NADPH metal inhibition

Although metal-catalyzed protein oxidation is undoubtedly a very effective oxidative process, the origin of free metal ions under in vivo conditions is still uncertain (see Chapter 21). However, protein oxidation can probably be initiated by metal-containing enzymes. Mukhopadhyay and Chatterjee [31] have shown that NADPH-stimulated oxidation of microsomal proteins was mediated by cytochrome P-450 and occurred in the absence of free metal ions. It is important that in contrast to metal ion-stimulated oxidation of proteins, ascorbate inhibited and not enhanced P-450-dependent protein oxidation reacting with the oxygenated P-450 complex. The following mechanism of P-450-dependent oxidation of the side chain protein amino acid residues has been proposed ... 

In 1988 Bast and Haenen [201] reported that both LA and DHLA did not affect iron-stimulated microsomal lipid peroxidation. However, Scholich et al. [202] found that DHLA inhibited NADPH-stimulated microsomal lipid peroxidation in the presence of iron-ADP complex. Inhibitory effect was observed only in the presence of a-tocopherol, suggesting that some interaction takes place between these two antioxidants. Stimulatory and inhibitory effects of DHLA have also been shown in other transition metal-stimulated lipid peroxidation systems [203,204]. Later on, the ability of DHLA (but not LA) to react with water-soluble and lipid-soluble peroxyl radicals has been proven [205], But it is possible that the double (stimulatory and inhibitory) effect of DHLA on lipid peroxidation originates from subsequent reactions of the DHLA free radical, capable of participating in new initiating processes. 

Thus, the mechanism of MT antioxidant activity might be connected with the possible antioxidant effect of zinc. Zinc is a nontransition metal and therefore, its participation in redox processes is not really expected. The simplest mechanism of zinc antioxidant activity is the competition with transition metal ions capable of initiating free radical-mediated processes. For example, it has recently been shown [342] that zinc inhibited copper- and iron-initiated liposomal peroxidation but had no effect on peroxidative processes initiated by free radicals and peroxynitrite. These findings contradict the earlier results obtained by Coassin et al. [343] who found no inhibitory effects of zinc on microsomal lipid peroxidation in contrast to the inhibitory effects of manganese and cobalt. Yeomans et al. [344] showed that the zinc-histidine complex is able to inhibit copper-induced LDL oxidation, but the antioxidant effect of this complex obviously depended on histidine and not zinc because zinc sulfate was ineffective. We proposed another mode of possible antioxidant effect of zinc [345], It has been found that Zn and Mg aspartates inhibited oxygen radical production by xanthine oxidase, NADPH oxidase, and human blood leukocytes. The antioxidant effect of these salts supposedly was a consequence of the acceleration of spontaneous superoxide dismutation due to increasing medium acidity. 

In-vitro approach Data are available in abundance concerning metal effects on isolated chloroplasts (for a review, see Clijsters and Van Assche, 1985). All the metals studied were found to be potential inhibitors of photosystem 2 (PS 2) photosystem 1 (PS 1) was reported to be less sensitive. From the in-vitro experiments, at least two potential metal-sensitive sites can be derived in the photosynthetic electron transport chain the water-splitting enzyme at the oxidising side of PS 2, and the NADPH-oxido-reductase (an enzyme with functional SH-groups) at the reducing side of PS 1 (Clijsters and Van Assche, 1985). Moreover, in vitro, non cyclic photophosphorylation was very sensitive to lead (Hampp et al., 1973 b) and mercury (Honeycutt and Korgmann, 1972). Both cyclic and non-cyclic photophosphorylation were proven to be inhibited by excess of copper (Uribe and Stark, 1982) and cadmium (Lucero et al, 1976). 

In this model system, as contrasted with the simple ferric ion reductase activity of the flavoprotein (38S), the metal is not the ultimate electron acceptor but presumably serves the dual role of oxygen activation and electron carrier. The reaction may involve superoxide anion since it is inhibited by superoxide dismutase (erthrocuprein) (394). Xanthine plus xanthine oxidase can also serve as electron donor, and this latter model system is also inhibited by superoxide dismutase (5P5). Superoxide dismutase also inhibits the menadione-mediated NADPH oxidase activity of NADPH-cytochrome P-450 (396) as well as the reconstituted benz-phetamine bydroxylation system (397). The involvement of NADPH-cytochrome P-450 reductase in microsomal lipid peroxidation has been confirmed by the demonstration that the reaction in microsomes is totally inhibited by antibody to tbe purified reductase (374). It has been suggested that lipid peroxidation by microsomes requires another component, in addition to the reductase, which takes the place of the ferric ion chelate in the model system (57. ). 

The E. coll enzyme can reduce nitrite and hydroxylamine to ammonia at the expense of NADPH (339). However, with the use of N-nitrite it was shown that hydroxylamine was not an intermediate in the reduction of nitrite. No cofactor requirements were shown for the E. coli enzyme, but similar to other flavin and metal requiring nitrite reductases it was inhibited by cyanide and mercurials. 

Although this review concerns those reactions catalyzed by iminium ion formation, it is important to note that there are enzymatic reactions that could logically be catalyzed by iminium ion formation but which are not. Yeast aldolase, for example, is the best known case [26] (see Ch. 6). This enzyme is metal ion dependent, does not demonstrate the loss activity in the presence of both substrate and borohydride, and is sensitive to inhibition by EDTA. The reaction catalyzed by this enzyme is identical to that catalyzed by the imine-forming enzyme, and even has evolved to exhibit the same retention stereochemistry. Another example is A -3-oxosteroid reductase which is responsible for the NADPH-dependent reduction of the enone double bond to the corresponding dihydrosteroid [124]. Even though iminium ion formation would increase the reactivity of this substrate toward the -hydride addition, a demonstrated lack of the required oxygen exchange proves that this does not occur. 

In plants, the cw-hydroxylase system is responsible for synthesis of cw-hydroxy fatty acyl components of cutin and suberin (Section 1.9 and 2.11). The reaction has been studied in preparations from Vida faba with NADPH and oxygen as the required cofactors (Kolattukudy, 1980). The true substrate for o>-hydroxylation of palmitate is the free acid and the active subcellular preparation is the microsomal fraction. The reaction showed the properties of a classic mixed-function oxidase, being inhibited by o-phenanthroline, 8-hydroxyquinoline (metal ion chelators), sodium azide and thiol-directed reagents. The involvement of cytochrome P-450 in the V.faba system is unproven. Although the hydroxylation is inhibited by carbon monoxide, this inhibition was not reversed by light at 420-460 nm. Thus if a cytochrome P-450 is involved in the system it must have unusual properties when compared to other cyto-... 

There are a number of mechanisms that can be affected by Cd " to increase ROS levels displacement of Fenton metals [472], inhibition of the mitochondrial electron transport chain [344], decrease of antioxidant enzyme activities [182], reduction of GSH levels [491], and activatiMi of NADPH oxidases [492]. The formation of ROS is integral to downstream signaling pathways, which are implicated in all types of stress and cell death, and could very well be one of the first responses in the cell following Cd entry. 

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