IUBMB Life, 55(7): 375–385, July 2003
Review Article
Redox Reactions and Electron Transfer Across the Red Cell Membrane
Eleanor C. Kennett and Philip W. Kuchel
School of Molecular and Microbial Biosciences, University of Sydney, Australia
Summary
Plasma membrane electron transport systems appear to be
ubiquitous. These systems are implicated in hormone signal
transduction, cell growth and differentiation events as well as
protection from oxidative stress. The red blood cell is constantly
exposed to oxidative stress; protection against the reactive species
generated during this process may be the main role of its membrane
electron transport systems. Membrane redox activity has been
studied for over three-quarters of a century, and yet many questions
remain regarding its identity and likely roles: are electron transfers
by distinct and specific mechanisms; what are the physiological
donors and acceptors; and how do these systems affect metabolism?
Current evidence suggests that the human erythrocyte membrane
contains a number of distinct electron transfer systems, some of
which, at least, involve membrane proteins, and NADH or ascorbate
as electron donors. The activity of these systems appears to be
closely related to the metabolic state of the cell, suggesting that
mediation of reducing equivalents across the plasma membrane
allows redox buffering of each environment, intra- and extracellular,
by the other. We have decided to study this from a new perspective,
NMR spectroscopy the area of our own technical expertise, hence
this review is slanted towards this more recent analysis.
IUBMB Life, 55: 375–385, 2003
Keywords Plasma membrane oxidoreductase; ascorbate; nuclear
magnetic resonance; NMR; erythrocyte; oxidative stress.
Received 28 April 2003; accepted 3 June 2003
Address correspondence to Prof Philip W. Kuchel, School of
Molecular and Microbial Biosciences, University of Sydney, NSW
2006, Australia. E-mail: p.kuchel@mmb.usyd.edu.au
Abbreviations: AA, ascorbic acid, vitamin C; AFR, monodehy-
droascorbate free radical; CoQ, coenzyme Q, ubiquinone; cyt b561,
cytochrome b561; DABS, diazobenzene sulfonate; DCIP, 2,6-dicho-
lorophenol-indophenol; DHA, dehydroascorbate; EPR, electron
paramagnetic resonance; GAPDH, glyceraldehyde-3-phosphate de-
hydrogenase; ghosts, isolated erythrocyte membranes; GSH, reduced
glutathione; GSSG, oxidised glutathione; NBT, nitroblue tetrazo-
lium; NEM, N-ethylmaleimide; NMR, nuclear magnetic resonance;
pCMB, p-chloromercuribenzoate; pCMBS, p-chloromercuribenzene
sulfonic acid; PPP, pentose phosphate pathway; RBC, human
erythrocyte (red blood cell); a-TH, a-tocopherol; a-TQ, a-tocopher-
olquinone; WST-1, water soluble tetrazolium-1[2-(4-iodophenyl)-3-
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
salt].
ISSN 1521-6543 print/ISSN 1521-6551 online # 2003 IUBMB
DOI: 10.1080/15216540310001592843
INTRODUCTION
The human erythrocyte (RBC)1, through its role as the
body’s oxygen and carbon dioxide transporter, is under
constant exposure to possible sources of reactive oxygen
species and oxidative stress. Extracellular antioxidant capa-
city, reduction of extracellular oxidants via transmembrane
electron transport, allows the RBC to respond to such stress.
The mobility of the RBC makes it an ideal antioxidant not
only for its own membrane and local environment, but also as
an oxidant scavenger throughout plasma-accessible parts of
the body.
In 1925 Voegtlin et al. (1), through their investigation of the
relation of redox states to cancer, provided the initial evidence
for membrane redox activity. The discovery that cells in
isolation reduce extracellular cell-impermeable oxidants, such
as ferricyanide (2), led to further study of this phenomenon,
providing evidence for transmembrane electron transport to
enable the reduction of extracellular oxidants. Membrane
proteins involved in electron transport have been isolated and
purified from a number of cell types (3). However, the variety
and non-physiological nature of the electron acceptors
identified (Fig. 1) suggests a non-specific or indirect mechan-
ism of action.
Ferricyanide (2, 4 – 8), 2,6-dichlorophenol indophenol
(DCIP) (5, 9, 10), nitroblue tetrazolium (NBT) (11 – 13)
and water soluble tetrazolium-1 (WST-1), can all accept
electrons from transmembrane electron transport. Physiolo-
gical acceptors however, are still largely unknown and may
vary between cell types (3). Oxygen can act as an electron
acceptor for transmembrane redox activity in some cell
lines and may be a physiological electron acceptor; but
RBC do not appear to possess this activity (14). Non-
physiological electron acceptors, chosen experimentally
based on their redox capability and the measurability of
their redox state, may not reflect the true nature of
physiological acceptors. Despite this, valuable information
on how RBC respond to oxidative stress, both at the
substrate and whole cell metabolic level, have been gained
from studying electron transport systems with these
acceptors. Some of the greatest challenges ahead lie in
establishing the true physiological acceptors for these
376 KENNETT AND KUCHEL

Figure 1. Electron acceptors (and reduced partners) for membrane redox systems. Ferricyanide is also called hexacyanoferrate;
DCIP and the other acronyms are defined in the abbreviations list.

systems and under what in vivo conditions these systems are
active.
EVIDENCE FOR ELECTRON TRANSPORT
Acquiring evidence for plasma membrane electron
transport has been difficult. Direct evidence is hard to
obtain, but through a range of techniques, sufficient
evidence for plasma membrane electron transport has been
gathered. Nuclear magnetic resonance spectroscopy (NMR)
enables real-time, non-invasive investigation of extra- and
intracellular events simultaneously, hence providing direct
insight into the metabolic effects of extracellular oxidative
stress and the reduction of the extracellular oxidant (Fig. 2)
(8, 15, 16). Membrane potential measurements (17) and
observations of free radicals with electron paramagnetic
resonance spectroscopy (EPR) (18) provide strong evidence
for electron transfer. These techniques, coupled to bio-
chemical assays of redox reactions and metabolites, have
enabled the elucidation of membrane electron-transfer
events.
Some similarities exist between plasma membrane redox
systems and the well-characterized mitochondrial electron
transport chain. Reduction of extracellular oxidants is
concomitant with proton efflux (4), however, in plasma
membrane redox, oxygen consumption does not increase and
it is not affected by traditional mitochondrial electron
transport inhibitors (4).
The localization of redox activity to the plasma membrane
was assisted by the discovery of NADH dehydrogenase
activity in isolated RBC membranes (ghosts) (5). The NADH:
(acceptor) oxidoreductase identified was capable of reducing
ferricyanide, cytochrome c and DCIP with specific activities of
568, 7.9 and 17.5 IU/mg protein, respectively. The enzyme
could not be released by membrane lysis or EDTA treatment
and so was thought to be transmembraneous. Subsequent
 ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 377

Figure 2. 13C NMR spectra of RBC suspensions (Hc *65%) in phosphate buffered saline (A), with 6.5 mM ferricyanide (B),
and with 6.5 mM ferrocyanide (C). The appearance of a ferrocyanide resonance in consecutive 13C NMR spectra (D) reflects the
rate of ferricyanide reduction. Reproduced from (8).

histochemical and membrane permeability results provided an
argument for an endofacial protein and reactive site (13).
Despite mounting evidence for transmembrane electron
transport, not all interpretations back this theory. Orringer
and Roer (6) observed the effect of ascorbic acid (AA) on
ferricyanide reduction and concluded that AA efflux from the
cell and non-enzymic reaction of AA with ferricyanide
explained the observed reduction, rather than transmembrane
electron transport. The ferricyanide reduction was saturable
for dehydroascorbate (DHA) and appeared to need NADH
formed by glyceraldehyde-3-phosphate dehydrogenase
(GADPH) to reduce intracellular DHA to AA. Investigations
into the rate of AA efflux and reaction with ferricyanide (7)
refuted these claims and an increasing body of evidence shows
that RBC plasma membranes do contain transmembrane
electron transport activity.
Manipulation of RBC membranes produces a variety of
surface topologies; open ghosts (sheets), sealed inside-out
vesicles, and right side out (resealed) vesicles (19). Using open
and closed-oriented ghosts the orientation of NADH:
ferricyanide oxidoreductase activity was clarified (9, 20).
Incubation of whole cells with diazobenzene sulfonate
(DABS), which binds to exposed exofacial proteins prior to
membrane isolation, produces open ghosts with 35% less
ferricyanide reductase activity than untreated controls, in-
dicating that at least some of the observed activity is
transmembraneous (9). All membrane orientations exhibit
ferricyanide reductase activity; transmembrane activity has
high affinity for NADH, Km of 90 mM, and ferricyanide, Km of
125 mM (9, 20), while low affinity sites for NADH and
ferricyanide have been found on each face of the membrane
with an additional high affinity NADH site situated on the
inside face. The high affinity endofacial activity, attributed to
NADH cytochrome b5 reductase, may contribute to the 65%
uninhibited activity observed with DABS (9, 20).
IDENTIFICATION OF ELECTRON TRANSPORT SYSTEMS
A number of different electron transport processes are
present in the RBC membrane (see below and Fig. 3). These
378 KENNETT AND KUCHEL

Figure 3. Enzymes and redox pathways involved in RBC membrane redox activity. Adapted from (3) and (15).

processes appear to be sensitive to their immediate environ-

ment and cellular redox state as large variations in oxido-
Table 1
reductase activities have been reported in the literature (Table
Reported activities for erythrocyte NADH: (acceptor)
1). This may reflect variations in donor source and therefore
oxidoreductases
redox capacity. Differences may also come from inadequate
Activity accounting for endofacial membrane redox activities
Oxidoreductasea (IU/mg protein) Reference such as NADH: cytochrome c oxidoreductase and NADH:
methaemoglobin reductase (related to NADH: cytochrome b5
NADH: ferricyanide 455 + 32 (20)
reductase (21)).
NADH: ferricyanide 568 + 46 (5)
NADH: ferricyanide 345 (9)
NADH: ferricyanide 241 + 33 (37) NADH: (Acceptor) Oxidoreductases
Cat NADH: ferricyanide 489 + 45 (37)
NADH: Ferricyanide Oxidoreductase. Ferricyanide re-
Dog NADH: ferricyanide 15 + 2 (37)
duction is readily measured by spectrophotometric techniques
NADPH: ferricyanide 0 (5, 20)
either directly at 420 nm (e = 1 mM71cm71) or through
NADH: cytochrome c 13 + 3 (20)
conjugated assay systems with phenanthroline derivatives,
NADH: cytochrome c 13 + 2 (37)
increasing the extinction coefficient (to *20 mM71cm71) and
NADH: cytochrome c 7.9 + 0.72 (5)
hence sensitivity. The NADH: ferricyanide oxidoreductase
NADH: indophenol 37 + 8 (20)
reaction time course is biphasic; with a poorly reproducible
NADH: DCIP 17.5 (5)
fast stage followed by a slower linear phase (7). The fast phase
a
Unless stated otherwise activities are for human erythrocyte isolated is removable by diluting the cell concentration. AA and DHA
membranes. stimulation of the activity is greater than could be explained
 ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 379

by AA efflux and non-enzymic reaction with ferricyanide,
furthering the case for electron rather than antioxidant
transport for the redox activity (7).
The effects of NADH: ferricyanide oxidoreductase activity
on RBC metabolism in near in vivo conditions can be
monitored by using 13C, 1H and 31P NMR, a non-invasive
technique that does not perturb the system (8). During
ferricyanide reduction, the ratio of reduced glutathione
(GSH) to oxidised glutathione (GSSG) did not decrease
significantly. Himmelreich et al. (8) interpreted this to mean
that reducing equivalents did not derive from NADPH or the
oxidative pentose phosphate pathway (PPP), a view supported
by the lack of NADPH: ferricyanide oxidoreductase activity
found in RBC membranes (20). May et al. (22) found that
GSH concentrations did not change and attributed this to
GSH recycling. However, they found that CO2 production
through the oxidative PPP was increased 4-fold above controls
when cells were treated with 1 mM ferricyanide, an effect
doubled by the presence of DHA.
NADH does not appear to be the sole source of electrons;
Himmelreich et al. (8) observed a minimal decrease in lactate
concentration and a 10% increase in pyruvate above controls;
this is in agreement with other investigators who obtained
minimal variation in NADH in glucose-supplied RBC exposed
to ferricyanide (22). Glucose-starved cells exposed to ferricya-
nide exhibited a reversal of the NAD + : NADH ratio
suggesting that although NADH may not be the major
electron donor it is necessary, especially under starvation
conditions, for coping with oxidative stress.
Enzyme activity varies between donors, depending on the
intracellular AA concentration (8), which may explain
differences in the membrane redox activities cited above. AA
supplementation increases donor’s ferricyanide reductase
capacity—clear evidence for a major role of AA in the
electron transport system (8).
results, extracellular reductants (16) have been claimed to be
involved in transmembrane thiol-disulfide exchange to in-
tracellular thiol-containing compounds through membrane
proteins rich in sulfhydryls.
Ascorbate: (Acceptor) Oxidoreductases
Vitamin C is an essential requirement for all cells. It is
involved in a number of different physiological functions and
is not synthesized by the human body. AA can undergo either
one or two electron oxidation to monodehydroascorbate free
radical (AFR), or DHA. Two molecules of AFR can
disproportionate to give one AA and one DHA. DHA is
unstable in aqueous media undergoing irreversible ring open-
ing, but can be recycled back to AA via other pathways before
degradation. This recycling appears to play an important role
in the protection of RBC from oxidative stress. DHA and AA
enter the RBC via the GLUT1 glucose transporter, however
the rate of entry for AA is substantially lower than that of
DHA (15, 23).
Ascorbate: Ferricyanide Oxidoreductase. AA reduction
of ferricyanide is thermodynamically favourable based on
redox potentials (Table 2). The compounds can react directly
(non-enzymatically) and indirectly through plasma membrane
electron transport systems. AA, and DHA which once inside
the cell is reduced to AA, both stimulate ferricyanide reductase
activity (8). AA mediation of ferricyanide reductase activity
may be through a mechanism distinct from NADH: ferricya-
nide oxidoreductase as each show differing sensitivities to
exofacial inhibitors such as sulfhydryl agents and proteolytic
Table 2
Standard redox potentials at pH 7 and half reactions for
molecules involved in membrane redox (47, 48).

NADH: NAD + Oxidoreductase. Recent 1H NMR results Redox half reaction E8 (mV)
from our laboratory provide evidence for NADH as an ½ O2 + 2H + 2e > H2O
+ 7
815
extracellular, as well as intracellular, electron donor for a-tocopheroxyl. + e7 > a-tocopherol 500
membrane electron transport systems. RBC membranes do Fe(CN)637 (ferricyanide) + e7 >
not seem to possess exofacial NADH oxidase activity, Fe(CN)647 (ferrocyanide) 360
however, we have shown that RBC are able to oxidise O2 (g) + 2H+ + 2e7 > H2O2 295
extracellular NADH by a transmembraneous mechanism, AFR + H+ + e7 > AA 282
which leads to reduction of intracellular NAD + (unpublished cytochrome c (Fe3+) + e7 > cytochrome c
data). The phenomenon has been observed in glucose-starved (Fe2 + ) 235
cells, in which carbon flux to pyruvate is from 2,3-bispho- DCIP (oxidised) + e7 > DCIP (reduced) 220
sphoglycerate, thus avoiding the cycling of NAD + from the DHA + e7 > AA 80a
LDH reaction via GAPDH. This activity may provide a Ubiquinone + 2H+ + 2e7 > H2O + ubiquinol 45
plausible means for a rebalance of intracellular redox buffers DHA + e7 > AFR 7174
after an extracellular oxidative stress. It seems unlikely that Pyruvate7 + 2H + + 2e7 > lactate7 7185
this reaction occurs as observed in vivo, as concentrations of ½ GSSG + e7 > GSH 7230
NADH used are 1000 fold greater than those thought to occur NAD+ + H+ + 2e7 > NADH 7320
in plasma. The capacity of cells to import as well as export
reducing equivalents is not a new claim. Based on 1H NMR a
E8 for pH 6.4
380 KENNETT AND KUCHEL
enzymes (24). Inhibition of the endofacial AA- but not
NADH-oxidation reactions by sulfhydryl agents indicates an
endofacial oxidation activity independent of NADH: cyto-
chrome b5 oxidoreductase.
The reduction of ferricyanide oxidises intracellular AA in a
one-electron oxidation, producing AFR (24) and causes efflux
of 14C-labelled DHA through GLUT-1 from 14C-AA loaded
cells (25). Similarly, DHA efflux is observed with hydrogen
peroxide as the oxidant. Greater DHA efflux is observed from
ghosts than whole cells due to the lower ability of ghosts to
recycle AA, possibly explaining the lack of DHA efflux
reported by Himmelreich et al. in their whole-cell NMR
experiments (15). AA appears to be the main electron donor.
Removal of AA from cells by oxidation to DHA with Tempol,
followed by washout, results in a 66% decrease in ferricyanide
reductase activity, suggesting that less than a third of the
enzyme activity uses NADH (25). Interestingly, this corre-
sponds with the 65% inhibition of ‘transmembrane’ NADH:
ferricyanide activity by DABS (25).
Ascorbate: Nitroblue Tetrazolium Oxidoreductase. The
cell-impermeable redox dye NBT (Fig. 1) forms an insoluble
monoformazan on 2-electron reduction. Whole cells and
ghosts can reduce extracellular NBT, in the presence of
intracellular AA, with the resultant monoformazan deposited
at the erythrocyte membrane (13, 26). NBT has limited
solubility in aqueous media hence dissolution in the membrane
before reduction may also occur (13). NBT in free solution
reacts slowly with AA, so compared to ferricyanide reduction
by AA background reduction is attenuated. The sulfhydryl
agents p-chloromercuribenzoate (pCMB), p-chloromercuri-
benzene sulfonic acid (pCMBS) and N-ethylmaleimide
(NEM), and also ferricyanide, inhibit NBT reduction, the
latter possibly through substrate competition for the same
transmembrane enzyme. Reduction of NBT correlates with
intracellular AA content and reduction results in DHA efflux,
although to a lesser extent than with ferricyanide reduction.
AA again proves to be a more effective donor than NADH
(26). Demehin et al. (12) found that like ferricyanide, NBT
reduction is biphasic and they attributed the initial rapid
reduction to reduced species within the membrane such as a-
tocopherol (a-TH) and those accessible to it like AA. Contrary
to electron transport theory, they attributed the slower phase
of NBT reduction to its interaction with membrane bound
haemoglobin, which can react directly with NBT. These
authors suggested that increased oxidative stress correlates
with increased membrane-haemoglobin interaction.
Ascorbate: Ascorbate Free Radical Oxidoreductase. RBC
can preserve extracellular AA concentrations by reduction of
AFR (17, 27, 28). AFR reduction, like ferricyanide reduc-
tion, depolarizes the membrane. The more hyperpolarized the
membrane, the faster the AFR reduction (17). Enhancement
of AFR reduction can also be achieved by increasing the
intracellular AA concentration (28). The reductase activity is
inhibited by NEM, which brings about a loss of available
GSH, presumably the basis of decreased intracellular AA
recycling (27). Unlike its effect on AA: ferricyanide
oxidoreductase activity, pCMBS does not affect the pre-
servation of extracellular AA. Van Duijn et al. (28) found
that low NADH concentrations resulted in higher rates of
AA oxidation to AFR, while the opposite was found for high
NADH concentrations, indicating that NADH is a necessary
requirement for AA regeneration from AFR. This group
speculated on the functional components of the electron
transport system claiming that an a-TH shuttle was an
unlikely candidate given the redox potential (Table 2) and
electrogenic nature of the transport. Coenzyme Q (CoQ)
involvement is also thought unlikely, as inhibitors of CoQ
(capsaicin and dicoumarol) fail to perturb the effect.
Involvement of a cytochrome in a protein or protein complex
was suggested as the most likely candidate for the electron
transport (discussed below).
WHAT IS THE INTRACELLULAR ELECTRON DONOR?
Electron donation appears to be reasonably specific; only
two main electron donors have been identified compared to
the long and growing list of electron acceptors. As discussed
above, both NADH and AA act as electron donors to
membrane redox systems, with AA as the main reducing
agent. More recently, other electron donors have been
identified (Fig. 4). Whether these compounds are direct
donors or act through an as yet unidentified intermediate is
unknown.
Flavinoids, which have iron chelating and antioxidant
properties, inhibit DHA transport. Contrary to expectation,
the flavinoids quercetin and myricetin, but not other flavinoids
that have less electron-donating ability, enhance rather than
attenuate ferricyanide reductase activity. RBC readily take up
both of these flavinoids. The degree of enhancement cannot be
explained by direct reaction with extracellular ferricyanide
(29). Therefore, these flavinoids may also act as intracellular
donors for this redox activity.
Ubiquinone (CoQ) is present in mitochondrial membranes
and is a major component of the mitochondrial electron
transport chain. It is also present in plasma membranes (14).
Extraction of CoQ from RBC membranes decreases NADH:
ferricyanide oxidoreductase activity by 80%. This can be
substantially restored by addition of 10 mM CoQ and
moderately restored by the addition of a-tocopherolquinone
(a-TQ) (14). CoQ can also reactivate ferricyanide reductase
after inhibition by CoQ analogues (14). NADH: oxygen
oxidoreductase (NADH: oxidase) activity in rat liver cells
requires CoQ for full activity. RBC membranes contain less
CoQ (0.01 nmol/mg protein) than a-TQ (1.2 nmol/mg pro-
tein). As stimulation of activity is proportional to CoQ and a-
TQ content, this may reflect the low NADH oxidase activity
 ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 381

Figure 4. Known electron donors and possible electron donors (*) for transmembrane electron transport shown alongside their
oxidised form(s).

observed in these membranes. Thus, CoQ may act, as in the
mitochondrial electron transport chain, as an electron shuttle
in the plasma membrane.
WHAT IS THE MECHANISM OF ELECTRON
TRANSPORT?
Is a Protein Involved?
The isolation and purification of dehydrogenase proteins
from cell membranes indicates that at least some component
of the redox activity is protein associated. An NADH:
(acceptor) oxidoreductase, distinct from NADH: cytochrome
b5 reductase, has been isolated and purified from RBC
membranes. It is a glycoprotein of 40 kDa apparent subunit
molecular weight and represents 1% of the total RBC
membrane protein mass (30). It is a transmembrane protein
with intracellular oxidation and extracellular reduction sites.
The enzyme, specific for NADH and able to reduce
ferricyanide, was prepared such that peripheral proteins were
removed in the isolation process.
NADH: DCIP oxidoreductases isolated from bovine and
rat synaptic membranes are complexes of at least three
different proteins, including peripheral proteins (10). The
isolation and purification techniques used by Wang and
Alaupovic (30), although producing a functional protein, may
have removed components which are associated in vivo.
Clearly, further experiments are required for full characteriza-
tion of these proteins.
382 KENNETT AND KUCHEL
Figure 5. Inhibitors of transmembrane electron transport.
Are Sulfhydryl Groups Involved?
Involvement of membrane thiols in electron transfer is clear
from the inhibition of NADH: ferricyanide oxidoreductase
activity (5, 7, 9) and AA-dependent ferricyanide reduction (24)
in whole RBC by pCMBS and its unsulfonated form pCMB
(Fig. 5). May and Qu (24) found that in resealed ghosts made
from pCMBS-treated whole cells AA-dependent ferricyanide
reduction was inhibited. Leaky ghosts made from pCMBS-
treated whole cells did not have inhibited NADH-dependent
ferricyanide reduction or AA: ferric citrate oxidoreductase
activities (24). The latter observation is more likely to reflect
endofacial NADH reductase activity or direct reduction of
ferricyanide by NADH, rather than a lack of inhibition of
NADH-dependent ferricyanide reductase activity. pCMBS
inhibits the flavinoid-enhanced ferricyanide reduction (29) but
not the fast phase of NADH-dependent ferricyanide reductase
activity (7); an indication that this phase might be mediated by
a different type of membrane reductant(s) rather than
membrane protein sulfhydryls. The variability in sensitivity
to sulfyhdryl agents is a good indication of the existence of a
number of different membrane electron transport systems
mediated by different processes, at least some involving
membrane proteins.
Other cellular thiols also appear to be involved in
membrane redox reactions. Glutathione, the most abundant
thiol-containing molecule in the RBC, is a major source of
reducing activity for reducing intracellular oxidised species
and is a cofactor for detoxifying enzymes such as glutathione
peroxidase and glutathione-S-transferase. Under conditions of
oxidative stress, flux through the oxidative PPP is increased.
While glucose-6-phosphate dehydrogenase activity was once
suggested to be a controlling factor in oxidatively stimulated
flux through the PPP we now know that hexokinase is the
principal determinant in such circumstances (31). Hence,
changes in the redox status of RBC glutathione will reflect the
cells’ response to oxidative stress rather than the flux through
the PPP.
The role of glutathione as an antioxidant may not be
limited to the cytosol; it may also be able to transfer its
reducing ‘tendency’ across the plasma membrane. Ciriolo et al.
(16), using 1H-Carr-Purcell-Meiboom-Gill-NMR investigated
the effect of 0.5 mM extracellular GSH on intracellular
glutathione. Intra- and extracellular glutathione peaks are
indistinguishable under normal conditions making identifica-
tion of changes in the appropriate glutathione population
difficult. On dilution of cell concentrations and the introduc-
tion of chemical shift reagents, the signals appeared to be
separable. Under these conditions Ciriolo et al. (16) observed
an increase in intracellular GSH when GSH was added
extracellularly and attributed this to the release of mixed
GSH-protein disulfides from the cytosolic face of the
membrane through transmembrane thiol/disulfide exchange.
Are Haems or Cytochromes Involved?
Inhibition of ferricyanide reductase activity by the lipid-
soluble iron reagent o-phenanthroline (7), but not by bath-
ophenanthroline sulfonate (a non-haem-iron protein inhibitor
(9)), suggests the involvement of a haem or metal centre in the
electron transfer. Calcium may also affect redox activity as
chlorpromazine, which affects Ca2 + function through calmo-
dulin and membrane structure, completely inhibits transmem-
brane ferricyanide-reductase activity in open and inside-out
ghosts, but not right-side-out ghosts (9).
Cytochrome b561 (cyt b561) facilitates AA: AFR oxidor-
eductase activity in chromaffin granules. To test the hypothe-
sised involvement of this and other cytochromes in AA: AFR
oxidoreductase activity (4, 32) Van Duijn et al. (33)
investigated the presence of cytochromes in the RBC
membrane. Reverse transcriptase-PCR on reticulocyte mRNA
transcripts and Western blotting of erythrocyte membrane
protein extracts did not provide evidence for either cyt b561
mRNA or protein. They concluded that cyt b561 is not present
in RBC membranes. Cytochrome P-420, a possible breakdown
product of cytochrome P-450, has previously been reported to
be present in RBC membranes (34), however, Van Duijn et al.
(33) found no evidence for either of these cytochromes in these
more recent studies. Spectrophotometric studies of erythrocyte
membranes suggest the presence of unknown cytochromes in
the RBC membrane, which could oxidise AA and may be
involved in this or other oxidoreductase activities.
How Does the Activity Relate to the Metabolic State of the
Cell?
ATP levels can be sustained in RBC by extracellular
ferricyanide, even in the presence of the ATP regeneration
inhibitor iodoacetate. This protection is enhanced by the
provision of adenosine as a metabolic substrate (4). This can
be explained by the observation that during oxidative stress,
such as that afforded by ferricyanide, RBC make more ATP
 ELECTRON TRANSFER ACROSS THE RBC MEMBRANE
per glucose molecule consumed than under normal conditions
through decreased flux through the Rapoport-Luebering 2,3-
bisphosphoglycerate shunt (8).
Ferricyanide exposure, like pyruvate exposure, increases
GAPDH activity (14) and its inhibition by iodoacetate results
in near complete inhibition of ferricyanide reductase activity
(4, 7, 8, 35). This inhibition is rendered reversible by adding
DHA at 6.4 mM (8). Vanadate, which inhibits GAPDH and
other glycolytic enzymes, also inhibits ferricyanide reductase
activity (7). These results indicate a functional link between
glycolysis, NADH formation and transmembrane redox
activity. The enolase inhibitor fluoride has a variable effect;
Himmelreich et al. (8) observed that fluoride and iodoacetate
exhibited similar levels of inhibition and recovery with AA,
while Schipfer et al. (7) saw no inhibition with fluoride. From
these results, it can be concluded that electron transport
depends on the metabolic state of the cell, and that both
NADH and AA are electron donors for the system.
The fact that hormone receptor binding can modify
NADH dehydrogenase activity is a possible indication of
coupling, direct or otherwise, of hormone response to redox
signals. Insulin (2, 35) and somatotrophin (35), at higher
than physiological concentrations, were found to stimulate
ferricyanide reductase activity in whole cells and ghosts; this
stimulation is attenuated by NEM, iodoacetate and vana-
date (35). In contrast, Crane et al. (20) found that
physiological concentrations of insulin inhibit membrane
redox activity. The b-adrenergic agonists adrenaline and
ritodrine stimulate redox activity in a pH- and concentra-
tion-dependent manner, while b- but not a-adrenergic
antagonists are inhibitory (36). Hormone enhanced redox
activity is susceptible to inhibition by sulfhydryl agents such
as pCMB(S) and NEM. The NEM inhibition is reversible
by GTP, most likely through a G-protein receptor coupling
to the oxidoreductase (36). Links between the hormone
activation of ferricyanide reductase activity and the Na + /
H + antiport activation are also suggested, since the
antiport inhibitor amiloride prevents redox stimulation by
insulin and somatotrophin (35).
A functional link between the cellular redox responses and
Ca2 + -activated K + channels has been suggested (32, 37).
Miner et al. (37) studied the activities of both proteins in
RBC from a number of different mammalian species. No
correlation between NADH: ferricyanide oxidoreductase
activity and Ca2 + -activated K + channel activity was found
in any of the species. Interestingly, both membrane proteins
have a similar sensitivity to Pb2 + , atebrin (quinacrine; a
known NADH: ferricyanide oxidoreductase inhibitor) and
menadione. Both proteins have variable activity depending
on the metabolic state of the cell. However, the redox activity
is not sensitive to the ion-channel activator propranolol (32).
The enzymes thus appear to be functionally distinct, but the
influence of redox activity on channel gating may yet be
shown to be important.
383
IS THERE ANY EVIDENCE OF LINKS TO DISEASE
STATES?
Diabetic neuropathy patients have decreased GSH and
increased ferricyanide reductase activity in their RBC com-
pared to controls. The increase is associated with changes in
plasma creatinine, urinary albumin and plasma thiols rather
than changes in metabolic factors (38). Chronic haemodialysis
patients also exhibit higher ferricyanide reductase activity than
controls (6). Again, the increase was related to plasma
components. (RBC from controls washed with haemodialysis
plasma also conferred higher ferricyanide reductase activity
due mainly to higher plasma AA concentrations through
supplementation). The observation that patients with
GAPDH deficiency exhibited normal ferricyanide reductase
activity (6) led the authors to conclude that NADH is not the
main electron donor for the system. However, this enzyme is
far from rate limiting in human glycolysis (39 – 41) so this
conclusion can not be drawn.
HOW ARE INTRACELLULAR AND MEMBRANE REDOX
COMPONENTS PRESERVED IN THE ACTIVE STATE?
Ascorbate Recycling
Himmelreich et al. (15) used 13C NMR to determine the site
of DHA reduction. Intra- and extracellular AA and DHA
produce separate peaks due to differences in hydrogen
bonding to water in the two environments; this is the so-
called ‘split-peak’ phenomenon (42). Their results indicated
that DHA is reduced extracellularly with reducing equivalents
coming from the intracellular oxidation of AA, the fate of this
extracellular AA, however, is not explained. Himmelreich et
al. (15) found that cytochalisin B, a GLUT1 inhibitor, did not
inhibit the transport of high concentrations of DHA into
metabolically active cells. From this, they suggested the
existence of an alternative DHA transport pathway that may
be able to reduce DHA during transport. The physiological
relevance of this finding could be challenged due to the long
time scale and high concentrations of DHA employed.
AA recycling can also occur through direct reaction of
DHA with GSH in a two-electron reduction by-passing AFR
formation (22). GSH-dependent reduction of DHA may also
be mediated by the enzymes glutaredoxin, protein disulfide
isomerase (43) and the seleno-enzyme NADPH-dependent
thioredoxin reductase (44). DHA reduction by GSH requires
glucose to maintain GSH in its reduced form, however in the
absence of glucose, reduction of DHA can occur from
intracellular NADH (11).
AFR can also be recycled at the RBC membrane. NADH:
AFR oxidoreductase activity, identified by May et al. (27), has
both endofacial and transmembrane orientations, and enables
RBC to maintain AA concentrations. The endofacial enzyme
has a high affinity for both AFR and NADH (Km 5 2 mM).
The transmembrane activity, which reduces extracellular
AFR, accounts for *12% of the total NADH-dependent
384 KENNETT AND KUCHEL
AFR reductase activity (23). This AFR reductase activity is
distinguishable from AA enhanced NADH: ferricyanide
reductase activity through its sensitivity to Triton X-100 and
insensitivity to digestion by cathespin D.
Protective Role of Ascorbate for RBC Membranes
Ferricyanide, as well as inducing intracellular oxidation,
causes peroxidation of membrane lipids. F2-isoprostanes, a
marker of lipid peroxidation, double in sealed ghosts treated
with ferricyanide (45). Ghosts that are resealed to contain
AA largely prevent F2-isoprostane formation and result in
oxidation of intravesicular AA, presumably through trans-
membrane electron transfer. A strong correlation between
this AA oxidation and membrane a-TH concentration is
found. The finding that a-TH oxidation by ferricyanide
decreases in the presence of AA, and increasing a-TH
concentrations enhance ferricyanide electron transfer, thus
suggests a role for a-TH in transmembrane electron
transport.
AA has been shown to play a protective role against
peroxidation of RBC membrane lipids and tocopherols by t-
butylhydroperoxide, preserving lipids by up to 92% and a-
and g-tocopherol by 50% and 65%, respectively (46). AA can
also help protect a-TH from depletion in membranes treated
with DABS (18), hence, protecting against lipid peroxidation
(which occurs when a-TH levels are depleted by 20%). This
protection is concomitant with transmembrane electron
transfer from AA as shown by EPR and formation of
intracellular AFR (18).
FUTURE DIRECTIONS OF RBC ELECTRON TRANSPORT
RESEARCH
Only limited success in the identification of electron
transport systems in the RBC and other-cell plasma
membranes has been achieved. Electron donors and
acceptors (many non-physiological) have been identified
(Figs. 1 and 4) and current evidence suggests that there are
multiple systems or different redox mechanisms within the
plasma membrane (Fig. 3). Hence, much needs to be done
for the full elucidation of the mechanisms of these plasma
membrane redox systems. Physiological acceptors and
donors must be conclusively identified before a full
understanding of the system can be achieved. The
involvement of membrane antioxidants such as CoQ and
a-TH needs to be clarified. Obviously, this area has a big
future with much still to be discovered before we under-
stand how membrane redox systems work and why they
are present at all.
ACKNOWLEDGEMENTS
This work was supported by grants from the Australian
Research Council and Medical Research Council to P.W.
Kuchel. E.C. Kennett received an Australian Commonwealth
Postgraduate Award. Drs B.E. Chapman and W.A. Bubb are
thanked for input in relation to NMR, and Mr W.G. Lowe for
expert technical assistance. We are also very appreciative of
numerous valuable discussions with Dr Alfons Lawen of
Monash University, and for information regarding WST-1
from Dr Michael Berridge of Wellington, NZ.
REFERENCES
1. Voegtlin, C., Johnson, J. M., and Dyer, H. A., Jr. (1925) Quantitative
estimation of the reducing power of normal and cancer tissue. J.
Pharmacol. Exp. Ther. 24, 305.
2. Dormandy, T. L., and Zarday, Z. (1965) The mechanism of insulin
action: the immediate electrochemical effects of insulin on red-cell
systems. J. Physiol. 180, 684 – 707.
3. Baker, M. A., and Lawen, A. (2000) The function of the plasma
membrane NADH-oxidoreductase system. A critical review of the
structural and functional data. Antioxid. Redox Signal. 2, 197 – 212.
4. Mishra, R. K., and Passow, H. (1969) Induction of intracellular ATP
synthesis by extracellular ferricyanide in human red blood cells. J.
Membr. Biol. 1, 214 – 224.
5. Zamudio, I., and Canessa, M. (1966) Nicotinamide-adenine dinucleo-
tide dehydrogenase activity of human erythrocyte membranes.
Biochim. Biophys. Acta 120, 165 – 169.
6. Orringer, E. P., and Roer, M. E. S. (1979) An ascorbate-mediated
transmembrane-reducing system of the human erythrocyte. J. Clin.
Invest. 63, 53 – 58.
7. Schipfer, W., Neophytou, B., Trobisch, R., Groiss, O., and Gold-
enberg, H. (1985) Reduction of extracellular potassium ferricyanide
by transmembrane NADH: (acceptor) oxidoreductase of human
erythrocytes. Int. J. Biochem. 17, 819 – 823.
8. Himmelreich, U., and Kuchel, P. W. (1997) 13C-NMR studies of
transmembrane electron transfer to extracellular ferricyanide in
human erythrocytes. Eur. J. Biochem. 246, 638 – 645.
9. Grebing, C., Crane, F. L., Löw, H., and Hall, K. (1984) A
transmembranous NADH-dehydrogenase in human erythrocyte
membranes. J. Bioenerg. Biomemb. 16, 517 – 533.
10. Bulliard, C., Zurbriggen, R., Tornare, J., Faty, M., Dastoor, Z., and
Dreyer, J.-L. (1997) Purification of a dichlorophenol-indophenol
oxidoreductase from rat and bovine synaptic membranes: tight
complex association of a glyceralehyde-3-phosphate dehydrogenase
isoform, TOAD64, enolase-g and aldolase C. Biochem. J. 324, 555 –
563.
11. May, J. M., Qu, Z., and Morrow, J. D. (2001) Mechanisms of ascorbic
acid recycling in human erythrocytes. Biochim. Biophys. Acta 1528,
159 – 166.
12. Demehin, A. A., Abugo, O. O., and Rifkind, J. M. (2001) The
reduction of nitroblue tetrazolium by red blood cells: a measure of red
cell membrane antioxidant capacity and hemoglobin-membrane
binding sites. Free Radical Res. 34, 605 – 620.
13. Zamudio, I., Cellino, M., and Canessa-Fischer, M. (1969) The relation
between membrane structure and NADH: (acceptor) oxidoreductase
activity of erythrocyte ghosts. Arch. Biochem. Biophys. 129, 336 – 345.
14. Sun, I. L., Sun, E. E., Crane, F. L., Morre, D. J., Lindgren, A., and
Löw, H. (1992) Requirement for coenzyme Q in plasma membrane
electron transport. Proc. Natl. Acad. Sci. USA 89, 11126 – 11130.
15. Himmelreich, U., Drew, K., Serianni, A. S., and Kuchel, P. W. (1998)
13
C NMR studies of vitamin C transport and its redox cycling in
human erythrocytes. Biochemistry 37, 7578 – 7588.
16. Ciriolo, A. R., Paci, M., Sette, M., De Martino, A., Bozzi, A., and
Rotilio, G. (1993) Transduction of reducing power across the plasma
membrane by reduced glutathione. Eur. J. Biochem. 215, 711 – 718.
 ELECTRON TRANSFER ACROSS THE RBC MEMBRANE
17. Van Duijn, M. M., Van Der Zee, J., and Van Den Broek, P. J. (2001)
The ascorbate-driven reduction of extracellular ascorbate free radical
by the erythrocyte is an electrogenic process. FEBS Lett. 491, 67 – 70.
18. May, J. M., Qu, Z., Morrow, J. D., and Cobb, C. E. (2000) Ascorbate-
dependent protection of human erythrocytes against oxidant stress
generated by extracellular diazobenzene sulfonate. Biochem. Pharma-
col. 60, 47 – 53.
19. Steck, T. L., and Kant, J. A. (1974) Preparation of impermeable
ghosts and inside-out vesicles from human erythrocyte membranes.
Meth. Enzymol. 31, 172 – 180.
20. Crane, F. L., Crane, H. E., Sun, I. L., Mackellar, W. C., Grebing, C.,
and Löw, H. (1982) Insulin control of a transplasma membrane
NADH dehydrogenase in erythrocyte membranes. J. Bioenerg.
Biomemb. 14, 425 – 433.
21. Choury, D., Wajcman, H., Boissel, J. P., and Kaplan, J. C. (1981)
Evidence for endogenous proteolytic solubilization of human red-cell
membrane NADH-cytochrome b5 reductase. FEBS Lett. 126, 172 –
174.
22. May, J. M., Qu, Z.-C., Whitesell, R. R., and Cobb, C. E. (1996)
Ascorbate recycling in human erythrocytes: role of GSH in reducing
dehydroascorbate. Free Radical Biol. Med. 20, 543 – 551.
23. May, J. M., Qu, Z., and Cobb, C. E. (2001) Recycling of the ascorbate
free radical by human erythrocyte membranes. Free Radical Biol.
Med. 31, 117 – 124.
24. May, J. M., and Qu, Z. C. (1999) Ascorbate-dependent electron
transfer across the human erythrocyte membrane. Biochim. Biophys.
Acta 1421, 19 – 31.
25. May, J. M., Qu, Z. C., and Whitesell, R. R. (1995) Ascorbic acid
recycling enhances the antioxidant reserve of human erythrocytes.
Biochemistry 34, 12721 – 12728.
26. May, J. M., Qu, Z. C., and Whitesell, R. R. (1995) Ascorbate is the
major electron donor for a transmembrane oxidoreductase of human
erythrocytes. Biochim. Biophys. Acta 1238, 127 – 136.
27. May, J. M., Qu, Z.-C., and Cobb, C. E. (2000) Extracellular reduction
of the ascorbate free radical by human erythrocytes. Biochem.
Biophys. Res. Commun. 267, 118 – 123.
28. Van Duijn, M. M., Tijssen, K., Vansteveninck, J., Van Der Broek, P.,
and Van Der Zee, J. (2000) Erythrocytes reduce extracellular
ascorbate free radicals using intracellular ascorbate as an electron
donor. J. Biol. Chem. 275, 27720 – 27725.
29. Fiorani, M., and Al, E. (2002) Intracellular flavinoids as electron
donors for extracellular ferricyanide reduction in human erythrocytes.
Free Radical Biol. Med. 32, 64 – 72.
30. Wang, C. S., and Alaupovic, P. (1978) Isolation and partial
characterization of human erythrocyte membrane NADH: (acceptor)
oxidoreductase. J. Supramol. Struct. 9, 1 – 14.
31. Thorburn, D. R., and Kuchel, P. W. (1985) Regulation of the human-
erythrocyte hexose-monophosphate shunt under conditions of oxida-
tive stress. A study using NMR spectroscopy, a kinetic isotope effect,
a reconstituted system and computer simulation. Eur. J. Biochem. 150,
371 – 386.
32. Fehlau, R., Grygorczyk, R., Fuhrmann, G. F., and Schwarz, W.
(1989) Modulation of the Ca2 + - or Pb2 + -activated K + -selective
channels in human red cells. II. Parallelisms to modulation of the
activity of a membrane-bound oxidoreductase. Biochim. Biophys. Acta
978, 37 – 42.
385
33. Van Duijn, M. M., Buijs, J. T., Van Der Zee, J., and Van Den Broek,
P. J. (2001) The ascorbate: ascorbate free radical oxidoreductase from
the erythrocyte membrane is not cytochrome b561. Protoplasma 217,
94 – 100.
34. Bruder, G., Bretscher, A., Franke, W. W., and Jarasch, E. D. (1980)
Plasma membranes from intestinal microvilli and erythrocytes contain
cytochromes b5 and P-420. Biochim. Biophys. Acta 600, 739 – 755.
35. Marques, F., Crespo, M. E., and Bicho, M. (1995) Control of NADH
ferricyanide reductase activity in the human erythrocyte by somato-
trophin and insulin. Redox Rep. 1, 113 – 117.
36. Marques, F., and Bicho, M. P. (1997) Activation of a NADH
dehydrogenase in the human erythrocyte by beta-adrenergic agonists:
possible involvement of a G protein in enzyme activation. Biol. Signals
6, 52 – 61.
37. Miner, C., Lopez-Burillo, S., Garcia-Sancho, J., and Herreros, B.
(1983) Plasma membrane NADH dehydrogenase and Ca2 + -depen-
dent potassium transport in erythrocytes of several animal species.
Biochim. Biophys. Acta 727, 266 – 272.
38. Matteucci, E., and Giampietro, O. (2000) Transmembrane electron
transfer in diabetic nephropathy. Diabetes Care 23, 994 – 999.
39. Mulquiney, P. J., Bubb, W. A., and Kuchel, P. W. (1999) Model of
2,3-bisphosphoglycerate metabolism in the human erythrocyte based
on detailed enzyme kinetic equations: in vivo kinetic characterization
of 2,3-bisphosphoglycerate synthase/phosphatase using 13C and 31P
NMR. Biochem. J. 342, 567 – 580.
40. Mulquiney, P. J., and Kuchel, P. W. (1999) Model of 2,3-bispho-
sphoglycerate metabolism in the human erythrocyte based on detailed
enzyme kinetic equations: equations and parameter refinement.
Biochem. J. 342, 581 – 596.
41. Mulquiney, P. J., and Kuchel, P. W. (1999) Model of 2,3-bispho-
sphoglycerate metabolism in the human erythrocyte based on detailed
enzyme kinetic equations: computer simulation and Metabolic
Control Analysis. Biochem. J. 342, 597 – 604.
42. Kirk, K., and Kuchel, P. W. (1988) Physical basis of the effect of
hemoglobin on the 31P NMR chemical shifts of various phosphoryl
compounds. Biochemistry 27, 8803 – 8810.
43. Wells, W. W., Xu, D. P., and Washburn, M. P. (1995) Glutathione:
dehydroascorbate oxidoreductases. Meth. Enzymol. 252, 30 – 38.
44. Mendiratta, S., Qu, Z. C., and May, J. M. (1998) Enzyme-dependent
ascorbate recycling in human erythrocytes: role of thioredoxin
reductase. Free Radical Biol. Med. 25, 221 – 228.
45. May, J. M., Qu, Z. C., and Morrow, J. D. (1996) Interaction of
ascorbate and alpha-tocopherol in resealed human erythrocyte ghosts
– transmembrane electron transfer and protection from lipid
peroxidation. J. Biol. Chem. 271, 10577 – 10582.
46. Mawatari, S., and Murakami, K. (1999) Effects of ascorbic acid on
peroxidation of human erythrocyte membranes by lipoxygenase. J.
Nutr. Sci. Vitamin. 45, 687 – 699.
47. Van Duijn, M. M. (2001) Ascorbate and its Interactions with Plasma
Membrane Redox Systems, University of Leiden, Leiden.
48. Voet, D., and Voet, J. G. (1995) Biochemistry. 2nd edn. John Wiley &
Sons, Inc., New York.