Unprecedented role of hydronaphthoquinone tautomers in biosynthesis

Article abstract of DOI:10.1002/anie.201404560

Quinones and hydroquinones are among the most common cellular cofactors, redox mediators, and natural products. Here, we report on the reduction of 2-hydroxynaphthoquinones to the stable 1,4-diketo tautomeric form of hydronaphthoquinones and their further reduction by fungal tetrahydroxynaphthalene reductase. The very high diastereomeric and enantiomeric excess, together with the high yield of cis-3,4-dihydroxy-1-tetralone, exclude an intermediary hydronaphthoquinone. Labeling experiments with NADPH and NADPD corroborated the formation of an unexpected 1,4-diketo tautomeric form of 2-hydroxyhydronaphthoquinone as a stable intermediate. Similar 1,4-diketo tautomers of hydronaphthoquinones were established as products of the NADPH-dependent enzymatic reduction of other 1,4-naphthoquinones, and as substrates for different members of the superfamily of short-chain dehydrogenases. We propose an essential role of hydroquinone diketo tautomers in biosynthesis and detoxification processes.

Full text of DOI:10.1002/anie.201404560

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DOI: 10.1002/anie.201404560
Biocatalysis
Hot Paper
Unprecedented Role of Hydronaphthoquinone Tautomers in
Biosynthesis**
Syed Masood Husain, Michael A. Schꢀtzle, Steffen Lꢁdeke, and Michael Mꢁller*
Abstract: Quinones and hydroquinones are among the most
common cellular cofactors, redox mediators, and natural
products. Here, we report on the reduction of 2-hydroxynaph-
thoquinones to the stable 1,4-diketo tautomeric form of
hydronaphthoquinones and their further reduction by fungal
tetrahydroxynaphthalene reductase. The very high diastereo-
meric and enantiomeric excess, together with the high yield of
cis-3,4-dihydroxy-1-tetralone, exclude an intermediary hydro-
naphthoquinone. Labeling experiments with NADPH and
NADPD corroborated the formation of an unexpected 1,4-
diketo tautomeric form of 2-hydroxyhydronaphthoquinone as
a stable intermediate. Similar 1,4-diketo tautomers of hydro-
naphthoquinones were established as products of the NADPH-
dependent enzymatic reduction of other 1,4-naphthoquinones,
and as substrates for different members of the superfamily of
short-chain dehydrogenases. We propose an essential role of
hydroquinone diketo tautomers in biosynthesis and detoxifi-
cation processes.
(Scheme 1). In 1985 when Wheeler and Stipanovic provided
a comprehensive summary of DHN melanin biosynthesis in
the fungus Wangiella dermatitidis, it became clear that fungal
Q
uinones and hydroquinones, together with their non-
oxidized precursors such as polyhydroxynaphthalenes, play
a central role as biosynthetic precursors for primary and
secondary metabolites and for polymers such as melanin.
Most fungal melanins and related metabolites are formed by
a polyketide route and are derived from the common
precursor molecule 1,8-dihydroxynaphthalene (DHN, 1).
The first known intermediate of the pathway is 1,3,6,8-
tetrahydroxynaphthalene (T4HN, 2). This metabolite is re-
duced by tetrahydroxynaphthalene reductase (T4HNR); the
product (R)-scytalone is then dehydrated to 1,3,8-trihydroxy-
naphthalene (T3HN, 3), which undergoes another reduction
to (R)-vermelone and dehydration to give DHN (1)
Scheme 1. Fungal DHN melanin biosynthesis (boxed) represents
a branching point towards several secondary metabolites (4–8).[1–6]
THT: trihydroxytetralone, DHT: dihydroxytetralone, T4HN: tetrahydroxy-
naphthalene, T4HNR: T4HN reductase, T3HN: trihydroxynaphthalene,
T3HNR: T3HN reductase, SD: scytalone dehydratase.
[*] Dr. S. M. Husain,[$] [+] Dr. M. A. Schꢀtzle,[+] Dr. S. Lꢁdeke,
Prof. Dr. M. Mꢁller
Institut fꢁr Pharmazeutische Wissenschaften
Albert-Ludwigs-Universitꢀt Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
E-mail: michael.mueller@pharmazie.uni-freiburg.de
[$] Present address: Centre of Biomedical Research
Raebareli Road, Lucknow 226 014, Uttar Pradesh (India)
[+] These authors contributed equally to this work.
DHN melanin is not formed by a straightforward pathway,
but rather a complex metabolic network.[1,2] Subsequently,
hundreds of metabolites, such as the dalmanols (e.g., 4),[3]
balticols (e.g., 5),[4] 3,4-dihydroxy-1-tetralones (e.g., 6),[1] 4-
hydroxy-1-tetralones (e.g., 7),[1] and spirodioxynaphthalenes
(e.g., 8),[5] have been shown to be derived from the
intermediate polyhydroxynaphthalenes 1–3. It has been
proposed that 4-hydroxy-1-tetralones and 3,4-dihydroxy-1-
tetralones are the products of the oxidation of polyhydroxy-
naphthalenes to naphthoquinones, followed by a double
reduction via hydronaphthoquinones.[1,2,6] Accordingly, it has
been suggested that the formation of 4-hydroxy-1-tetralone
derivatives represents a detoxification process.[1,2]
[**] Financial support of this work by the DFG (IRTG 1038) is gratefully
acknowledged. We thank O. Fuchs for technical support, V. Brecht
and S. Ferlaino for measurement of NMR spectra, Prof. A. Stolz,
University of Stuttgart, for providing NfsB,[15] and Prof. G. Fuchs,
University of Freiburg, for critically reading this paper. We
acknowledge the use of the computing resources provided by the
Black Forest Grid Initiative.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404560.
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Moreover, quinones are among the most common cellular
cofactors and natural products. From a traditional point of
view, “primary” quinones are widely distributed as electron
acceptors, while “secondary” quinoid metabolites play
a defense role by generating reactive oxygen species (ROS)
through redox cycling, which results in (nonspecific) super-
oxide toxicity. Redox cycling arises from the one-electron
reduction of the quinone to the activated semiquinone form
(toxification), or from the two-electron reduction to the
hydroquinone form, back-oxidation of which generates ROS.
Under the prevailing reduced intracellular redox state, two-
electron reduction normally leads to detoxification.[7,8] In
contrast, the producing cell in the “redox state”, which
constitutes all redox-active molecules, has to maintain cellular
homeostasis and the ability to deal with redox changes in
a highly regulated manner.[9] In this context, it has remained
unclear how organisms producing quinoid metabolites handle
potentially toxic intermediates and byproducts.[10,11]
We show herein that NADPH-dependent enzymatic
reduction of 2-hydroxynaphthoquinones, resulting in 3,4-
dihydroxy-1-tetralones, proceeds via the stable 1,4-diketo
tautomer of the hydronaphthoquinones. This is in contrast to
the well-known two-electron reduction of quinones resulting
in the respective hydroquinones. We propose that hydro-
naphthoquinone tautomers play an unprecedented and
essential role in the biosynthesis of many natural products
and are involved in breaking the redox cycle of quinones–
hydroquinones.
We recently reported the reduction of 2-hydroxy-1,4-
naphthoquinones to the corresponding cis-3,4-dihydroxy-1-
tetralones by the NADPH-dependent enzyme T4HNR from
the rice blast fungus Magnaporthe grisea.[12] This enzyme
plays an integral part in the biosynthesis of DHN melanin,
a virulence factor of many filamentous fungi.[13] To elucidate
the mechanism of these reactions, we employed lawsone (9)
as a model substrate (Scheme 2). Lawsone was reduced with
2 equivalents of NADPH to (3S,4R)-3,4-dihydroxy-1-tetra-
lone (10) by T4HNR with high diastereoselectivity (d.r.cis/trans
ꢀ 99:1), in high enantiomeric excess (> 99% ee), and in 90%
yield (see Scheme 2A and the Supporting Information).
In order to verify the involvement of hydronaphthoqui-
nones in such transformations, as proposed by Wheeler
et al.,[1,2] we synthesized the hydroquinoid 1,2,4-trihydroxy-
naphthalene (11) by reduction of lawsone (9) using sodium
dithionite, and applied it as a potential substrate of T4HNR.
The reaction was performed under anoxic conditions to
prevent back-oxidation of 11 to 9. Surprisingly, the expected
cis-ketodiol 10 was not formed, in contrast to the previous
proposal for analogous transformations.[1,2,6] Hence, the
putative two-step enzymatic formation of 10 from 2-hydroxy-
quinone 9 does not involve the hydroquinone 11.
To clarify the mechanism of the reduction of 9, we
performed deuterium-labeling experiments. The use of in situ
generated NADPD [[1-D]-d-glucose/glucose dehydrogenase
(GDH) cofactor regeneration system] resulted in double
incorporation of deuterium at positions 3 and 4 ([3,4-D2]-10,
Scheme 2B). In the inverse experiment, using NADPH
(d-glucose/GDH) in D2O buffer, no incorporation of deute-
rium at these positions occurred. This clearly demonstrates
that both hydrogen atoms are transferred in the enzymatic
reduction process using either NADPH or NADPD, rather
than being taken up from the solvent through keto–enol
tautomerism.
When the reaction was terminated before completion by
fast extraction of the reaction mixture, we observed hydro-
naphthoquinone 11 (by NMR spectroscopy) and, unexpect-
edly, the correspondding 1,4-diketo tautomer 12. Hydro-
naphthoquinone 11 is formed by aromatization of 12 and, as
expected, it was shown to be the product of redox cycling of 9.
Such redox cycling is generally considered to be the cause of
quinone toxicity;[14] here, the cycle is the result of catalytic
activity of crude extracts from E. coli, the host organism used
for heterologous expression (Scheme 2C).[15] In contrast, the
diketo tautomer 12 was formed only in the presence of
T4HNR and its surprising stability suggested that aromatiza-
tion proceeds sufficiently slow, allowing for draining the cycle
through subsequent reduction to ketodiol 10.
To prove formation of the supposedly reactive 1,4-diketo
tautomers of hydroquinones, we optimized the reaction
conditions in order to isolate sufficient amounts of the
putative intermediate 12 to be used as a substrate. Conversion
of 12 (synthesized by monoreduction of 9 with NADPH and
T4HNR), using NADPD and T4HNR under anoxic condi-
tions, resulted in the formation of the monodeuterated cis-
ketodiol [4-D1]-10 (Scheme 2D). Similarly, conversion of
[2-D1]-12 (synthesized by monoreduction of 9 with NADPD
and T4HNR), using NADPH and T4HNR under anoxic
conditions, resulted in the formation of the monodeuterated
cis-ketodiol [3-D1]-10 (see the Supporting Information).
Hence, our results suggest an unprecedented mechanism
for the conversion of 2-hydroxy-1,4-naphthoquinone into the
vicinal ketodiol. This pathway proceeds as follows: 1) initial
reduction of the 2-hydroxynaphthoquinone at the enolic
position generates the nonaromatic 1,4-diketo tautomer of
the hydronaphthoquinone, either by Michael addition of
hydride or by tautomerism to the 2-ketone before keto
reduction; 2) a second reduction at the benzylic position of
the 1,4-diketo tautomer results in the vicinal diol; and
3) partial aromatization of the 1,4-diketone forms the hydro-
Scheme 2. Overview of the reactions performed for elucidation of the
mechanism of vicinal cis-ketodiol formation by T4HNR. Reduction of
lawsone (9) with 2 equivalents of NADPH results in the vicinal cis-
ketodiol 10 (A), while reduction with 2 equivalents of NADPD leads to
double deuteration (B). Redox cycling of lawsone (9) is catalyzed by
crude extracts from E. coli (C), and by aromatization of intermediate
12. The reduction of 12 with NADPD yields [4-D1]-10 (D).
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naphthoquinone, whose back-oxidation to the quinone sub-
strate enables complete conversion. In contrast to the well-
established two-electron reduction of quinones resulting in
hydroquinones, themselves prone to (nonenzymatic) reoxi-
dation to the quinones, this new mechanism proceeds via
a two-step transformation with decisive consequences for this
redox cycle: although the first enzymatic reduction product,
the 1,4-diketo tautomer, can aromatize to the hydronaphtho-
quinone, alternatively it can be reduced in a second NADPH-
dependent enzymatic reduction, eventually resulting in
a drain of the quinone–hydroquinone compounds. If the
mechanism established for the T4HNR-catalyzed reduction of
2-hydroxynaphthoquinone 9 proves applicable to other
quinone–enzyme combinations, it would challenge the gen-
eral notion of quinone–hydroquinone transformations as the
only underlying equilibrium reactions.
The T4HNR-catalyzed reduction was next tested with
other related metabolites as substrates. The reduction of
flaviolin (13), a putative intermediate in the biosynthesis of
several monomeric and oligomeric polyketide metabolites, to
the naturally occurring cis-(3S,4R)-4-hydroxyscytalone (14)[12]
and isolation of the nonaromatic, monoreduced 1,4-diketo
tautomer 15 strongly suggests the general applicability of
these results for DHN melanin biosynthesis (Scheme 3A). In
addition, when lower enzyme concentrations were used,
naturally occurring (R)-5-hydroxyscytalone (16)[1,16] was
formed as a side product (Scheme 3B).
16 as the main product, flaviolin (13) was reduced in situ with
sodium dithionite under a nitrogen atmosphere. Subsequent
conversion with T4HNR gave 16 in 24% yield.
Tautomers are different isomers of “one” compound in
the same oxidation state: due to the mostly rapid intercon-
version, they are commonly considered to be the same
chemical compound. The formation of tetralones 14 and 16
nicely demonstrates that different tautomers can be regio-
and stereoselectively reduced by one and the same enzyme.
This not only corroborates our proposed new mechanism, but
also sheds light on the diversity-oriented biosynthesis of
melanins: here, starting from one substrate, namely flaviolin
(13), the three distinctly different products 14, 15, and 16 are
regio- and enantioselectively formed by the action of one
enzyme.
Our results also provide insight into the role of 4-hydroxy-
1-tetralones, another group of metabolites observed in the
DHN melanin pathway. It has been suggested that such
compounds are the products of hydronaphthoquinone reduc-
tion by polyhydroxynaphthalene reductases.[1,2,6] However,
the 1,4-diketo tautomers, and not the hydronaphthoquinones,
might just as well represent the basic substrates for keto
reduction, even though the course of their generation in
biosynthesis remains unknown. Accordingly, we tested chemi-
cally synthesized 1,4-diketo tautomers of the hydronaphtho-
quinones as putative substrates of T4HNR. 2,3-Dihydronaph-
thalene-1,4-dione (20a) was indeed reduced by T4HNR (21%
yield) to (S)-4-hydroxy-1-tetralone (21) (Figure 1). (S)-4,8-
Compound 16 represents the product of the reduction of
the tautomeric forms 17 and 18 of flaviolin hydroquinone
(19), either by Michael addition of hydride to the 1-ketone 17
or by hydride addition to the 2-ketone 18. Again, the
hydroquinone 19 is formed by aromatization of 15 and/or
quinone reduction by crude extracts from E. coli. Moreover,
the phenolic and ketonic forms are in equilibrium, as
demonstrated by the reduction to 16, and back-oxidation to
13 competes with enzymatic reduction. Therefore, to obtain
Figure 1. Reduction of the 1,4-diketo tautomers of hydronaphthoqui-
nones. 2,3-Dihydronaphthalene-1,4-diones (20) were reduced by
T4HNR and GDH leading to 4-hydroxy-1-tetralones 7 and 21. n.d.: not
determined.
Dihydroxy-1-tetralone [(S)-7] was obtained by T4HNR-cata-
lyzed reduction (9% yield) of 5-hydroxy-2,3-dihydronaph-
thalene-1,4-dione (20b). Again, in both cases, the respective
hydronaphthoquinone showed no conversion, but was
observed as a side product. Thus, it can be assumed that the
installation of a 1,4-diketo-tautomer-forming process results
in complete conversion, as observed for the vicinal ketodiols
10 and 14.
The results demonstrate that DHN melanin biosynthesis
constitutes a diversity-oriented metabolic network, compris-
ing vicinal ketodiol and 4-hydroxy-1-tetralone biosyntheses as
branching points. Presumably, polyhydroxynaphthalene
reductases are involved in promiscuous reduction reactions,
reflecting the idea of a matrix biosynthetic pathway.[17,18]
Scheme 3. Enzymatic reduction of flaviolin (13) by T4HNR. A) Reduc-
tion of 13 by T4HNR results in the vicinal cis-ketodiol cis-(3S,4R)-14.[12]
B) As a side product, (R)-5-HS (16) was observed, the product of
reduction of the tautomeric forms of hydroquinone 19. The final
products are highlighted with gray boxes. HS: hydroxyscytalone, PHN:
pentahydroxynaphthalene.
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These results directed us towards a group of fungal
secondary metabolites with a great variety of biological
activities: namely, the spirodioxynaphthalenes, in which
naphthalene and dihydronaphthalene units are bridged by
a spiroketal linkage (Figure 2A). The monomers putatively
are also involved in the formation of these derivatives and we
tested 2,3-epoxy-1,4-naphthoquinones as substrates of
T4HNR.
Due to partial epoxide opening by crude extracts from
E. coli, His-tagged and purified T4HNR-his was used in these
experiments. 2,3-Epoxy-1,4-naphthoquinone (26a) was re-
duced by T4HNR-his to trans-(2S,3S,4R)-27a in 74% yield,
whereas menadione epoxide (26b) showed only a poor
conversion (< 10%, mainly cis-27b, Figure 2B). Hence, the
present work suggests also the involvement of polyhydroxy-
naphthalene reductases in spirodioxynaphthalene biosynthe-
sis; the details of which metabolic steps occur before and after
formation of the spiroketal linkage are yet to be elaborated.
T4HNR is a member of the short-chain dehydrogenases/
reductases (SDR) enzyme family, which constitutes one of the
largest enzyme superfamilies.[19] We hypothesized that other
SDR should behave in a similar manner to T4HNR, thus being
able to reduce carbonyls, aromatic hydroxyls (e.g., poly-
hydroxynaphthalenes), 2-hydroxyquinones, and/or 1,4-diketo
tautomers of hydroquinones. This view was strongly sup-
ported by the serendipitous observation that glucose dehy-
drogenase (GDH) indeed showed activity for (2-hydroxy)-
1,4-diketones: when glucose/GDH was used as a regeneration
system for NADPH in the T4HNR-catalyzed reduction of 12
(see Scheme 2), the ketodiol 10 was obtained as a mixture of
diastereomers (d.r.cis/trans ꢁ 92:8). This lower diastereoselec-
tivity is due to a background reaction caused by GDH. When
the cofactor regeneration system was changed to l-malic acid/
malic enzyme, the d.r. was restored to d.r.cis/trans > 99:1 (see
above), clearly showing that 12 is a substrate for GDH.
In addition, reduction of 20a by GDH gave 21 in 14%
yield, while 20b as substrate gave virtually racemic 7 in 26%
yield (Figure 1).[20] GDH also reduced epoxyquinone 26a,
leading to cis-(2S,3S,4S)-27a in 64% yield. Menadione
epoxide (26b) was reduced by GDH to cis-(2S,3S,4S)-27b in
40% yield, along with cis-(2S,3S,4S)-27c in 9% yield
(Figure 2B).
Previously, it was shown that 17b-hydroxysteroid dehy-
drogenase from Cochliobolus lunatus (a homologue of
T4HNR) and T3HNR catalyze reductions of polyhydroxy-
naphthalenes with a high degree of regio- and stereocon-
trol.[20] Due to their close relationship to T4HNR we propose
that these and other SDR, such as GDH, will behave in
a diversity-oriented manner similar to T4HNR, resulting, for
instance, in cis- and trans-3,4-dihydroxy-1-tetralones.
The quinone–hydroquinone equilibrium is of utmost
importance for many biological processes. The selective
formation of cis-3,4-dihydroxy-1-tetralones in the T4HNR-
catalyzed reduction of 2-hydroxyquinones challenged the
generally accepted mechanism of NADPH-dependent qui-
none reductions. The elucidation of the mechanism of the
enzymatic reduction of the model substrate lawsone (9)
resulted in the identification and isolation of the unexpectedly
stable 1,4-diketo tautomer 12 of hydronaphthoquinone 11.
The involvement of such 1,4-diketo tautomers in enzymatic
redox reactions of quinones was subsequently demonstrated
for several compounds. The stability of the 1,4-diketo
tautomers was sufficient for their isolation and application
in asymmetric transformations. In addition to obvious bio-
Figure 2. Reduction of 2,3-epoxy-1,4-naphthoquinones. Spirodioxynaph-
thalenes often exhibit a 2,3-epoxy-4-hydroxy-1-tetralone subunit[5] (A)
which can be obtained by reduction of the corresponding 2,3-epoxy-
1,4-naphthoquinone (B). [a] The absolute configuration was deter-
mined by VCD spectroscopy.
originate from fungal DHN melanin biosynthesis and often
resemble a highly variable 1,4-naphthoquinone-derived back-
bone, such as in palmarumycin CP2 (8) (see Scheme 1), CJ-
12,371 (22), palmarumycin C2 (23), bipendensin (24), and
preussomerin C (25). The generation of different oxidation
states has been proposed to occur after formation of the
spiroketal linkage of two DHN units by oxidative phenol
coupling.[5] However, our findings from T4HNR-catalyzed
reductions show that this heterogeneity may well be intro-
duced at the monomer stage, prior to formation of the
spiroketal linkage by acetalization (see, for example, the 1,4-
diketonic substructures in 8 and 25, and the 4-hydroxy-1-
tetralone substructure in 22). In addition, a variety of
spirodioxynaphthalenes show a highly variable 2,3-epoxy-4-
hydroxy-1-tetralone-derived backbone (e.g., 24). Accord-
ingly, we assumed that polyhydroxynaphthalene reductases
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catalytic and nonenzymatic[21–23] transformations,[24] these
results may have major implications for the biosynthesis and
the physiological role of primary and secondary quinone
metabolites.
Our findings contribute to the understanding of melanin
biosynthesis. In the “classical” DHN melanin pathway, the
equilibrium of the phenolic and ketonic tautomers facilitates
the chemoenzymatic reduction.[25] In contrast, we have
demonstrated that 1,4-diketonic tautomers represent true
intermediates of reduction and not of tautomerism of the
hydronaphthoquinones. This represents a new unifying con-
cept for the fungal metabolism of polyhydroxynaphthalenes,
which is achieved simply by enzyme promiscuity.
Received: May 1, 2014
Published online: July 22, 2014
Keywords: dearomatization · diversity-oriented synthesis ·
.
enzyme catalysis · hydroquinones · reaction mechanisms
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The results presented herein might have a broader
significance regarding quinoid pathways. Vicinal ketodiols
and 4-hydroxy-1-tetralones, such as 7,[1,6,26] 21,[27] catalpo-
nol,[28] isoshinanolone,[29] and juglanoside A,[27] are common
among fungi, but have also been isolated from plants. We
postulate that diketo tautomers often do not represent the
product of tautomerism, but rather emerge as true inter-
mediates in biosynthesis. Accordingly, a 1,4-diketo tautomer
has been described in actinorhodin biosynthesis,[30] and
diketonic dihydrofusarubins have been suggested as repre-
senting the physiological metabolites.[31] Other diketones of
reduced pyranonaphthoquinone antibiotics have been iso-
lated[32] and the leuco forms of anthrahydroquinones have
been described as products of enzymatic quinone reduc-
tion.[33,34] For menaquinone biosynthesis, two metabolic steps
have even been proposed to proceed via diketo tautomeric
forms.[35,36] We have shown that the keto tautomers of emodin
hydroquinone represent the true substrates for enzymatic
reduction by a T4HNR homologous enzyme in the biosyn-
thesis of monodictyphenone; the hydroquinone was not
observed.[37]
Finally, the generation and reduction of 1,4-diketo tau-
tomers of hydronaphthoquinones represents a strategy to
bypass potentially toxic byproducts in biosynthesis. The redox
cycling of (undirected) quinoid metabolites such as 1,4-
naphthoquinones and 2-hydroxy-1,4-naphthoquinones as
byproducts in biosyntheses poses a potential risk to the
producing cell. It has been suggested previously that the
formation of 4-hydroxytetralone derivatives represents
a detoxification process.[1,2] Hence, our finding of the reduc-
tion of nonaromatic hydroquinone tautomers indicates a pos-
sibility to break the redox cycle. Moreover, increasing
evidence suggests that these “secondary” metabolites also
directly influence redox signaling; for example, stress horm-
esis of naphthoquinones has been described recently.[38] In all
cases, upon accumulation of the metabolites, indirect effects
occur by changes in the glutathione/oxidized glutathione and
NAD(P)H/NAD(P)+ ratios, as well as by a direct action of
reactive oxygen species. However, redox cycling of lawsone
(9) and of anticancer drugs, such as b-lapachone, cannot be
arrested in the hydroquinone state by two-electron reduction
via DT-diaphorase [NAD(P)H:quinone reductase].[7,8] There-
fore, we conclude that diketo tautomers emerge as true
intermediates in biosynthesis and that their formation breaks
the (redox) cycle, thus protecting the cell from stress-related
redox events.
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