Cytochrome isolation

One important finding from purification studies as well as cloning and expressing of individual isoforms is that the lack of substrate specificity of microsomes for monooxygenase activity is not an artifact caused by the presence of several specific cytochromes. Rather, it appears that many of the cytochromes isolated are still relatively nonspecific. The relative activity toward different substrates does nevertheless vary greatly from one CYP isoform to another even when both are relatively nonspecific. This lack of specificity is illustrated in Table 7.2, using human isoforms as examples. 

Giudici-Orticoni MT, Leroy G, Nitschke W, Bmschi M (2000) Characterization of a new dihemic c(4)-type cytochrome isolated from Thiobacillus ferrooxidans. Biochem 39 7205-7211 Gustafsson JP (2001) The surface chemistry of imogolite. Clays Clay Minerals 49 73-80 Guthrie GD, Bish DL, Reynolds RC (1995) Modeling the X-ray diffraction pattern of opal-CT. Am Mineral 80 869-872... 

Subsequently Prince, Cogdell and Crofts also examined the reaction between Rp. sphaeroides reaction centers and mammalian cytochrome c as well as native cytochrome isolated from the same cells. They confirmed the electrostatic nature of the interaction between the Rp. sphaeroides reaction centers and mammalian cytochrome c. However, unlike the Rp. sphaeroides reaction center/mammalian cytochrome c system, the reaction with cytochrome isolated from Rp. sphaeroides is independent of pH between pH 4 and 10, and the effect of changing ionic strength on reaction rate is negligible at all pH values. The pH-independent behavior in this case has been attributed to the fact that the reaction-center complex and cytochrome have essentially the same isoelectric points, and thus similar overall charges at all pHs. 

Fig. 8. Difference spectra of the three spinach cytochromes isolated by imposing appropriate redox potentials In the sample and reference. See text for details. Figure source DS Bendall, HE Davenport and R Hill (1974) Cytochrome components of the higher plants. Methods in Enzymology 23 341.

Giudici-Orticoni M-T, Leroy G, Nitschke W, Bruschi M (2000) Characterization of a new dihemic O-type cytochrome isolated from Thiobacillus ferrooxidans. Biochemistry 39 7205-7211... 

A microsomal subfraction was dissolved in deoxycholate and resolved by gel filtration into two protein fractions (Holloway Katz, 1972). The first fraction designated P3 was almost devoid of lipid and cytochrome bs.which were eluted later in the second fraction. Neither fraction alone had any desaturase activity but a combination of the two had activity. The second fraction could be replaced by cytochrome b5 isolated by detergent procedure, plus lipid vesicles. As shown in Table 1 cytochrome b5 isolated by a trypsin procedure was unable to replace the cytochrome isolated by the detergent procedure. Desaturation was also stimulated by NADH-cytochrome reductase, in agreement with our earlier demonstration that this protein was essential for desaturase activity (Holloway Wakil, 1970). Similar requirements were established by Sato and co-workers at the same time, although, they used different resolution procedures (Shimakata et al., 1972). 

C. Cytochrome Isolated from Heart Muscle (Sekuzu et of., 1960)... 

L-lactate-cytochrome c-oxidoreductase (flavocytochrome was isolated for the first time from the thermo-tolerant yeast H. polymorpha. The mentioned above enzyme preparations were used for construction of the biorecognition elements of electrochemical sensors. 

No region of the cytochrome penetrates the membrane nevertheless, the cytochrome subunit is an integral part of this reaction center complex, held through protein-protein interactions similar to those in soluble globular multisubunit proteins. The protein-protein interactions that bind cytochrome in the reaction center of Rhodopseudomonas viridis are strong enough to survive the purification procedure. However, when the reaction center of Rhodohacter sphaeroides is isolated, the cytochrome is lost, even though the structures of the L, M, and H subunits are very similar in the two species. 

Sometimes it is stated that the extrusion of two protons from the matrix is associated with the oxidation of one molecule of ubiquinol by complex III and four with the oxidation of two molecules of reduced cytochrome c by complex IV (Hinkle et al., 1991). For the oxidation of ubiquinol by complex III in isolation (ubiquinolxytochrome c reductase) the reaction is thought to be... 

Fatal infantile cytochrome c oxidase (CCO) deficiency is characterized by total absence of catalytic activity in skeletal muscle. This often occurs within the context of the Fanconi syndrome, or less commonly in association with a cardiomyopathy. Although the deficiency is global in skeletal muscle, with all fibers affected, only isolated scattered fibers show abnormal aggregations of mitochondria (ragged-red fibers). Multiple affected siblings within one family are frequently encountered and suggest autosomal recessive inheritance. The condition normally proves fatal before the age of six months and is characterized by worsening intractable lactic acidemia. 

Enzyme-mediated chiral sulfoxidation has been reviewed comprehensively in historical context [188-191]. The biotransformation can be mediated by cytochrome P-450 and flavin-dependent MOs, peroxidases, and haloperoxidases. Owing to limited stability and troublesome protein isolation, a majority of biotransformations were reported using whole-cells or crude preparations. In particular, fungi have been identified as valuable sources of such biocatalysts and the catalytic entities have not been fully identified in all cases. 

In 1964, Rieske and co-workers reported the observation of an EPR signal around g = 1.90 in the cytochrome bci complex (1). They succeeded in the isolation of the iron sulfur protein that gave rise to the EPR signal and showed that it contained a [2Fe-2S] cluster. Over the... 

In be complexes bci complexes of mitochondria and bacteria and b f complexes of chloroplasts), the catalytic domain of the Rieske protein corresponding to the isolated water-soluble fragments that have been crystallized is anchored to the rest of the complex (in particular, cytochrome b) by a long (37 residues in bovine heart bci complex) transmembrane helix acting as a membrane anchor (41, 42). The great length of the transmembrane helix is due to the fact that the helix stretches across the bci complex dimer and that the catalytic domain of the Rieske protein is swapped between the monomers, that is, the transmembrane helix interacts with one monomer and the catalytic domain with the other monomer. The connection between the membrane anchor and the catalytic domain is formed by a 12-residue flexible linker that allows for movement of the catalytic domain during the turnover of the enzyme (Fig. 8a see Section VII). Three different positional states of the catalytic domain of the Rieske protein have been observed in different crystal forms (Fig. 8b) (41, 42) ... 

The use of direct electrochemical methods (cyclic voltammetry Pig. 17) has enabled us to measure the thermodynamic parameters of isolated water-soluble fragments of the Rieske proteins of various bci complexes (Table XII)). (55, 92). The values determined for the standard reaction entropy, AS°, for both the mitochondrial and the bacterial Rieske fragments are similar to values obtained for water-soluble cytochromes they are more negative than values measured for other electron transfer proteins (93). Large negative values of AS° have been correlated with a less exposed metal site (93). However, this is opposite to what is observed in Rieske proteins, since the cluster appears to be less exposed in Rieske-type ferredoxins that show less negative values of AS° (see Section V,B). 

At least six isoforms of cytochrome P450 are present in the endoplasmic reticulum of human hver, each with wide and somewhat overlapping substrate specificities and acting on both xenobiotics and endogenous compounds. The genes for many isoforms of P450 (from both humans and animals such as the rat) have been isolated and smdied in detail in recent years. 

Miller RE, Guengerich FP. 1983. Metabolism of trichloroethylene in isolated hepatocytes, microsomes, and reconstituted systems containing cytochrome P-450. Cancer Res 43 1145-1152. 

Harder PA, DP O Keefe, JA Romesser, KJ Leto, CA Omer (1991) Isolation and characterization of Strepto-myces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism. Mol Gen Genet 227 238-244. 

A cytochrome P450 has been purified from Saccharomyces cerevisiae that has benzo[a]pyrene hydroxylase activity (King et al. 1984), and metabolizes benzo[fl]pyrene to 3- and 9-hydroxybenzo[fl]pyrene and benzo[fl]pyrene-7,8-dihydrodiol (Wiseman and Woods 1979). The transformation of PAHs by Candida Upolytica produced predominantly monohydroxyl-ated products naphth-l-ol from naphthalene, 4-hydroxybiphenyl from biphenyl and 3- and 9-hydroxybenzo[fl]pyrene from benzo[fl]pyrene (Cerniglia and Crow 1981). The transformation of phenanthrene was demonstrated in a number of yeasts isolated from littoral sediments and of these, Trichosporumpenicillatum was the most active. In contrast, biotransformation of benz[fl]anthracene by Candida krusei and Rhodotorula minuta was much slower (MacGillivray and Shiaris 1993). 

Organic peroxides such as cumene hydroperoxide and t-butyl hydroperoxide have extensively been used as experimental agents. They provoke lipid peroxidation in hepatocytes, probably by the generation of alkoxyl and peroxyl radical intermediates after reaction with cytochrome P450. Other cytotoxic mechanisms are probably involved including protein thiol and non-protein thiol oxidation and deranged calcium homeostasis (Jewell et al., 1986). In fact, the addition of cumene hydroperoxide to isolated bUe duct cells, devoid of cytochrome P450 activity, still results in cell death but lipid peroxidation is not detectable (Parola et al., 1990). 

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