Ubiquinone-cytochrome c reductase

Reported applications of DST include the crosslinking of ubiquinone cytochrome C reductase (Smith et al., 1978), characterization of the cell surface receptor for colony-stimulating factor (Park et al., 1986), investigation of the Ca+2-, Mg+2-activated ATP of E. coli (Bragg and Hou, 1980), and characterization of human properdin polymers (Farries and Atkinson, 1989). 

Smith, R. J., Capaldi, R. A., Muchmore, D., and Dahlquist, F. (1978) Cross-linking of ubiquinone cytochrome c reductase (complex III) with periodate-cleavable bifunctional reagents. Biochemistry 17, 3719—3723. 

The classic cytochrome h, expressed sometimes as cytochrome hn, b-561, or b-562, has a single symmetric a band at 561-562 nm in the reduced minus oxidized difference spectrum at room temperature at liquid nitrogen temperature (77°K) it has an a band at 558-559.5 nm, a /3 band at 529 nm, and a Soret band at 428 nm. Cytochrome b is readily reduced by succinate and NAD-linked substrate in both coupled and uncoupled mitochondria. This cytochrome is associated with complex III (ubiquinone-cytochrome c reductase) 24). Figure 1 shows the absorption spectra of reduced cytochrome b at different temperatures between liquid helium and room temperature 26). 

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. 

There are other variations on this type of experiment. Protoplasts from Para-coccus denitrificans showed a similar type of response [204] except that there was a higher level of endogeneous cytochrome c reduction. Respiration was tapped oxidatively at + 395 mV under conditions of which anaerobiosis was marked by a sharp increase in current. Further increase was obtained by additions of succinate, which is a reductant for cytochrome c via the sequence of membrane-bound enzymes succinate dehydrogenase (Complex II) and ubiquinone-cytochrome c reductase (Complex III). By contrast, respiratory coupling could not be observed with protoplasts of Escherichia coli, an organism in which the terminal oxidase system is not reduced via cytochrome c. 

The respiratory chain can be separated by various techniques into three multienzyme complexes (Figure 16.3). Complex I is NADH-ubiquinone reductase. Complex III is known as ubiquinone-cytochrome c reductase and contains cytochromes b and Ci. Complex IV is cytochrome oxidase. (Succinate dehydrogenase is referred to as Complex II). Ubiquinone and cytochrome c are small molecules which do not form part of these complexes. Reconstitution of the isolated Complexes I-IV with cytochrome c and ubiquinone leads to recovery of the activity of the respiratory chain. 

The enzyme ubiquinone-q/ tochrome c reductase catalyzes this very compUcated reaction. The enzyme is another gigantic protein complex with a molecular mass of about 500,000. Three different cytochromes and a protein containing an iron—sulfur complex are components of ubiquinone-cytochrome c reductase. 

Weiss, H., and Kolb, H. J., 1979, Isolation of mitochondrial succinate ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent, Eur. J. Biochem. 99 139nl49. 

Weiss, H., and Leonard, K., 1987, Structure and function of mitochondrial ubiquinol cytochrome c reductase and NADH ubiquinone reductase, Chemica Scripta 27B 73n 81. 

Yu, L., and Yu, C. A., 1982, The interaction of arylazido ubiquinone derivative with mitochondrial ubiquinol-cytochrome c reductase, J. Biol. Chem. 257 10215910221. 

Li L. Zheng LX. Y ang FY. Effect of propensity of hexagonal II phase formation on the activity of mitochondrial ubiquinol-cytochrome c reductase and H(-i-)-ATPase. Chem Phys Lipids 1995 76 135-144. Maggio B. Diplock AT. Lucy J A. Interactions of tocopherols and ubiquinones with monolayers of phospho-... 

Cadenas E, Boveris A, Ragan CI, Stoppani AOM (1977) Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef heart mitochondria. Arch Biochem Biophys 180 248-257 Han D, Williams E, Cadenas E (2001) Mitochondrial respiratory chain-dependent generation of superoxide anion and its release to the intermebrane space. Biochem J 353 411-416... 

The respiratory chain is composed of four multiple-subunit complexes, NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV, CcO) (7). The four complexes, located in the inner mitochondrial membrane of eukaryotes and the inner cytoplasmic membrane of prokaryotes, are electronically connected by ubiquinone and cytochrome c, which transfer electrons through complex I or complex II to complex III, and finally to complex IV, where molecular oxygen is reduced to water. Concurrently, protons are pumped across the inner mitochondrial membrane of eukaryotes or the cytoplasmic membrane of prokaryotes. The proton gradient is utilized by ATP synthase (complex V) to synthesize ATP. In many organisms, the respiratory complexes and complex V are assembled into supercomplexes which have been... 

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). 

Because other liposoluble vitamins (K and E) have often been implicated in the electron transport chain, the effects of vitamin A on mitochondria and mitochondrial enzymes have also been investigated. Although the mitochondrial respiration rate may be decreased in vitamin A-deficient animals, there is no change in the activity of succinoxidase or the enzymes of the Krebs cycle. Some investigators claim that cytochrome c reductase activity is decreased in tissues of vitamin A-deficient animals. A decrease in trans-hydrogenase activity of liver mitochondria and an increase in NADPH cytochrome c reductase has also been described in vitamin A deficiency. Ubiquinone is consistently in excess in deficient rats but not in chickens. 

Vitamin E is in the NADH cytochrome c reductase system. When the system was extracted with isoectane, NADEl oxidation was blocked normal oxidation rates can be restored by adding a-tocopherol, but many other lipid substances are also active in that respect e.g., vitamin K, ubiquinone, phytol. Of course, such observations do not exclude the possibility that in vivo vitamin E is in fact the only substance capable of maintaining the integrity of the NADH cytochrome c reductase system. 

It has been found that a cytochrome-c-reductase preparation, deactivated by extraction with isodctane, can be reactivated by addition of vitamine E and derivatives (Nason and Lehman, 1956). As described in Section VII, 2, other substances with an isoprene-like side chain, such as ubiquinone, vitamin Ki, and K were found to be active. Additionally it could be shown that this side chain— and not the redox system—is responsible for the reactivation (Weber el at., 1958a,b). 

A criterion for a substance to be a member of the respiratory chain is its ability to reactivate an enzymatic system from which this component has been removed. Such a system has been described by Nason and Lehman (1956), who used a rat skeletal muscle cytochrome c reductase inactivated by extraction with isooctane. In such a test vitamin E (Nason and Lehman, 1956) and vitamin K (Deul et ah, 1958) were found to be active. In an extensive study Weber et ah (1958a, b) showed, however, that this reactivation is not related to the redox system but to the isoprene-like side chain. Esterified tocopherol and the diacetates of the dihydro forms of vitamin Ki, Kj, and of ubiquinone(50) are as active as the free forms. Moreover, isolated side chains, such as phytol and squalene, showed exactly the same activity as the corresponding menadione derivatives, compared on a molar basis. The authors speculate that in this experiment a special function of the isoprene-like side chain, namely the binding of this vitamin to the fat material of the mitochondria, is demonstrated. 

One very active NADHj-cytochrome c reductase from mitochondria recently was separated into two components, a NADHr-ubiquinone reductase and an ubihydroquinone-cytochrome c reductase (see below under Quinone Catalysis ). In addition, non-heme-bound iron was found (about 15 moles/mole of flavin). Martius prepared a highly purified phylloquinone reductase, which contains flavin-adenine dinuoleotide (FAD) and which reduces ubiquinone besides phylloquinone (= vitamin K). The hydroquinone is assumed to be reoxidized to the quinone by cytochrome b. 

Recently, with the application of low-temperature (<77°K) ESR techniques in combination with potentiometric titration, an ESR signal from a second iron-sulfur center, designated as center S2 (Ohnishi et al, 1973), was detected and characterized in particulate preparations such as succinate-ubiquinone reductase or in succinate-cytochrome c reductase, in addition... 

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