Transplasma membrane electron transport

Review

Transplasma membrane electron transport: enzymes involved and biological function Jennifer D. Ly, Alfons Lawen Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne, Victoria, Australia

The notion of transmembrane electron transport is usually associated with mitochondria and chloroplasts. However, since the early 1970s, it has been known that this phenomenon also occurs at the level of the plasma membrane. Ever since, evidence has accumulated for the existence of a plethora of transplasma membrane electron transport enzymes. In this review, we discuss the various enzymes known, their molecular characteristics and their biological functions.

INTRODUCTION The existence of membrane redox systems is usually associated with the enzymes in the inner mitochondrial and thylacoid membranes. The importance of such systems in other cellular membranes is often under-rated by scientists and perhaps remains controversial in some contexts. It is well established, however, that a transplasma membrane electron transport (TPMET) system or plasma membrane redox system (PMRS) is expressed in every living cell, including bacteria, cyanobacteria, yeasts, algae and all kinds of plant and animal cells.1–3 In fact, PMRSs are not a simple curiosity, but there is increasing experimental evidence for their direct involvement in several vital biological functions. Many different specialized PMRSs have been described including the: (i) NADH:ascorbate free radical (AFR) oxidoreductase (ii) NADH:ubiquinone (CoQ) oxidoreductase; (iii) superoxide generating NADPH oxidase of defense; (iv) TPMET system in nonphagocytic cells; (v) superoxide generating NADPH oxidases of fertilization; (vi) ferric reductase; (vii) NADH: dichlorophenol-indophenol (DCIP) reductase; (viii) NADH oxidase; (ix) doxorubicin-inhibitable NADH:ferricyanideReceived 9 July 2002 Accepted 7 October 2002 Correspondence to: Alfons Lawen, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Building 13D, 100 Wellington Road, Melbourne 3800, Victoria, Australia Tel: +61 3 9905 3711; Fax: +61 3 9905 3726; E-mail: [email protected] Redox Report, Vol. 8, No. 1, 2003 DOI 10.1179/135100003125001198

reductase; and (x) the NADH:ferricyanide-reductase (Table 1). This review is dedicated to the critical analysis of the evidence for these transplasma membrane electron transport enzymes and provides a discussion of their biological functions.

BIOENERGETICS AND REDOX HOMEOSTASIS

Maintenance of appropriate cytoplasmic NADH/NAD+ ratios The two basic mechanisms responsible for cellular ATP production are cytosolic glycolysis and mitochondrial respiration. Glycolysis involves the metabolism of glucose to pyruvate, coupled to the reduction of NAD+ to NADH. In order for this pathway to remain operative, and thereby sustain ATP levels, NAD+ must continually be regenerated. One source for this constant supply of NAD+ is mitochondrial respiration, which employs oxygen as a ‘redox sink’ coupled to the re-oxidation of NADH. The textbook view is that, under normal conditions, most of the NAD+ would be derived from this mechanism. However, the presence of a functional respiratory chain is not a prerequisite for the survival of cultured cells. This has been demonstrated in human r0 cells (devoid of mitochondrial DNA) in which the mitochondria are no longer capable of fully functioning.4–6 The growth media of r0 cells is usually supplemented with pyruvate and uridine. Therefore, the pyruvate/lactate couple can apparently maintain the NADH/NAD+ balance under conditions where mitochondrial respiration is © W. S. Maney & Son Ltd

–

EC 1.6.5.4

NADH:ascorbate free radical (AFR) oxido-reductase

NADH:DCIP reductase

EC 1.16.1.2 O54902

–

NADH diferric transferrin reductase DCT1, (or Nramp2 or DMT1)

Glutathione-dependent ferric reductase (GSH-FR)

EC 1.6.99.13 P21373 P40088 P49573 P38636

Rat, human, yeast

Animals

Bacteria

Saccharomyces cerevisiae

Various plants

–

Ferric reductase: standard and turbo

Ferrireductase system FRE1 and FRE2 gene UTR1 gene FTR1 gene CTR1 gene ATX1 gene

Archaeoglobus fulgidus

–

Ferric reductase

Escherichia coli

–

Human, pig

Human

Rat Human

FepA

Q9NR02

–

Dual oxidase-1, Duox1 (or ThOX1)

Dual oxidase-2, Duox2 (or p138Tox or ThOX2)

–

Doxorubicin-inhibitable NADH:ferricyanide reductase

Mouse Rabbit

Human

–

Constitutive NADH oxidase, CNOX Q925G2 –

Human

–

Aging-related NADH-oxidase, arNOX

Dcytb (Mouse) Cytochrome b558 ferric/cupric reductase (Rabbit)

Species

EC number or primary accession number (swissprot)

Transplasma membrane electron transport enzyme

Table 1. Transplasma membrane electron transport enzymes

Tumor cell growth

Protection against reactive oxygen species-induced damage

Iron uptake

Iron uptake

Iron uptake

Iron uptake

Iron uptake

Iron uptake

Unknown

Unknown

Cell growth

Iron uptake

Cell growth and enlargement

Unknown

Proposed function

6,136,137,138

30,31,32–35,39

10,98,108,109,118,119, 120,121,122,123

102

104–107,113,114,117

99,100,112

103

101

79,80

79,80

139

110,111,124

131

128

Reference

4 Ly, Lawen

Human, rat

Q9Y5S8

Enzymes in bold have been cloned and/or purified.

Q9UH82

Human

Human, rat

–

Transplasma membrane NADH oxidase system (tpmNOX)

Tumor-associated NADH oxidase, tNOX

Human

–

Transplasma membrane electron transport (TPMET) in macrophages

Human, bovine

–

Human, mouse

Human, mammalian

Human, mouse

Transplasma membrane electron transport (TPMET) in endothelial cells

–

Q9HAM8

NADPH oxidase of sperm (NOX5)

PM NADH:ferricyanide-reductase/voltage-dependent anion channel-1 (VDAC-1)

Q9JHI8

Human

–

NOX3

NOX4 (or KOX-1)

Human, mouse, bovine

–

NADPH oxidase of phagocytes (NOX2)

NADPH oxidase of smooth muscle cells (NOX1 or Mox1)

Sea urchin

–

NADPH oxidase of oocytes

Rat

Species

–

EC number or primary accession number (swissprot)

NADH:ubiquinone oxidoreductase

Transplasma membrane electron transport enzyme

Table 1 (continued)

61

59,60

9–11,164,165 (submitted for publication)

69,78,89

69

69

40,43,50,54–56

62,63,65–67

71,72,73,77

36,37,38

Reference

Tumor cell proliferation

126,130,132

Cell growth and apoptosis; bioenergetics 6,8,16,125,155,162

LDL oxidation

Defense

Redox homeostasis

Drive capacitation for fertilisation

Tumor cell proliferation

Tumor cell proliferation

Production of reactive oxygen species against pathogens

Cell proliferation and cell defense

Block to polyspermy upon fertilisation

Protection against reactive oxygen species-induced damage

Proposed function

Transplasma membrane electron transport 5

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impaired (r0 cells) or under partial anaerobic conditions as observed in tumors. However, other compensatory enzyme systems, such as the plasma membrane NADH-oxidoreductase (PMOR), may play a major role in the survival of such cells. Growth of r0 cells can be maintained if culture media are supplemented with cell-impermeant redox compounds in the absence of pyruvate.7,6 The PMOR of mammalian systems has at least two enzyme activities: an NADH:ferricyanide-reductase and an NADH-oxidase.8–10 Ferricyanide, being impermeant to the cell’s plasma membrane (PM) due to its large anionic and hydrophilic nature (reviewed by Baker & Lawen11), is capable of maintaining cell survival in serum-free media.12 Ferricyanide is reduced to ferrocyanide12,13 and changes in the nucleotide pool (NADH/NAD+) can be observed during the growth of cells.14 This was one of the first times an inorganic compound was shown to be able to replace important biological growth factors and support the growth of r0 cells in vitro, thus presenting the idea that ferricyanide must have been functioning at the level of the PM to maintain cell survival.15,16 Reduction of extracellular ferricyanide leads to oxidation of intracellular NADH, maintaining the NADH/NAD+ ratio, and enabling cell viability and growth of cells.6,17 Consequently, the PMOR has the ability to control the cytoplasmic redox state within the cell and maintain homeostasis of intracellular NADH/NAD+ levels. These nucleotides would ultimately lead to the production of ATP, and hence maintain cell survival and growth. Maintenance of high aerobic glycolytic fluxes by (up-regulation of) the PMRS in cells deficient or devoid of mitochondrial electron transport chain may explain, at least in part, why energy-deficient cells (observed in aging and tumor progression) may still survive.6 The textbook view that resting cells produce most of their energy by mitochondrial respiration may be oversimplified, as many cells (especially lymphocytes) seem to not increase their glucose uptake whilst up-regulating their PMRS when made r0.18 Proton extrusion and control of internal pH A PMRS appears to be expressed in every living cell.1–3 In the cells tested, PMRS activity is accompanied by an acidification of the medium, suggesting it may act as, or be linked to, a proton pump.19 In animal cells, evidence supports a correlation between the PMRS and a Na+/H+ antiport.1,3 Upon the addition of ferricyanide to the medium, the PM NADH:ferricyanide-reductase begins to oxidize cytosolic NADH, and the Na+/H+ antiporter pumps the protons formed upon oxidation from the cytosol out of the cell.20 This is demonstrated in Ehrlich ascites tumor cells where proton extrusion by a transplasma membrane ferricyanide reductase is accompanied by an alkalization of

the cytoplasm and acidification of the medium;21 this, in turn, supposedly provides a mitogenic signal for growth of the tumor cells.22 Another consequence of the flux of electrons through the PMOR is the modulation of PM potential and membrane resistance. Under steady state conditions, proton influx and efflux remain constant and no difference in PM potential would occur. However, accumulation of protons in the cytosol results in hyperpolarization of the PM whereby the PM potential increases. Alternatively, depolarization of the PM occurs when cytosolic protons are exported out of the cells into the media leading to a decrease in the PM potential.23 Such a depolarization is consistent with a role for the transplasma membrane ferricyanide reductase and the observation of cytosolic alkalization as described above. Furthermore, in plant cells, the establishment of this electrochemical gradient of protons provides the energy required for solute transport and, accordingly, provides the amino acids necessary for the growth of plants.24,25

DEFENSE

Cellular defense against stress is another key function in which the PMRS is involved and reactive oxygen species (ROS) play a double underlying role in that they can be produced by some of the PMRS enzymes and be protected against by others. Antioxidant properties: protective role of PMRS against ROS ROS are generated in cells by both enzymatic and nonenzymatic reactions as by-products of redox reactions. ROS are potentially very destructive to cells due to their highly reactive nature – they can react with lipids, proteins, or nucleic acids giving rise to detrimental cell damage. The main mechanism of protection and elimination against ROS at the PM is excising off these radical chain reactions by small molecules. These include the ubiquinol/ubiquinone (CoQH2/CoQ) redox pair and a-tocopherol (vitamin E)26 inside the lipid bilayer, and ascorbate (vitamin C)27 at the interphase – whereby maintenance of their proper redox state is dependent upon the PMRS.28 The presence of a constitutive PMRS helps to maintain adequate antioxidant levels into and around membranes. 28 Two PMRS enzymes, namely an NADH:ascorbate free radical (AFR) oxidoreductase and an NADH:ubiquinone (CoQ) oxidoreductase, have been shown to drive electrons to a semi-oxidized form of ascorbate, ascorbate free radical (AFR), through CoQ, resulting in stabilization of ascorbate.

Transplasma membrane electron transport NADH:AFR oxidoreductase Ascorbate can donate either one or two electrons in redox reactions. Loss of the first electron results in the AFR, which is not very reactive. Upon reaction with mild oxidants such as ferricyanide, the second electron is removed and AFR is converted to a less stable form, dehydroascorbic acid (DHA). Nevertheless, studies lean towards an NADH:AFR oxidoreductase to provide a mechanism for cells to regenerate efficiently extracellular ascorbate from the AFR.29 The PM NADH:AFR oxidoreductase appears to be distinct from the PM NADH:ferricyanide reductase (described above), implicating the different levels of transplasma membrane electron transport systems that exist for discrete functions.30 The NADH:AFR reductase serves to protect cellular components from free radical-induced damage by a direct quenching of soluble free radicals or scavenging those radicals that initiate lipid peroxidation.31 Membrane-bound tocopheroxyl radicals are reduced by ascorbate to tocopherol, which is a protective agent against peroxidation of polyunsaturated membrane lipids by reducing lipid hydroperoxyl radicals to hydroperoxides.32,33 Anti-oxidant recycling of a-tocopherol by ascorbate has been observed in liposomes, cellular organelles and erythrocytes.34,35 The NADH:AFR reductase has a high apparent affinity for both NADH and the AFR. In open erythrocyte ghosts, the reductase is comprised of an inner membrane activity (both substrate sites are on the cytosolic membrane face) and a transmembrane activity that mediates extracellular AFR reduction using intracellular NADH.31 NADH:CoQ oxidoreductase The NADH:CoQ oxidoreductase is a 34 kDa protein with an internal fragment sequence identical to cytochrome b5 reductase.36 The precise participation of CoQ in transplasma membrane electron transport has been described. Using PMs from the deletion mutant yeast strain coq3D, which is defective in CoQ6 biosynthesis, Santos-Ocaña’s group37 provided for the first time genetic evidence for the participation of CoQ in the NADH:AFR reductase, as a source of electrons for transmembrane ascorbate stabilization. At the PM interphase, CoQ would maintain the antioxidant property of ascorbate using cytoplasmic NADH as a unique electron source. Conversely, the NADH:CoQ reductase catalyzes a NADH-driven one-electron reduction of CoQ to its semiquinone radical via a superoxide-dependent process. This in turn causes the reduction of phenoxyl radicals of a-tocopherol, regenerating a-tocopherol. 38 Thus, CoQ can also function as a free radical chainexcising antioxidant, due to its capacity to regenerate tocopherol and to scavenge peroxyl radicals in its hydroquinone form.26 Thus reduced CoQ acts as a carrier between an internal NADH:CoQ oxidoreductase 38,39 and an external side final acceptor, ascorbate.

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Pro-oxidant properties: production of ROS by PMRS As a first defensive weapon against pathogens, ROS are also generated at the cell surface of certain lineages of cells – a phenomenon known as a respiratory burst.40 The professional phagocytes of the immune system have the ability to produce ROS as microbicidal agents against pathogens. The enzymes responsible for the production of ROS are a multicomponent inducible NADPH oxidase, that requires assembly at the PM to function as an oxidase, and a myeloperoxidase. Also known as ‘respiratory burst oxidases’, the activated NADPH oxidases generate tightly controlled and localized O2•– anions. This respiratory burst is accompanied by increased oxygen consumption41 due to the activity of the oxidase, which catalyzes the reaction: NADPH + 2O2 ® NADP+ + 2O2•– + 2H+

Eq. 1

Furthermore, the O2•– anion rapidly dismutates to hydrogen peroxide (H2O2) and water. The H2O2 can be transformed by other membrane enzyme systems into other more reactive ROS (such as hydroxyl radical and singlet oxygen). The most prominent of these enzymes is neutrophil myeloperoxidase, which generates hydrochloric acid through the oxidation of Cl– by H2O2 (for a recent review, see Klebanoff 42). The NADPH oxidase of phagocytes (NOX2) is a special and inducible form of ubiquitous PMRS (Fig. 1). It is a member of a family of enzymes comprising NOX1 to NOX5, and Duox1 and Duox2 (Table 1). NOX2 is a transplasma membrane heterodimeric cytochrome b558, composed of a small a-subunit (p22phox) and a larger bsubunit (gp91phox), associated with two proteins, p47phox and p67phox, located in the cytoplasma of unstimulated cells.43 gp91phox functions as an electron transport chain containing four NADPH binding regions, an FAD binding site, and two heme groups anchored by four histidines.44 NOX1, NOX3, NOX4, NOX5, Duox1 and Duox2, which are present in non-phagocytic tissues (discussed in the subsequent sections), are all homologs of the larger subunit, gp91phox of NOX2. In addition to the two subunits (p22phox and gp91phox) and their associated proteins (p47phox and p67phox), at least five other components are required for complete NADPH oxidase activity in phagocytes, specifically for the activation of the electron flow: 1. Rac1, a GTPase, serves as a membrane-targeting molecule for p67phox.45 2. Rac2, a cytosolic guanine nucleoside-binding protein required for oxidase activation. Rac2 interacts with p67phox through its ‘effector region’, with the PM through its C-terminus, and with the cytochrome b558 through its ‘insert region’.46,47

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3. p40phox, a protein that enhances the activity of the system. In fact, it can bind both p47phox and p67phox; however, after activation, p40phox only binds to the Cterminus of p67phox.48 4. A H+ channel, which is essential for the activity of the oxidase, providing efflux of H+ ions, which is accompanied by the efflux of electrons and the necessary charge compensation. 49 5. Rap1A, which is a small membrane guanine nucleotide-binding protein involved in the transitional states of the oxidase (for a recent review, see del Castillo-Olivares et al.50). During activation of the oxidase, the two main cytosolic factors (p47phox and p67phox) are phosphorylated by protein kinase C-dependent pathways, and are translocated to the

PM.51 The translocation of p47phox precedes, and is necessary for, the translocation of p67phox.51 Furthermore, the SH3 domain of p47phox interacts directly with the proline-rich region of p22phox,52 whereas the C-terminal SH3 domain of p67phox associates with the proline-rich region of the p47phox.53 Thus the p22phox subunit serves as a docking station for the cytosolic factors (Fig. 1). Recently, Henderson54 demonstrated that the protein gp91phox is capable of acting as the NADPH oxidaseassociated voltage-gated H+ conductance channel in a stably transfected Chinese hamster ovary (CHO) cell line, implicating its role in providing efflux of H+. Maturana’s group55 also described similar observations; they indicated that the gp91phox proton channel is activated upon release of heme from its His-115 ligand. These data suggest that changes in heme co-ordination and/or spin state during the activation of the oxidase

Fig. 1. The inducible NADPH oxidase of defense (NOX2) requires assembly at the plasma membrane to function as an oxidase, generating superoxide anions. It is a transplasma membrane heterodimeric cytochrome b558, composed of a small-subunit (p22phox) and a larger-subunit (gp91phox), associated with two proteins, p47phox and p67phox, located in the cytoplasma of unstimulated cells. Upon activation, p47phox and p67phox translocate to the plasma membrane and associate with cytochrome b558. In addition, at least five other components are required for complete NADPH oxidase activity: Rac1, Rac2, p40phox, a H+ channel and Rap1A. Figure adapted from del Castillo-Olivares et al.50

Transplasma membrane electron transport complex might thereby functionally couple electron and proton transport. A quantum leap of research into the phagocytic NADPH oxidase has recently been achieved by reconstituting the active enzyme, by expressing gp91phox, p22phox, p47phox and p67phox in COS-7 cells, which express Rac1.56 This system will allow for further analysis of the role of individual components of the enzyme. TPMET systems in non-phagocytic cells Endothelial cells In addition to participating in bacterial killing, ROS, which have recently been shown to be produced enzymatically by non-phagocytic cells, have been implicated in inflammation and tissue injury. Oxidation of low density lipoprotein (LDL) in the arterial intima (the space between endothelial cells that form the inner surface of the artery wall and the surrounding smooth muscle cells) has been implicated in artherogenesis (the underlying process of cardiovascular disease). The major anti-oxidant in LDL is a-tocopherol, present at 6–12 molecules per LDL particle, which is raised by high dietary intake of vitamin E.57 However, without sufficient supply of electrons from other antioxidants, a-tocopherol is actually a pro-oxidant (reviewed by de Grey58). Macrophages in the arterial wall are able to modify LDL oxidatively; however, the exact mechanism of macrophage-mediated LDL oxidation remains unclear. Nevertheless, it has been established that macrophages are able to reduce extracellular copper and iron, and the contribution to this reduction of a ubiquitous TPMET system in macrophages has been suggested.59 TPMET activity reduces extracellular substrates by using electrons from intracellular NADH to blood-borne electron acceptors. Thus, these TPMET systems are uniquely situated to influence blood composition and vascular and organ function. In fact, several endothelial cell TPMET systems have been identified on the basis of different electron acceptor and/or donor specificities. Since none of these systems is molecularly defined, it is not clear as yet whether the various research groups are dealing with different or identical enzymes. One such system, thiazine reductase in pulmonary arterial endothelial cells, can utilize a PM-impermeant thiazine electron acceptor, toluidine blue-O-polyacrylamide (TBOP). It has been demonstrated that in this system, TPMET activity is sensitive to the cytoplasmic redox status as reflected in the poise of the reduced/oxidized pyridine nucleotides.60 Thus it would appear that the intracellular supply of reducing equivalents is an important factor controlling the rate of electron transport to the extracellular electron acceptor. This is consistent with the observation that TPMET activity could be significantly enhanced by pre-

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loading cells with ascorbate, which might act as an additional electron donor for the TPMET in macrophages, thereby increasing their ability to oxidise LDL.61 However, the exact mechanism of these TPMET systems is less well defined than in other cell types, and requires an in-depth investigation into identifying the cofactors and the carriers involved. Smooth muscle cells Smooth muscle proliferation and migration is also dependent on redox-sensitive activation of specific signaling pathways involving ROS production. 62,63 In smooth muscle cells, O2•– anion is produced mainly by an NAD(P)H oxidase distinct from the phagocytic NADPH oxidase (NOX2).64–66 However, it does share some similarity to the phagocytic NADPH oxidase. The smaller a-subunit p22phox, p47phox and Rac1 are also present in smooth muscle cells. The larger b-subunit gp91phox, responsible for electron transfer during the phagocytic respiratory burst, is undetected, though three homologs of this subunit expressed at higher levels have been identified: NOX1,66,67 NOX368 and NOX4.66 NOX1, NOX3 and NOX4 are 65 kDa in size.68 NOX1 (or Mox1), possibly in association with p22phox, may contribute to the large production of O2•– anion for the initial smooth muscle cell proliferation. As always with ROS, their concentration has to be tightly regulated. Thus, when NOX1 is overexpressed in NIH3T3 cells, O2•– production results in marked tumorigenicity and cell transformation. 67 NOX3 is expressed in the fetal kidney and to a lesser extent in the liver, lung and spleen,69 whereas NOX4 (or KOX-1) is expressed in the fetal kidney and in the adult pancreas.69 The biological function of NOX3 and NOX4 is currently unknown. It is speculated that NOX3 and NOX4 may contribute at a later phase of smooth muscle cell proliferation by blocking the growth inhibitory functions of nitric oxide via mediating a steady production of low amounts of O2•– anion.66 Thus, aberrant expression or regulation of both NOX3 and NOX4 may account for the increased ROS generation seen in some cancer cells, leading to uncontrolled cell proliferation, 69 which may account for the neoplastic growth of tumor cells.69 Confirmation of the exact role of these homologs in regulating mechanisms responsible for smooth muscle proliferation is thus required.

FERTILIZATION

NADPH oxidase of fertilization: block to polyspermy Upon fertilization, metazoan oocytes alter their extracellular protein coats to provide a structural block to polyspermy.70 In the case of a sea urchin egg, the ‘respiratory burst oxidase of fertilization’ on the PM is a crucial

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participant in these structural alterations. In a ‘respiratory burst’, within minutes of fertilization, sea urchin eggs consume oxygen to produce H2O2 as an extracellular oxidant to cross-link their protective surface envelopes. The egg generates H2O2 via an NADPH-specific oxidase that requires protein kinase C for activation.71 Oxidative ‘hardening’ reaction during a respiratory burst converts the fertilization envelope into a single macromolecular structure. The biochemical basis for this hardening reaction is the formation of O,O-dityrosine cross-links between closely apposed polypeptides of the fertilization envelope. 72 The cross-linking reaction is catalyzed by ovoperoxidase, a 70 kDa heme protein that bears spectroscopic similarities to lactoperoxidase72,73 – an enzyme involved in host defense against antimicrobial activity.74 Using H2O2 generated by the fertilized egg, ovoperoxidase induces the formation of long-lived tyrosyl radicals that undergo phenolic coupling.75 Furthermore, it has been demonstrated that ovoperoxidase possesses O2•– degrading activity, implying that it may play a role in protecting the developing embryo from oxidants derived from O2•–.76 The respiratory burst of fertilization requires both Ca2+ and MgATP2+ for activation and is sensitive to Zn2+ ions. It catalyzes the direct reduction of molecular oxygen to H2O2, and uses NADPH, but not NADH, as a cofactor as shown in Equation 2: NADPH + O2 + H+ ® H2O2 + NADP+

Eq. 2

Heinecke and Shapiro77 proposed a model for the regulation of early events in the echinoderm oocyte activation. Following the union of an egg and sperm, an early transient membrane depolarization establishes an electrical block to polyspermy; this is followed by the apparent activation of a phospholipase C, liberating diacylglycerol and inositol trisphosphate from phosphatidylinositol-4,5-bisphosphate. Inositol trisphosphate stimulates release of Ca2+ from intracellular stores, producing the necessary ionic signal for many of the early events in egg activation, including the calmodulin-dependent stimulation of NAD kinase and cortical granule exocytosis. NAD kinase converts NAD+ to NADP+, while hexose monophosphate shunt activity accelerates, reducing NADP+ to NADPH. NADPH provides substrate for the respiratory burst oxidase as well for reduction of ovothiol (OSH), an aromatic mercaptohistidine, which scavenges H2O2 that enters the egg. The increase in Ca2+ together with diacylglycerol stimulates protein kinase C, activating H2O2 synthesis by the membrane-associated NADPH oxidase. Many of the mechanisms employed in sea urchin fertilization have been conserved by mammalian gametes during evolution.70 Therefore, peroxidative pathways appear to play an important role in the early developmental program of the mammalian embryo.

NADPH oxidase of sperm: NOX5 Recently, Bánfi’s group78 identified a novel PM O2•– anion-producing respiratory burst NADPH oxidase in sperm, NOX5. Upon Ca2+ activation, NOX5 generates large amounts of O2•– anion, and functions as a proton channel, presumably to compensate charge and pH alterations due to electron export.78 Thus NOX5 fulfils a dual role: transport of electrons and proton conductance, similar to the NADPH oxidase of phagocytes (NOX2). NOX5 is distantly related to the larger b subunit, gp91phox of the phagocyte NADPH oxidase with conserved regions crucial for electron transport (NADPH, FAD and heme binding sites). However, NOX5 has an N-terminal extension that contains three EF hand motifs similar to Duox1, Duox2 and plant NOX.69 Duox1 (also called ThOX1) and Duox2 (also called p138Tox or ThOX2) are expressed in the thyroid and are larger than the other NOX isoforms, varying in size from 175–180 kDa.79,80 These homologs contain a C-terminal domain that is homologous to the NOX isoforms, but also contain an N-terminal domain that is homologous to peroxidases, giving rise to the terminology Dual oxidase or Duox.68,80 Additionally, located at the extreme N-terminus on the cytosolic side of the membrane of NOX5 is a Pro-Argrich sequence. This could serve as a binding sequence for SH3 domains in cytosolic regulatory proteins (analogous to the p22phox subunit of the NADPH oxidase of phagocytes), or it may interact with negatively charged membrane phospholipids. 69 Mammalian spermatozoa were among the first cells in which a respiratory burst was detected.81 It was later shown by Aitken’s group that generation of ROS by spermatozoa is activated by intracellular Ca2+ elevations, suggesting the presence of an Ca2+-activated NADPH oxidase.82 Due to its high level of expression in the testis (and in lymphoid organs), NOX5 is likely to play a role in sperm cells (and in T- and B-lymphocytes, respectively).78 Thus, the identification of NOX5 suggests that this Ca2+-activated NADPH oxidase in spermatozoa is identical to the enzyme previously described by Aitken’s group. 82 However, as yet, a definitive role for NOX5 in sperm cells (and T- and B-lymphocytes) is not resolved. While gp91phox of the phagocyte NADPH oxidase needs to assemble several other subunits for its activation (described above), it appears that in NOX5 the regulatory and catalytic modules are combined within a single protein. This may be reflected by the fundamental differences between sperm and phagocytes in terms of their need for activation. In phagocytes, an activation mechanism is imposed on the oxidase in order to ensue that O2•– anion is not constantly generated in these cells, but is only produced during the protection against pathogens. In contrast, spermatozoa have a chronic need for ROS production in order to stimulate redox-regulated signal transduction

Transplasma membrane electron transport cascades that drive capacitation83,84 (a priming event that renders mammalian spermatozoa responsive to signals originating from the cumulus–oocyte complex), 85 which take many hours to complete.83 The sensitization of spermatozoa to such calcium signals from the oocyte during capacitation involves a complex array of changes. Most notable is the spontaneous increase in tyrosine phosphorylation that is an absolute precondition for the attainment of a capacitated state86,87 whereby the involvement of cAMP in the control of tyrosine phosphorylation is redox regulated and stimulated by ROS.88,89 Also conceivable is that ROS generation by NOX5 may be an important mediator of the acrosome reaction and the sperm–oocyte fusion during fertilization.90 Thus, NOX5 might couple Ca2+ elevations during sperm activation to spermatozoa effector functions. Similarly, NOX5 might play a role as a bridge between B-cell and T-cell receptor activation and the proliferation and differentiation of B- and T-lymphocytes whereby Ca2+ and the production of ROS play an essential role.78 A role for the smaller a subunit, p22phox in NOX5 activity cannot be excluded as Bánfi’s group78 observed low levels of p22phox mRNA in all three cell lines (HEK293, COS-7 and HeLa cells) they used for their transfection experiments. Likewise, the existence of hitherto undefined, NOX5-interacting proteins cannot be excluded as well. Further elucidation and characterization of the structure of this novel NADPH oxidase may have important implications for our understanding of the fundamental cellular mechanisms regulating sperm function and maturation. Oxidative stress in human spermatozoa: male infertility Although, ROS generation appears to be essential for signaling in the mature spermatozoa, oxidative stress induced by ROS can also have detrimental effects on the spermatozoa, particularly in male infertility. However, the factors responsible for the excessive generation of ROS by the spermatozoa of infertile men have not yet been established. In some cases, it may be defects in the cellular mechanisms that most normally regulate free radical generation by these cells. The most important may be a defect in Sertoli cell function – failing to remove sufficient residual cytoplasm in the sperm midpiece before spermatozoa are discharged from the germinal epithelium.91 Subsequently, the presence of excess residual cytoplasm is then thought to enhance the free radical generating system of the spermatozoa. This may be such that the presence of excess glucose-6-phosphate dehydrogenase in the cytoplasm enhances the cellular generation of NADPH, which in turn fuels the generation of free radicals by the sperm NADPH oxidase (NOX5).92 The resulting oxidative stress causes motility loss in mammalian spermatozoa through the induction of

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peroxidative damage to the sperm PM. Human spermatozoa are particularly vulnerable to such stress because their PMs are highly enriched with unsaturated fatty acids, particularly docosahexaenoic acid with 6 double bonds per molecule,93 to give the PM the fluidity it needs to engage in the membrane fusion events associated with fertilization. Therefore, when ROS attack the double bonds, a lipid peroxidative chain reaction, catalyzed by transition metals such as iron and copper93 is initiated leading to the loss of sperm function due to the failure of membrane fluidity. In other cases, oxidative stress also attacks the integrity of the DNA carried in the sperm nucleus and mitochondria, such as DNA fragmentation. 94 This can decrease the fertilization capacity of the sperm, or can give rise to mutations after fertilization with the oocyte and be responsible for infertility and even cancer in the offspring (reviewed by Visconti et al.86). The reduced levels of antioxidants also form part of the oxidative stress that depicts male infertility. These include antioxidant enzymes, glutathione peroxidase (GPx5) and superoxide dismutase (SOD), and small molecular mass free radical scavengers such as ascorbate, a-tocopherol, tyrosine, hypotaurine and uric acid.95 Thus, a tight regulation of ROS generation as well as scavenging of ROS in spermatozoa is required to reduce the risk of male infertility.

IRON UPTAKE

Iron is essential for a large number of biological processes, serving as co-factors in numerous biochemical reactions. However, free iron can be extremely toxic, especially in the presence of molecular oxygen. Iron can convert oxygen to ROS via a series of reactions. Sequential one electron reduction of oxygen yields the O2•– anion, H2O2 and the hydroxyl radical.96 Biological targets of these oxidants are membrane lipids, proteins and DNA as discussed previously.97 Thus, there is a requirement for biological regulation to provide organisms with sufficient iron and prevention against iron toxicity. The role of the PMRS on ferric iron reduction has been established since 1985 through the work of Crane and colleagues.98 Four systems for iron uptake have been characterized: (i) the ‘standard’ and inducible ‘turbo’ reductase in plant cells;99,100 (ii) ferrireductase in bacteria;101–103 (iii) ferrireductase in yeast;104–107 and (iv) NADH:diferric transferrin reductase in animal cells.98,108–111 Ferric reductase system in plants and bacteria Ferric ions in the soil may be solubilized by means of chelators, including those that are excreted by bacteria

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and fungi, or by roots of the plants.99 However, for some plants, the reduction of ferric ion to ferrous ion is a prerequisite for uptake of iron across the root PM.99 Two types of ferric reductase activities have been identified in plant PMs (for a recent review see Lüthje et al.112). One, termed ‘standard’ reductase, is a constitutive, ubiquitous, PM reductase that is present in all kinds of cells.100 However, the true nature of components participating in electron transport and their organization in the PM is not known. The other, inducible under iron deficiency (stress) and relatively more active, is the ‘turbo’ reductase.100 The electron transport is associated with proton release and uses intracellular NAD(P)H as substrate. The electron flow leads to changes in intracellular redox status, pH, and metabolic energy and regulation of ion transport.

Ferric reductases have also been described for iron uptake in some bacteria. Some of these have been molecularly characterised including a glutathione-dependent ferric reductase (GSH-FR),102 an Archaeoglobus fulgidus ferric reductase (A. fulgidus FeR)103 and a FepA from Escherichia coli101 (Table 1). The crystal structures of the latter two have been solved. However, in this review, focusing on mammalian systems, an in-depth discussion of these enzymes will not be given. Ferrireductase system of the yeast Saccharomyces cerevisiae Studies of mutants of the yeast S. cerevisiae have led to the identification of genes required for high affinity iron uptake. Reduction of ferric iron outside the cell is

Fig. 2. Iron uptake in the yeast S. cerevisiae requires a ferrireductase system and ferrous transport system. The ferrireductase system is a multicomponent system composed of Fre1p, Fre2p, Utr1p and NADPH dehydrogenase for reduction of ferric iron outside the cell (A). Ferrous iron transport from the exterior to the interior of the cell occurs by means of a ferrous transport system. This is composed of multi-copper-containing oxidase, Ftr1p and an iron permease, Fet3p (B). It is suggested that the copper uptake system may have an underlying role in ferrous iron uptake, whereby the Ctr1p and Atx1p deliver copper to Fet3p (C or D). The mammalian homologs for the ferrireductase system and ferrous transporter system are in square parentheses.

Transplasma membrane electron transport accomplished by means of PM ferric reductases encoded by the ferric reductase transmembrane components, FRE1 and FRE2 gene (Fig. 2A), which have significant sequence homology with the larger b-subunit, gp91phox of the phagocyte NADPH oxidase (NOX2).104,105 These systems resemble one another in the direction of the movement of reducing equivalents from cytoplasm to an extracellular acceptor and the single-electron nature of the reduction.113 It also appears that this ferrireductase system is a multicomponent system, requiring a cytosolic factor, the product of the unknown transcript 1, UTR1 gene and an NADPH dehydrogenase, which would act synergistically with the FRE1 gene product to increase cell ferrireductase activity.106,107 The UTR1 protein (UTR1p) is an NAD+ kinase, consisting of six identical subunits with a molecular mass of 60 kDa each.114 UTR1p is suggested to contribute to the ferrireductase system through the supply of NADP+. Furthermore, inhibitory effects of NADP+ and NADPH, especially NADPH, on the NAD+ kinase activity of UTR1p may indicate that the intrinsic function of UTR1p is not only in the supply of NADP, but also in the regulation of the ferrireductase system.114 High-affinity ferrous iron transport from the exterior to the interior of the cell occurs by means of system which is not yet molecularly characterized (Fig. 2B). The transport process requires the activity of a Fe(II) transport complex composed of an intracellular multicopper-containing oxidase encoded by the FET3 gene, which has significant homology to ascorbate oxidase,115 and the PM iron permease, FTR1 gene product. 106 The exact mechanism and interaction between the two components is at present unknown. However, it is postulated that copper uptake is indirectly required for ferrous iron uptake. It is envisaged that high-affinity copper uptake mediated by the PM copper transport protein encoded by CTR1 gene is required to provide the FET3 protein with copper.115,116 Delivery of the copper from the CTR1 protein transporter to the FET3 protein is achieved by the protein encoded by the metal homeostasis factor, ATX1 gene (Fig. 2C,D).117 The mechanisms of iron uptake described for yeast may be conserved in some form in complex eukaryotes, and may provide some insights into dissecting out the pathways in iron uptake in mammals, such as in man. NADH:diferric transferrin reductase in animal cells In animals, transferrin is the predominant iron-carrying protein in serum. Iron uptake by cells from transferrin has been proposed to be carried out by two alternative convergent mechanisms: (i) specific binding of diferric transferrin to its receptor followed by endocytosis of the complex, release of iron at acidic pH in the endosome

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and re-cycling of the protein; or (ii) reduction of diferric transferrin at the PM and transport of ferrous ions.98 It is however, speculated that the two mechanisms may be activated simultaneously. Moreover, the transmembranous electron transport system required in the endosome to channel electrons from cytoplasmic reducing agents to ferric ions, is actually the same as that present in the PM from which the endosome originates.10 However, the basic mechanism underlying transferrinindependent iron transport involves the activities of a ferrireductase and an Fe(II) transmembrane transport system similar to that described for yeast. A divalent cation transporter, DCT1 (also known as Nramp2 and DMT1)118,119 has been shown to be responsible for the uptake of ferrous iron from the lumen into the mucosa of the small intestine. However, because most dietary iron is in the form of ferric iron complexes, these must be reduced to yield ferrous ions before iron can be successfully transported by DCT1. The most complete model for mammalian transmembrane reduction of iron has come form the work of Glass and colleagues in their studies on the transport of iron out of reticulocyte endosomes and in transformed human intestinal epithelial (Caco-2) cells.108,109 These results indicated that Caco-2 cells reduce apical ferric (Fe3+) by two parallel mechanisms – a PM NADH:ferrireductase and by the secretion of reductants of either cellular or basolateral origin.120 These data support a model for Fe3+ intestinal absorption in which cell-mediated Fe3+ reduction occurs before cellular Fe2+ uptake.121 Similar studies using human chronic myelogenous leukemia K562 cells also support this model of transferrin-independent iron uptake. K562 cells have a unique ferricyanide reductase that is neither involved in growth nor is responsive to insulin, but is necessary for iron uptake.122 Ferricyanide, competing with the iron-binding site in the ferricyanide reductase, completely inhibits iron uptake of [55Fe]-nitriloacetic acid in these cells.123 Other animal ferric reductases Recently, McKie’s group110 identified the gene encoding a 31.5 kDa mammalian PM b-type cytochrome with ferric reductase activity from mouse duodenal mucosa, termed Dcytb (Fig. 2A) by using a subtractive cloning strategy aimed to identify intestinal genes involved in iron absorption. Located in the duodenual brush border (the intestinal region most active in the absorption of dietary iron), it was demonstrated to function as a ferric reductase. Dcytb micro-injected into Xenopus oocytes and transfected into intestinal HuTu-80 and Caco-2 cells resulted in elevated reduction of ferric iron complexes. Furthermore, Dcytb mRNA and protein levels were upregulated by several independent stimulators of iron

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absorption, including chronic anemia, iron deficiency and hypoxia. Sequence analysis reveals that Dcytb shares 45–50% similarity to the cytochrome b561 family of membrane reductases.110 However, despite this sequence similarity, it is still unclear how Dcytb may act as a TPMET system. Cytochrome b561 functions as a transplasma membrane electron shuttle between the cytoplasm and the inside of chromaffin granules, where ascorbate and semi-DHA act as reducing co-factor and electron acceptor, respectively.124 Since Dcytb appears to lack any conventional NADH, NADPH, or flavin binding motifs that would allow these co-factors to act as intracellular electron donors, a more plausible electron donor would be ascorbate110 or glutathione. Also, like the gp91phox of NADPH oxidase of phagocytes, Dcytb may associate with several other proteins to form an active complex. The recent characterization of a rabbit homologue of Dcytb, a cytochrome b558 ferric/cupric reductase111 may resolve some uncertainties underlying the mechanisms of a ferric reductase in mammalian intestinal iron absorption. This protein is a cytochrome b558 with an apparent molecular weight of 33 kDa. It was demonstrated to stimulate ascorbate-driven copper and iron reduction in vitro and led to a model for duodenal reduction of iron. The authors suggest that the cytochrome b558 may shuttle electrons from intracellular ascorbate across the membrane to reduce luminal dehydroascorbate (as for cytochrome b561). The ascorbate generated reduces luminal iron (or copper) for transport. Transporter DCT1 can then take up ferric iron, and protons are recycled in an Fe2+/H+ symport mechanism (Fig. 2B).111

CONTROL OF CELL GROWTH AND APOPTOSIS

Plasma membrane NAD(P)H oxidoreductase and cell growth Diverse lines of evidence are accumulating which show that the redox state of the cell and, accordingly, PM electron transport contribute to control of cell growth, development and apoptosis. Initial studies demonstrated that external redox compounds such as ferricyanide that are reduced by the PMOR system stimulate cell growth in serum-limiting media, where NADH is the electron donor.16,125 Unfortunately, nomenclature in this area has not been standardized yet. Whereas the NADPH-oxidases are now referred to as NOX1 to NOX5, and Duox1 and Duox2, the NADH-oxidases have also been referred to as NOX especially by the Morré group with various prefixes to distinguish the various enzymes. Thus far, only the a-isoform of the tumor-associated tNOX126 has been cloned (see below). Morré’s group has also described a

constitutive NOX (CNOX),127 an aging-related NOX (arNOX)128 and a plant auxin (2,4-D)-induced NOX (dNOX).129 Furthermore, there is also evidence for a transplasma membrane NOX, which we have termed here in analogy tpmNOX (discussed later). Tumor-associated NADH-oxidase (tNOX) NOX proteins appear to exist in several forms, one of which, tNOX a 34 kDa protein, is tumor-associated and is an ectoenzyme that can be released into the extracellular space/serum. tNOX has been cloned126 and described as having two activities – a hydroquinone (NADH) oxidation and a protein disulfide–thiol interchange activity, which is susceptible to thiol reagents.130 The two enzymatic activities are supposed to alternate to generate a regular period length of about 22–23 min,131 although these data have yet to be reproduced in other laboratories. Furthermore, it has been described to have properties of a prion (i.e. resistance against proteolysis and cyanogen bromide digestion) and the ability to form amyloid fibers.132 tNOX activity was measured by NADH oxidation and is anticancer-drug responsive (quinone-site inhibitory analogs, capsaicin, adriamycin and antitumor sulfonylureas) and has been found to be present in most types of transformed cultured cells as well the sera of cancer patients.133,134 A unique characteristic of tNOX is that the protein is shed from the unprocessed tNOX possibly via proteolytic cleavage and released into culture media135 and sera of cancer patients.134 Its existence was described by Morré’s laboratory to correlate tightly with unregulated growth and loss of differentiated characteristics that are generally linked to cancer phenotypes.134 Therefore, it might serve as a diagnostic device or as a therapeutic target for cancer. Using a tetrazolium dye, which cannot cross the cell membrane (WST-1),136 Berridge’s laboratory was able to observe a similar activity, which was capable of reducing the dye in the presence of NADH. This activity, like tNOX, is shed into the medium but, in contrast to tNOX, is resistant to (or activated by) capsaicin. Like tNOX, this activity can be inhibited by pCMBS and was found to be enhanced in the sera of some cancer patients, especially colon cancer patients (M. Berridge, personal communication). It is not clear as yet whether these two activities represent the same enzyme nor whether they are involved in TPMET (for which they could constitute a terminal oxidase). Also, the role for these membrane redox systems in cell transformation is still elusive. Nevertheless, the coupling of PM electron transport to cellular growth in both the proliferative and transformed states marked a big step forward and will provide a benchmark for continued research. Constitutive NADH-oxidase (CNOX) Another form of NOX is constitutive (CNOX) and is described to be present in non-cancer cells and tissues.127

Transplasma membrane electron transport According to Morré’s laboratory, it also has an oxidase activity as well as protein disulfide–thiol interchange activity as described for tNOX.126 CNOX is described as a 24 kDa protein that is refractory to inhibition by putative quinone site inhibitors (capsaicin or the antitumor sulfonylurea, LY181984) and has a period length of 24 min.127,131 However, antibodies directed against tNOX did not cross-react with CNOX, raising doubt about whether these two enzymes really represent isozymes. Aging-related NADH-oxidase (arNOX) Recently, Morré’s group reported on yet another NOX enzyme, the aging-related NOX (arNOX).128 This protein was described as generating O2•– at the surface of individuals older than 70 years and to be shed similar to tNOX.128 arNOX may also act as a terminal oxidase in a PMOR electron transport chain. However, the function of the claimed specific age-related expression remains obscure. It has been suggested by Morré’s group to link accumulating lesions in the mitochondrial DNA to accumulations of ROS. NADH-dichlorophenol-indophenol (DCIP) reductase The presence of an NADH-DCIP reductase in the PM was first identified by Zurbriggen and Dreyer137 in a neuroblastoma (NB41A3) cell line. The enzyme was shown to account for greater than a third of the total cellular diaphorase and was responsible for cell growth of the neuroblastoma cells. In fact, later studies by this group characterized the function of this enzyme during cell proliferation and differentiation. Using FACS analysis and specific cell-cycle inhibitors such as a-amanitin and DMSO (both blockers of the G1 phase) and taxol (M phase blocker), they demonstrated that the enzyme is highly activated at the G1 phase and G2/M phases of the cell cycle.138 Moreover, they demonstrated that the enzyme was switched off after cell differentiation. However, the molecular mechanisms by which this activation and deactivation of the NADH-DCIP reductase occurs still remain to be elucidated. The same group also has described the enzyme to be an isozyme of glyceraldehyde dehydrogenase. 139 Plasma membrane doxorubicin-inhibitable NADHquinone (coenzyme Q-0):ferricyanide reductase Recently, a novel TPMET enzyme was identified and characterized. Although redox enzymes have been purified previously from PMs of rat liver, pig liver, Ehrlich tumor cells, K562 cells, HeLa cells and human erythrocytes (as discussed above), none of these enzymes has yet been characterized molecularly. Morré’s group140 reported the purification and characterization of a doxorubicin-inhibited NADH-quinone (CoQ):ferricyanide reductase from rat liver PMs which has cross-reactivity with K562 cells. This enzyme has an apparent molecular

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mass of 57 kDa, and a doxorubicin-inhibitable NADH quinone reductase activity. Doxorubicin is an anthracycline anti-tumor drug and, since it can inhibit the PM redox system, this redox enzyme system can be implicated in the control of cell proliferation and growth. Furthermore, fluorescence microscopy indicated that the reaction was with the external surface of the PM. The enzyme also had an NADH-CoQ reductase activity (or NADH:external acceptor (quinone) reductase activity), suggesting that the substrate for this enzyme may be CoQ.37 The nature, potential components and mechanisms of this electron transport system are unknown. NAD(P)H:quinone oxidoreductase-1 (NQO1) NQO1 (or DT-diaphorase) was originally described by Ernster’s group. 141,142 It is a 60 kDa homodimeric, ubiquitous, cytosolic/membrane flavoprotein that catalyzes the two-electron reduction of quinones to hydroquinones (without accumulating the associated semiquinone), and that can spontaneously produce a one-electron reduction of molecular oxygen to O2•–.141,143 However, the precise localization of NQO1 is poorly defined. NQO1 has also been shown to function physiologically as an antioxidant enzyme, generating antioxidant forms of CoQ and atocopherol. 144 Furthermore, it can redox couple with and reduce membrane CoQ, protecting membranes from damage by free radicals,144 implicating its potential involvement in TPMET. NQO1 expression itself can be up-regulated by H2O2 and some threshold expression of NQO1 appears to be necessary for cell proliferation. 145 In addition, it has been suggested to generate ROS, possibly driving constitutive activation of the transcriptional factor NF-kB in malignant melanoma cells.143 Constitutive NF-kB activation has been recently identified to be important for proliferation of malignant melanoma, including activation of Jun growth signaling pathways.143 Thus, these results raise the possibility that ROS produced endogenously by mechanisms involving NQO1 can constitutively activate NF-kB and stimulate tumor cell proliferation. This may also explain why NQO1 is overexpressed in many solid tumors.146,147 This was corroborated recently when Ross’s group149 confirmed the presence of NQO1 in the nucleus (and not PM localization) of human non-small cell lung (H661) and colon (HT29) carcinoma cells using confocal, immunoelectron microscopy and cell fractionation techniques. These results illustrate NQO1’s role in the nucleus as a target for DNA damaging bioactivated antitumor quinones and chemoprotection,150,151 but not as a TPMET enzyme. However, judgement on NQO1 involvement in physiological events is often made by assessment of the NQO1 inhibitor sensitivity of the effect analyzed. Although dicoumarol is routinely used as an NQO1 inhibitor, other actions of dicoumarol such as inhibition of the NADH:ferricyanide reductase148 activity have also

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been described. Therefore, some of the actions attributed to NQO1 may actually be those of the NADH:ferricyanide reductase, such that a requirement for reassessment of NQO1’s biological effects may be necessary. Transplasma membrane electron transport enzymes and apoptosis PMOR NADH:oxidase (tpmNOX) The PMOR of mammalian cells is a multi-enzyme complex that transfers electrons from cytoplasmic NADH via CoQ to external electron acceptors such as O2, ferricyanide, transferrin and ascorbate. As discussed above, the PMOR of mammalian systems has at least two enzyme activities: an NADH:ferricyanide-reductase activity and an NADH-oxidase activity.8–10 It has been accepted that the PMOR system plays an important role in the regulation of internal redox equilibrium in response to external stimuli. In fact, activation of the PMOR system by addition of growth factors or extracellular electron acceptors stimulates cellular proliferation.6,8–10 The PM of eukaryotic cells contains an NADH-oxidase, which transfers electrons across the membrane. This oxidase has been shown to be activated by several ligands, including epidermal growth factor, platelet-derived growth factor and hormones.8 Oncogenes such as Ha-ras152 and Nmyc153 as well as natural serum components such as diferric transferrin and ceruloplasmin that can stimulate proliferation can also stimulate activity of the oxidase.125,154 The exact nature of the enzyme involved is still elusive, but our evidence would suggest that enzyme to be a transplasma membrane NADH-oxidase (tpmNOX), different from the NOX enzymes discussed above in that this enzyme cannot be stimulated by extracellular NADH but requires intracellular NADH to function. Flavin and CoQ are apparent possible electron carriers for this TPMET. CoQ stimulates cell growth155 and analogs such as capsaicin reversibly inhibit growth and transmembrane electron transport,133 at concentrations where they specifically inhibit the tpmNOX activity.156 The function of CoQ in PM electron transport may be a crucial aspect of message generation for gene activation. There are two approaches to CoQ function: first, the redox state may act to control a tyrosine kinase; or the auto-oxidation of CoQ semiquinone in the membrane could generate H2O2,157 which would then act to activate protein tyrosine kinases158 or phosphatases159 or to generate gene activation signals by interacting directly with response elements at the DNA.160 Activation of protein kinases leads to generation of second messenger functions and, ultimately, to early nuclear gene activation and cell proliferation. 155 Furthermore, the putative involvement of membrane redox activities leads to the modulation of signaling, such as proton release,20 Ca2+

efflux,161 protein phosphorylation 162 or on the NAD+/NADH ratios in the cytosol125 – all of which contribute to control of cell growth and differentiation. Inhibitors of the tpmNOX activity (vanilloids, chloroquine and retinoic acid) have been demonstrated to induce apoptosis in different tumor cell lines.156 It was also demonstrated that the PMOR alters membrane calcium fluxes and signals for apoptosis through hypergeneration of ROS and activation of calcineurin;163 this induction of apoptosis was almost completely inhibited by Bcl-2 overexpression. 156 Furthermore, it has been shown that capsaicin inhibition of the PMOR system induces apoptosis only in activated and transformed cells.163 These results indicate an alteration in intracellular redox equilibrium of cells undergoing apoptosis and, therefore, demonstrate the importance of membrane redox systems in cell proliferation and cell death. PMOR NADH:ferricyanide-reductase (or voltagedependent anion channel-1, VDAC-1) VDAC-1 (or porin) is the predominant protein in the outer mitochondrial membrane, and it has been suggested to be also involved in the pore forming for cytochrome c release during apoptosis.164 However, VDAC-1 was described to be localized to the PM165 and we have evidence that it possesses transplasma membrane NADH:ferricyanide-reductase activity (data submitted for publication). PM VDAC-1 has a molecular mass of 35 kDa, and contains two cysteine residues, at least one of which appears to be involved in its redox activity (specifically for electron transfer) since addition of sulfhydryl binding reagents inhibits ferricyanide reduction in whole cells.166 Furthermore, analysis of the amino acid sequence of VDAC-1 reveals a putative NAD+ binding motif. We propose that PM VDAC-1 may function to maintain cellular redox homeostasis, in particular the ratio of NADH/NAD+. Thus stimulation of VDAC-1 would lead to a decrease in oxidative stress, by decreasing the NADH/NAD+ ratio and leading to cell survival. Conversely, inhibition of VDAC-1 would upset cellular redox levels of NADH/NAD+, and lead to the induction of apoptosis. Indeed, we have demonstrated that in PM VDAC-1 overexpressing cells, induction of apoptosis by two inhibitors (capsaicin and resiniferatoxin) of the PMOR NADH-oxidase, tpmNOX, was significantly inhibited. This finding was also reproduced with the anticancer drugs didemnin B167 and etoposide,168 which may induce apoptosis by causing oxidative stress in the context of intracellular NAD+/NADH imbalance. Thus, redox imbalance can be restored by stimulation of the PM NADH:ferricyanide-reductase activity and, therefore, rescue cells from oxidative stress-induced apoptosis. Redox control by PM VDAC-1 may be, therefore, a novel, critical determinant in apoptosis regulation and constitutes a novel function of VDAC-1.

Transplasma membrane electron transport CONCLUSIONS AND OUTLOOK Although much has been learned over the last decade on transplasma membrane electron transport, the systems responsible are still largely ill-defined. As evidence is accumulating for the biological importance of these strategically well-localised systems in cell growth, apoptosis and iron uptake, the time has arrived to define finally the enzymes and mechanisms involved, using the biochemical and molecular biological tools available today. It seems to us that mainstream research thus far has tended to overlook PMOR systems. With the emerging importance of PMOR systems in the regulation of cell proliferation, cell growth and apoptosis, researchers should change this attitude. An understanding of how the various enzymes involved in the PMOR system maintain redox homeostasis within the cell may have implications and future prospects for treatment of various diseases.

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