Flavoprotein reduction product

The hydroxylase that converts 2,4-dichlorophenol into 3,5-dichlorocatechol (Figure 3.14a) before ring fission has been purified from a strain of Acinetobacter sp. (Beadle and Smith 1982), and ixomAlcaligenes eutrophus IMP 134 (Don et al. 1985 Perkins et al. 1990). The reductant is NADPH, the enzyme is a flavoprotein containing FAD, and in the presence of compounds that are not substrates, NADPH and O2 are consumed with the production OfH202. 

Photodegradation of DDT by the protease-liberated flavo-protein from TX-20 resulted in the formation of TDE as the major product in addition to three other minor compounds. It has been well established that DDT conversion to TDE, anaerobically, is a reductive process involving replacement of a chlorine atom by hydrogen. On the other hand, it has been suggested that photo-lytic reactions involve a charge transfer from an amine to DDT and a subsequent pickup of a proton. Thus there is a possibility that the photochemical reaction involving flavoproteins undergoes a similar reaction scheme. Much more data are, however, needed to confirm this point. 

Tertiary amine oxides and hydroxy la mines are also reduced by cytochromes P-450. Hydroxylamines, as well as being reduced by cytochromes P-450, are also reduced by a flavoprotein, which is part of a system, which requires NADH and includes NADH cytochrome b5 reductase and cytochrome b5. Quinones, such as the anticancer drug adriamycin (doxorubicin) and menadione, can undergo one-electron reduction catalyzed by NADPH cytochrome P-450 reductase. The semiquinone product may be oxidized back to the quinone with the concomitant production of superoxide anion radical, giving rise to redox cycling and potential cytotoxicity. This underlies the cardiac toxicity of adriamycin (see chap. 6). 

The biochemical importance of flavin coenzymes ap-pears to be their versatility in mediating a variety of redox processes, including electron transfer and the activation of molecular oxygen for oxygenation reactions. An especially important manifestation of their redox versatility is their ability to serve as the switch point from the two-electron processes, which predominate in cytosolic carbon metabo-lism, to the one-electron transfer processes, which predomi-nate in membrane-associated terminal electron-transfer pathways. In mammalian cells, for example, the end products of the aerobic metabolism of glucose are C02 and NADH (see chapter 13). The terminal electron-transfer pathway is a membrane-bound system of cytochromes, nonheme iron proteins, and copper-heme proteins—all one-electron acceptors that transfer electrons ultimately to 02 to produce H20 and NAD+ with the concomitant production of ATP from ADP and P . The interaction of NADH with this pathway is mediated by NADH dehydrogenase, a flavoprotein that couples the two-electron oxidation of NADH with the one-electron reductive processes of the membrane. 

DAAO is one of the most extensively studied flavoprotein oxidases. The homodimeric enzyme catalyzes the strictly stere-ospecihc oxidative deamination of neutral and hydrophobic D-amino acids to give a-keto acids and ammonia (Fig. 3a). In the reductive half-reaction the D-amino acid substrate is converted to the imino acid product via hydride transfer (21). During the oxidative half-reaction, the imino acid is released and hydrolyzed. Mammalian and yeast DAAO share the same catalytic mechanism, but they differ in kinetic mechanism, catalytic efficiency, substrate specificity, and protein stability. The dimeric structures of the mammalian enzymes show a head-to-head mode of monomer-monomer interaction, which is different from the head-to-tail mode of dimerization observed in Rhodotorula gracilis DAAO (20). Benzoate is a potent competitive inhibitor of mammalian DAAO. Binding of this ligand strengthens the apoenzyme-flavin interaction and increases the conformational stability of the porcine enzyme. 

The catalytic cycles of flavoenzymes can usually be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized flavoprotein is reduced by the first substrate, resulting in reduced flavoenzyme. In the oxidative half-reaction, a second substrate oxidizes the reduced flavoprotein (Scheme 1), usually after the product of the reductive half-reaction dissociates, so flavoenzymes frequently have ping-pong kinetic mechanisms. Even when the first product remains bound during the oxidative... 

The formation of superoxide is the result of one electron transfer by several coenzymes in ETS, including flavins, flavoproteins, quinones, and iron sulfur proteins. This product has a longer half-life than other intermediates and is toxic to anaerobic bacteria. Peroxidase is formed by two electron transfers and mediated by flavoproteins. Peroxidase is further reduced to the hydroxyl radical with the addition of one electron followed by subsequent reduction to water by the addition of another electron. The oxidative effect of these intermediates can result in the destruction of cells. The aerobic bacteria have enzyme systems such as superoxidase dismutase, peroxidase, and catalase to reduce the toxic levels of these intermediates. 

NQOl was first characterized as DT diaphorase, with diaphorase being an older term for an enzyme that catalyzes electron transfer from pyridine nucleotides. DT indicated that this enzyme could accept electrons from NADH (formerly termed DPNH) or NADPH (formerly TPNH). Some of the other early work was unclear but the location of the enzyme is cytosolic (Huang et al., 1979). This is a flavoprotein that reduces substrates. In contrast to NADPH-P450 reductase, most NQO reactions are 2-electron reductions, avoiding the generation of radicals. However, the product hydroquinones may react with O2 to generate 2 . ... 

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