**1.2. Function of mitochondrial complex II**

(Ip), cytochrome *b* large subunit (CybL), and cytochrome *b* small subunit (CybS). The Fp and Ip subunits comprise the catalytic domain of the enzyme. The Fp subunit has a flavin adenine dinucleotide (FAD) as a prosthetic group and contains the dicarboxylate-binding site, which serves as the binding site for succinate and fumarate. The Ip subunit generally contains three iron-sulfur clusters [2Fe-2S]2+,1+, [4Fe-4S]2+,1+, and [3Fe-4S]1+,0 as prosthetic groups. Subunits CybL and CybS, with heme *b* as the prosthetic group, form the anchor domain of the enzyme. This anchors the catalytic domain to the inner mitochondrial membrane and also forms the

In 2003, our laboratory revealed that human mitochondrial complex II exists in two isoforms, which differ in the Fp subunit. These two human Fp subunits, which are referred as Fp I and Fp II, differ only in two amino acids in the C-terminal of the protein-Tyr629Phe and Val657Ile [8, 9]. The majority of human tissues have shown expression of both isoforms of complex II with predominant expression of type I Fp. However, some cancer cell lines and fetal tissues have shown predominant expression of type II Fp also [4]. Furthermore, type II Fp expression has been found to increase in cultured cells under ischemic conditions [10]. Moreover, our laboratory has revealed that Fp subunit of human complex II undergoes posttranslational modifications by phosphorylation of its tyrosine, threonine, and serine residues under tumor mimicking microenvironments (hypoxic and hypoglycemic conditions), which affects its

**Figure 1.** Schematic representation of the mitochondrial complex II. The four subunits of the complex II, namely the flavoprotein subunit, iron sulfur subunit, cytochrome *b* large subunit, and cytochrome *b* small subunit are labeled as Fp, Ip, CybL, and CybS, respectively. The prosthetic groups that participate in the electron transfer are the flavin adenine dinucleotide (FAD), three iron sulfur clusters 2Fe-2S, 3Fe-4S, 4Fe-4S, and heme *b*. Dicarboxylate-binding site in the Fp subunit serves as the catalytic site for succinate oxidation/fumarate reduction. Quinone-binding site comprised of Ip,

CybL, and CybS subunits serves as the catalytic site for quinone reduction/quinol oxidation.

quinone (Q)-binding site, together with the Ip subunit [6, 7] (**Figure 1**).

enzymatic activity [11].

88 Mitochondrial Diseases

In human cells, this enzyme plays dual roles as an indispensable enzyme in the TCA cycle as well as in the aerobic respiratory chain (**Figure 2**). It oxidizes succinate to fumarate in the TCA cycle and reduces quinone to quinol in the aerobic respiratory chain [6, 12]. This is in contrast to the function of the mitochondrial complex II of the anaerobic organisms such as *Ascaris suum,* which oxidizes quinol into quinone that is coupled with reduction of fumarate to succinate (**Figure 3**) [13]. However, our laboratory has revealed distinct fumarate reductase (FRD) activity in the human cancer cells, which have significantly increased upon exposure to hypoxia and hypoglycemia. In these cells, mitochondrial complex II is reported to acquire FRD activity through phosphorylation of its Fp subunit [11]. This finding is further supported

**Figure 2.** Schematic representation of the function of the mitochondrial complex II in the aerobic respiratory chain and the TCA cycle. Functioning as a component of the TCA cycle, mitochondrial complex II catalyzes the oxidation of succinate to fumarate with transfer of electrons to FAD. Functioning as a component of the aerobic respiratory chain, the electrons on FAD are transferred through Fe-S clusters and heme *b* to reduce the ubiquinone. For simplicity of the representation, the subunit composition of the respiratory chain complexes I, III, IV, and V is not shown.

**1.3. ROS production from mitochondrial complex II**

mutated into hydrogen peroxide (H2

there is accessibility to oxygen (**Figure 5**).

*1* mutation demonstrating O2

H2 O2

Major ROS produced by the respiratory chain is superoxide (O2

O2

Reactive oxygen species (ROS) are inevitable byproducts of the aerobic respiratory chain.

Differential Effect of Atpenin A5 on ROS Production from Wild-Type Mitochondrial Complex II…

are known to contribute to physiological and pathological redox signaling [15, 16].

However, conventionally, mitochondrial complex II is not identified as a significant source of ROS. But, two potential ROS-producing sites can be identified in mitochondrial complex II when its sequence of electron transport is considered together with the spatial arrangement of its redox centers. When one molecule of succinate is oxidized by complex II, FAD is reduced with two electrons, but subsequent transfer of electrons through Fe-S clusters occurs one at a time. Hence, when one electron is delivered to the proximal Fe-S cluster from FAD, a flavin radical is formed until the second electron is delivered. Similarly, after sequential transfer of single electrons through Fe-S clusters, quinone is reduced with two electrons. Hence, when one electron arrives at the Q site, until another electron arrives from Fe-S clusters, a quinone radical is formed. These two radicals have the potential to generate ROS, if

During the past two decades, complex II has emerged as an important source of ROS. On the one hand, ROS produced from mutated complex II has been proposed to be the underlying cause of complex II–associated human diseases. Thus, numerous studies have been conducted to understand ROS production from human complex II. Most of these studies have been conducted on model organisms such as *Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans* [17–20]. A hallmark in these studies is the finding that the *mev-1*

and oxygen hypersensitivity [19, 20]. Later on, this finding was further reinforced by a study using a transgenic mouse cell line with the equivalent mutation in the *CybL* gene as the *mev-*

esis [21]. More recently, mutations in the Ip subunit in the *C. elegans* have also been found to display enhanced ROS production [22]. Site-directed mutagenesis of CybS subunit in *E. coli* [23] and the Ip and CybS subunits in *S. cerevisiae* [18, 24] has also shown enhanced ROS production providing further support for the hypothesis that ROS underlies the complex II mutations-associated carcinogenesis. Indeed, some studies have been conducted on human cell lines using pharmacological and genetic interventions to mimic complex II mutations, but the results are controversial. For example, ROS production from human mitochondrial complex II has been shown by Guzy et al. [25] in intact cells of the hepatoma cell line Hep3B by inhibiting the complex II activity by the Q site inhibitor theonyl trifluoroacetone (TTFA) and by RNA interference of Ip subunit. In contrast, RNA interference of the same subunit of complex II in the same cell line by Cervera et al. have failed to detect ROS production [26].

On the other hand, ROS produced from mitochondrial complex II has received considerable attention as a mediator of "mitocans" (a novel class of mitochondrial targeted anticancer drugs) induced cancer cell death [5]. Interestingly, complex II Q site inhibiting mitocan, ɑ-tocopheryl succinate (ɑ-TOS) has been found to induce ROS only in complex II competent

overproduction from mitochondria that leads to tumorigen-

mutation in the mitochondrial complex II of *C. elegans* results in enhanced O2

−

−

) by superoxide dismutase (**Figure 4**). Both O2

). It is subsequently dis-

http://dx.doi.org/10.5772/intechopen.71638

−

production

− and 91

**Figure 3.** A schematic representation of the function of the mitochondrial complex II in the anaerobic respiratory chain. Electrons originating from NADH oxidation in complex I reduce the rhodoquinone (RQ) to rhodoquinol (RQH2) at its Q site. Rhodoquinol is subsequently oxidized to rhodoquinone at the Q site of the complex II. The resulting electrons are transferred via the Fe-S clusters and FAD to reduce fumarate to succinate. Fumarate is the terminal electron acceptor of the anaerobic respiratory chain.

by recent metabolic profiling studies also on gastric and colon carcinomas [14]. In addition to its role in energy metabolism, mitochondrial complex II has been identified to have a novel function as a tumor suppressor in certain tissues. Mutations in its subunits are reported to cause cancers such as pheochromocytoma (tumors of the chromaffin cells in the adrenal medulla) and paraganglioma (extra adrenal tumors of sympathetic or parasympathetic origin). Moreover, breast, thyroid, and renal carcinomas are also known to be associated with complex II mutations. More recently, mutations in the complex II assembly factors, succinate dehydrogenase assembly factor 1 (SDHAF1) and succinate dehydrogenase assembly factor 2 (SDHAF2), are also reported to be linked with infantile leukoencephalopathy and paraganglioma (for a recent review, see [2]).
