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A hypothesis for the minimal overall structure of the mammalian
plasma membrane redox system
Running head: A. de Grey: A hypothesis for the structure of the mammalian PMRS A. D. N. J. de Grey*Department of Genetics, University of Cambridge, Cambridge.
*Correspondence and reprints: Department of Genetics, University of Cambridge, Downing Street,Cambridge CB2 3EH, UK. Email: ag24@gen.cam.ac.uk Summary
After a long period of frustration, many components of the mammalian plasma membrane redox
system are now being identified at the molecular level. Some are apparently ubiquitous but are
necessary only for a subset of electron donors or acceptors; some are present only in certain cell
types; some appear to be associated with proton extrusion; some appear to be capable of superoxide
production. The volume and variety of data now available have begun to allow the formulation of
tentative models for the overall network of interactions of enzymes and substrates that together
make up the plasma membrane redox system. Such a model is presented here. The structure
discussed here is of the mammalian system, though parts of it may apply more or less accurately to
fungal and plant cells too. Judging from the history of mitochondrial oxidative phosphorylation, it
may be hoped that the development of models of the whole system—even if they undergo
substantial revision thereafter—will markedly accelerate the pace of research in plasma membrane
redox, by providing a coherent basis for the design of future experiments.
Keywords: plasma membrane redox; proton pumping; superoxide; Q cycle; cancer; mammals. Abbreviations: PMRS plasma membrane redox system; Q ubiquinone; QH ubiquinol; CoQ coenzyme Q (either Q or QH ); NQO1 NAD(P)H-ubiquinone oxidoreductase 1 or DT-diaphorase; CNOX constitutive NADH oxidase; PDI protein thiol-disulphide interchange; pCMBS p-chloromercuriphenyl sulphonate; FeCN ferricyanide or Fe(CN) 3-; PHOX phagocyte NADPH oxidase; NC5R NADH-cytochrome b oxidoreductase; AFR ascorbate free radical; TBOP toluidine Introduction
The term “plasma membrane redox system” is used to denote the machinery by which cells oxidise
electron donors, typically NADH and/or NADPH, and transfer the resulting electrons to
extracellular acceptors. Historically the term “plasma membrane oxidoreductase” has also been
widespread, but in this author’s opinion it should now be avoided, in view of its connotation that
this activity is performed by a single enzyme, when in fact a network of enzymes is involved.
The appreciation that the plasma membrane redox system (PMRS) is so complex has come about asa result of studies in several laboratories and spanning two decades. These experiments haveyielded a considerable body of information on the properties of the system in various cell typesspanning the entire eukaryotic domain (Crane et al. 1985, Baker and Lawen 2000, Böttger 1998).
However, only in recent years has sufficient molecular information come to light on the PMRS’sindividual constituents to allow us to begin to propose how they fit together. Major aspects of thenecessary data are still lacking, so in this paper attention will be restricted to the “minimal”mammalian PMRS, while recognising that information from cell types (mammalian or otherwise)possessing additional PMRS components will surely be of value in testing and/or elaborating themodel proposed here.
An exception to the above characterisation of work on the PMRS occurs in phagocytic lymphocytes(phagocytes). Here there is an enzyme with some of the same properties as the PMRS but alsoimportant differences, whose molecular structure has now been resolved in detail (e.g. Abo et al.
1992, Dang et al. 2002). This enzyme, phagocyte NADPH oxidase (PHOX), has homologues inother cell types (Meier et al. 1991, 1993) that have recently been discussed systematically(Lambeth et al. 2000) using the terminology “NOX”. This family of enzymes is not the main topicof this paper, except insofar as to distinguish it from other activities. The PMRS that will bediscussed here will be termed the “classical” PMRS when a distinction is not clear from context.
This article is in two parts: a survey of the major types of experimental data that must beaccommodated by any viable model of the PMRS, followed by a description of the proposedstructure and an explanation of how it might fit these observations. A number of experimental testsof the proposal are readily apparent and are briefly noted.
Categories of data to be accommodated
Here we summarise the essential properties of the PMRS that have been determined over the past
two decades or more and which the model presented in the following section is intended to possess.
Trans-plasma membrane electron transport
The physiological role of the PMRS is to reduce extracellular electron acceptors when the electron
donor is cytosolic. This activity can most directly be demonstrated by the use of electron acceptors
that are not able to enter the cell, either because they carry multiple negative charges (such as the
classical acceptor ferricyanide (Crane and Löw 1976)) or because of their very large molecular
weight (such as TBOP, toluidine blue O polyacrylamide (Audi et al. 2001)). This suffices because
the electron donor is typically either NADH or NADPH, both of which are also unable to cross
membranes. It should be noted that the classical PMRS exhibits a generally higher affinity for
NADH as donor than NADPH (Crane et al. 1985), the reverse of the phagocyte enzyme.
Proton pumping into the extracellular space
It has long been known (Sun et al. 1984, 1988) that trans-plasma membrane electron transport is
accompanied by superstoichimetric proton efflux—that is, proton pumping. The H+/e- export ratio
varies considerably depending on the electron acceptor, exceeding 100 in some circumstances
when transferrin is used (Sun et al. 1988). Much of the activity is inhibited by amiloride (Sun et al.
1995), demonstrating a role for the Na+/H+ antiporter, but much is amiloride-insensitive (Sun et al.
1995). This activity may be one means by which cancer cells acidify the extracellular medium in
vivo (Gillies et al. 1994), so its biomedical relevance may be considerable. This argument is
strengthened by the finding that many anti-cancer drugs inhibit the PMRS (discussed in more detail
below). It is found that inhibitors of electron transport also inhibit proton efflux and vice versa,
with the sole exception of retinoic acid (Crane et al. 1991). A key feature of transmembrane
proton-pumping is that it generates a transmembrane proton gradient, which is a means to conserve
the large free energy of oxidation of NADH. A physiological function for this process, which may
be involved in the life-extending action of caloric restriction, was proposed recently (de Grey
2001). The H+/e- export ratio of the PMRS in various circumstances is summarised in Table 1.
Proton export is another major difference between the properties of the PMRS and of the phagocyteNADPH oxidase. On initiation of electron transport into the phagosome, the pH of the phagocytecytosol falls as a result of the excess of protons left there (Henderson et al. 1988). This issubsequently reversed, with alkalisation of the cytosol and considerable acidification of thephagosome, but that is the result of proton pumping not by PHOX but by the vacuolar ATPase(Lukacs et al. 1990). PHOX itself does possess a proton channel or wire (Henderson et al. 1987),but not a pump—it can only translocate protons at the same rate as electrons, not faster (Mankelowet al. 2002).
Oxidation of extracellular NAD(P)H
Much of the work on the PMRS has been performed using electron donors added to the
extracellular medium, rather than cytosolic ones. This may at first seem uninformative, since it is
non-physiological: there is essentially no NADH or NADPH in the extracellular space in vivo.
However, the simple fact that the PMRS can perform cell surface redox chemistry is useful, since it
provides more options for the dissection and characterisation of the PMRS. Nonetheless, it is
critical to remain aware that two essentially distinct activities are being assayed when the substrate
is provided on different sides of the membrane and that the nature and extent of their commonality
at the structural level is a hypothesis to be tested, not a given.
Protein disulphide/thiol interchange activity
It was relatively recently discovered (Pogue et al. 2000) that the NADH oxidation activity of
certain cell surface proteins alternates with a completely different redox activity: PDI, protein
thiol/disulphide interchange. This has been proposed to act on membrane-bound proteins and may
have pleiotropic regulatory functions.
Superoxide production
A key feature of the PMRS, which may have even greater biomedical relevance than its proton-
pumping activity (de Grey 1998, 2000, 2002, Morré et al. 2000), is its ability to use molecular
oxygen as an electron acceptor and reduce it to superoxide. This has been demonstrated in the case
of both transmembrane and extracellular electron transfer; in the latter case the rate of superoxide
production can be as much as 2 fmol/min per cell (O’Donnell and Azzi 1996), comparable to that
of the “professional” superoxide generator PHOX. In vivo, it is very likely that the major electronacceptor for the PMRS is indeed molecular oxygen, since no other reducible substrate is present incomparable quantity. However, it is presumed that this oxygen is mainly reduced all the way towater. A clear similarity is thus suggested with the mitochondrial respiratory chain, which mainlyperforms four-electron reduction of oxygen but also generates a small amount of superoxide. Sincedisulphide/thiol interchange is a two-electron redox activity, there is also the possibility that thePMRS reduces oxygen via a hydrogen peroxide intermediate.
The Morré labs have so far characterised three enzymes with the above combination of activities(Chueh et al. 2002, Morré et al. 2000, 2002). Only one, which they have termed “constitutiveNADH oxidase” (CNOX), will be considered here, since the others are present only in a subset ofcell tyes and should thus be omitted from the “minimal” structure discussed in this paper.
Dependence on coenzyme Q
Among the common electron acceptors used in assays of the PMRS is the partially oxidised form of
vitamin C, ascorbate free radical (AFR). Cells readily reduce AFR to ascorbate. However, it has
been shown (Villalba et al. 1995) that this capacity is dependent on the presence of coenzyme Q in
the membrane: Q-depleted membranes lose AFR reductase activity. This is in contrast to cytosolic
redox activity, which is largely unaffected by Q depletion (Villalba et al. 1995).
The role of known NAD(P)H dehydrogenases
Two well-studied enzymes have been implicated in the transfer of electrons from cytosolic
NAD(P)H to the plasma membrane. Both are best known for roles elsewhere in the cell, however.
NADH-cytochrome b oxidoreductase (NC5R) participates in fatty acid desaturation; curiously,
though it plays a major role in the PMRS, its usual electron acceptor, cytochrome b , is evidently not involved (Villalba et al. 1997). NAD(P)H-quinone oxidoreductase (NQO1), also known as DT-diaphorase, is usually considered a cytosolic enzyme and is probably a general-purpose maintainerof Q pools in the reduced state in various membranes. A third enzyme, so far unnamed, has ahigher affinity for NADPH than NADH but otherwise performs the same two-electron redoxreaction as NQO1 (Kishi et al. 1999).
Inhibitor specificity and dependence on substrate
It was noted previously that the PMRS, in contrast to PHOX, has a higher affinity for NADH than
NADPH as electron donor. This is true of both the extracellular and transmembrane activities. The
difference can be accentuated by addition of DPI, diphenyleneiodonium, which inhibits the
NADPH-dependent activity to a greater degree than the NADH-dependent activity (Morré 2002).
Many agents, including capsaicin, resiniferatoxin and N-ethylmaleamide, exert a marked inhibitoryeffect on the transmembrane activity but are largely without effect on the extracellular activity(Berridge and Tan 2000). Many such compounds are coenzyme Q analogues; an exception is theantitumour sulfonylurea LY181984 (Morré et al. 1995), but it too appears to bind to quinone-binding sites (Schloss et al. 1988). By contrast, the mercury-containing thiol reagent pCMBSinhibits the extracellular activity more than the transmembrane activity (Berridge and Tan 2000).
The proposed structure
The data reviewed in the previous section may be summarised as implying the following properties
of the PMRS:
1) It can accept electron from either intracellular or extracellular two-electron donors, including 2) It can perform one-, two- and probably four-electron reduction of various substrates.
3) It pumps protons out of the cell much faster than electrons, via multiple pathways. The proton and electron transport activities are affected similarly by all known inhibitors except retinoicacid.
4) It exhibits Q-dependence varying with the electron acceptor.
These properties can, it will be argued, be fulfilled economically by a system such as that depictedin Figure 1.
This proposed structure possesses three core novel features: (1) the production of superoxide isproposed to occur via the NAD(P) radical; (2) the stimulation of amiloride- and transferrin-sensitive proton extrusion is proposed to be mediated via a PDI-based interaction; and (3) the one-electron redox and (in part) proton-pumping properties of the PMRS are proposed to result from aQ-cycle mechanism, in which electrons are passed from a location at the cell surface to one at thecytosolic face of the plasma membrane via enzyme-bound redox centres.
Why are these features plausible? Let us first consider the Q cycle proposal. (Strictly it should betermed an inverted Q cycle, since it incorporates a merging of electron pathways at the inner face ofthe relevant membrane, whereas the prototypical Q cycle at mitochondrial Complex IIIincorporates a bifurcation of electron paths at the outer face. The effect is the same, however,namely capture of protons on the inside and their release on the outside.) Its main purpose is toprovide the amiloride-insensitive proton pumping observed by Sun et al. (1995), something thatcannot be achieved by a simple two-electron reduction of Q to QH at the cytosolic face and its reoxidation at the cell surface since then no charge separation takes place. Additionally, theintrinsic involvement of single-electron transfers between redox centres allows, as at mitochondrialComplex III, for escape of electrons to free one-electron acceptors such as ferricyanide and (withlow affinity) molecular oxygen. The proposed sites of action of pCMBS and doxorubicin areconsistent with their specificity for internal versus external electron supply from NAD(P)H, butother locations are also possible. The cytosolic component of the Q-cycling enzyme complex isproposed to be NC5R, whose involvement in the PMRS is well-established; the recipient of theelectrons that pass through NC5R is unclear, since cytochrome b itself appears not to be needed here (Villalba et al. 1997), but the fact that cytochrome b is a single-electron carrier implicates the oxidoreductase as occupying this position in the proposed Q cycle. Moreover, the fact that NC5R’sprototypical electron acceptor is a cytochrome is circumstantially supportive of the possibility thatit physically interacts with other proteins in the plasma membrane as suggested here. The externalcomponent of this proposed complex, which is suggested to interact with FeCN, may be theenzyme recently reported by Kim et al. (2002). In the present proposal, electrons pass singly fromthis enzyme to NC5R, providing the charge separation necessary for proton pumping; when NC5Ris absent, such as after treatment with cathepsin D (Kim et al. 2002), these electrons may beaccepted by Q. The validity of this general structure can be tested more readily now that candidatesfor components of this complex are molecularly characterised, since its predictions regarding theinteraction with various inhibitors are clear.
Now let us examine the proposal for the involvement of NAD(P) radical. This is not a widely-studied species, but it is known to react with molecular oxygen (forming superoxide) at close todiffusion-controlled rates (Halliwell and Gutteridge 1999), so its production on the cell surfacecould readily explain the extremely fast superoxide production observed by O’Donnell and Azzi (1996), which is otherwise highly paradoxical. The location labelled “Q ” in Figure 1 would possess a high reduction potential, but not necessarily high enough to preclude acceptance of anelectron from NAD(P)H, creating the required NAD(P) radical. Electrons would then be removedfrom Q by the Q cycle and eventually reduce oxygen at CNOX. It is not necessarily implausible that such a dangerous reaction might be permitted here, when one recalls that the extracellularmedium in vivo is almost devoid of NADH or NADPH. It may be straightforward to establish (e.g.
by electron paramagnetic resonance) whether NAD(P) radicals are present in such circumstances.
Finally, there must be some form of communication between the PMRS and the Na+/H+ antiporterin order to explain the large component of PMRS-associated proton extrusion that is amiloride-sensitive (Sun et al. 1995). The antiporter has inter-subunit disulphide bonds. There is evidencethat the redox state of these disulphides has a regulatory role (Ismailov et al. 1996), though theexistence of a family of such antiporters with varying tissue-specificity complicates interpretationat this point. The transferrin receptor also contains inter-subunit disulphide bonds (Rutledge et al.
1987), so the massive stimulation of proton efflux by diferric transferrin (Sun et al. 1988) mayresult from a similar interaction. If tNOX (tumour-associated NADH oxidase, Chueh et al. 2002) isa terminal electron transfer protein that augments CNOX in transformed cells, this would explainthe inhibition of proton as well as electron efflux in such cells by Q analogues (Sun et al. 1992).
The organisation suggested above also has the potential to explain a remarkable finding of Berridgeand Tan (2000): that the rate of superoxide production by intact cells (measured as SOD-inhibitablereduction of the tetrazolium WST-1, see Figure 1) is inversely related to oxygen tension. Thismight result if at low oxygen tension the plasma membrane Q pool, and hence Q , is highly reduced due to slow turnover of CNOX, allowing frequent formation of the NAD radical, which isprevented at high oxygen tension because CNOX keeps the Q pool adequately oxidised.
It must be stressed that the arrangement described above is intended as a minimal structure for thePMRS, which may be elaborated in many different ways in certain cell types. Perhaps the clearestexample of this is tumour cells, which, as noted above, have been found (Wilkinson et al. 1992) toexpress a capsaicin-sensitive protein, tNOX, with many functional similarities to CNOX. It wasnoted long ago (Warburg 1924) that tumour cells do not revert to an oxidative metabolism evenwhen released from the hypoxic environment of the tumour core; perhaps the additional action oftNOX helps to stimulate the PMRS to export more protons, which may be of physiological benefitto the tumour (e.g. as a defence against the immune system). Similarly, the startling finding thatVDAC1, the protein that forms the pore in the mitochondrial outer membrane, is also a plasmamembrane NADH-ferricyanide oxidoreductase (Baker et al. 2002) must be incorporated into atleast a version of this model, since the enzyme reported by Kim et al. (2002) is not VDAC1. Afurther embellishment may be required in order to accommodate the exceptional effects of retinoicacid in inhibiting the PMRS’s electron but not proton transport activities (Crane et al. 1991).
Finally, more information is urgently needed on the PMRS of intact untransformed mammaliancells; for example, several cell types, especially erythrocytes, express a PMRS whose maincytosolic electron donor is ascorbate and which does not involve NC5R (May and Qu 1999).
Conclusion
The study of a complex system in biology generally proceeds by three stages, not necessarily
entirely sequential: analysis of its overall “black-box” behaviour, molecular identification of its
components, and determination of how those components are juxtaposed and interact. Progress in
the first of these areas has been steady for over two decades; progress in the second has markedly
accelerated in recent years; the paucity of work in the third is, therefore, becoming increasingly
conspicuous. An attempt has been made here to provide a theoretical basis for future experimentalwork aimed at elucidating the overall structure of the PMRS. Tests of the model’s variouscomponents and predictions might falsify it altogether, but might instead serve to refine it into arobustly tested structure in the fairly near future.
The lack of a comprehensive model for the cell surface electron- and proton-translocatingmachinery is a considerable barrier to its study. While the proposal presented here is claimed onlyto be a first step towards such a model, it is hoped that it will form the basis for the relatively rapiddevelopment of a more detailed structure, and thereby lead to a better understanding of the role ofthe PMRS in cellular metabolism in general.
Acknowledgements
I am much indebted to Jim and Dorothy Morré, Mike Berridge and Alfons Lawen for their
comments on an early version of the model proposed here and for sharing pre-publication data that
contributed considerably to its development.
References
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Figure 1. Proposed minimal structure of the mammalian PMRS. For details, see text.
Abbreviations not used elsewhere in this paper: NAD• NAD radical; NFeOR NADH-ferricyanide
oxidoreductase; PMS phenazine methosulfate; WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium. Movement of “2H” within the membrane denotes movement of
QH and opposite movement of Q. Parentheses around O indicate low affinity. Sites denoted Q ,
Q are CoQ binding sites; other redox centres are denoted R , R .
Tables
Electron acceptor, inhibitor
Range of H+/e- ratios
References
Table 1. Summary of the effects of some inhibitors and acceptors on the electron- and proton-translocating activities of the PMRS.

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