**2. SDH structure**

212 Dehydrogenases

**Figure 1.** TCA cycle.

dehydrogenase, a mitochondrial NAD+-specific one and two NADP+-dependent ones (one mitochondrial and the other cytosolic). A number of pieces of evidence point to the role of mitochondrial NAD+-specific isocitrate dehydrogenase in the regulation of the rate of mitochondrial assembly besides its specific role in the TCA cycle [2]. The formation of SDH is a heterotetramer composed of flavoprotein (Fp) of about 70 kDa, an iron-sulfur protein (Ip) of about 30 kDa, and two hydrophobic anchoring subunits of 7-17 kDa. The Fp contains the active site and the unusual cofactor, an 8α-*N*(3)-histidyl-FAD linked at a conserved histidine residue (Figure 2).

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 Ip subunit contains three different iron-sulfur clusters: [2Fe-2S], [3Fe-4S], and [4Fe-4S] (Figure 3).

These clusters are coordinated by conserved cysteine residues: [2Fe-2S] (Cys-67, Cys-72, Cys-75, Cys-87); [4Fe-4S] (Cys-159, Cys-162, Cys-165, Cys-226) and [3Fe-4S] (Cys-169, Cys-216, Cys-222) [10].

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 215

Pro160 of second subunit B. These residues, along with Il209, Trp163, and Trp164 of second subunit B, and Ser27 (C atom) of third subunit, form the hydrophobic environment of the quinine binding pocket. The succinate binding site and ubiquinone binding site are connected by a chain of redox centres including FAD and the iron-sulfur clusters. This chain extends over 40 Å through the enzyme monomer with an edge-to-edge distance between the

In the place for heme *b*, the N 2 atom of Sdh3p His106 and the S atom of Sdh4p Cys78 are correctly oriented to form coordinating bonds with the central iron atom of the heme. The distance between the iron atom and the N 2 atom is 2.21 Å, but the S atom of Cys78, the putative second heme axial ligand, is 2.96 Å away from the heme Fe. UQ can be docked into two spatially separated sites with an edge-to-edge distance of 25.4 Å. The new amino acid residues that may determine the structural or catalytic properties of each of the two quinone binding sites were identified. The model also provided insight into the unusual use of a cysteine (Sdh4p Cys78) as the second heme ligand instead of the histidine residues [10].

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

centres is less that 14 Å limit for physiological electron transfer [8, 11].

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

**3. SDH function and regulation** 

the respiratory chain itself (3) [13] (Figure 4).

**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 Pro160 of second subunit B. These residues, along with Il209, Trp163, and Trp164 of second subunit B, and Ser27 (C atom) of third subunit, form the hydrophobic environment of the quinine binding pocket. The succinate binding site and ubiquinone binding site are connected by a chain of redox centres including FAD and the iron-sulfur clusters. This chain extends over 40 Å through the enzyme monomer with an edge-to-edge distance between the centres is less that 14 Å limit for physiological electron transfer [8, 11].

In the place for heme *b*, the N 2 atom of Sdh3p His106 and the S atom of Sdh4p Cys78 are correctly oriented to form coordinating bonds with the central iron atom of the heme. The distance between the iron atom and the N 2 atom is 2.21 Å, but the S atom of Cys78, the putative second heme axial ligand, is 2.96 Å away from the heme Fe. UQ can be docked into two spatially separated sites with an edge-to-edge distance of 25.4 Å. The new amino acid residues that may determine the structural or catalytic properties of each of the two quinone binding sites were identified. The model also provided insight into the unusual use of a cysteine (Sdh4p Cys78) as the second heme ligand instead of the histidine residues [10].
