerobic respiration in anaerobic organisms revealed the presence of NADH-fumarate reductase system as Complex II. Fumarate reductase couples the reduction of fumarate to succinate to the oxidation of quinol to quinone [10]. By investigating the possible presence of this system in mammalian mitochondria, it was shown that in the normoxic respiratory chain, Complex II functions as succinate dehydrogenase and produces fumarate. Under hypoxic and glucose-deprived conditions or under tumour microenvironment conditions, complex II can function as fumarate reductase and produces succinate [11,