Cytochrome c reductase activity

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. 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

The microsome fractions see Fig. 1) that were prepared from mulberry cortical parenchyma cells were fractionated to 24 or 25 fractions using the 15-50% sucrose linear density gradient centrifugation see Fig. 2). Profiles of the marker enzymes and the protein content are described in Fig. 3. In general, the antimycin A-insensitive cytochrome c reductase activity is exhibited at a lower density than are those of the marker enzymes. The fraction that exhibited the highest antimycin A-insensitive cytochrome c reductase activity for each month was used as the ER-enriched fraction. 

Cytochrome c reductase activity and cytochrome P-450 were determined by methods outlined by Mazel (41J and the method of AFB conversion to AFL was carried out by the method of Loveland et al. (42). 

These findings are at variance with reported increases in cytochrome P-450 content of rats fed increasing levels of casein (45, 46, 47, 49, 50). Only slight differences were observed in cytochrome c reductase activities between the diets, with the highest activity of 20nmoles cytochrome c reduced/min/mg protein occurring in fish fed the 32% casein diet (Table III). 

Nitromethane was administered intraperitoneally (200 mg/kg bw) to male Wistar rats (three months of age) as a 10% solution in olive oil. The effects of nitromethane in the liver were detected only 48 h after administration and included a decrease in NADPH-cytochrome c reductase activity with proliferation of the smooth endoplasmic reticulum. Nitromethane also caused an increase in brain acid proteinase (4 h after injection) and acetylcholine esterase activities (4, 24 and 48 h after injection) (Zitting etal, 1982). 

Benzoate 1,2-dioxygenase was shown to catalyze the conversion of benzoate to l,2-dihydro-l,2-dihydroxybenzoic acid (DHB) in the presence of NADH and oxygen by Reiner et al.205, 206). Yamaguchi et al.61> have demonstrated that the enzyme system consists of two components (A and B),both of which are required for benzoate 1,2-dioxygenase activity. Component A shows NADH-cytochrome c reductase activity and component B appears to be dioxygenase, thus the following reaction scheme is suggested [Eq. (25)]. 

Cooper KO, Witmer CM, Witz G. 1987. Inhibition of microsomal cytochrome c reductase activity by a series of alpha, beta-unsaturated aldehydes. Biochem Pharmacol 36 627-631. 

The influence of mercurials on the NADPH-cytochrome c reductase activity is complex. The activity in microsomes is stimulated about 50% by p-mercuribenzoate (11). Mersalyl inhibits the NADPH-cytochrome c reductase activity (S87). 

Information regarding the involvement of flavin and iron in enzyme catalysis is not available. Rothschild et al. (258) have reported that dialysis of rat liver particles resulted in the loss of choline-cytochrome c reductase activity, which could be restored by addition of FAD but not FMN. However, these results have not been substantiated by others (255). Singer has stated that the difference spectrum of the enzyme shows bleaching by substrate in both the flavin and the iron regions (227). This spectrum has not been published. 

Ferricyanide appears to accept electrons from both the flavin and the heme (299-302), and it is believed that heme is required for cytochrome c reduction. Forestier and Baudras (302) have reported that, by treatment with guanidinium chloride, preparations of cytochrome 62 could be rendered partially deficient in flavin and heme. Thus, enzyme preparations were obtained which contained 65-75% flavin and variable amounts of heme from about 12 to 100%. The low heme preparations showed considerably greater loss of cytochrome c reductase than ferricyanide reductase activity. When preparations with increasing content of heme relative to flavin were tested, both the ferricyanide and the cytochrome c reductase activities increased as a linear function of heme to flavin ratio (up to heme flavin =1), but the increase in the heme content had a much greater effect on the cytochrome c reductase activity of the enzyme. The apoenzyme of cytochrome 62 has been prepared. However, reconstitution with FMN, heme, and FMN plus heme in all cases resulted in extremely... 

Campbell, W. H., 1992, Expression in Escherichia coli of cytochrome c reductase activity from a maize NADH nitrate reductase complementary DNA, Plant Physiol. 99 693fi... 

FMN, the first acceptor in the electron-transfer pathway, has been clearly shown to be essential for lactate dehydrogenase activity (105). What is the role of the heme Forestier and Baudras found a linear relationship between heme content and enzyme activity extrapolation to zero heme content indicated zero cytochrome c reductase activity and a lowered ferricyanide reductase activity (106). Thus electron... 

As mentioned in Section V,A, heme-deficient (dehemo) flavocytochrome 2 has no cytochrome c reductase activity (126), but retains some ferricyanide reductase activity. These results are consistent with 2-heme being essential for cytochrome c reduction and also indicate that ferricyanide and cytochrome c are reduced by different mechanisms. 

A very important conclusion was reached based on the effect of p-mer-curibenzoate on the NADPH oxidase and the NADPH-cytochrome c reductase activities of microsomes, namely, that the natural acceptor might be a component reactive with oxygen and involved in hydroxyla-tions or demethylations (11). It was found that in the absence of cytochrome c, the oxidase activity was largely inhibited by p-mercuri-benzoate. In the presence of cytochrome c, NADPH oxidation exceeded cytochrome c reduction in the absence of p-mercuribenzoate and the two rates equaled each other in the presence of p-mercuribenzoate. Thus, a mercurial sensitive oxidase distinct from the reductase was indicated, and this component was hypothesized to be connected with hydroxylation and/or demethyl-ation (11). 

Only those flavoproteins where Oi- is being formed are able to display cytochrome-c reductase activity which can be inhibited by erythrocuprein (Table 10). However, it cannot be fully excluded that erythrocuprein may react with the above-mentioned intermediate... 

Table 10. Effect of erythrocuprein on cytochrome-c reductase activity of flavoproteins. The cytochrome-c reductase activity was measured in air-equilibrated solutions containing 0.1 M pyrophosphate, pH 8.5, in the presence of 3.33 X 70-5 M cytochrome c and 10 fig bovine catalase. The concentration of erythrocuprein in this assay mixture was 0.62 fiM. The temperature was 25° (150)...

T. cruzi contains NADPH-cytochrome c reductase activity in both the microsomal and cytosolic-cell fractions (29). The cytosolic enzyme may act as an oxygenase, protecting the cell in times of hydrogen peroxide production, since trypanosomes are deficient in catalase (30). It may also metabolize drugs directly, possible conferring resistance to antitrypanosomal drugs (31). The particulate enzyme form probably functions in microsomal electron transport associated with trypanosomal cytochrome P450. 

---