Transplasma membrane electron transport comes in two ﬂavors · elusive. In order for iron citrate to be taken up by a cell, iron has to be ﬁrst reduced from iron (III) to iron

BioFactors 34 (2009) 191–200 191DOI 10.3233/BIO-2009-1072IOS Press

Transplasma membrane electron transportcomes in two flavors

Darius J. R. Lane and Alfons Lawen∗

Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, MonashUniversity VIC 3800, Australia

Abstract. All tested cells possess transplasma membrane electron transfer (tPMET) systems that are capable of reducingextracellular electron acceptors at the cost of cytosolic electron donors. In mammals, classically NAD(P)H- and NADH-dependent systems have been distinguished. The NADH-dependent system has been suggested to be involved in non-transferrin-bound iron (NTBI) reduction and uptake. Recently we reported that transplasma membrane ascorbate/dehydroascorbatecycling can promote NTBI reduction and uptake by human erythroleukemia (K562) cells (D.J.R. Lane and A. Lawen, J BiolChem 283 (2008), 12701–12708). This system, involves i) cellular import of dehydroascorbate, ii) intracellular reduction ofdehydroascorbate to ascorbate using metabolically-derived reducing equivalents, iii) export of ascorbate down its concentrationgradient, iv) direct reduction of low molecular weight iron chelates by ascorbate, and v) uptake of iron (II) into the cell. We herepropose the consideration of this system as a novel form of tPMET which shares with classical enzyme-mediated tPMET systemsthe net transfer of reducing equivalents from the cytoplasmic compartment to the extracellular space, but lacks the involvementof the plasma membrane oxidoreductases responsible for the latter. Thus, transplasma membrane electron transfer can anddoes occur at two mechanistically distinct levels: i) enzyme-mediated transmembrane electron transfer and ii) transmembranemetabolite shuttling/cycling.

Keywords: Astrocytes, dehydroascorbate, K562 cells, non-transferrin-bound iron, Vitamin C

Abbreviations: AFR, ascorbate free radical; DHA, dehydroascorbate; GLUT, facilitative glucose transporter; NTBI, non-transferrin-bound iron; tPMET, transplasma membrane electron transport; SVCT, sodium-ascorbate co-transporter; VSOAC,volume-sensitive osmolyte and anion channel

1. Introduction

Transplasma membrane electron transport (tPMET) in eukaryotes is now well established [8,10,15,44,59,61,68]. The concept of tPMET arose from the observation that cell-impermeant dyes [43,87] can bereduced by tissue slices [97]. tPMET activities have since been related to the regulation of vital cellularprocesses including cellular bioenergetics [53,86], growth control and differentiation [8,15,68], apopto-sis [54,55,70,101], pH control and mitogenesis [8,68], cell signal transduction [68], antioxidation [61,85], and iron/copper metabolism [8,15,67,68,102]. Accordingly, deregulation of tPMET has been linkedto various human conditions including aging and neurodegeneration [36,37], macrophage-mediated LDLoxidation in atherogenesis [9], diabetic nephropathy [60] and glycolytic cancer progression [31–33].

∗Address for correspondence: Alfons Lawen, Department of Biochemistry and Molecular Biology, School of BiomedicalSciences, Monash University, VIC 3800, Australia. Tel.: +61 3 9905 3711; Fax: +61 3 9905 3726; E-mail: [email protected].

0951-6433/09/$17.00 2009 – IUBMB/IOS Press and the authors. All rights reserved

192 D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors

Classically a distinction was made between NAD(P)H- and NADH-dependent systems, the former ofwhich includes the members of the Nox and Duox families [59], while the latter – often referred to as theplasma membrane NADH:oxidoreductase system or PMOR – is suggested to include at least an NADHoxidase and an NADH:ferricyanide reductase activity [8,15,59].

Several enzymes have been suggested to be responsible for the plasma membrane NADH:ferricyanidereductase activity: a 57 kDa NADH-quinone oxidoreductase from rat liver plasma membranes [46]; aplasma membrane localized voltage-dependent anion channel (VDAC) isoform 1 [7]; and a membrane-bound form of cytochrome b5 reductase in neuronal plasma membranes [79]. Moreover, a multi-component, quinone-dependent tPMET system is also well-described [31] that is capable of reducingcell-impermeant water-soluble tetrazolium salts (e.g. WST-1) or extracellular dioxygen at the expenseof intracellular NADH [10,31].

Iron is vital for cellular survival: without it, every cell will die. Iron is a cofactor for oxidative phos-phorylation, neurotransmitter and nucleotide synthesis, nitric oxide metabolism and oxygen transport.However, iron can also catalyze the formation of reactive oxygen species [21,75,103]. Since too muchand too little iron can compromise cell viability, cellular iron homeostasis has to be tightly controlled.

In its physiological form, extracellular iron is complexed by biological chelators, the most importantof which are transferrin and citrate. Whereas the uptake of iron from transferrin is reasonably wellunderstood, the mechanism of non-transferrin-bound iron (NTBI) uptake by mammalian cells remainselusive. In order for iron citrate to be taken up by a cell, iron has to be first reduced from iron (III) toiron (II) as almost all cellular iron uptake can be inhibited by iron (II) chelators [18,30,38,39,41,76,91].Ferrous iron is then taken up by ferrous-specific transporters (e.g., DMT1 [5,104] and Zip14 [58]) in theplasma membrane.

Soon after its discovery, the transplasma ferricyanide reductase activity was suggested to be responsiblefor the reduction of NTBI prior to uptake as Fe2+ through the plasma membrane by divalent metal iontransporters [3,15,17,39,41]. Several enzymes have been suggested to be responsible for NTBI reductionbefore uptake, including duodenal cytochrome b561 (Dcytb [67]) and voltage dependent anion-selectivechannel, isoform 2 (VDAC2 [94]). The involvement of Dcytb in NTBI uptake, however, has since beenquestioned [27] and work in our own laboratory was not successful in linking either VDAC1 or VDAC2to NTBI uptake.

Ascorbate is known to promote the bioavailability of iron from numerous food sources in vivo andin vitro [22,29,30]. An ascorbate-stimulatede ferricyanide reductase has been described, but remains tobe molecularly identified. Moreover, ascorbate supplementation was shown to stimulate extracellularferricyanide reduction by several cell types, including K562 cells [51,82,83], HL-60 cells [95] and humanerythrocytes [44,61]. These data prompted us to ask the question of whether the ascorbate-stimulatedferricyanide reductase is involved in iron reduction for the uptake of iron from iron citrate.

2. The biochemistry of ascorbate

The physiologically prevalent monovalent ascorbate anion can undergo sequential one-electron oxi-dations under physiological conditions of pH, temperature and oxygen tension [61,78] (Fig. 1). The firstoxidation yields the relatively long-lived and electrochemically stable ascorbate free radical (AFR; alsoknown as semi- or mono-dehydroascorbate; E ′

0 = + 330 mV) [72]. This first oxidation of ascorbate isenhanced by low levels of circulating redox-active transition metals, such as iron and copper [78,84,88,89]. As AFR is relatively unreactive with dioxygen [78] – unlike many other free radicals [13,57] – andtends to decay mainly by disproportionation [11], the formation of AFR by reaction of ascorbate with

D.J.R. Lane and A. Lawen / Transplasma membrane electron transport comes in two flavors 193

Fig. 1. The oxidation products of vitamin C. The undissociated, fully reduced form of vitamin C (ascorbic acid) undergoes amonoprotic ionization at the carbon-3 hydroxyl with a pKa of 4.2, so that the ascorbate monoanion is the predominant species atphysiological pH. Ascorbate can undergo a thermodynamically favorable and reversible one-electron oxidation to the ascorbatefree radical (AFR). AFR is stabilized by resonant distribution of the resultant unpaired electron over the ring structure. AFRcan undergo a subsequent reversible one-electron oxidation to form the two-electron oxidized form, dehydroascorbate (DHA).DHA is highly unstable under physiological conditions and undergoes an essentially irreversible hydrolytic ring opening to2,3-diketogulonic acid (DKG) with a half-life of several minutes.

reactive radical species tends to inhibit free radical-induced oxidative chain reactions [13,57]; especiallyin the face of rapid AFR reduction back to ascorbate. AFR can undergo a further monoelectronic ox-idation to DHA (E′

0 = − 210 mV) [72] in the presence of mild oxidants such as ferricyanide [61,96]and/or NTBI species [14,88,89]. In the absence of such oxidants, however, two AFR molecules willrapidly disproportionate to one ascorbate and one DHA molecule [11,63] (Fig. 1). Though oxidation(or disproportionation) of AFR to DHA effectively allows utilization of the two-electron reducing ca-pacity of ascorbate, DHA is a structurally labile species that rapidly undergoes an irreversible hydrolyticring-opening to 2,3-diketogulonic acid under conditions found in plasma with a half-life of severalminutes [48,49].

Degradation of DHA results in an irrevocable loss of the vitamin from mammalian systems [42,48,49] – a point that is particularly pertinent in the case of species lacking gulono-γ-lactone oxidaseactivity [92]. In order to cope with this tendency for ascorbate loss, the vitamin must be maintainedpredominantly in the two-electron reduced form (i.e. ascorbate) in both intra- and extracellular biologicalfluids [74]. Consistent with this observation, mammalian cells possess a variety of conservative reductivemechanisms for maintaining both intra- and extracellular ascorbate [61,66,78,99]. Even cultured cells –which may be chronically ascorbate-deficient due to lack of supplementation under standard cultureconditions [35,90] – still maintain an extraordinary ability for ascorbate regeneration.

3. The ascorbate-stimulated plasma membrane ferricyanide reductase

Human erythrocytes possess a tPMET activity that utilizes intracellular ascorbate as the major electrondonor to reduce extracellular ferricyanide [44,61]. It is not clear at this stage whether ascorbate is a


(a) (b)

Fig. 2. DHA uptake by K562 cells occurs by GLUT-mediated transport. To assess the involvement of facilitative glucosetransporters in DHA uptake by K562 human erythroleukemia cells, cells previously grown to 6–8 × 106 cells/ml in RPMI +10% fetal bovine serum at 37◦C, 5% CO2 and 95% air were initially washed three times with MOPS-buffered saline (MBS,137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3). (a) Washed cells were then exposed to increasing concentrations ofthe GLUT-inhibitor cytochalasin B (CB) or the non-GLUT inhibiting structural analog, dihydrocytochalasin B (H2CB) 10 minprior to, and during incubation with 400 µM dehydroascorbate (DHA) for 30 min at 37◦C. Cells were then washed three timesin 100 volumes in cold MBS and their intracellular ascorbate determined essentially according to Lane and Lawen [52]. AsDHA uptake is inhibitable by CB, but not H2CB, GLUT-mediated DHA uptake is implied. (b) Alternatively, washed cells wereexposed to increasing concentrations of the GLUT-transportable, but non-metabolizable glucose analog 3-O-methyl-D-glucose(3-OMG) or the non-GLUT-transportable glucose stereoisomer L-glucose prior to incubation with DHA as in panel A. Againthe results indicate GLUT involvement in DHA import as inhibition of intracellular ascorbate accumulation occurs only in thepresence of the GLUT-transportable glucose analog.

significant electron donor for tPMET systems in other cells. When we tested human chronic myeloidleukemia (K562) cells, we observed a significant stimulation of the plasma membrane ferricyanide re-ductase activity after increasing intracellular ascorbate by preloading with dehydroascorbate [51]. Thisstimulation is not affected by addition of ascorbate oxidase (i.e. at a concentration that oxidizes allextracellular ascorbate to DHA and consequently inhibits direct reduction of ferricyanide by ascorbate),indicating that intracellular ascorbate can serve as an electron donor for extracellular ferricyanide reduc-tion (Lane et al., data not shown). The stimulation of the reductase activity could not be reproduced whenascorbate was directly added to the cells, suggesting that these cells do not express significant levels ofsodium-ascorbate co-transporters (SVCTs) [81]. Similar results were obtained with primary mouse andrat astrocytes (Lane et al., data not shown), indicating that these phenomena are not restricted to K562cells.

4. Cellular DHA uptake

The majority of mammalian cells maintain intracellular ascorbate concentrations that are markedlyhigher (e.g. up to 30-fold in some cases [47]) than those in the extracellular fluid or plasma [61,78,99]. Though many cells maintain this outward-facing concentration gradient by SVCT-mediatedascorbate import [81,99], the facilitated diffusion of DHA through low-affinity, high-capacity GLUTsis also a significant contributor [99]. With respect to DHA uptake by cells, an inward-facing DHAgradient is maintained by the rapid reduction of imported DHA back to ascorbate, the latter of whichis a poor substrate for GLUT-mediated transport [34,77,99]. K562 cells showed elevated intracellularascorbate levels after loading with DHA [52] that was inhibitable by cytochalasin B, suggesting response-dependence on DHA uptake via GLUTs, as previously suggested [77,78,93,99]. The involvement ofGLUTs in DHA uptake by K562 cells is further supported by two pharmacological observations thatintracellular ascorbate accumulation in response to extracellular DHA is inhibited by: i) low micromolar


Fig. 3. Ascorbate/DHA shuttling in mammalian NTBI uptake. Recent evidence suggests that NTBI ferrireduction may occurby transplasma membrane Asc cycling in which i) extracellular Asc reacts directly with NTBI, forming both DHA and Fe2+.The latter is then imported into the cell putatively via ferrous-selective transporters (e.g. DMT1 and/or Zip 14). ExtracellularAsc is subsequently regenerated for further ferric reduction events by ii) DHA import via glucose transporters (GLUTs), iii)intracellular reduction of DHA to Asc by an unspecified redox couple (‘R/O’; e.g. GSH/GSSG or NADPH/NADP+), followedby release of Asc through as yet unidentified Asc transporters (Anion Channel) in the plasma membrane (PM).

concentrations of cytochalasin B, but not the structural analog dihydrocytochalasin B (Fig. 2a), the latterof which shares with cytochalasin B its inhibition of cellular motile processes but not that of facilitatedglucose transport [56]; and ii) millimolar concentrations of the transportable (but non-metabolizable)D-glucose analog 3-O-methyl-D-glucose, but not the non-transportable glucose stereoisomer L-glucose(Fig. 2b). Again, primary astrocytes demonstrate similar behavior (Lane et al., data not shown).

5. Iron uptake and the ascorbate/DHA shuttle

Cellular uptake of NTBI is well documented, but less well understood than the classical transferrin-dependent iron import pathway [2,16,19,28,38,39,41,50,64,71]. The former may be particularly relevantin iron overload diseases such as hereditary hemochromatosis, hypotransferrinemia, and thalassemia [4,12,18,23], in which plasma iron presents in excess of transferrin-binding capacity [6]. When we analyzedthe ascorbate-mediated stimulation of NTBI reduction and uptake by human erythroleukemia (K562)cells we found that DHA loading of cells stimulated both processes (viz. 12- and 2-fold, respectively),yet – unlike the reduction of ferricyanide – remained inhibitable by extracellular ascorbate oxidase [52].Furthermore, as cells were able to import iron in a manner inhibitable by cell-impermeant ferrous ionchelators, the ascorbate-stimulated iron uptake is clearly dependent on the initial adoption of the ferrousstate, as previously observed [18,30,38,39,41,69,76,91].

Our data suggest that ascorbate released from cells – following uptake and reduction of DHA – mediatesdirect reduction of ferric to ferrous iron, the latter of which is then imported (Fig. 3). Subsequent additionof DHA to control or loaded cells resulted in a dose-dependent stimulation of both iron reduction and


uptake that was inhibitable by cytochalasin B, suggesting response-dependence on DHA uptake viaGLUTs (see Section 4). Again, these results are basically reproducible with primary astrocyte cultures(Lane et al., data not shown).

Several plausible candidates for the cellular export of ascorbate have been proposed [99], includ-ing exocytosis of ascorbate-containing vesicles [98,99], ascorbate-ascorbate homeoexchangers [24,45],connexin hemi-channels [1] and volume-sensitive osmolyte and anion channels (VSOACs) [47,62,99,100]. VSOAC permeability [80] and ascorbate efflux from cells [47,99] can be inhibited by generic anionchannel inhibitors, such as 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), suggesting that a significant proportion of ascorbaterelease occurs via this pathway. The observation that DIDS inhibits ascorbate release, ferrireduction andiron uptake to a similar degree in K562 cells [52] supports this conclusion.

6. ‘Shuttle-based’ tPMET systems – conclusions

Historically, research focused on enzymatic tPMET systems; however several examples of ‘shuttle-based’ tPMET systems have been documented as well, including ascorbate/DHA [30,52,64], dihy-drolipoic acid/α-lipoic acid [40,65], reduced glutathione/cysteine [20,25] and superoxide/dioxygen [26,73] shuttles. As with classical enzyme-mediated tPMET systems, these ‘shuttle-based’ systems resultin the net transfer of metabolically-derived reducing equivalents from the cytoplasmic compartment tothe extracellular space. Once in the extracellular space, however, the fate of these reducing equivalentswill depend on the particular redox couple involved. A canonical example of shuttle-based tPMET istransplasma membrane ascorbate/DHA cycling, which was the focus of this brief review. Our sugges-tion is that tPMET can and does come in two ‘flavors’: i) enzyme-mediated transmembrane electrontransfer and ii) transmembrane metabolite shuttling/cycling. In the latter reducing equivalents derivedfrom cellular metabolism are transferred – via transmembrane metabolite shuttling – to the extracellularspace for participation in extracellular redox events. Transplasma membrane ascorbate/DHA cyclingmay contribute significantly to NTBI ferric reduction prior to ferrous uptake.

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Transplasma membrane electron transport comes in two ﬂavors · elusive. In order for iron citrate to be taken up by a cell, iron has to be ﬁrst reduced from iron (III) to iron