Succinate oxidation

As indicated in Sections 1 and 2, succinate is an electron donor widely utilized for NAD(P) reduction by phototrophic purple bacteria. The membrane-bound enzyme responsible for succinate oxidation has been solubilized and partially characterized in the purple non-sulfur bacteria R. rubrum [73,74] and Rhodopseudo-monas sphaeroides (recently renamed Rhodobacter sphaeroides) [57]. In situ characterization of the iron-sulfur centers likely to be associated with succinate dehydrogenase has been accomplished for Rps. capsulata [59] and C. vinosum [51]. Of particular interest is the presence of a succinate-reducible [51,57,58,73] and fu-marate-oxidizable [51] iron-sulfur cluster with near +50 mV that, like center S-3 [60,61,75,76] of mitochondrial succinic dehydrogenase (Complex II), is paramagnetic in the oxidized state. The enzyme in phototrophic bacteria also appears to have one or two ferredoxin-like (i.e., paramagnetic in the reduced state) iron-sulfur centers that correspond to centers S-1 (succinate-reducible, EJ ranging from 

---

This complex consists of four subunits, all of which are encoded on nuclear DNA, synthesized on cytosolic ribosomes, and transported into mitochondria. The succinate dehydrogenase (SDH) component of the complex oxidizes succinate to fumarate with transfer of electrons via its prosthetic group, FAD, to ubiquinone. It is unique in that it participates both in the respiratory chain and in the tricarboxylic acid (TC A) cycle. Defects of complex II are rare and only about 10 cases have been reported to date. Clinical syndromes include myopathy, but the major presenting features are often encephalopathy, with seizures and psychomotor retardation. Succinate oxidation is severely impaired (Figure 11). 

By 1949 low temperature spectroscopy had been introduced. With this technique Keilin and Hartree detected a further component in the electron transfer chain which had a sharp band at 552 nm. They later showed it to be identical with cytochrome cj, which had first been observed by Yakushiji and Okunuki (1940) during succinate oxidation by cyanide-inhibited beef heart muscle. As the oxidation of cytochrome C was accelerated by cytochrome c, Okunuki and Yakushiji (1941) had placed C] in the chain in the order... 

Further support for the cyclical nature of the chain came from observations of malonate-inhibited muscle slices. In such preparations the addition of any of the intermediates of the chain led to accumulation of succinate. Even the addition of fumarate, the immediate product of succinate oxidation, led to accumulation of succinate. 

Melnick RL, Schiller CM. 1985. Effect of phthalate esters on energy coupling and succinate oxidation in rat liver mitochondria. Toxicology 34 13-27. 

Most anaerobically functioning mitochondria use endogenously produced fumarate as a terminal electron-acceptor (see before) and thus contain a FRD as the final respiratory chain complex (Behm 1991). The reduction of fumarate is the reversal of succinate oxidation, a Krebs cycle reaction catalysed by succinate dehydrogenase (SDH), also known as complex II of the electron-transport chain (Fig. 5.3). The interconversion of succinate and fumarate is readily reversible by FRD and SDH complexes in vitro. However, under standard conditions in the cell, oxidation and reduction reactions preferentially occur when electrons are transferred to an acceptor with a higher standard redox potential therefore, electrons derived from the oxidation of succinate to fumarate (E° = + 30 mV) are transferred by SDH to ubiquinone,... 

Conditions Complex II was inactivated by 20 min incubation at pH 9.3 and readjusted to pH 7.6 (protein 11.25 mg/ml). Succinate dehydrogenase was prepared and stored as an ammonium sulfate-precipitated pellet for 1 day at —70°. It was dissolved in 50 mM Tris-HCl (pH 8.0) containing 20 mAf succinate and 5 mAf di-thiothreitol before using (protein 21.1 mg/ml). Alkali-treated complex II (0.5 ml) and succinate dehydrogenase (0.6 ml) were mixed together, assayed, and centrifuged for 60 min at 49,000 rpm. The supernatant and the pellet were then separated, the latter was suspended in Tris-succinate-dithiothreitol buffer and both fractions were assayed as indicated. Activities shown are expressed as micromoles of succinate oxidized X min X mg of total protein at. 38°. 

The second type of reconstitution is demonstrated by the work of Bruni and Racker (179). These investigators reconstituted a succinate-ubiquinone reductase system from a King-type preparation of succinate dehydrogenase (2.6 nmoles flavin per mg protein), a preparation of cytochrome b (24-27 nmoles heme per mg protein) solubilized and purified with the use of bile salts and SDS, and mitochondrial or soybean phospholipids. The highest succinate-ubiquinone reductase activity achieved was 980 moles succinate oxidized per minute per mole of succinate dehydrogenase flavin. While this activity is only 10% of the turnover number of complex II, it is still quite appreciable for this type of reconstitution. Since the preparations of succinate dehydrogenase and cytochrome b used were not pure, it is not known what is the minimum number of components needed for reconstitution of succinate-ubiquinone reductase activity. The role and the exact nature of the b-type cytochrome used in these experi-... 

The two subunits resolved by Davis and Hatefi (143, 166) by the use of chaotropes and freeze-thawing were inactive, separately and in combination, for succinate oxidation or fumarate reduction. However, the possibilities have not been fully explored. Nor has this sort of resolution been performed on the cyanide-treated enzyme to see whether one or the other subunit can be preferentially modified. Further work in these areas might... 

The rate with the quinone is less than 1% of the rate with TMPD so that it would be interesting to determine whether the in-vivo rate of quinone turnover during succinate oxidation is consistent with the operation of the electron transport system. Although the Sulfolobus enzyme is composed of four subunits, there is no evidence that all are associated with the enzyme other than that they copurify with enzyme activity. The succinate dehydrogenase from eucarya and bacteria have two subunits whose M, are approximately 70000 and 28 000 [112]. They appear to be analogous to the M, 66000 and 31 000 subunits from the Sulfolobus succinic dehydrogenase. The M, 66 000 subunit from Sulfolobus is catalytically active by itself, and like the Mr 70000 subunit contains covalently-bound FAD. Antisera against the Mr 66000 Sulfolobus subunit cross-reacts with an Mr 67 000 constituent in membranes from T. acidophilum, S. solfataricus, beef-heart submitochondrial particles, and Bacillus subtilis. 

Fig. 2.4. Further variants of the proton circuit, a, reversed electron transfer in Complex I driven by Aft from succinate oxidation, b, reversed electron transfer in Complex I driven by Afin from ATP hydrolysis.

Besides non-enzymatic 02 generation. Of can be enzymatically formed as a result of the ApH+-consuming reverse electron transfer from succinate to O2. In fact, standard redox potential of fumarate/succinate is slightly positive whereas that of OfOf is negative. It was found that ApH+ generated by succinate oxidation via Complexes III and IV can be used to reduce O2 to 02 (eq. 4) ... 

D. If the cytochromes are inhibited, ATP production, the electrochemical potential, heat production from NADH oxidation, and succinate oxidation all decrease. 

Cadmium has been shown to impair many plant cellular functions such as photophosphorylation, succinate oxidation, ATP synthesis, mitochondrial... 

Leger. C., Heflfron, K., Pershad, H.R., Maklashina, E., Luna-Chavez, C., Cecchini, G., Ackrell, B.A.C., and Armstrong, F.A. (2001) Enzyme electrokinetics energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase. Biochemistry, 40. 11234-11245. 

---