Cytochrome bacterial sources

There is a third class of peroxidases isolable from the bacterial sources such as P. aeruginosa and Pseudomonas stutzerii, which oxidize cytochrome C551, or azurin. This protein contains two heme protoporphyrin IX groups covalently bound to a single polypeptide side chain. In this enzyme one heme group is oxidized from Fe(III) to Fe(IV)=0 and the second heme, from Fe(II) to Fe(III). The oxidizing equivalents are directed to two centers with very different redox potentials (75). 

A comparative study of the metal centers in cytochrome c oxidase from several bacterial sources, including Thermus thermophilus and P. denitrificans, using EPR and MCD spectroscopy has established that in both cases cytochrome a is liganded by two histidine oxidases and the Cua center is identical to that in bovine cytochrome c oxidase (105, 106). The properties of the cytochrome Os/Cub dimer have not been established to be identical, although ferrocytochrome 03 is high-spin ferrous, as expected. Recent studies of the MCD properties of the Cua center in cytochrome c oxidase and a copper center in nitrous oxide reductase (107,108) show that the two centers are virtually identical. The evidence from the EPR hyperfine structure of the copper center in nitrous oxide reductase suggests that the center in this enzyme is a mixed-valence Cu(I)/Cu(II) dimer, which raises the interesting prospect that the Cua center in cytochrome c oxidase is also a dimeric copper species. 

Current interest in the use of cytochrome P-450 enzyme systems for commercial syntheses or biosensor development is restrained not only by the need for expensive cofactors, but also by the limited supplies of isolated and purified enzyme which until a few years ago was known to be produced by only mammalian cells. Now, bacterial sources of P-450 offer the possibility to produce larger quantities of the active enzyme. The known bacterial sources of cytochrome P-450 has been summarized by Sligar... 

Such an involvement of an amino acid side-chain ligand switch within each catalytic cycle was a novel proposal and as such needs to be scrutinized by a variety of experimental procedures as well as analysis in the context of information known for cytochrome cd nitrite reductase from another source (see later discussion). However, it is interesting to note that something similar has been proposed for the protocate-chuate 3,4-dioxygenase enzyme from Pseudomonas putida (15). On the other hand, bacterial cytochrome c peroxidase offers an example where ligand switching seemingly relates only to an activation phenomenon. 

Blom was the first to demonstrate, in 1928, the formation of HA by an unknown mixture of bacteria which utilized nitrate as their sole nitrogen source to produce ammonia , an observation substantiated by Lindsey and Rhines who generalized this reaction to a diverse set of microorganisms capable of producing NH3 by reduction of both nitrites and nitrates. The mechanism of the 6-electron reduction of nitrite to ammonia (i.e. conversion of the [N + 02] species to by bacterial cytochrome c nitrite reductase... 

Cytochromes from bacterial, yeast, and mammalian sources have been investigated by Mossbauer spectroscopy (114—117). Horseheart cytochrome c and the c-type cytochrome from T. utilis show spectra characteristic of low-spin Fe(III) in the oxidized form of the protein and low-spin Fe(II) for the reduced form of the protein. Lang et al. (115) have analyzed the Mossbauer data in terms of a low-spin Hamiltonian in some detail. Cooke and Debrunner (116) present quadrupole data on dehydrated forms of oxidized and reduced cytochrome c the quadrupole splittings for hydrated and dehydrated forms of the reduced protein are quite similar in contrast to a difference of the oxidized form. No spin-state change is reported for either form of cytochrome c. 

Cytochrome c peroxidase can oxidize a number of reducing agents such as ascorbate, pyrogallol, guaiacol, hydroquinone, and ferrocyanide as well as mammalian and yeast ferrocytochrome c (1, 4, 14)- Ferrocytochromes b, be, and Ci from mammalian sources and a majority of bacterial ferrocytochrome c are not oxidized. The molecular activity of cytochrome c peroxidase for mammalian and yeast ferrocytochrome c (fcs = 10 sec" ) is at least two orders of magnitude larger than those for other reductants. Thus, this enzyme has a high substrate specificity toward ferrocytochrome c. Alkylhydroperoxides, such as methyl-, ethyl-, and propylhy-droperoxides, can be effectively utilized as Si by this enzyme. 

Significant differences in the equilibrium constants for carbon monoxide binding to cytochromes P450 from bacterial, liver microsomal, and adrenal cortex microsomal sources, different isozymes of the liver microsomal proteins, and for substrate-free and substrate-bound enzymes, have been observed and have been related to similar factors that affect O2 and CO binding in oxygen transport and storage heme proteins. The importance of the cis and tmns effects, that is electronic effects associated with the porphyrin... 

The successful solution of the many bacterial cytochrome crystal structures was largely due to the simple fact that they are soluble. Mammalian microsomal P450s, on the other hand, are membrane bound and hence insoluble. The A -terminal region of the microsomal isoforms is believed to form a single membrane-spanning a-helix that tethers it close to the other protein in the system, the NADP/H dependent cytochrome reductase. This reductase is the source of electrons for the catalytic mechanism described earlier. 

Fig. 6. Photochemical cycles showing coupling of electron transfer to proton transfer, cytochrome oxidation and quinone exchange in (A) native reaction centers where two Cyt c are oxidized in the cycie, (B) reaction centers where uptake of the first proton is inhibited, and (C) reaction centers where uptake ofthe second proton is inhibited (shading indicates the quinone pool). Figure source (A) Paddock, Rongey, McPherson, Juth, Feher and Okamura (1994) Pathway of proton transfer in bacterial reaction centers role of aspartate-L21Z in proton transfers associated with reduction of quinone to dihydroquinone. Biochemistry 33 734 (B) Okamura and Feher (1992) Proton transfer in reaction centers from photosynthetic bacteria. Annu Rev Biochemistry. 61 868 (C) Feher, Paddock, Rongey and Okamura (1992) Proton transfer pathways in photosynthetic reaction centers studied by site-directed mutagenesis. In A Pullman, J Jortner and B Pullman (eds) Membrane Proteins Structures, Interactions and Models, p 485. Kluwer.

Fig. 4. Absorbance-change kinetics of photooxidation due to the primary eiectron donor and its decay (re-reduction) [upper paneis] and the oxidation of a c-type cytochrome [iower panels] in C. vinosum (left) and Rp. viridis [right panels]. The C. vinosum sample was poised at a redox potential so that Cyt c555 ("Cyt c422 ) is reduced before flash excitation the ambient redox potential in Rp. viridis was -250 mV, so that only Cyt c5S8 is present in the reduced state before excitation. Figure source left panels (C. vinosum) from Parson (1968) The role of P870 in bacterial photosynthesis. Biochim Biophys Acta 153 254 right panels (Rp. viridis) from Shopes, Levine, Molten and Wraight (1987) Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis. Photosynthesis Res 12 167.

Cytochrome P-450 Catalyzed Reactions - Studies with 02 have established that the cytochrome P-450 mediated hydroxylation of camphor by the bacterial enzyme and the enzyme purified from rat liver results in the incorporation of atmospheric oxygen in the 5-exo-hydroxylation product. Cumene [ 02]hydroperoxide will transfer its peripheral oxygen atom to a variety of compounds which serve as substrates for mammalian cytochrome P-450. As expected, 02 served as the oxygen source for the bacterial cytochrome P-450 catalyzed epoxidation of 5,6-dehydrocamphor.N-Hydroxy-methylcarbazole, formed by the cytochrome P-450 catalyzed oxidation of N-raethylcarbazole, incorporates 0 exclusively from dioxygen. Under anaerobic conditions cytochrome P-450 may catalyze the intramolecular transfer of oxygen present in tertiary amine N-oxides. Mechanistic studies on S-dealkylation and S-oxidation reactions also have used tracer methods. ... 

Not fully understood. Sulfamethoxazole is a known inhibitor of the cytochrome P450 isoenzyme CYP2C9, by which S-warfarin in predominantly metabolised. The finding that co-trimoxazole caused a modest 22% increase in S-warfarin levels supports this mechanism. Acenocoumarol and phenprocoumon are also metabolised by CYP2C9 and might be expected to be similarly affected. Plasma protein binding displacement has been suggested as a mechanism, but on its own it does not provide an adequate explanation because the interaction is sustained. Sulfonamides can drastically reduce the intestinal bacterial synthesis of vitamin K, but this is not normally an essential source of the vitamin unless dietary sources are exceptionally low, see also Coumarins + Antibacterials , p.365. 

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