Cytochrome differences

Besides heme P-460 of hydroxylamine oxidoreductase, cytochrome P-460 has been isolated from N. europaea (Erickson and Hooper, 1972 Miller et al., 1984 Numata et al., 1990). Cytochrome P-460 shows a weak activity of hydroxylamine oxidoreductase, and its molecule is composed of 3 subunits of 18 kDa. As the amino acid sequence of the cytochrome differs from that of hydroxylamine oxidoreductase and DNA encoding the cytochrome is found to be different from that of the oxidoreductase, the cytochrome is not a proteolytic fragment of the oxidoreductase (Bergman and Hooper, 1994b). Indeed, cytochrome P-460 has recently been crystallized and its spatial structure has been determined (Pearson et al., 2007). Its heme is a modified heme C in which lysine residue links to... 

Recall How do the cytochromes differ from hemoglobin and myoglobin in terms of chemical activity ... 

Reflect and Apply Why do the electron-transfer reactions of the cytochromes differ in standard reduction potential, even though all the reactions involve the same oxidation-reduction reaction of iron ... 

Among redoxases, the role of cytochrome C in evolution has received special attention. Cytochrome C contains 104-108 amino acid units per molecule (depending upon species) as well as covalently bonded heme as the prosthetic group. There are structural differences among species. Thus, cytochrome C from man differs from that of the rhesus monkey by only one of the 104 amino acids, while the further species are from one another phylogenetically, the more the various cytochromes differ from one another. The difference from human cytochrome C can be as much as, for example. 

Even with respect to such ubiquitous enzymes as the cytochromes, differences among mitochondria are apparent (Table III). Particulate fractions of muscle tissue from the intestinal helminth Ascaris luntbri-coides contain no cytochrome oxidase, although succinate dehydrogenase. 

A detailed example recording of a cytochrome difference spectrum (reduced minus oxidized)... 

As a class of compounds, the two main toxicity concerns for nitriles are acute lethality and osteolathyrsm. A comprehensive review of the toxicity of nitriles, including detailed discussion of biochemical mechanisms of toxicity and stmcture-activity relationships, is available (12). Nitriles vary broadly in their abiUty to cause acute lethaUty and subde differences in stmcture can greatly affect toxic potency. The biochemical basis of their acute toxicity is related to their metaboHsm in the body. Following exposure and absorption, nitriles are metabolized by cytochrome p450 enzymes in the Hver. The metaboHsm involves initial hydrogen abstraction resulting in the formation of a carbon radical, followed by hydroxylation of the carbon radical. MetaboHsm at the carbon atom adjacent (alpha) to the cyano group would yield a cyanohydrin metaboHte, which decomposes readily in the body to produce cyanide. Hydroxylation at other carbon positions in the nitrile does not result in cyanide release. 

Clotrimazole and other azole derivatives have a different mode of action than the polyenes, eg, amphotericin B. The latter biad to the ergosterol present ia the membranes of yeasts and fungi, but azole derivatives inhibit the cytochrome P-450 dependent biosynthesis of ergosterol (8—11). This inhibition not only results in a reduction of ergosterol, but also in an accumulation of C-14 methyl sterols. They disturb membrane permeabiUty, inhibit cell rephcation, and are basically responsible, in combination with the reduction of ergosterol levels, for the antifungal action. 

Cytochromes c (Cyt c) can be defined as electron- transfer proteins having one or several haem c groups, bound to the protein by one or, more commonly two, thioether bonds. Cyt c possesses a wide range of properties and function in a large number of different redox processes. 

The L and M subunits show about 25% sequence identity and are therefore homologous and evolutionarily related proteins. The H subunit, on the other hand, has a completely different sequence. The fourth subunit of the reaction center is a cytochrome that has 336 amino acids with a sequence that is not similar to any other known cytochrome sequence. 

The L and the M subunits are firmly anchored in the membrane, each by five hydrophobic transmembrane a helices (yellow and red, respectively, in Figure 12.14). The structures of the L and M subunits are quite similar as expected from their sequence similarity they differ only in some of the loop regions. These loops, which connect the membrane-spanning helices, form rather flat hydrophilic regions on either side of the membrane to provide interaction areas with the H subunit (green in Figure 12.14) on the cytoplasmic side and with the cytochrome (blue in Figure 12.14) on the periplasmic side. The H subunit, in addition, has one transmembrane a helix at the car-boxy terminus of its polypeptide chain. The carboxy end of this chain is therefore on the same side of the membrane as the cytochrome. In total, eleven transmembrane a helices attach the L, M, and H subunits to the membrane. 

The different pore sizes and exclusion limits of each TSK-GEL SW column will have a substantial effect on the resolution of a biomolecule mixture. G2000SW packing, which has the smallest pores, provides the best resolution for smaller proteins such as myoglobin and cytochrome c (16,900 and 12,400 Da, respectively, Rs = 1.01). Resolution of the proteins myoglobin... 

FIGURE 7.6 Effect of column length on the separation efficiency. Two different Fractogel EMD BioSEC columns (A 600 X 16 mm, B 1000 X 16 mm) were tested using BSA, ovalbumin, and cytochrome c (S/S/3 mg/ml) as sample (20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2 0.5 ml/min). Better resolution can be achieved using longer columns. 

The electron transport protein, cytochrome c, found in the mitochondria of all eukaryotic organisms, provides the best-studied example of homology. The polypeptide chain of cytochrome c from most species contains slightly more than 100 amino acids and has a molecular weight of about 12.5 kD. Amino acid sequencing of cytochrome c from more than 40 different species has revealed that there are 28 positions in the polypeptide chain where the same amino acid residues are always found (Figure 5.27). These invariant residues apparently serve roles crucial to the biological function of this protein, and thus substitutions of other amino acids at these positions cannot be tolerated. 

Furthermore, as shown in Figure 5.28, the number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived. The cytochrome c in humans and in chimpanzees is identical human and another mammalian (sheep) cytochrome c differ at 10 residues. The human cytochrome c sequence has 14 variant residues from a reptile sequence (rattlesnake), 18 from a fish (carp), 29 from a mollusc (snail), 31 from an insect (moth), and more than 40 from yeast or higher plants (cauliflower). 

FIGURE 6.28 Examples of protein domains with different numbers of layers of backbone strnctnre. (a) Cytochrome c with two layers of a-helix. (b) Domain 2 of phosphoglycerate kinase, composed of a /3-sheet layer between two layers of helix, three layers overall, (c) An nnnsnal five-layer strnctnre, domain 2 of glycogen phosphorylase, a /S-sheet layer sandwiched between four layers of a-helix. (d) The concentric layers of /S-sheet (inside) and a-helix (outside) in triose phosphate isomerase. Hydrophobic residnes are bnried between these concentric layers in the same manner as in the planar layers of the other proteins. The hydrophobic layers are shaded yellow. (Jane Richarelson)... 

In the third complex of the electron transport chain, reduced coenzyme Q (UQHg) passes its electrons to cytochrome c via a unique redox pathway known as the Q cycle. UQ cytochrome c reductase (UQ-cyt c reductase), as this complex is known, involves three different cytochromes and an Fe-S protein. In the cytochromes of these and similar complexes, the iron atom at the center of the porphyrin ring cycles between the reduced Fe (ferrous) and oxidized Fe (ferric) states. 

FIGURE 21.9 Typical visible absorption spectra of cytochromes, (a) Cytochrome c, reduced spectrum (b) cytochrome c, oxidized spectrum (c) the difference spectrum (a) minus (b) (d) beef heart mitochondrial particles room temperature difference (reduced minus oxidized) spectrum (e) beef heart submitochondrial particles same as (d) but at 77 K. a- and /3- bauds are labeled, and in (d) and (e) the bauds for cytochromes a, h and c are indicated. 

It should be emphasized here that the four major complexes of the electron transport chain operate quite independently in the inner mitochondrial membrane. Each is a multiprotein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the four complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the four complexes. 

Cytochrome P450 monooxygenases are characterized through the presence of the heme (protoporphyrin IX) prosthetic group (Scheme 10.1) that is coordinated to the enzyme through a conserved cysteine ligand. They have obtained their name from the signature absorption band with a maximum near 450 nm in the difference spectrum when incubated with CO. The absorption arises from the Soret Jilt transition of the ferrous protoporphyrin IX-CO complex. 

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