**3. SDH function and regulation**

214 Dehydrogenases

**Figure 2.** The covalent bond between FAD and succinate dehydrogenase.

**Figure 3.** The 2Fe-2S cluster of succinate dehydrogenase [9].

The hydrophobic anchoring subunits are integral membrane proteins and interact with quinone substrates. The yeast and mammalian SDH also contains a *b*-type heme. Oyedotun et al. [11] demonstrated the presence of an amount of cytochrome b562 steichiometric to covalent FAD. Together, the Fp and Ip form a catalytic dimer that is attached to the membrane by the anchoring subunits, thereby composing the holoenzyme. In yeast, the SDH Fp, Ip, and two anchoring subunits are encoded by the nuclear genes, *SDH1*, *SDH2*, *SDH3*, and *SDH4*, respectively, which have all been cloned and sequenced. The SDH subunits are translated in the cytoplasm, targeted to mitochondria by cleavable aminoterminal presequences, translocated across both mitochondrial membranes, and finally assembled with each other and their respective co-factors into a functional complex [10].

The quaternary structure model of the SDH for different cells, e.g. *S. cerevisiae* and mammalian, was described in study conducted by Oyedotun and Lemire [10] and Yankovskaya et al. [12]. First subunit of SDH provides the binding site for the oxidation of succinate. The side chains Thr254, His354, and Arg399 stabilize the molecule while FAD oxidizes and carries the electrons to the first of the iron-sulfur [2Fe-2S] clusters. Whereas, ubiquinone binding site is located is in a gap composed of three SDH subunits. Ubiquinone is stabilized by the side chains of His207 of second subunit, Ser27 and Arg31 of third subunit C, and Tyr83 of fourth subunit. The quinine ring is surrounded by Ile28 of third subunit and Succinate dehydrogenase is a key enzyme in intermediary metabolism and aerobic energy production in living cells. This enzymes catalyses the oxidation of succinate into fumarate in the Krebs cycle (1), derived electrons being fed to the respiratory chain complex III to reduce oxygen and form water (2). This builds up an electrochemical gradient across the mitochondrial inner membrane allowing for the synthesis of ATP. Alternatively, electrons can be diverted to reduce the ubiquinone pool (UQ pool) and provide reducing equivalents necessary to reduce superoxide anions originating either from an exogenous source or from the respiratory chain itself (3) [13] (Figure 4).

**Figure 4.** The functions of the succinate dehydrogenase in the mitochondria [13].

In the reaction of oxidation of succinate to fumarate, two hydrogen atoms are removed from substrate by flavin adenine dinucleotide (FAD), a prosthetic group that is tightly attached to succinate dehydrogenase (Figure 5).

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 217

Electronic paramagnetic resonance measurements of SDH components indicated that at least three separate centres are present. The S-3 centre (E'o = +60 mV) is a high potential Fe-S protein and is probably identical with the 4Fe-rS centre of low molecular weight subunit. Centres S-1 and S-2 (E'o = +15 mV and -260 mV respectively), are of the 2Fe-2S ferredoxin type and are probably associated with a larger, flavin containing subunit. Thus, electron transfer from succinate to ubiquinone probably occurs in the sequence: FAD, S-1, S-3. The redox potential of S-2 is rather too low to allow this centre to be catalytically active in the

The catalytic activity of succinate dehydrogenase is modulated both by post translational phosphorylation/acetylation and active site inhibition. For example, phosphorylation of the Sdh1 subunit leads to attenuate activity of SDH. The activity of this enzyme may be also modulated by Krebs cycle intermediates including oxaloacetate or malonate which are strong inhibitors. Mechanisms of inhibition by these compounds differ significantly because oxaloacetate, a competitive inhibitor of succinate dehydrogenase, bounds with a sulfhydryl group of the enzyme to abolish the enzyme activity [16]. It is known that SDH is sensitive to different thiol-binding reagents. Inhibition of the enzyme by these kinds of reagents resulted from the modification of a sulfhydryl group located at the active site. This thiol, although not essential for substrate binding or catalysis, could influence the binding of dicarboxylates, probably by steric hindrance when a larger group or a charged group were attached to it. The inhibition of SDH by histidine specific reagents was also reported, and the participation of an imidazole ring in the initial step of succinate oxidation was suggested. The inactivation of SDH by phenylglyoxal and 2,3 –butanedione showed the presence of an arginine-residues that interacts with dicarboxylate to form the primary

SDH is not only known to catalyse a unique reaction, which requires the participation of its four subunits, but deleterious mutations in any of the SDH genes should invariably result in a decreased SDH activity. Therefore, the striking phenotypic differences associated with mutations in the four subunits raise puzzling questions. SDH also plays a specific role in the maintenance of the mitochondrial UQ pool reduction. Ubiquinone, beside its function as an electron carrier mediating electron transfer, is admittedly working as a powerful antioxidant in biological membranes. Then, only a portion of the UQ pool may be actually involved in electron transfer depending on dehydrogenases involved. Accordingly, the measurable redox status of the UQ pool should result from the reducing activity of the different dehydrogenases, the oxidising activity of complex III and the kinetic equilibrium in the pool. The UQ pool therefore represents an electron sink and, when reduced, an antioxidant reservoir in the mitochondrial inner membrane. However, UQ is a double-faced compound, possibly working as either an antioxidant when fully reduced to ubiquinol, or a pro-oxidant when semi-reduced to the unstable ubisemiquinone form. Possibly together with reduced cytochrome b, semi-reduced quinones constitute the prominent source of superoxides. Finally, when defective, the abnormal amount of superoxides can be produced, e.g. flavin radicals of complex I. Delivering electrons for the full reduction of UQ to UQH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria.

forward direction [8,15].

enzyme-substrate complex [17].

**Figure 5.** The succinate dehydrogenase reaction.

Two electrons from the reduced SDH-FADH2 complex are then transferred to ubiquinone (Q), a soluble component of the electron transport system complex II. Ubiquinone is then reduced to ubiquinol ( QH2).

Flavin adenine dinucleotide (FAD) is an essential cofactor for SDH enzyme. The generation of adenosine triphosphate (ATP) in mitochondria is coupled to the oxidation of nicotinamide adenine dinucleotide (NADH) and FADH2 and reduction of oxygen to water within the respiratory chain and a three-dimensional structure of the mitochondrial respiratory membrane protein complex II. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate [9]. The substrate analog malonate is a competitive inhibitor of the succinate dehydrogenase complex. Malonate, like succinate, is a dicarboxylate that binds to cationic amino acid residues in the active site of the succinate dehydrogenase complex. However, malonate cannot undergo oxidation because it lacks the -CH2 - CH2- group necessary for dehydration. To study the effects of a competitive inhibitior on the activity of succinate dehydrogenase, malonate will be added to a reaction mixture; malonate is sufficiently different from succinate that it cannot de dehydrogenated, i.e. malonate is not metabolized [8, 14].

SDH is a difficult enzyme to extract from respiratory membrane whilst still retaining its *in vivo* properties. Most of the extraction procedures used in early work were rather drastic and yielded soluble preparations of rather dubious integrity. However, the recent introduction of a more gentle method, involving disruption of the membrane with chemotropic agents, has yielded an active and nearly homogeneous enzyme of relatively low molecular weight (97,000). This enzyme can be separated by freezing and thawing into two inactive subunits. One of these, an iron sulphur flavoprotein of molecular weight 70,000, contains one mole of FAD and four moles each of iron and labile sulphide per mole of protein; The other, an iron-sulphur protein of molecular weight 27,000, also contains four moles each of iron and labile sulphide. It was determined that the large subunit of SDH is essential for catalytic activity, but the function of the small subunit, be it catalytic or regulatory [8].

Electronic paramagnetic resonance measurements of SDH components indicated that at least three separate centres are present. The S-3 centre (E'o = +60 mV) is a high potential Fe-S protein and is probably identical with the 4Fe-rS centre of low molecular weight subunit. Centres S-1 and S-2 (E'o = +15 mV and -260 mV respectively), are of the 2Fe-2S ferredoxin type and are probably associated with a larger, flavin containing subunit. Thus, electron transfer from succinate to ubiquinone probably occurs in the sequence: FAD, S-1, S-3. The redox potential of S-2 is rather too low to allow this centre to be catalytically active in the forward direction [8,15].

216 Dehydrogenases

succinate dehydrogenase (Figure 5).

**Figure 5.** The succinate dehydrogenase reaction.

i.e. malonate is not metabolized [8, 14].

regulatory [8].

reduced to ubiquinol ( QH2).

In the reaction of oxidation of succinate to fumarate, two hydrogen atoms are removed from substrate by flavin adenine dinucleotide (FAD), a prosthetic group that is tightly attached to

Two electrons from the reduced SDH-FADH2 complex are then transferred to ubiquinone (Q), a soluble component of the electron transport system complex II. Ubiquinone is then

Flavin adenine dinucleotide (FAD) is an essential cofactor for SDH enzyme. The generation of adenosine triphosphate (ATP) in mitochondria is coupled to the oxidation of nicotinamide adenine dinucleotide (NADH) and FADH2 and reduction of oxygen to water within the respiratory chain and a three-dimensional structure of the mitochondrial respiratory membrane protein complex II. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate [9]. The substrate analog malonate is a competitive inhibitor of the succinate dehydrogenase complex. Malonate, like succinate, is a dicarboxylate that binds to cationic amino acid residues in the active site of the succinate dehydrogenase complex. However, malonate cannot undergo oxidation because it lacks the -CH2 - CH2- group necessary for dehydration. To study the effects of a competitive inhibitior on the activity of succinate dehydrogenase, malonate will be added to a reaction mixture; malonate is sufficiently different from succinate that it cannot de dehydrogenated,

SDH is a difficult enzyme to extract from respiratory membrane whilst still retaining its *in vivo* properties. Most of the extraction procedures used in early work were rather drastic and yielded soluble preparations of rather dubious integrity. However, the recent introduction of a more gentle method, involving disruption of the membrane with chemotropic agents, has yielded an active and nearly homogeneous enzyme of relatively low molecular weight (97,000). This enzyme can be separated by freezing and thawing into two inactive subunits. One of these, an iron sulphur flavoprotein of molecular weight 70,000, contains one mole of FAD and four moles each of iron and labile sulphide per mole of protein; The other, an iron-sulphur protein of molecular weight 27,000, also contains four moles each of iron and labile sulphide. It was determined that the large subunit of SDH is essential for catalytic activity, but the function of the small subunit, be it catalytic or The catalytic activity of succinate dehydrogenase is modulated both by post translational phosphorylation/acetylation and active site inhibition. For example, phosphorylation of the Sdh1 subunit leads to attenuate activity of SDH. The activity of this enzyme may be also modulated by Krebs cycle intermediates including oxaloacetate or malonate which are strong inhibitors. Mechanisms of inhibition by these compounds differ significantly because oxaloacetate, a competitive inhibitor of succinate dehydrogenase, bounds with a sulfhydryl group of the enzyme to abolish the enzyme activity [16]. It is known that SDH is sensitive to different thiol-binding reagents. Inhibition of the enzyme by these kinds of reagents resulted from the modification of a sulfhydryl group located at the active site. This thiol, although not essential for substrate binding or catalysis, could influence the binding of dicarboxylates, probably by steric hindrance when a larger group or a charged group were attached to it. The inhibition of SDH by histidine specific reagents was also reported, and the participation of an imidazole ring in the initial step of succinate oxidation was suggested. The inactivation of SDH by phenylglyoxal and 2,3 –butanedione showed the presence of an arginine-residues that interacts with dicarboxylate to form the primary enzyme-substrate complex [17].

SDH is not only known to catalyse a unique reaction, which requires the participation of its four subunits, but deleterious mutations in any of the SDH genes should invariably result in a decreased SDH activity. Therefore, the striking phenotypic differences associated with mutations in the four subunits raise puzzling questions. SDH also plays a specific role in the maintenance of the mitochondrial UQ pool reduction. Ubiquinone, beside its function as an electron carrier mediating electron transfer, is admittedly working as a powerful antioxidant in biological membranes. Then, only a portion of the UQ pool may be actually involved in electron transfer depending on dehydrogenases involved. Accordingly, the measurable redox status of the UQ pool should result from the reducing activity of the different dehydrogenases, the oxidising activity of complex III and the kinetic equilibrium in the pool. The UQ pool therefore represents an electron sink and, when reduced, an antioxidant reservoir in the mitochondrial inner membrane. However, UQ is a double-faced compound, possibly working as either an antioxidant when fully reduced to ubiquinol, or a pro-oxidant when semi-reduced to the unstable ubisemiquinone form. Possibly together with reduced cytochrome b, semi-reduced quinones constitute the prominent source of superoxides. Finally, when defective, the abnormal amount of superoxides can be produced, e.g. flavin radicals of complex I. Delivering electrons for the full reduction of UQ to UQH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria. Therefore, the SDH, thanks to its unique redox properties, may be a key enzyme to control UQ pool redox poise under these conditions [13].

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 219

cyanide to the reaction mixture. These poisons inhibit the transfer of electrons from cytochrome a3 to the final electron acceptor, oxygen, thus electrons cannot be passed along by the preceding cytochromes and coenzyme Q. Instead, the electrons from SDH-FADH2 can be picked up by an artificial electron acceptor, such as the dye 2,6dichlorophenolindophenol (DCIP). The reduction of DCIP can be followed spectrophotometrically since the oxidized form of the dye is blue and the reduced form is

SDH-FADH2 + DCIPoxid. → SDH-FAD + DCIPred. + 2H+ The change in absorbance, measured at 600 nm, can be used to follow the reaction over time [21]. To use an artificial electron acceptor, the normal path of electrons in the electron transport chain must be blocked. This is accomplished by adding either potassium cyanide or sodium azide to the reaction mixture. The rate of the disappearance of the blue color is proportional to the concentration of enzyme. The change in absorbance of the mixture is measured as a function of time and the enzyme concentration is determined from these data. Enzymatic reactions in yeasts are usually studied in cell-free extracts which requires disruption of cells and as consequence, inactivation of particular enzymes often can be observed. Generally we can conclud that determination of SDH enzyme activity has proved to be a difficult enzyme to extract from respiratory membrane whilst still retaining its *in vivo*  properties. Most of the described extraction procedures were rather drastic and yielded

In recent years quantitative histochemical procedures has been proved to be a powerful research tool, especially in microphotometric assessment *in situ* of the specific activity of dehydrogenases in individual cells. These assays are simple and valid alternative to conventional biochemical techniques. Methods *in situ* can provide the cellular resolution necessary to determine enzyme-specific activities not only in whole cell preparations but

Reduction of various tetrazolium salts by dehydrogenases of metabolically active cells leads to production of highly colored end products – formazans (Figure 7). The history of the tetrazolium salts and formazans goes back 100 years, to when Friese (1875) reacted benzene diazonium nitrate with nitromethane, to produce a cherry-red "Neue Verbindung". This was the first formazan. Nineteen years later, Von Pechmann and Runge (1894) oxidised a

colorless. This reaction can be summarized as

soluble preparations of rather dubious integrity [8].

also in distinct subcellular compartments [19].

formazan to produce the first tetrazolium salt [21].

**Figure 7.** Tetrazolium salt and its coloured formazan.

Also mutations in genes encoding SDH subunits lead to reduced activity of SDH enzyme. The yeast cells disrupted in *SDH2* (*sdh2*Δ) showed dramatically accumulate succinate resulting in inhibition of at least two α-ketoglutarate dependent enzymes that generate succinate as a by-product. Disruption of complex II activity should alter TCA cycle metabolite levels in the mitochondrial matrix. It was found that neither *sdh1*Δ, nor s*dh2*Δ cells have measurable SDH activity. The succinate accumulates to 8-fold higher levels in *sdh2*Δ cells relative to wild-type cells. Furthermore, complex II + III activity was completely abolished in both SDH mutants without a corresponding compensation in NADH dehydrogenase activity. As a result, complex IV activity was decreased in the SDH mutants [18] (Figure 6).

**Figure 6.** Relative concentration of main metabolities of TCA cycle for *sdh1*Δ, nor s*dh2*Δ yeast cells [18].
