**2. Key mitochondrial dehydrogenases and their regulation**

For the purpose of this review, we will focus on the following dehydrogenases:

*Glutamate dehydrogenase* (EC 1.4.1.4): The presence of this enzyme in eukaryotes is essential for urea synthesis in the urea cycle, in that it converts glutamate to α-ketoglutarate, and vice versa. However, in addition to having a very low Km for ammonia (~1 mM), at equilibrium the reaction catalyzed by this enzyme favors ammonia and α-ketoglutarate production. Conversion of α-ketoglutarate to glutamate does occur in brain mitochondria as a result of local NAD<sup>+</sup> :NADH/H<sup>+</sup> ratio [9]. In humans, the activity of the enzyme is regulated through ADP-ribosylation, but also by caloric restriction or hypoglycemia, as either of the latter two conditions increase glutamate dehydrogenase activity to increase the amount of α-ketoglutarate produced. In turn, α-ketoglutarate is used to provide energy thought the citric acid cycle, ultimately generating ATP. The activity of the enzyme does not appear to be regulated by an increase in the levels of Ca2+ and Mg2+, which can accumulate to a significant extent within mitochondria under physiological conditions. On the other hand, Zn2+ has been reported to act as an allosteric regulator of the enzyme, together with ATP and possibly GTP.

these studies, the utilization of various experimental approaches resulted in the identification of a variety of enzymes and proteins localized either within the mitochondrial matrix or associated with the inner mitochondrial membrane or the inter-membraneous space, as well as the presence of mitochondrial DNA [for an historical overview see [6] and the refs. therein]. Because the reduplication of mitochondrial DNA occurs following the reduplication of cellular DNA, and mDNA only accounts for less than half of all the mitochondrial enzymes and proteins, sophisticated biological mechanisms are in place to coordinate the import and proper allocation of mitochondrial enzymatic components within the organelle following their cellular synthesis. Furthermore, mitochondria are not static organelles but undergo constant fusion and division (fission) to form ever changing tubular networks in most eukaryotic cells. These changes are important for the normal physiology of the cell, and

From the functional stand-point, mitochondria are regarded as the source of energy for the cell, in that they produce a steady flow of ATP that is utilized in a variety of cellular functions and signaling events. In addition to producing ATP, mitochondria play a major role in signaling, cell differentiation and growth, cell cycle, cell death (apoptosis), and in the production of

While certain mitochondrial functions are present almost exclusively in specific cells (e.g. ammonia detoxification in hepatic mitochondria), ATP production represents the predominant and ubiquitous function of the organelle in all eukaryotic cells. The chemiosmotic coupling of proton movement out of specific sites of the electron transport chain to their re-entry

by Mitchell [8] in 1966, is now generally accepted, although some details of the process need further refinement. The generated ATP is then extruded in a 1ADPin: 1ATPout ratio into the cytoplasm through the adenine nucleotide translocase (AdNT), one of the most abundant

Despite our current understanding of the mechanisms responsible for ATP synthesis within the mitochondria and its extrusion across the organelle membrane, the specific role of various mitochondrial dehydrogenases and their regulation in modulating ATP synthesis to maintain a stable and viable flow of energetic 'currency' for the cell based on the available substrate is

The present review does not have the pretense of being comprehensive in addressing the role and regulation of all mitochondrial dehydrogenases. Rather, it will focus on the regulation of some of them, and the dysregulation occurring under specific pathological conditions, which

*Glutamate dehydrogenase* (EC 1.4.1.4): The presence of this enzyme in eukaryotes is essential for urea synthesis in the urea cycle, in that it converts glutamate to α-ketoglutarate, and

: 1ATP ratio, as proposed

may dramatically affect cell behavior under pathological conditions.

through the F0-F1 ATPase, with associated ATP production in a 3H<sup>+</sup>

ultimately impacts the proper functioning of specific organs or tissues.

**2. Key mitochondrial dehydrogenases and their regulation**

For the purpose of this review, we will focus on the following dehydrogenases:

proteins present in the mitochondrial membrane.

reactive oxygen species (ROS) [7].

126 Secondary Metabolites - Sources and Applications

still not completely elucidated.

*α-ketoglutarate dehydrogenase* (1.2.4.2): Also known as oxoglutarate dehydrogenase complex (OGDC), this enzymatic complex is known for its role in the citric acid cycle. Three different forms of this complex can be identified based on the specific substrate of interaction. One form is specific for pyruvate, a second one is specific for 2-oxoglutarate, and a third one is specific for branched-chain α-keto-acids. Because the same dehydrogenase subunit is utilized, the three forms of the complex utilized the same coenzymes: i.e. TTP, CoA, lipoate, FAD and NAD. Functionally, this dehydrogenase is involved in lysine degradation and tryptophan metabolism in addition to playing a key control point in the TCA cycle. In this contest, the dehydrogenase controls the level of reducing equivalents, such as NADH, generated in the cycle which, in turn increase the electrons flux through the mitochondrial electron transport chain, enhancing oxidative phosphorylation and ultimately ATP synthesis [10]. In a classic enzymatic inhibitory feedback, the activity of α-ketoglutarate dehydrogenase is inhibited by the reaction by-products succinyl-CoA and NADH, as well as by a high energy status (elevated ATP and NADH levels) within the cell, and by CoA-SH. Conversely, an increase in ADP level acts as an allosteric activator of the dehydrogenase. Calcium [10] and magnesium [11] ions also act as allosteric activators of the enzyme. The effects of Ca2+ and Mg2+ on the enzyme activity appear to be additive when the concentrations of free Mg2+ in the matrix is <1 mM. The presence of the divalent cations markedly decrease the Km for α-ketoglutarate from ~4–5 mM in the absence of Ca2+ and Mg2+, to 2.2 mM in the presence of Ca2+ alone, to 0.3 mM in the presence of both cations [11]. The effect of Mg2+ is only observed in the presence of thiamine pyrophosphate (TPP), suggesting that the enzyme requires both TPP and Mg2+ for maximal activity. The presence of both cations also decreases the affinity of the dehydrogenase for NAD<sup>+</sup> [11]. The modulatory effect of both Ca2+ and Mg2+ are observed at concentrations that are well within the range reported to occur in mitochondria for these cations under stimulatory conditions, e.g. by catecholamine or other hormones, thus supporting the likelihood that these regulatory effects can occur under in vivo conditions.

A side product of a-ketoglutarate activity is the generation of free radicals, which can lead to oxidative stress if accumulating in high levels due to increased production and/or reduced detoxification. Due to its ability to generate free radicals, this dehydrogenase is considered de facto a mitochondrial redox sensor [12] in that it can modulate the rate of mitochondrial functioning and consequently the level of oxidative stress, thereby limiting the associated damage [12]. Under conditions in which the level of free radicals becomes extremely elevated, the enzyme can undergo a fully reversible oxidative inhibition [13]. This temporary inhibition appears to occur through the reversible glutathionylation of the E2-lipoic acid domain of the dehydrogenase [14], thereby protecting the E2 domain, and the catalytic site of the dehydrogenase, from damaging oxidative stress. As the flux of electrons through the electron chain decreases, so does the production of free radicals, optimizing the conditions for the mitochondrial detox systems to scavenger these toxic agents.

1) it converts succinate to fumarate as part of the citric acid cycle while converting FAD to

; 2) electrons from FADH2 are transferred to the iron–sulfur clusters of the SdhB subunits as part of the respiratory chain function, and 3) the electrons are ultimately transferred to ubiquinone Q via the hydrophobic subunits o the complex. Inhibition of the succinateubiquinone activity results in the transfer of the electrons flowing through the SdhB subunit

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, thereby generating reactive oxygen species (e.g. superoxide). Accumulation of reactive oxygen species stabilizes the production of HIF-1α, and facilitating its interaction with HIF-1b to form a critical heterodimeric complex that induces the expression of anti-apoptotic genes but also tumor-causing genes (see section 3 for further details). Under conditions in which succinate dehydrogenase activity is inhibited, succinate accumulates within the mitochondria and then diffuses into the cytoplasm where it inhibits the physiological hydroxylation of HIF-1α in the cytosol by prolyl-hydroxylase (PHD). Inhibition of HIF-1a hydroxylation works in conjunction with the accumulation of reactive oxygen species occurring through the succinate dehydrogenase complex to stabilize HIF-1a and promote the formation of a stable and active HIF complex that promotes the expression of tumor-inducing genes [19]. Because PHD activity requires oxygen, α-ketoglutarate as substrates, and ferrous iron and ascorbate as co-factors, increasing α-ketoglutarate levels could represents a viable therapeutic approach

*Pyruvate dehydrogenase* (1.2.4.1): This enzyme is the first component of the pyruvate dehydrogenase complex (PDC), which is responsible for transforming pyruvate to acetyl-CoA via pyruvate decarboxylation. The Acetyl-CoA generated by the reaction then enters the citric acid cycle, contributing to cell respiration. As a result, pyruvate dehydrogenase links glycolysis to the citric acid cycle and the release of energy via NADH. The complex is constituted by the pyruvate dehydrogenase (E1) component, a dihydrolipoamide acetyltransferase (E2) component, a pyruvate dehydrogenase kinase (PDK) and a pyruvate dehydrogenase phosphatase. The reaction operated by E1 uses thiamine pyrophosphate (TPP) as a required cofactor, and it is considered to be the rate-limiting step for the whole pyruvate dehydrogenase complex (PDHc) activity. Phosphorylation of E1 by PDK inactivates E1 and consequently the whole complex. Pyruvate is a natural inhibitor of PDK, thereby resulting in a higher level of active, unphosphorylated PDH [20]. Alternatively, the phosphorylation state of E1 is reversed by the activity of pyruvate dehydrogenase phosphatase, which is stimulated by insulin, phospho-enol-pyruvate, and AMP, and competitively inhibited by ATP, NADH, and Acetyl-CoA. Physiological fluctuations in mitochondrial Ca2+ and Mg2+ levels, as observed following insulin stimulation, also stimulate the phosphatase activity, maintaining E1 in the

Cancer cells are characterized by increased glycolytic ATP production as a result of decreased mitochondria effectiveness. Inhibition (or decreased activity) of the citric acid cycle is considered to be one of the main causes forcing the cells to generate ATP through anaerobic glycolysis [21]. This reprogramming results from oncogene activation or inhibition of tumor suppressors [21]. Consistent with this observation, inhibition of glycolysis by dichloroacetate,

to limit tumor development and growth under SDH deficiency.

**3. Mitochondrial dehydrogenases and cancer**

FADH<sup>2</sup>

to O<sup>2</sup>

active state [20].

*Isocitrate Dehydrogenase* (1.1.1.42 and 1.1.1.41): This enzyme catalyzes the oxidative decarboxylation of isocitrate to generate a-ketoglutarate and CO<sup>2</sup> in the citric acid cycle. The reaction is a two-step process, supported by the conversion of NAD<sup>+</sup> to NADH. The mitochondrial isoform of this enzyme, IDH3, is a heterotetramer composed by two alpha, one beta, and one gamma subunits (2α1β1γ) Two other isoforms (IDH1 and IDH2) of the enzyme are known in humans. They catalyze the same reaction in the cytosol and in peroxisomes converting NADP<sup>+</sup> to NADPH in the process.

Owing to the large negative free energy change involved in the reaction, the step catalyzed by this dehydrogenase represents one of the irreversible reactions in the citric acid cycle. Hence, tight regulation is required to avoid unnecessary depletion of isocitrate. The reaction is promoted by substrate availability and the presence of cofactors such as NAD<sup>+</sup> and Mg2+ (or Mn2+), which both bind specific active sites on the IDH structure, and inhibited by ATP levels [15]

*Succinate dehydrogenase* (1.3.5.1): This enzyme participates in two key cycles within the mitochondrion. It is part of the respiratory complex II (whereby it is also defined as succinatecoenzyme Q reductase) and as such is key in controlling the electron flux through the electron transport chain [16]. In addition, it participates in the citric acid cycle, in which it catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol.

Structurally, the succinate dehydrogenase is composed of 2 hydrophilic and 2 hydrophobic subunits. The two hydrophilic subunits possess a covalently attached flavin adenine dinucleotide (FAD) cofactor and the succinate binding site (subunit SdhA) and three iron–sulfur clusters (SdhB). The subunit SdhA provides the binding site for the oxidation of succinate. The two hydrophobic subunits SdhC and SdhD act as membrane anchors. Human mitochondria contain 2 distinct SdhA isoforms (FpI and FpII), which form a membrane bound cytochrome b complex with 1 heme b group and 1 ubiquinone binding site. More specifically, the ubiquinone binding site is located in an enclosure formed by SdhB, and the two hydrophobic subunits SdhC and SdhD. These two subunits also present one cardiolipin and one phosphatidylethanolamine molecule attached [17].

Functionally, the succinate-binding site in SdhA and the ubiquinone-binding site in the pocket formed by the three other subunits are connected by a chain of redox centers that include FAD and the three iron–sulfur clusters [17]. Due to its localization, the succinate dehydrogenase participates to both the citric acid cycle and the respiratory chain, as indicated previously. Electron removed from succinate during its oxidation to fumarate are transferred through the iron–sulfur clusters present in SdhB to the ubiquinone molecule, and ultimately to the two hydrophobic subunits anchoring the complex to the mitochondrial membrane. The acceptance of the electrons by the ubiquinone occurs in a two-step process whereby, after the first electron transfer, the ubiquinone is converted to a semiquinone radical species. The transfer of the second electron fully converts this intermediate to ubiquinol [18]. Hence, SdhA acts as an intermediate in the enzymatic activity of the succinate dehydrogenase complex in that: 1) it converts succinate to fumarate as part of the citric acid cycle while converting FAD to FADH<sup>2</sup> ; 2) electrons from FADH2 are transferred to the iron–sulfur clusters of the SdhB subunits as part of the respiratory chain function, and 3) the electrons are ultimately transferred to ubiquinone Q via the hydrophobic subunits o the complex. Inhibition of the succinateubiquinone activity results in the transfer of the electrons flowing through the SdhB subunit to O<sup>2</sup> , thereby generating reactive oxygen species (e.g. superoxide). Accumulation of reactive oxygen species stabilizes the production of HIF-1α, and facilitating its interaction with HIF-1b to form a critical heterodimeric complex that induces the expression of anti-apoptotic genes but also tumor-causing genes (see section 3 for further details). Under conditions in which succinate dehydrogenase activity is inhibited, succinate accumulates within the mitochondria and then diffuses into the cytoplasm where it inhibits the physiological hydroxylation of HIF-1α in the cytosol by prolyl-hydroxylase (PHD). Inhibition of HIF-1a hydroxylation works in conjunction with the accumulation of reactive oxygen species occurring through the succinate dehydrogenase complex to stabilize HIF-1a and promote the formation of a stable and active HIF complex that promotes the expression of tumor-inducing genes [19]. Because PHD activity requires oxygen, α-ketoglutarate as substrates, and ferrous iron and ascorbate as co-factors, increasing α-ketoglutarate levels could represents a viable therapeutic approach to limit tumor development and growth under SDH deficiency.

*Pyruvate dehydrogenase* (1.2.4.1): This enzyme is the first component of the pyruvate dehydrogenase complex (PDC), which is responsible for transforming pyruvate to acetyl-CoA via pyruvate decarboxylation. The Acetyl-CoA generated by the reaction then enters the citric acid cycle, contributing to cell respiration. As a result, pyruvate dehydrogenase links glycolysis to the citric acid cycle and the release of energy via NADH. The complex is constituted by the pyruvate dehydrogenase (E1) component, a dihydrolipoamide acetyltransferase (E2) component, a pyruvate dehydrogenase kinase (PDK) and a pyruvate dehydrogenase phosphatase. The reaction operated by E1 uses thiamine pyrophosphate (TPP) as a required cofactor, and it is considered to be the rate-limiting step for the whole pyruvate dehydrogenase complex (PDHc) activity. Phosphorylation of E1 by PDK inactivates E1 and consequently the whole complex. Pyruvate is a natural inhibitor of PDK, thereby resulting in a higher level of active, unphosphorylated PDH [20]. Alternatively, the phosphorylation state of E1 is reversed by the activity of pyruvate dehydrogenase phosphatase, which is stimulated by insulin, phospho-enol-pyruvate, and AMP, and competitively inhibited by ATP, NADH, and Acetyl-CoA. Physiological fluctuations in mitochondrial Ca2+ and Mg2+ levels, as observed following insulin stimulation, also stimulate the phosphatase activity, maintaining E1 in the active state [20].
