Succinate-Cytochrom c reductase

Such a process is supposed to occur within the limits of Q-cycle mechanism (Figure 23.2). In accord with this scheme ubihydroquinone reduced dioxygen in Complex III, while superoxide producers in Complex I could be FMN or the FeS center [12]. Zhang et al. [24] also suggested that the Q-cycle mechanism is responsible for the superoxide production by the succinate-cytochrome c reductase in bovine heart mitochondria and that FAD of succinate dehydrogenase is another producer of superoxide. Young et al. [25] concluded that, in addition to Complex III, flavin-containing enzymes and FeS centers are also the sites of superoxide production in liver mitochondria. 

Abnormalities of the respiratoiy chain. These are increasingly identified as the hallmark of mitochondrial diseases or mitochondrial encephalomyopathies [13]. They can be identified on the basis of polarographic studies showing differential impairment in the ability of isolated intact mitochondria to use different substrates. For example, defective respiration with NAD-dependent substrates, such as pyruvate and malate, but normal respiration with FAD-dependent substrates, such as succinate, suggests an isolated defect of complex I (Fig. 42-3). However, defective respiration with both types of substrates in the presence of normal cytochrome c oxidase activity, also termed complex IV, localizes the lesions to complex III (Fig. 42-3). Because frozen muscle is much more commonly available than fresh tissue, electron transport is usually measured through discrete portions of the respiratory chain. Thus, isolated defects of NADH-cytochrome c reductase, or NADH-coenzyme Q (CoQ) reductase suggest a problem within complex I, while a simultaneous defect of NADH and succinate-cytochrome c reductase activities points to a biochemical error in complex III (Fig. 42-3). Isolated defects of complex III can be confirmed by measuring reduced CoQ-cytochrome c reductase activity. 

Defects of complex II. These have not been fully characterized in the few reported patients, and the diagnosis has often been based solely on a decrease of succinate-cytochrome c reductase activity (Fig. 42-3). However, partial complex II deficiency was documented in muscle and cultured fibroblasts from two sisters with clinical and neuroradiological evidence of Leigh s syndrome, and molecular genetic analysis showed that both patients were homozygous for a point mutation in the flavoprotein subunit of the complex [17]. This was the first documentation of a molecular defect in the nuclear genome associated with a respiratory chain disorder. 

Ohnishi, T., and Trumpower, B. L., 1980, Differential effects of antimycin on ubisemiquinone bound in different environments in isolated succinate cytochrome c reductase complex, J. Biol. Chem. 255 3278iB284. 

The high affinity of Con A for cell surface oligosaccharides has also facilitated the immobilization of various cells including those of yeast [131], red blood cells [126, 128] and Trichosporon cutaneum [132]. An early study has also described the co-immobilization of enzymes and living cells using Con A [133]. More recently Habibi-Rezaei and Nemat-Gorgani [134] immobilized submitochondrial particles prepared from beef liver mitochondria on Con A support for continuous catalytic transformations involving succinate-cytochrome c reductase. 

Figure 3. DeDuve plots of relative specific activities (specific activity/specific activity in PNS) in various fractions. Key to fractions PNS, postnuclear supernatant SOL, soluble MIT, mitochondria MIC, microsomes and Nl, N2, N3, and N4, the discontinuous gradient fractions (see Figure 2). Key to activities A, 5 -nucleotidase B, succinate-cytochrome c reductase and C, NADPH-cytochrome...

The second cytochrome b, designated as C3dochrome 6t or as 5-565 or 5-566, has a characteristically split a band at 565-566 and 557-559 nm in the reduced minus oxidized difference spectrum at room temperature (cf. footnote 5 of Table III). At liquid nitrogen temperature it has a double a band at 562.5 and 554-555 nm, 0 bands at 535 and 528 nm, and a 7 band at 430 nm (see Fig. 2,). This cytochrome 5t is fully reduced by succinate and NAD-linked substrate in intact mitochondria in the presence of ATP but not fully in uncoupled mitochondria. In the presence of antimycin succinate reduces cytochrome 5t fully in mitochondria and fragmented succinate-cytochrome c reductase when oxygen or other electron acceptors are present (27,28). The reported value of Eo is... 

The spectral and potentiometric characteristics of type b cytochromes in a succinate-cytochrome c reductase, prepared from pigeon breast muscle mitochondria using a mixture of ionic and nonionic detergents, have shown a close resemblance to those in intact mitochondria 8,34,37). The succinate-cytochrome c reductase may be fractionated into two complexes, succinate-ubiquinone reductase and ubiquinone-cytochrome c reductase, so-called complexes II and III, respectively. Complex III contains type b cytochromes, cytochrome c, and nonheme iron protein in a stoichiometry of 2 1 1 (38). One of the type b cytochromes shows an a peak at 562 nm (559.5 nm at 77°K) by the reduction with succinate and is identified as cytochrome b. The other, with the a peak at 566 nm (562.5 and 554 nm at 77°K), is reduced by succinate only in the presence of antimycin or by dithionite and is identified as cytochrome bi- These two cytochromes do not combine with CO. 

Finally, it may be useful to note that the effect of a-tocopherol in restoring the activity of isooctane-extracted DPN- and succinate-cytochrome c reductase systems (Nason and Lehman, 1956) can be duplicated by several members of the ubiquinone series (Weber et al., 1958). 

Hydroxybutyrate dehydrogenase a-Ketoglutarate dehydrogenase Succinate-cytochrome c reductase Succinate dehydrogenase Rotenone-sensitive NADH dehydrogenase Matrix... 

Similar results have been obtained with mitochondrial fractions from eggs and whole embryos of the surf clam Spisula solidissima. Specific activities of cytochrome oxidase, succinate-cytochrome c reductase, and NADH-cytochrome c reductase were found to be constant during the period from fertilization through the development of swimming stages. Cell division and differentiation apparently entailed neither a significant increase in the total content of. . . mitochondria nor a differentiation of these particles with respect to (their) enzymatic components (Stritt-... 

Fig. 2. Specific activities (Mmoles substrate/minute/mg protein) of cytochrome oxidase and succinate-cytochrome c reductase of mitochondria from embryos of Xenopus laevis. Numerals above abscissa denote Nieuwkoop and Faber stages.

The insect fat body is considered to be the functional analog of the vertebrate liver, and it is of interest, therefore, to note that mitochondrial fractions from the fat body of the cockroach Blaherus discoidalis reveal increases in enzymic activity during development similar to those reported for mammalian liver (Keeley, 1972). Cytochrome oxidase and succinate-cytochrome c reductase activities do not increase in parallel. Immediately after the nymphal-adult ecdysis, succinate-cytochrome c reductase increases in activity and continues to rise for about 10 days when it plateaus at the adult level. Cytochrome oxidase activity remains constant during the first 5 days of this period, but, after the fifth day. 

The role of the mitochondrial genome in the synthesis of succinate cytochrome c reductase an inner mitochondrial membrane enzyme, is unclear. The activity of this enzyme decreases in suspension cnltnres of HeLa cells and L cells in the presence of CAP (King et al., 1972). Thus, this enzyme may be added to the list of proteins potentially regulated by the mitochondrial genome. 

Further studies showed that, in addition to cytochromes a and b, cytochrome c, was lacking, and that the cells contained no NADH or succinate cytochrome c reductase activity, but did contain the primary... 

When air is admitted to an anaerobic yeast culture, respiration reaches normal values within about 4 hours [21]. The cytochrome bands at 556 mfi and 580 m/i are replaced by the bands of cytochromes c, b, and a [19]. Additionally, succinate-cytochrome c reductase activity rises. Catalase and cytochrome peroxidase activities increase over 30-fold, as do NADH-cytochrome c reductase, lactate-cytochrome c reductase, and a-glycerophosphate-cytochrome c reductase. Enzymes of the tricarboxylic acid cycle also increase in activity [21]. 

DNA is probably the most typical of all reference compounds more than 90% of DNA is in the nuclear pellet, and the small amount in other fractions results from contamination. Eighty per cent of cytochrome oxidase or succinic cytochrome c reductase is associated rith the mitochondrial fraction. By counting the mitochondrria in the nuclear fraction, Schneider and Hogeboom [19] demonstrated that the cytochrome oxidase activity measured in this pellet could be accounted for by contaminating mitochondria. 

In all these reactions, the flavin nucleotide combines with a protein to form a flavoprotein. In the reactions involved in electron transport, the flavoprotein acts as a hydrogen carrier and plays an important role in the transfer of electrons from pyridine nucleotides to cytochromes. Among the flavoproteins of interest in the electron transport chain are (1) coenzyme 1-diaphorase, (2) the Warburg flavoprotein, (3) NADH cytochrome c reductase, (4) NADPH cytochrome c reductase, (5) NADH cytochrome reductase, (6) NADH 2-methyl-l,4maphthoquinone reductase, and (7) succinic cytochrome c reductase. The most important among these are succinic cytochrome c reductase and NADH cytochrome c reductase. The following discussion of these two flavoproteins may serve as an example for the others. 

Flavoproteins, NADH, cytochrome c reductase, and succinate cytochrome c reductase must occupy the first step of the electron transport chain because they are reacting with their specific substrates in isolated form (see Fig. 1-20). 

Cytochrome Ci acts before cytochrome c because (1) cytochrome c is oxidized more rapidly than cytochrome Cl (2) the oxidation-reduction potential of cytochrome Ci falls between the oxidation-reduction potential of cytochrome c and that of cytochrome b (3) antimycin prevents the oxidation of cytochrome Cj and (4) NADH or succinate cytochrome c reductase reduces cytochrome Ci in the absence of cytochrome c. 

Isolated oligoenzyne complexes of the respiratory chain of mitochondria - cytochrome oxidase, succinate-cytochrome c reductase, and NADH-CoQ reductase -catalyze the transfer of charges between water and octane that can be recorded from the change in the potential shift at the octane/water phase separation boundary by the vibrating plate method. A necessary condition for the appearance of this effect has proved [18,62] to be the presence of the corresponding enzymes in the oxidation substrates in the aqueous phase and also the presence of a charge acceptor in the octane phase [10, 18, 59]. 

Fig. 15. Dependence of the displacement of the Volta potential in the octane/water system catalyzed by succinate-cytochrome c reductase on the concentration of succinate (a) and of cytochrome c (b). Incubation medium 0.05 M tris-HCl (pH 7.4) 0.5 mM cytochrome c, 0.1 mM MNQ, and 0.4 mg of sucdnate-cytochrome c reductase protein per ml (for b the incubation medium also contained 4 mM succinate)...

The half-saturation of the system with cytochrome c was achieved at a concentration of about 10 M (Fig. 15a, b). In all cases, the effect was reversed and prevented by antimycin - an inhibitor of the succinate-cytochrome c reductase activity of this enzyme complex - but was not suppressed by cyanide. In concluding this series of experiments, we investigated the isolated NADH-CoQ reductase complex of the respiratory chain of mitochondria. As can be seen from the results given in Fig. 16, an increase in the concentration of the enzyme complex was accompanied by an increase in the negative charge of the octane phase. 

Fig. 18. Catalysis of the transfer of positive charges from water into octane by NADH-CoQ reductase (1) and by succinate-cytochrome c reductase (2) as a function of the concentration of DNP. Incubation medium l)20mU tris-HCl (pH 7.4) 0.2 mM NADH and 25 ig of NADH-CoQ reductase protein per ml 2)20 mM tris-HCl (pH 4.7), 7 mM succinate, 0.2 mM cytochrome c, and 40 pg of succinate-cytochrome c reductase protein per ml. In both experiments, the medium contained 1 mM ferricyanide [18]...

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