Preparation of cytochrome

It has been shown recently that the mitochondrial electron transport system contains at least three different fe-type cytochromes 178). Two of these cytochromes are found in complex III, and under appropriate conditions are reducible with substrates. The third 6-type cytochrome was discovered by Davis et al. 178), and shown to fractionate exclusively into complex II. At 77°K, the cytochrome 6 of complex II exhibits a double a band at 557.5 and 550 nm, a prominent band at 531 nm, and a Soret band at 422 nm (Fig. 29). Cytochrome 6557.5 appears to have a low reduction potential. It is not detectably reduced by succinate in either complex II or respiratory particles, but its dithionite reduced form is rapidly oxidized by either fumarate or ubiquinone. The role of this cytochrome in mammalian mitochondria is not known. Davis et al. 178) have suggested that it might be an electron entry point for an unknown ancillary tributary of the respiratory chain. Further, Bruni and Racker 179) have shown that a preparation of cytochrome 6 is required for reconstitution of succinate-ubiquinone reductase activity (see below). 

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-... 

Cytochrome 62 is found as a soluble protein in the autolysates of Sac-charomyces cerevisiae. The crystalline preparations of Appleby and Morton 278) were shown to sediment as a single peak in the ultracentrifuge. Minimum molecular weight based on amino acid analysis and a heme extinction coefficient of 232 mM cm was calculated to be 53,000 283). The heme extinction coefficient was then corrected to 183 mM- cm-, and the minimum molecular weight per mole of heme recalculated to be 58,600 284). It was concluded that cytochrome 6 is a tetrameric structure. This conclusion agreed with the results of X-ray diffraction studies on type I and type II crystals, which indicated molecular weights of 235,000 10,000 and 234,000 8,000, respectively, for these two preparations of cytochrome 2 285). The oxidized and reduced spectral bands of cytochrome ba are given in Table XIV. 

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... 

In 1965, Yonetani and Ray (14) obtained a highly purified preparation of cytochrome c peroxidase in an excellent yield using DElAE-cellulose ion exchange chromatography. Shortly thereafter, Yonetani et al. (15) crystallized this enzyme by isoelectric dialysis. Subsequently, Yonetani and co-workers (16-40) carried out a series of extensive investigations... 

The studies referred to were performed with crude preparations of cytochrome P-450. In a series of papers Wikvall, Hansson and collaborators have reported studies on 7a-hydroxylation of cholesterol in highly purified preparations of cytochrome P-450 from rats and rabbits [82,84,85], They showed that electrophoretically homogenous cytochrome P-450 LM4 isolated from cholestyramine-treated rabbits catalyses 7 -hydroxylation of cholesterol as well as some other hydroxylations. Chromatography of a cytochrome P-450 LM4 fraction on octylamine-Sepharose resulted in 2 subfractions, cytochrome P-450 LM4 I and cytochrome P-450 LM4 II, with different catalytic properties. Cytochrome P-450 LM4 I was unable to catalyse 7a-hydroxylation of cholesterol, but catalysed 12a- and 25-hydroxylations. Cytochrome P-450 LM4 II efficiently catalysed cholesterol 7a-hydroxylation. It also catalysed the other hydroxylations, although at lower rates than the original cytochrome P-450 LM4. 

Imai, Y. and R. Sato (1974). A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits. Biochem. Biophys. Res. Commun. 60, 8-14. 

Purified preparations of cytochrome oxidase are unstable and researchers have to deal, as a rule, with submitochondrial particles including, together with cytochrome oxidase, a part of the lipid membrane. The enzyme contains heme and copper in equimolar quantities as prosthetic groups. It apparently reacts with cytochrome c due to electrostatic interaction interaction with oxygen is limited by the latter s rate of diffusion. 

Purified preparations of cytochrome oxidase in the oxidized state display an absorption band (maximum 830 nm) in the near-infrared region which disappears on reduction and reappears on reoxidation at rates commensurate with those of the band at 605 nm (Wharton and Tzagoloflf, 1964). This chromophore can be gradually and irreversibly destroyed by treating the oxidase with the copper-specific chelator bathocuproinesulfonate in the presence of acetate buffer below pH 5 or by dialyzing... 

The heme determination indicates that the cytochrome oxidase system contains five molecules of heme per molecule of protein. This evidence is not conclusive for at least two reasons. First, the purified preparation of cytochrome oxidase has very low catalytic activities, and therefore its true relationship with the native mitochondrial enzyme remains unsettled. Second, the data do not exclude the possibility that cytochrome oxidase is formed by the intimate complexion of two proteins, cytochromes a and a. ... 

In vitro, oxidised preparations of cytochrome oxidase and cytochrome c are reduced by PH3. The observed changes in the absorption spectra are similar to those produced by dithionite, suggesting reduction at the haem site The possibility that PH3 can bind directly to Fe at these haem sites must also be considered (see Sect. 4.5). 

Racker and his group have obtained normal electron transfer from succinate to oxygen by combining several soluble mitochondrial components, Mg", and phospholipids. However, they found it essential to add a sedimentable preparation of cytochrome b, which in the electron microscope appeared to consist of vesicles this particulate preparation could conceivably provide the structural material on which the carriers must be properly organized before a normal electron flow can be obtained. 

The particulate preparations which are loosely referred to as preparations of cytochrome oxidase contain in addition to the oxidase the entire electron transfer system other than the cytochromes and variable amoimts of the various cytochrome components. Such preparations will readily oxidize reduced cytochrome c by molecular oxygen, though whether reduced cytochrome c is oxidized by a sequence as shown above or directly by cytochrome oxidase is indeterminate at present. 

The crude preparation of cytochrome oxidase obtained after this chromatographic step was further purified by chromatography on DEAE-cellulose in a Tween-80 solution. The final yield was 10%. The crude preparation of cytochrome b was further purified by means of chromatography on DEAE-cellulose and gel filtration on Sephadex G-75, all steps being performed in a bile acid solution. The final yield was 2-4%. ... 

Two preparations of cytochrome b from beef heart mitochondria have been reported which resemble our preparation with regard to degree of purity. Molecular weights of 28,000 and 22,400 were calculated on the basis of the specific heme contents. As these preparations were obtained in highly polymeric states, a possible quaternary structure for cytochrome b was not considered. Cytochrome b preparations from cells with proteins selectively labeled as described above (so as to allow their attribution to one of the two protein-synthesizing systems) have not yet been described by other authors. 

B. Isolation, Purification, and Crystallization of Cytochrome c Regarding the preparation of cytochrome c, see Okunuki (1959). 

Second Ammonium Sulfate Fractionation. The combined main fraction was treated with ammonium sulfate to obtain a fraction precipitating between 35 and 55% saturation. This was dissolved in 0.5% cholic acid in 0.1 M phosphate buffer, pH 7.4. By repeating the above fractionation two or three times, a highly purified preparation of cytochrome c was obtained. The fraction described in the foregoing section, containing... 

Eichel et al. (1950), Green and Crane (1957), and Okunuki et al. (1957) have reported that their cytochrome oxidase and cytochrome a preparations contain high concentrations of copper as well as of heme iron. On the other hand, many investigators (Cohen and Elvehjem, 1934 Yoshikawa, 1937 Gallagher, 1956) have observed from dietary experiments that copper-deficient tissues and yeast have a low cytochrome oxidase activity and a decreased heme a content. From the above results, it has been considered that the copper in the cytochrome oxidase of typical cytochrome systems plays an important role in the cytochrome oxidase reaction. However, there is no direct evidence to support the above role of copper. In order to determine whether the copper participates in the cytochrome oxidase reaction, the iron and copper contents of various preparations of cytochrome a were first analyzed. The results are summarized in Table IV. 

As mentioned in the previous sections, a purified preparation of cytochrome a is not autoxidizable, but combines with oxygen. In the... 

---