Free Radical-Containing Enzymes

The majority of the calculations employ the B3LYP functional which has been shown to give very satisfactory results. In many cases, the enzyme environment is described via a dielectric continuum with 4 and, with some models, certain atoms are fixed at their X-ray structure coordinates. Although the models used are quite sophisticated and have the advantage over experimental methods that short-lived species such as transition states (TSs) can be studied just as easily as longer-lived species, the authors caution that the error in calculated reaction barriers is of the order of 3-5 kcal mol Nevertheless, it is often possible to exploit theoretical calculations at least to dismiss energetically unreasonable mechanisms and to suggest via- 

Class I ribonucleotide reductase Fe2 Uses O2 to generate tyrosyl radical 

Coenzyme Bi2-dependent enzymes Co Homolytic Co-C bond cleavage to generate Co plus radical 

Water oxidation by Photosystem II Mn Mononuclear Mn complex as initial model 

Amine oxidase Cu Initial species Cu(II)-superoxo-tyrosyl radical 

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For GAOX, structural analysis is particularly complicated, because of the existence of multiple states of the enzyme differing essentially only in the number of electrons, i.e., the oxidation state of the metalloprotein complex. Three distinct oxidation states can be prepared, each with properties and reactivities dramatically different from the others, as indicated in Fig. 10 (Whittaker and Whittaker, 1988). When isolated from culture medium, GAOX is a mixture of two of these states a blue, one-electron reduced, catalytically inactive form (lAGO) that contains a Cu(II) ion and no radical and a green form that is catalytically active (AGO) and contains both Cu(II) and a free radical. The enzyme may be converted to... 

Peroxisomes are present in almost every cell type and contain many degradative enzymes, in addition to fatty acyl CoA oxidase, that generate hydrogen peroxide. H202can generate toxic free radicals. Thus, these enzymes are confined to peroxisomes, where the H2O2 can be neutralized by the free radical defense enzyme, catalase. Catalase converts H2O2 to water and O2. 

Copper is an essential trace element. It is required in the diet because it is the metal cofactor for a variety of enzymes (see Table 50—5). Copper accepts and donates electrons and is involved in reactions involving dismu-tation, hydroxylation, and oxygenation. However, excess copper can cause problems because it can oxidize proteins and hpids, bind to nucleic acids, and enhance the production of free radicals. It is thus important to have mechanisms that will maintain the amount of copper in the body within normal hmits. The body of the normal adult contains about 100 mg of copper, located mostly in bone, liver, kidney, and muscle. The daily intake of copper is about 2—A mg, with about 50% being absorbed in the stomach and upper small intestine and the remainder excreted in the feces. Copper is carried to the liver bound to albumin, taken up by liver cells, and part of it is excreted in the bile. Copper also leaves the liver attached to ceruloplasmin, which is synthesized in that organ. 

This enzyme catalyzes the conversion of pyruvate to formate and acetyl CoA and is a key enzyme in the anaerobic degradation of carbohydrates in some Enterobacteriaceae. Using an enzyme selectively C-labeled with glycine, it was shown by EPR that the reaction involves production of a free radical at C-2 of glycine (Wagner et al. 1992). This was confirmed by destruction of the radical with O2, and determination of part of the structure of the small protein that contained an oxalyl residue originating from gly-734. 

The theory underlying the pathophysiology of ischaemia-reperfusion injury, and the role of free radicals in this process has been discussed in detail above. The human colon contains relatively little XO (Parks and Granger, 1986) and so the arguments supporting a role for this enzyme in the pathogenesis of small bowel... 

Laccase is one of the main oxidizing enzymes responsible for polyphenol degradation. It is a copper-containing polyphenoloxidase (p-diphenoloxidase, EC 1.10.3.2) that catalyzes the oxidation of several compounds such as polyphenols, methoxy-substituted phenols, diamines, and other compounds, but that does not oxidize tyrosine (Thurston, 1994). In a classical laccase reaction, a phenol undergoes a one-electron oxidation to form a free radical. In this typical reaction the active oxygen species can be transformed in a second oxidation step into a quinone that, as the free radical product, can undergo polymerization. 

There are numerous in vitro and in vivo studies, in which the damaging free radical-mediated effects of iron have been demonstrated. Many such examples are cited in the following chapters. However, recent studies [170,171] showed that not only iron excess but also iron deficiency may induce free radical-mediated damage. It has been shown that iron deficiency causes the uncoupling of mitochondria that can be the origin of an increase in mitochondria superoxide release. Furthermore, a decrease in iron apparently results in the reduction of the activity of iron-containing enzymes. Thus, any disturbance in iron metabolism may lead to the initiation of free radical overproduction. 

Several compounds can be oxidized by peroxidases by a free radical mechanism. Among various substrates of peroxidases, L-tyrosine attracts a great interest as an important phenolic compound containing at 100 200 pmol 1 1 in plasma and cells, which can be involved in lipid and protein oxidation. In 1980, Ralston and Dunford [187] have shown that HRP Compound II oxidizes L-tyrosine and 3,5-diiodo-L-tyrosine with pH-dependent reaction rates. Ohtaki et al. [188] measured the rate constants for the reactions of hog thyroid peroxidase Compounds I and II with L-tyrosine (Table 22.1) and showed that Compound I was reduced directly to ferric enzyme. Thus, in this case the reaction of Compound I with L-tyrosine proceeds by two-electron mechanism. In subsequent work these authors have shown [189] that at physiological pH TPO catalyzed the two-electron oxidation not only L-tyrosine but also D-tyrosine, A -acetyltyrosinamide, and monoiodotyrosine, whereas diiodotyrosine was oxidized by a one-electron mechanism. 

LOXs are proteins containing a single atom of nonheme iron in catalytic center, with the ferric enzyme in an active form. The free radical-mediated mechanism of LOX-catalyzed process may be presented as follows (see also Figure 26.1) ... 

Copper is part of several essential enzymes including tyrosinase (melanin production), dopamine beta-hydroxylase (catecholamine production), copper-zinc superoxide dismutase (free radical detoxification), and cytochrome oxidase and ceruloplasmin (iron conversion) (Aaseth and Norseth 1986). All terrestrial animals contain copper as a constituent of cytochrome c oxidase, monophenol oxidase, plasma monoamine oxidase, and copper protein complexes (Schroeder et al. 1966). Excess copper causes a variety of toxic effects, including altered permeability of cellular membranes. The primary target for free cupric ions in the cellular membranes are thiol groups that reduce cupric (Cu+2) to cuprous (Cu+1) upon simultaneous oxidation to disulfides in the membrane. Cuprous ions are reoxidized to Cu+2 in the presence of molecular oxygen molecular oxygen is thereby converted to the toxic superoxide radical O2, which induces lipoperoxidation (Aaseth and Norseth 1986). 

We begin this overview of manganese biochemistry with a brief account of its role in the detoxification of free radicals, before considering the function of a dinuclear Mn(II) active site in the important eukaryotic urea cycle enzyme arginase. We then pass in review a few microbial Mn-containing enzymes involved in intermediary metabolism, and conclude with the very exciting recent results on the structure and function of the catalytic manganese cluster involved in the photosynthetic oxidation of water. 

The number of molecules with single electron orbitals, and therefore suitable for ESR, is limited due to the electron-sharing feature of the usual covalent bond. This tends to restrict its use to compounds containing transition metals and reactions involving free radicals. However, this does make ESR very useful for monitoring reactions involving metallo-enzymes or free radicals. 

All aerobic organisms contain substances that help prevent injury mediated by free radicals, and these include antioxidants such as a-tocopherol and the enzymes superoxide dismutase and glutathione peroxidase. When the protective effect of the antioxidants is overwhelmed by the production of reactive oxygen species, the intracellular milieu becomes oxidative, leading to a state known as oxidative stress (Halliwell and Gutteridge, 1999). Thus the balance between the generated free radicals and the efficiency of the protective antioxidant system determines the extent of cellular damage. 

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