Biochemistry of extramitochondrial ubiquinone

As a result of its antioxidant function, CoQ becomes oxidized. However, a substantial amount is maintained in its reduced state in the plasma membrane and endomembranes,14 and in plasma lipoproteins as well.7 Furthermore, natural ubiquinones become reduced after dietary uptake.34 Although it is generally accepted that oxidized CoQ is the final product of its biosynthetic pathway, the de novo synthesis of the hydroquinone has also been proposed to contribute, at least partially, to the high levels of CoQH2 observed in vivo.34,35 However, de novo synthesis cannot explain how CoQH2 is maintained during oxidative challenge. Thus, it is clear that some enzymatic systems must operate for CoQH2 regeneration from its oxidized or semioxidized quinone. In the mitochondria, this function is linked to the electron transport chain dehydrogenases and other enzymes as well.2 35 36 Current evidence supports that, similar to the inner mitochondrial membrane, CoQH2 regeneration in extramitochondrial membranes is accomplished by the electron transport linked to oxidation of pyridine nucleotides by flavodehydrogenases.

Several enzymes have been reported to function as CoQ reductases outside mitochondria; most of them were previously characterized as flavoenzymes.2,37 They fall into two categories according to the reduction mechanism of the quinone: one-electron CoQ-reductases, which reduce CoQ in two successive electron transfers via a semiquinone intermediate (as is the case for mitochondrial NADH-CoQ reductase), and two-electron CoQ-reductases, which reduce CoQ directly via simultaneous transfer of two electrons to the quinone.

5.3.1 One-Electron Ubiquinone Reductases

Two extramitochondrial enzymes able to reduce ubiquinone through a one-electron mechanism have been described. Both are integral membrane proteins whose participation in microsomal electron chains has been long recognized, namely the NADH-cytochrome b5 reductase and the NADPH-cytochrome P450 reductase.

The NADH-cytochrome b5 reductase has been found in the cytosolic side of all endomembranes and the plasma membrane, and in the erythrocyte cytosol.9,11,38,39 The reductase was known primarily by its function in the reduction of microsomal cytochrome b5 to transfer electrons to the nonheme iron fatty acid desaturase,40 and as a methemoglobin reductase.39 This enzyme has also been related to the detoxification based on cytochrome P450 as an alternative source of electrons.10 In addition, cytochrome b5 reductase reduces a variety of quinones by a one-electron mechanism to generate the corresponding semiquinones and hydroquinones in the absence of cytochrome b5,10 and has been proposed to be involved in CoQH2 regeneration in the plasma membrane.9,11,41 Incubation of pig liver plasma membranes with NADH results in the reduction of endogenous CoQ10 to CoQ10H2, an activity attributed to the NADH-cytochrome b5 reductase.42

As a CoQ reductase, the enzyme displays maximal activity with CoQ0, a hydrophilic CoQ analogue. Reduction of natural hydrophobic homologues with long isoprenoid side-chain such as CoQ10 requires reconstitution into phospholipid liposomes.11 The quinone moiety of CoQ10 is freely movable in the lipid bilayers and thus, it can orient itself toward both sides of membranes,43 (see [43] and the balance of this book). At the cytosolic layer, CoQ10 might be accessible to the catalytic domain of NADH-cytochrome b5 reductase, similar to the reaction between CoQH2 and cytosolic myoglobin.44

NADPH-cytochrome P450 reductase can also reduce quinones through a one-electron mecha-nism.10 Evidence for the putative role of this enzyme as a CoQ-reductase arises from the ability of NADPH-driven electron transport to inhibit lipid peroxidation, possibly through antioxidant recycling in a process requiring CoQ.36

5.3.2 Two-Electron Ubiquinone Reductases

Two enzymes have been reported to reduce ubiquinone through a two-electron mechanism, both residing in the cytosol: DT-diaphorase,12,45 and a distinct NADPH-dependent ubiquinone reductase.13,46,47

Cytosolic DT-diaphorase is an inducible enzyme that can reduce a great variety of quinones and other substrates as well, as it is very sensitive to low concentrations of dicumarol. Much is known about its biochemistry, mechanism, and molecular biology,37,48,49 although its physiological role has still not been defined. Since this enzyme is particularly active in the reduction of hydrophilic quinones, but displays little reactivity towards hydrophobic ones, its putative role in reduction of extramitochon-drial CoQ10 was not initially considered.37 However, it has been recently reported that DT-diaphorase can maintain the reduced state of hydrophobic ubiquinones in phospholipid liposomes, thus promoting antioxidant function. Consequently, the suggestion was made that this could represent its actual role in vivo.12,45 Although DT-diaphorase is a soluble protein, its assay requires detergent to achieve maximal activity, which might indicate that the enzyme can interact with membrane components located at the membrane-cytosol interphase.12 In addition, a minor portion is usually associated with membranes,37 which may be relevant for protection of extramitochondrial membranes against oxidative stress.21

In addtion to DT-diaphorase, a novel cytosolic NADPH-ubiquinone reductase has been recently described.13,46,47 This enzyme can be distinguished from microsomal and mitochondrial enzymes, as well as DT-diaphorase, and its activity remains insensitive to low concentrations of dicumarol.13,46 In vitro studies have demonstrated that this enzyme reduces CoQ in liposomes and microsomes and also inhibits lipid peroxidation in these membranes, acting preferentially on long-chain CoQ analogues, like CoQ10.13,47 Further studies have shown that this enzyme also functions in antioxidant regeneration in animals treated with carbon tetrachloride.50

5.3.3 Extramitochondrial Ubiquinone Oxidases

Ubiquinone participates as an electron carrier in transplasma membrane electron transport mediating the reduction of extracellular oxidants such as ferricyanide, diferric transferrin, and the ascorbate free radical.9,51 The exact mechanism for electron transfer from plasma membrane ubiquinol to extracellular oxidants has not been fully defined. Current evidence suggests that, unlike the direct chemical reaction observed between ubiquinol and the tocopheroxyl radical inside the membrane,8,36 the reduction of extracellular oxidants requires additional enzymatic components facing the external side of the plasma membrane.52-55 NADH-oxidase activity partially purified from the surface of HeLa cells has been recently reported by Kishi et al.56 to exhibit CoQH2-oxidase activity. Whether this protein is a terminal oxidase of CoQ-dependent transplasma membrane electron transport remains to be elucidated.

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