Evidence for NQO1 and NQO2 catalyzed reduction of ortho-and para-quinone methides

Article abstract of DOI:10.3109/10715762.2013.847527

NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinones and thereby prevent generation of toxic radicals. Quinone methides (QMs) covalently react with cellular macromolecules to form DNA adducts and/or protein conjugates resulting in toxicity and carcinogenesis. Based on similar structural features of quinones and QMs, it is logical to assume that NQO1 and/or NQO2 could also catalyze the two-electron reduction of QMs. However, hitherto the reduction of QMs, as both endogenous and/or exogenous biological substrates, by either NQO1/NQO2 has never been demonstrated. Here we show for the first time that both NQO1 and NQO2 can catalyze the reduction of electrophilic ortho-/para-QMs. The involvement of the enzyme in the reduction of p-cresol quinone methide (PCQM) and o-cresol quinone methide (OCQM) was demonstrated by reappearance of NQO1/NQO2-FAD peak at 450 nm after addition of the QMs to the assay mixture. Further reduction of methides by NQO1/NQO2 was confirmed by analyzing the assay mixture by tandem mass spectrometry. Preliminary kinetic studies show that NQO2 is faster in reducing QMs than its homolog NQO1, and moreover, ortho-QMs are reduced faster than para-QMs. Enzyme-substrate docking studies showed results consistent with enzyme catalysis. Thus, NQO1/NQO2 can play a significant role in deactivation of QMs.

Full text of DOI:10.3109/10715762.2013.847527

Just Accepted by Free Radical Research
Evidence for NQO1 and NQO2 Catalyzed
Reduction of ortho- and para-Quinone Methides
Heidi R. Kucera, Merricka Livingstone,
Carlos G. Moscoso and Nilesh W. Gaikwad
10.3109/10715762.2013.847527
Abstract
NAD(P)H:quinone oxidoreductase (NQO1) and NRH:quinone
oxidoreductase 2 (NQO2) catalyze the two-electron reduction of
quinones and thereby prevent generation of toxic radicals. Quinone
methides (QM) covalently react with cellular macromolecules to
form DNA adducts and/or protein conjugates resulting in toxicity and
carcinogenesis. Based on similar structural features of quinones and
QM’s, it is logical to assume that NQO1 and/or NQO2 could also
catalyze the two-electron reduction of QM’s. However, hitherto the
reduction of QM’s, as both endogenous and/or exogenous biological
substrates, by either NQO1/NQO2 has never been demonstrated.
Here we show for the first time that both NQO1 and NQO2 can
catalyze the reduction of electrophilic ortho-/para-QM’s. The involve-
ment of the enzyme in the reduction of p-cresol-quinone-methide
(PCQM) and o-cresol-quinone-methide (OCQM) was demonstrated
by reappearance of NQO1/NQO2-FAD peak at 450 nm after addition
of the QM’s to the assay mixture. Further reduction of methides
by NQO1/NQO2 was confirmed by analyzing the assay mixture by
tandem mass spectrometry. Preliminary kinetic studies show that
NQO2 is faster in reducing QM’s than its homolog NQO1, and
moreover, ortho-QM’s are reduced faster than para-QM’s.
Enzyme-substrate docking studies showed results consistent with
enzyme catalysis. Thus, NQO1/NQO2 can play a significant role
in deactivation of QM’s.
© 2013 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and
full text (HTML) versions will be made available soon.
DISCLAIMER:The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or
the journal.The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained
in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be
administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care
professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted
articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published
in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the
Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.

Evidence for NQO1 and NQO2 Catalyzed
Reduction of ortho- and para-Quinone Methides
Heidi R. Kucera2, Merricka Livingstone4, Carlos G. Moscoso3 and Nilesh W. Gaikwad1,2*
1Department of Nutrition, 2Department of Environmental Toxicology, and 3Department of
Molecular and Cellular Biology, University of California, Davis, CA 95616, USA, 4Department
of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Short title: NQO1 and NQO2 are quinone methide reductase
Correspondence should be addressed to: Nilesh W. Gaikwad, Ph.D.
Departments of Nutrition and Environmental Toxicology, University of California,
One Sheilds Ave., Davis, CA, 95616, Tel.: (530) 752-5255, FAX: (530)-752-8966,
E-mail: ngaikwad@ucdavis.edu
1

Abstract
NAD(P)H:quinone
oxidoreductase
(NQO1)
and
NRH:quinone
oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinones and
thereby prevent generation of toxic radicals. Quinone methides (QM) covalently
react with cellular macromolecules to form DNA adducts and/or protein conjugates
resulting in toxicity and carcinogenesis. Based on similar structural features of
quinones and QM’s, it is logical to assume that NQO1 and/or NQO2 could also
catalyze the two-electron reduction of QM’s. However, hitherto the reduction of
QM’s, as both endogenous and/or exogenous biological substrates, by either
NQO1/NQO2 has never been demonstrated. Here we show for the first time that
both NQO1 and NQO2 can catalyze the reduction of electrophilic ortho-/para-QM’s.
The involvement of the enzyme in the reduction of p-cresol-quinone-methide
(PCQM) and o-cresol-quinone-methide (OCQM) was demonstrated by
reappearance of NQO1/NQO2-FAD peak at 450 nm after addition of the QM’s to
the assay mixture. Further reduction of methides by NQO1/NQO2 was confirmed by
analyzing the assay mixture by tandem mass spectrometry. Preliminary kinetic
studies show that NQO2 is faster in reducing QM’s than its homolog NQO1, and
moreover, ortho-QM’s are reduced faster than para-QM’s. Enzyme-substrate
docking studies showed results consistent with enzyme catalysis. Thus,
NQO1/NQO2 can play a significant role in deactivation of QM’s.
Key words: NQO1; NQO2; ortho-quinone methide; para-quinone methide; mass
spectrometry; enzyme kinetics; substrate binding
Abbreviations: NQO1, NAD(P)H:quinone oxidoreductase 1; BNAH,
benzyldihydronicotinamide riboside; NQO2, NRH:quinone oxidoreductase 2; NRH,
dihydronicotinamide riboside; PCQM, p-cresol quinone methide; OCQM, o-cresol
quinone methide; MD,Menadione
Introduction
NAD(P)H:quinone
oxidoreductase
(NQO1,
DT-diaphorase)
and
NRH:quinone oxidoreductase 2 (NQO2) enzymes have unique ability to catalyze
the two-electron reduction of quinones to hydroquinones, thereby prevents
generation of toxic semiquinone radicals, reactive oxygen species and alkylation [1-
5]. Recently expression levels or single nucleotide polymorphisms in NQO1 have
been shown to be associated with numerous cancers, viz. breast [6], prostate [7],
colorectal [8], esophageal [9], gastric [10], osteocarcinoma [11], skin [12], acute
myeloid leukemia (AML) [13] as well as cardiovascular diseases [14], Parkinson’s
disease [15] and diabetes [16]. Similarly, expression levels or single nucleotide
polymorphisms in NQO2 are shown to be associated with AML [13], breast [17],
prostate [18] and esophageal [9] cancer as well as cardiovascular diseases [14],
2

Parkinson’s disease [19] and schizophrenia [20]. Furthermore, both NQO1 and
NQO2 are proposed as strong prognostic and predictive indicators of breast cancer
[21]. NQO1 has been extensively studied since 1960 when it was first identified by
Ernster et al. and named as DT-diaphorase [22]. NQO2 was discovered by Liao and
Williams-Ashman in 1961, but its exact biological role is still being determined [23].
NQO1 and its homolog NQO2 are ubiquitous among various tissues, including liver,
kidney, brain, heart, and lung [24, 25]; in addition, many solid tumors including
thyroid, adrenal, breast, ovarian, cornea, colon, liver, and non-small cell lungs
cancers express NQO1. The high level of sequence homology between NQO1 and
NQO2 suggests that NQO2 may have overlapping substrate specificities and similar
functions to that of NQO1 [26, 27]. NQO2 is different from NQO1, however, in its
cofactor requirements, using a metabolic product of NAD(P)H, dihydronicotinamide
riboside (NRH), rather than NAD(P)H as an electron donor. Another major
difference between NQO2 and NQO1 is that NQO2 is resistant to typical inhibitors
of NQO1, such as dicumarol. The crystal structure analysis of NQO2 shows that it
contains a specific metal binding site that is not present in NQO1 [28, 29].
Gathering evidence suggest that in addition to two-electron reduction of quinone
compounds, NQO1 is involved in scavenging of superoxide anion radicals,
maintenance of endogenous antioxidants, 20S proteasome pathway and
stabilization of p53 protein [21]. Owing to their broad substrate specificity, NQO1
and NQO2 play an important role in the detoxification of various endogenous and
exogenous quinones [30-32], including estrogen quinones [33, 34] which is
consistent with their association with many diseases. However, hitherto the
reduction of quinone methides (QM), as both endogenous and exogenous biological
substrates, by either NQO1 or NQO2 has never been demonstrated.
Quinone methides are structurally analogous to quinones, with one of the
carbonyl groups replaced by a methylene group. Due to this, QM’s are more
polarized and reactive than quinones, with rearomatization being a key driving
force. QM’s are known to interact with biological macromolecules resulting in
formation of DNA adducts and/or protein conjugates (Fig. 1) [35-38]. Like quinones,
QM’s are also known to undergo redox-cycling and generate reactive oxygen
species. QM intermediates react with glutathione, resulting in glutathione depletion,
which can cause cell death. Previous studies with p-cresol, o-cresol, and tamoxifen
have demonstrated the enzyme-catalyzed formation of QM’s [36-38], which
covalently interact with DNA resulting in formation of DNA adducts. Due to their
cytotoxicity, molecules that have the QM moiety or that can produce QM
intermediates upon biological activation are being synthesized or extracted from
natural products and are tested as potential chemotherapeutic drugs against cancer
[39].
Taking into account the similar structural features of quinones and quinone
methides, it is logical to assume that NQO1 and/or NQO2 could also catalyze the
two electron reduction of endogenous or exogenous QM’s. In fact QM’s have never
3

been investigated as substrates for NQO1 and NQO2. In this study, we used
strategies to ascertain the role of NQO1 and NQO2 in catalyzing the reduction of
ortho- and para-quinone methides. Both o-cresol and p-cresol have minimal
structural features needed to form the basic backbone of ortho- and para-quinone
methide respectively, and hence they were used in this study. For the first time, we
have established that QM’s are biological substrates of NQO1 and NQO2.
EXPERIMENTAL PROCEDURES
NQO1 (expressed in E. coli, lyophilized powder), NQO2 (expressed in E.
coli), NADH, menadione (MD), p-cresol and o-cresol were purchased from Sigma
Chemical Co. (St. Louis, MO). BNAH was purchased from TCI America (Portland,
OR). Activated MnO2 was obtained from Aldrich Chemical Co. (Milwaukee, WI). All
solvents used were HPLC grade and all other chemicals were of the highest grade
available. p-cresol quinone methide (PCQM) and o-cresol quinone methide
(OCQM) were freshly synthesized using a reported procedure [36, 37]. The identity
of both quinone methides was confirmed by UV and MS/MS (Fig. 2).
Determination of NQO1 and NQO2 activity using UV-Vis spectroscope
The anaerobic reduction and oxidation of protein-bound FAD were
monitored at 450 nm in the absence and presence of the cofactor, and substrate
using a UV-Vis spectrophotometer (Evolution 300, Fisher Scientific). Nitrogen gas
was bubbled continuously for 2 h in the buffer, and organic solvents were degassed
to ensure anaerobic conditions. The reduction of protein-bound FAD was monitored
between 280 nm and 700 nm. First, the protein-bound FAD was reduced by NADH,
and then the reduced flavin was used to carry out the reduction of the substrate.
10U of NQO1 was added to 1mL buffer (25 mM Tris-HCl, pH 6.8) containing
0.7 mg/ml BSA in a 1.5 ml cuvette, and a spectrum was recorded. This was
followed by the addition of 0.9 eq NADH to the same cuvette, and then the
reoxidation of the protein-bound flavin was carried out by the addition of PCQM or
OCQM (1.2eq). Spectra were recorded after each addition. Exactly same procedure
was followed for NQO2 assay, except BNAH was used as the cofactor. The assay
mixtures were passed through 5000MW cutoff filters and directly injected into the
mass spectrometer for product analysis.
Control spectra were recorded separately of NADH (0.9 eq), BNAH (0.9 eq),
and 1.2 eq of PCQM and OCQM added at room temperature to 1mL buffer (25 mM
Tris-HCl, pH 6.8) containing 0.7 mg/ml BSA in a 1.5ml cuvette. As a control, to
determine whether p-cresol or o-cresol regenerates the protein-bound reduced
FAD, a separate assay was performed with the cresol isoforms as the substrates.
4

MS/MS analysis of quinone methide reduction
Xevo-TQ triple quadrupole mass spectrometer (Waters, Milford, MA, USA)
recorded MS/MS spectra using Electron Spray Ionization (ESI) in positive mode,
capillary voltage of 3.0 kV, extractor cone voltage of 3V and detector voltage of 500
V. Desolvation gas flow was maintained at 600L/h. Source temperature and
desolvation temperatures were set at 150 and 350° C, respectively. The collision
energy was varied to optimize daughter ions. The acquisition range was 20-500 Da.
The test samples, pure standards of ortho-/ para-cresol, both QM’s and the enzyme
assay mixtures, were introduced to the source at a flow rate of 5µl/min by using
methanol: water (1:1) and 0.1% formic acid mixture as the carrier solution and the
MS and MS/MS spectra were recorded.
Kinetic studies
Kinetic measurements were carried out at 25°C in 1 ml of 25 mM Tris-HCl,
pH 6.8, containing 0.7 mg/ml BSA in
a 1.5-ml cuvette using a UV
spectrophotometer. Reduction of the substrate, PCQM, OCQM and MD, by reduced
NQO1/NQO2 was assayed at 450 nm. In these cases, the kinetic studies were
carried out by assaying 0.4 μM NQO1/NQO2 at increasing concentrations of
PCQM, OCQM, MD. Km values were calculated with Origin software (Origin 8.5.0,
Northampton, MA).
Substrate docking study
Substrate docking was performed with SwissDock [40, 41] using PDB’s for
NQO1 (1D4A) [42] and NQO2 (1QR2) [28] along with PCQM (CID: 136328) [43]
and OCQM (CID: 13265823) [44] as inputs for docking. Zinc atoms were deleted
from NQO2 PDB. Only one subunit of NQO1 PDB was utilized for docking.
Solutions were chosen based on reported energy values and proximity to and
correct alignment of the substrate with the FAD isoalloxazine ring and orientation of
the alkene and N-5, based on previously reported crystal structure of menadione
with NQO2 (PDB 2QR2, [28]). Docking results were visualized with UCSF Chimera
[45].
Results
We have investigated the NQO1- and NQO2-catalyzed reduction of both
ortho- and para-quinone methides by using UV and tandem mass spectrometry.
Determination of methide reductase activity of NQO1 & NQO2
We took advantage of the ping-pong mechanism of enzyme catalysis [33,
34], and divided the assay into two steps. First, FAD-NQO1/ FAD-NQO2 were
reduced with either NADH or BNAH, and second, the reduced enzyme was further
used to carry out the reduction of the substrate. The reduction of methide
substrates by the NQO1/ NQO2 enzymes was studied by monitoring the redox
5

cycle of FAD in the active site of NQO1/ NQO2. The UV-Vis absorption spectrum of
native (oxidized) NQO1 is shown in Fig.3. The stoichiometry of cofactor, NADH was
adjusted to 0.9 eq to avoid spontaneous reduction of the substrate by excess
cofactor. Upon addition of NADH to the assay mixture containing NQO1 the FAD
absorption peak at 450 nm completely disappeared, due to its reduction to FADH2
(Fig. 3 Ab & Bb). Next, 1.2 eq of PCQM or OCQM was added to the assay mixture,
which caused the peak at 450 nm to reappear (Fig. 3 Ac & Bc). Excess PCQM and
OCQM (1.2 eq) was used in the assay so that it could outnumber any unreacted
NADH and sufficient molecules would be available for enzyme catalysis. Control
experiments were performed in which excess p-cresol or o-cresol was added to
assay mixture containing reduced NQO1 (Fig. 3 C & D). Addition of either p-cresol
or o-cresol did not result in reappearance of peak at 450 nm confirming that NQO1
used only methides as a substrates and reduction of methide was indeed catalyzed
by NQO1.
Similarly, The UV-Vis absorption spectrum of native (oxidized) NQO2 is
shown in Fig.4. The stoichiometry of cofactor, BNAH, was adjusted to 0.9 eq to
avoid spontaneous reduction of the substrate by excess cofactor. Upon addition of
BNAH to the assay mixture containing NQO2, the FAD absorption peak at 450 nm
completely disappeared, due to its reduction to FADH2 (Fig. 4 Ab & Bb). Next, 1.2
eq of PCQM and OCQM was added to the reaction mixture, which caused the peak
at 450 nm to reappear (Fig. 4 Ac & Bc). Like the NQO1 assay, excess PCQM and
OCQM (1.2 eq) was used in the NQO2 assay so that it could outnumber any
unreacted BNAH and sufficient molecules would be available for enzyme catalysis.
Control experiments were performed in which excess p-cresol or o-cresol was
added to assay mixture containing reduced NQO2 (Fig. 4 C & D). Addition of either
p-cresol or o-cresol did not result in reappearance of peak at 450 nm confirming
that NQO2 used only methides as a substrates and reduction of methide was
indeed catalyzed by NQO2.
Both these observations are consistent with the regeneration of FAD from
FADH2 after transfer of hydride. However, it is reasonable to assume that an
increase in the peak at 450 nm reports on the reduction of the substrate, because it
is the immediate product of hydride transfer from FADH2. At the completion of the
substrate reduction, the spectrum of FAD was completely restored in both NQO1
and NQO2 (Fig. 3&4).
Furthermore, to confirm the reduction of PCQM and OCQM by NQO1, the
assay mixtures were passed through a filter and subjected to MS and MS/MS
analysis. Assay mixture containing PCQM/OCQM and NQO1 showed the presence
of MS peak at 108.9 (Fig. 5B) and 109 (Fig. 5E) m/z respectively, which is
consistent with the M+1 of molecular mass of p-cresol or o-cresol (Fig. 2A & 2D).
Further parent peaks, 108.9 and 109 m/z, were fragmented resulting in daughter
ions at 92.7, 56.6 and 92.8, 56.3 m/z respectively (Figure 5B &5E). The
fragmentation patterns matched well with that of standard p-cresol or o-cresol. To
6

confirm the reduction of PCQM and OCQM by NQO2, the assay mixtures were
passed through a filter and subjected to MS and MS/MS analysis. Assay mixture
containing PCQM/OCQM and NQO2 showed the presence of MS peak at 108.8
and 109 m/z respectively, which is consistent with the M+1 of molecular mass of p-
cresol or o-cresol (Fig. 2A & 2D). Further parent peaks, 108.8 and 109 m/z, were
fragmented resulting in daughter ions at 93, 56.2 and 92.8, 56.4 m/z respectively
(Figure 5C &5F). The fragmentation patterns matched well with that of standard p-
cresol or o-cresol.
Kinetic studies
The Km values for reduction of PCQM, OCQM and MD were determined to
be 0.474, 0.242, and 0.403 μM respectively for NQO1 mediated catalysis and
0.242, 0.192, and 0.354 μM respectively for NQO2 mediated catalysis (Fig. 6). As
expected in enzyme catalyzed reaction, the graph of velocity against substrate
concentration was non-linear for all above assays (Fig. 6). When compared among
themselves the rates of the catalysis were following order NQO2-OCQM > NQO1-
OCQM > NQO2-PCQM > NQO2-MD > NQO1-MD > NQO1-PCQM.
Substrate docking study
The outputs from these four combinations appeared in proximity to the FAD
isoalloxazine ring, (Fig. 7), and were oriented to align the site of nucleophilic attack
(alkene) and N-5 of the FAD isoalloxazine ring. The chosen docking solutions had
NQO1 with OCQM had a total Energy = 13.66, ∆G = -5.49, and a distance of 1.87 Å
NQO1 with PCQM had a total Energy = 12.52, ∆G = -5.2, and a distance of 2.05 Å.
NQO2 with OCQM had a total Energy = 11.98, ∆G = -5.66, and a distance of 2.42
Å. NQO2 with PCQM had a total Energy = 10.55, ∆G = -5.48, and a distance of 4.71
Å. The crystal structure of NQO2 and MD (PDB 2QR2, [28]) had a distance of 4.23Å
(Table.1, Fig. 7).
In summary, UV-Vis and mass spectrometry as well as the docking studies
of the enzyme–substrate complex revealed the ability of NQO1 and NQO2 to
reduce the ortho- as well as para- quinone methide backbones. Kinetic studies
revealed that NQO1 and NQO2 reduced para-quinone methides slower than ortho-
quinone methides.
7

Discussion
For the first time, we have demonstrated the NQO1- and NQO2-catalyzed
reduction of both ortho- and para-quinone methides by using UV-Vis spectroscopy
and tandem mass spectroscopy. These studies were further supported by docking
studies of the enzyme–substrate complex. The NQO1 and NQO2 activity assays
were carried out in two stages. First, in the absence of oxygen, NQO1/NQO2 was
stoichiometrically reduced with NADH/BNAH, which transferred hydride to the FAD
bound to the enzyme (Fig. 8). It has been suggested that the negative charge
generated by hydride transfer is delocalized to the oxygen of the isoalloxazine of
FAD, which takes a proton from the neighboring amino acid, tyrosine. After hydride
transfer, the resulting NAD+ leaves and makes the binding pocket available for
substrate binding [46]. Fig. 3 & 4 reflect that the NQO1/NQO2 enzymes are readily
reduced by NADH and BNAH respectively, irrespective of a possible need for the
presence of the substrate as an activating effector. In the next step, hydride transfer
from reduced flavin to ortho-/para-quinone methide is carried out (Fig. 8). This
transfer is primarily dependent upon the correct orientation and approach of the C-
6/8 of the quinone methide, PCQM/OCQM, and N-5 of the isoalloxazine ring. The
reoxidation of FAD after addition of PCQM/OCQM, confirming the reduction of
PCQM/OCQM in the binding pocket of NQO1/NQO2, is shown in Fig. 3 & 4. After
this reduction, the resulting hydro-quinone methide tautomerizes to methylphenol,
p-cresol/o-cresol (Fig. 8) and the isoalloxazine of FAD returns to the oxidized
quinoid form. The regeneration of FAD implies that PCQM/OCQM has reached the
binding pocket and did not alkylate the binding site. It is possible that some part of
QM’s, due to its high reactivity, could covalently interact with minor portion of
NQO1/NQO2, rendering them inactive. However, assay conditions such as almost
instantaneous recording of the UV spectrum after addition of substrate keeps the
major portion of the enzyme intact, which is reflected in Mechalis –Menton kinetics.
Subsequent tandem mass spectrometry analysis of the reaction mixtures that were
used for UV assays were performed to investigate the catalytic products of NQO1
and NQO2 enzyme reductions. MS/MS results clearly demonstrates the presence
of p-cresol/o-cresol in the respective assay mixtures confirming the reduction of
PCQM/OCQM (Fig. 5).
To provide additional evidence for enzyme involvement in the reduction and
to understand the comparative activities of NQO1 and NQO2, we used the ping-
pong mechanism of enzyme catalysis [33, 34] to determine the Km values for
PCQM, OCQM, and MD. Our results suggest that NQO2-catalyzed reaction is
slightly faster for all three substrates, PCQM, OCQM, and MD, than NQO1 (Fig. 6).
The binding pockets of NQO1 and NQO2 are similar to each other with the
exception of three residues, residue # 126, 128, and 131, which allow the NQO2
binding pocket to be slightly bigger and hydrophobic than that of NQO1 [28, 47].
The larger size and hydrophobicity of the NQO2 binding site might explain the
8

higher affinity of all three substrates, (PCQM, OCQM, and MD) for NQO2 than for
NQO1. The substrate with the highest affinity for both enzymes was OCQM (Fig. 6),
which is not surprising because OCQM is more electrophilic than PCQM based on
strain in ring structure due to the ortho- positioned alkene. Both NQO1 and NQO2
reduced para-quinone methides slower than ortho-quinone methides. The para-
containing substrates, PCQM and MD, had similar affinities for NQO1 and NQO2.
The results show that NQO2 has a slightly higher affinity for PCQM than MD.
However, NQO1 appears to have a higher affinity for MD than PCQM (Fig. 5).
These results also suggest that for NQO2, QM’s appears to be better substrates
than quinones, whereas for NQO1 the distinction is not clear.
NQO1 and NQO2 both contain one FAD binding site per subunit [28, 42].
The FAD sits in the floor of the binding pocket, and the isoalloxazine ring of the FAD
is responsible for the stacking of planar aromatic substrates that insert into the
active site [48, 49]. The hydrophobic pocket along with π-stacking of the
isoalloxazine ring of NQO1 and NQO2 could provide a region with high affinity for
ligands such as PCQM and OCQM (Fig. 7) [19]. Docking studies show that both
PCQM and OCQM are aligned over the isoalloxazine ring in the binding pocket of
NQO1/NQO2, which is consistent with previous reports [14, 19]. There were no
direct contacts observed between the substrate and enzyme, following previous
reports in the menadione NQO2 crystal structure [28]. We chose docking outputs
that oriented the alkene group of both PCQM and OCQM near the N-5 of the
isoalloxazine ring in order to facilitate reduction by respective 1,6 [36] and 1,8 [50]
Michael’s addition reaction (Fig. 8). The optimal distance for direct hydride transfer
from the N-5 of the isoalloxazine ring of FAD to the substrate has been shown to be
within 10 Å [51]. For example, the distance between NADH and N-5 of NQO1 has
been previously reported to be 4.02 Å [19, 42] and the distance between MD and
the N-5 of NQO2 was found to be 4.23 Å (Table 1, Fig. 7, [28]). In the current study
the distance between PCQM and N-5 of NQO1 was found to be 4.71 Å, whereas it
was 1.87 Å in case of NQO2. For OCQM the distance was found to be 2.05 Å for
NQO1 and 2.42 Å in the case of NQO2. All these distances fall within needed 10 Å
range, suggesting that these docking solutions position the N-5 at a favorable
distance for hydride transfer (Table 1, Fig. 7). Additionally, a similar planar
orientation over the isoalloxazine ring π-system has been observed, though with
some slight deviations for some solutions (Fig. 7). Due to their reasonably wide
binding site, both NQO1 and NQO2 could accommodate structurally diverse
endogenous and exogenous quinone methides.
It has been well documented that alkyl phenol group present in endogenous
molecules, environmental toxicants, carcinogens and chemotherapeutic drugs could
undergo enzymatic oxidation by peroxidases and P450 enzymes to form transient
radical species, which can further oxidize to quinone methides [39]. Quinone
methides are known electrophilic species that covalently react with cellular
macromolecules to form DNA adducts and/or protein conjugates [35-39]. The
9

covalent binding of quinone methides have been shown to lead to various forms of
cytotoxicity, immunotoxicity, and carcinogenesis [35-39]. The UV spectra obtained
indicate that both NQO1 and NQO2 accept QM’s a substrate (Fig. 3&4). Our results
suggest that NQO1/NQO2 can catalyze reduction of a variety of both ortho- and
para-quinone methides and thereby provide protection against these reactive
species.
On the contrary, the distribution of NQO1 and NQO2 in many tumors could
have a potential impact on the development and efficacy of QM generating or
containing anticancer agents. Many solid tumors including thyroid, adrenal, breast,
ovarian, cornea, colon, liver, and non-small cell lungs cancers express the NQO1
gene [52- 53]. Expression of the NQO2 gene is high in normal human liver and
skeletal muscle [24, 56, 57], and minimal in the kidney, heart, pancreas, brain, and
red blood cells [58]. There is growing interest in the possible use of QM containing
molecules as promising chemotherapeutic drugs [39]. Recently, numerous synthetic
QM DNA alkylating agents that can form nucleobase adducts have been developed
as probable antitumor agents [39, 59]. The ability of NQO1 and NQO2 to reduce
QM’s may have significant implications against the efficacy of potential QM based
anticancer drugs currently under development.
In conclusion, ortho- and para-QM’s are substrates of NQO1 as well as
NQO2, and NQO1/NQO2 can play significant role in deactivation of ortho- and
para-QM’s that are formed from endogenous molecules, environmental toxicants,
and carcinogens that might lead to toxicity. These findings may also have broad
implications for the efficacy of potential QM based anticancer drugs that are
currently under development.
Acknowledgments
This research was supported by Hellman grant, University of California,
Davis and NIFA, USDA grant # CA-D-NTR-2104-H.
References
[1]
Dinkova-Kostova, A. T.; Talalay, P. NAD(P)H:quinone acceptor
oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and
exceptionally versatile cytoprotector. Archives of biochemistry and
biophysics 501:116-123; 2010.
[2]
[3]
Ross, D.; Siegel, D. NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-
diaphorase),
functions
and
pharmacogenetics.
Methods
in
enzymology 382:115-144; 2004.
Iyanagi, T. On the mechanisms of one- and two- electron transfer by
flavin enzymes. . Chem. Scr 27A:31-36; 1987.
10

[4]
[5]
[6]
Leung, K. K. K.; Litchfield, D. W.; Shilton, B. H. Flavin adenine
dinucleotide content of quinone reductase 2: Analysis and
optimization for structure–function studies. Analytical Biochemistry
420:84-89; 2012.
Dinkova-Kostova, A. T.; Talalay, P. Persuasive evidence that
quinone reductase type 1 (DT diaphorase) protects cells against the
toxicity of electrophiles and reactive forms of oxygen. Free Radical
Biology and Medicine 29:231-240; 2000.
Jamshidi, M.; Bartkova, J.; Greco, D.; Tommiska, J.; Fagerholm, R.;
Aittomaki, K.; Mattson, J.; Villman, K.; Vrtel, R.; Lukas, J.; Heikkila,
P.; Blomqvist, C.; Bartek, J.; Nevanlinna, H. NQO1 expression
correlates inversely with NFkappaB activation in human breast
cancer. Breast cancer research and treatment 132:955-968; 2012.
Stoehr, C. G.; Nolte, E.; Wach, S.; Wieland, W. F.; Hofstaedter, F.;
[7]
[8]
[9]
Hartmann, A.; Stoehr, R. NAD(P)H:Quinone Oxidoreductase
1
(NQO1) P187S Polymorphism and Prostate Cancer Risk in
Caucasians. International journal of molecular sciences 13:10959-
10969; 2012.
Chen, J.; Lin, Y.; Zhang, R.; Huang, Z. J.; Pan, X. G. Contribution of
NAD(P)H
quinone
oxidoreductase
1
(NQO1)
Pro187Ser
polymorphism and risk of colorectal adenoma and colorectal cancer
in Caucasians: a meta-analysis. Archives of medical research 43:58-
66; 2012.
Malik, M. A.; Zargar, S. A.; Mittal, B. Role of NQO1 609C>T and
NQO2 -3423G>A gene polymorphisms in esophageal cancer risk in
Kashmir valley and meta analysis. Molecular biology reports
39:9095-9104; 2012.
[10] Zhang, Y.; Wang, Z. T.; Zhong, J. Meta-analysis demonstrates that
the NAD(P)H: quinone oxidoreductase 1 (NQO1) gene 609 C>T
polymorphism is associated with increased gastric cancer risk in
Asians. Genetics and molecular research: GMR 11:2328-2337; 2012.
[11] Windsor, R. E.; Strauss, S. J.; Kallis, C.; Wood, N. E.; Whelan, J. S.
Germline genetic polymorphisms may influence chemotherapy
response and disease outcome in osteosarcoma: a pilot study.
Cancer 118:1856-1867; 2012.
[12] Patrick, B. A.; Jaiswal, A. K. Stress-induced NQO1 controls stability
of C/EBPalpha against 20S proteasomal degradation to regulate p63
expression with implications in protection against chemical-induced
skin cancer. Oncogene 31:4362-4371; 2012.
[13] Dunna NR, A. C., Vure S, Sailaja K, Surekha D, Raghunadharao D,
Rajappa
S,
Vishnupriya
S.
NQO1*2
[NAD(P)H:
quinone
11

oxidoreductase 1 polymorphism and its influence on acute leukemia
risk. Biology and Medicine 3:19-25; 2011.
[14] Zhu, H.; Li, Y. NAD(P)H: quinone oxidoreductase 1 and its potential
protective role in cardiovascular diseases and related conditions.
Cardiovascular toxicology 12:39-45; 2012.
[15] van Muiswinkel, F. L.; de Vos, R. A.; Bol, J. G.; Andringa, G.; Jansen
Steur, E. N.; Ross, D.; Siegel, D.; Drukarch, B. Expression of
NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian
substantia nigra. Neurobiology of aging 25:1253-1262; 2004.
[16] Ramprasath, T.; Murugan, P. S.; Kalaiarasan, E.; Gomathi, P.;
Rathinavel, A.; Selvam, G. S. Genetic association of Glutathione
peroxidase-1 (GPx-1) and NAD(P)H:Quinone Oxidoreductase
1(NQO1) variants and their association of CAD in patients with type-
2 diabetes. Molecular and cellular biochemistry 361:143-150; 2012.
[17] Hubackova, M.; Vaclavikova, R.; Ehrlichova, M.; Mrhalova, M.;
Kodet, R.; Kubackova, K.; Vrana, D.; Gut, I.; Soucek, P. Association
of superoxide dismutases and NAD(P)H quinone oxidoreductases
with prognosis of patients with breast carcinomas. International
journal of cancer. Journal international du cancer 130:338-348;
2012.
[18] Mandal, R. K.; Nissar, K.; Mittal, R. D. Genetic variants in
metabolizing genes NQO1, NQO2, MTHFR and risk of prostate
cancer:
a
study from North India. Molecular biology reports
39:11145-11152; 2012.
[19] Fu, Y.; Buryanovskyy, L.; Zhang, Z. Quinone reductase 2 is a
catechol quinone reductase. The Journal of biological chemistry
283:23829-23835; 2008.
[20] Harada, S.; Tachikawa, H.; Kawanishi, Y. A possible association
between an insertion/deletion polymorphism of the NQO2 gene and
schizophrenia. Psychiatric genetics 13:205-209; 2003.
[21] Fagerholm, R.; Hofstetter, B.; Tommiska, J.; Aaltonen, K.; Vrtel, R.;
Syrjakoski, K.; Kallioniemi, A.; Kilpivaara, O.; Mannermaa, A.;
Kosma, V. M.; Uusitupa, M.; Eskelinen, M.; Kataja, V.; Aittomaki, K.;
von Smitten, K.; Heikkila, P.; Lukas, J.; Holli, K.; Bartkova, J.;
Blomqvist, C.; Bartek, J.; Nevanlinna, H. NAD(P)H:quinone
oxidoreductase 1 NQO1*2 genotype (P187S) is a strong prognostic
and predictive factor in breast cancer. Nature genetics 40:844-853;
2008.
[22] Ernster, L.; Lindberg, O. Animal mitochondria. Annual review of
physiology 20:13-42; 1958.
12

[23] Liao, S.; Williams-Ashman, H. G. Enzymatic oxidation of some non-
phosphorylated derivatives of dihydronicotinamide. Biochemical and
biophysical research communications 4:208-213; 1961.
[24] Long, D. J., 2nd; Jaiswal, A. K. NRH:quinone oxidoreductase2
(NQO2). Chemico-biological interactions 129:99-112; 2000.
[25] Nosjean, O.; Nicolas, J. P.; Klupsch, F.; Delagrange, P.; Canet, E.;
Boutin, J. A. Comparative pharmacological studies of melatonin
receptors: MT1, MT2 and MT3/QR2. Tissue distribution of MT3/QR2.
Biochemical pharmacology 61:1369-1379; 2001.
[26] Jaiswal, A. K.; Burnett, P.; Adesnik, M.; McBride, O. W. Nucleotide
and deduced amino acid sequence of a human cDNA (NQO2)
corresponding to
a
second member of the NAD(P)H:quinone
oxidoreductase gene family. Extensive polymorphism at the NQO2
gene locus on chromosome 6. Biochemistry 29:1899-1906; 1990.
[27] Wu, K.; Knox, R.; Sun, X. Z.; Joseph, P.; Jaiswal, A. K.; Zhang, D.;
Deng, P. S.; Chen, S. Catalytic properties of NAD(P)H:quinone
oxidoreductase-2 (NQO2), a dihydronicotinamide riboside dependent
oxidoreductase. Archives of biochemistry and biophysics 347:221-
228; 1997.
[28] Foster, C. E.; Bianchet, M. A.; Talalay, P.; Zhao, Q.; Amzel, L. M.
Crystal structure of human quinone reductase type 2,
a
metalloflavoprotein. Biochemistry 38:9881-9886; 1999.
[29] AbuKhader, M.; Heap, J.; De Matteis, C.; Kellam, B.; Doughty, S. W.;
Minton, N.; Paoli, M. Binding of the anticancer prodrug CB1954 to
the activating enzyme NQO2 revealed by the crystal structure of
their complex. Journal of medicinal chemistry 48:7714-7719; 2005.
[30] Ross, D.; Siegel, D. NAD(P)H:Quinone Oxidoreductase 1 (NQO1,
DT-Diaphorase), Functions and Pharmacogenetics. In: Helmut, S.;
Lester, P., eds. Methods in enzymology: Academic Press; 2004: 115-
144.
[31] Kiyohara, C.; Yoshimasu, K.; Takayama, K.; Nakanishi, Y. NQO1,
MPO, and the risk of lung cancer: a HuGE review. Genetics in
medicine: official journal of the American College of Medical
Genetics 7:463-478; 2005.
[32] Bianco, N. R.; Chaplin, L. J.; Montano, M. M. Differential induction of
quinone reductase by phytoestrogens and protection against
oestrogen-induced DNA damage. The Biochemical journal 385:279-
287; 2005.
[33] Gaikwad, N. W.; Rogan, E. G.; Cavalieri, E. L. Evidence from ESI-
MS for NQO1-catalyzed reduction of estrogen ortho-quinones. Free
radical biology & medicine 43:1289-1298; 2007.
13

[34] Gaikwad, N. W.; Yang, L.; Rogan, E. G.; Cavalieri, E. L. Evidence for
NQO2-mediated reduction of the carcinogenic estrogen ortho-
quinones. Free radical biology & medicine 46:253-262; 2009.
[35] Thompson, D. C.; Thompson, J. A.; Sugumaran, M.; Moldeus, P.
Biological and toxicological consequences of quinone methide
formation. Chemico-biological interactions 86:129-162; 1993.
[36] Gaikwad, N. W.; Bodell, W. J. Formation of DNA adducts by
microsomal and peroxidase activation of p-cresol: role of quinone
methide in DNA adduct formation. Chemico-biological interactions
138:217-229; 2001.
[37] Gaikwad, N. W.; Bodell, W. J. Formation of DNA adducts in HL-60
cells treated with the toluene metabolite p-cresol: a potential
biomarker for toluene exposure. Chemico-biological interactions
145:149-158; 2003.
[38] Gaikwad, N. W.; Bodell, W. J. Peroxidase-mediated dealkylation of
tamoxifen, detected by electrospray ionization-mass spectrometry,
and activation to form DNA adducts. Free radical biology & medicine
52:340-347; 2012.
[39] Dufrasne, F.; Gelbcke, M.; Neve, J.; Kiss, R.; Kraus, J. L. Quinone
methides and their prodrugs: a subtle equilibrium between cancer
promotion, prevention, and cure. Current medicinal chemistry
18:3995-4011; 2011.
[40] Grosdidier, A.; Zoete, V.; Michielin, O. Fast docking using the
CHARMM force field with EADock DSS. Journal of computational
chemistry; 2011.
[41] Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a protein-small
molecule docking web service based on EADock DSS. Nucleic acids
research 39:W270-277; 2011.
[42] Faig, M.; Bianchet, M. A.; Talalay, P.; Chen, S.; Winski, S.; Ross, D.;
Amzel, L. M. Structures of recombinant human and mouse
NAD(P)H:quinone oxidoreductases: species comparison and
structural changes with substrate binding and release. Proceedings
of the National Academy of Sciences of the United States of America
97:3177-3182; 2000.
[43] National Center for Biotechnology Information. PubChem Compound
Database; CID=136328, h. p. n. n. n. g. s. s. c. c. l. e. r. a. N.,
2012).
[44] National Center for Biotechnology Information. PubChem Compound
Database; CID=13265823, h. p. n. n. n. g. s. s. c. c. l. e. r. a. N.,
2012).
[45] Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera--a
14

visualization system for exploratory research and analysis. Journal
of computational chemistry 25:1605-1612; 2004.
[46] Li, R.; Bianchet, M. A.; Talalay, P.; Amzel, L. M. The three-
dimensional structure of NAD(P)H:quinone reductase, a flavoprotein
involved in cancer chemoprotection and chemotherapy: mechanism
of the two-electron reduction. Proceedings of the National Academy
of Sciences of the United States of America 92:8846-8850; 1995.
[47] Vella, F.; Ferry, G.; Delagrange, P.; Boutin, J. A. NRH:quinone
reductase 2: an enzyme of surprises and mysteries. Biochemical
pharmacology 71:1-12; 2005.
[48] Calamini, B.; Santarsiero, B. D.; Boutin, J. A.; Mesecar, A. D.
Kinetic, thermodynamic and X-ray structural insights into the
interaction of melatonin and analogues with quinone reductase 2.
The Biochemical journal 413:81-91; 2008.
[49] Bianchet, M. A.; Erdemli, S. B.; Amzel, L. M. Structure, function, and
mechanism of cytosolic quinone reductases. Vitamins and hormones
78:63-84; 2008.
[50] Liu, Y.; Rokita, S. E. Inducible alkylation of DNA by a quinone
methide-peptide nucleic acid conjugate. Biochemistry 51:1020-1027;
2012.
[51] Basner, J. E.; Schwartz, S. D. Donor−Acceptor Distance and Protein
Promoting Vibration Coupling to Hydride Transfer:ꢀ A Possible
Mechanism for Kinetic Control in Isozymes of Human Lactate
Dehydrogenase. The Journal of Physical Chemistry B 108:444-451;
2003.
[52] Belinsky, M.; Jaiswal, A. K. NAD(P)H:quinone oxidoreductase1 (DT-
diaphorase) expression in normal and tumor tissues. Cancer
metastasis reviews 12:103-117; 1993.
[53] Joseph, P.; Xie, T.; Xu, Y.; Jaiswal, A. K. NAD(P)H:quinone
oxidoreductase1 (DT-diaphorase): expression, regulation, and role in
cancer. Oncology research 6:525-532; 1994.
[54] Jaiswal, A. K. Human NAD(P)H:quinone oxidoreductase2. Gene
structure, activity, and tissue-specific expression. The Journal of
biological chemistry 269:14502-14508; 1994.
[55] Siegel, D.; Ross, D. Immunodetection of NAD(P)H:quinone
oxidoreductase 1 (NQO1) in human tissues. Free Radical Biology
and Medicine 29:246-253; 2000.
[56] Jaiswal, A. K. Regulation of genes encoding NAD(P)H:quinone
oxidoreductases. Free radical biology & medicine 29:254-262; 2000.
[57] Strassburg, A.; Strassburg, C. P.; Manns, M. P.; Tukey, R. H.
Differential gene expression of NAD(P)H:quinone oxidoreductase
15

and NRH:quinone oxidoreductase in human hepatocellular and
biliary tissue. Molecular pharmacology 61:320-325; 2002.
[58] Graves, P. R.; Kwiek, J. J.; Fadden, P.; Ray, R.; Hardeman, K.;
Coley, A. M.; Foley, M.; Haystead, T. A. Discovery of novel targets
of quinoline drugs in the human purine binding proteome. Molecular
pharmacology 62:1364-1372; 2002.
[59] Zhou, Q.; Qu, Y.; Mangrum, J. B.; Wang, X. DNA alkylation with N-
methylquinolinium quinone methide to N2-dG adducts resulting in
extensive stops in primer extension with DNA polymerases and
subsequent suppression of GFP expression in A549 cells. Chemical
research in toxicology 24:402-411; 2011.
16

Figure Legends
Figure 1
Proposed biological mechanisms of quinone methide formation and toxic pathways.
17

Figure 2
ESI-MS/MS spectra of synthesized PCQM (A) and OCQM (B) substrates.
18

Figure 3
Spectral changes of NQO1 following addition of NADH and substrate, PCQM (A)
and OCQM (B). The absorption spectra were measured after sequential addition of
NQO1, NADH and substrate to 1 ml of 25 mM Tris-HCl, pH 6.8 at room
temperature. The concentration of the enzyme was 10 U NQO1 (spectra a). The
spectra after addition of 0.9 eq NADH (spectra b) and 1.2 eq substrate (spectra c),
in that order. The control spectra (C & D) were recorded by using p-cresol or o-
cresol as a substrates. The absorption spectra were measured after sequential
addition of 10 U NQO1 and 0.9 eq NADH to 1 ml of 25 mM Tris-HCl, pH 6.8 at room
temperature (spectra d). Addition of either 1.2 eq of p-cresol (C) or o-cresol (D) to
assay mixture did not cause reoxidation of NQO1 (spectra e).
19

Figure 4
Spectral changes of NQO2 following addition of BNAH and substrate, PCQM (A)
and OCQM (B). The absorption spectra were measured after sequential addition of
NQO2, BNAH and substrate to 1 ml of 25 mM Tris-HCl, pH 6.8 at room
temperature. The concentration of the enzyme was 10 U NQO1 (spectra a). The
spectra after addition of 0.9 eq BNAH (spectra b) and 1.2 eq substrate (spectra c),
in that order. The control spectra (C & D) were recorded by using p-cresol or o-
cresol as a substrates. The absorption spectra were measured after sequential
addition of 10 U NQO2 and 0.9 eq BNAH to 1 ml of 25 mM Tris-HCl, pH 6.8 at room
temperature (spectra d). Addition of either 1.2 eq of p-cresol (C) or o-cresol (D) to
assay mixture did not cause reoxidation of NQO2 (spectra e).
20

Figure 5
ESI-MS/MS spectra of p-cresol standard (A) and o-cresol standard (D). MS/MS
analysis of the assay mixture containing NQO1, NADH and PCQM/OCQM clearly
shows formation of p-cresol (B) and o-cresol (E) respectively. Similarly, MS/MS
analysis of the assay mixture containing NQO2, BNAH and PCQM/OCQM clearly
shows formation of p-cresol (C) and o-cresol (F) respectively.
21

Figure 6
Michaelis-Menten plots for the kinetics of NQO1 (A & B) and NQO2 (C & D)
catalysis. Reduction of PCQM (A), OCQM (B) and MD (E) by NQO1 enzyme and
reduction of PCQM (C), OCQM (D) and MD (F) by NQO2 enzyme, respectively.
22

Figure 7
Substrate binding with NQO1 (light grey) and NQO2 (dark grey). (A) PCQM
(orange) docking to NQO1. (B) OCQM (yellow) docking to NQO1. (C) PCQM
docking with NQO2. (D) OCQM docking to NQO2. (E) MD (green) bound to NQO2
(28). All substrate and enzymes docking was performed using Swissdock online
docking (11), and created using Chimera (45).
23

Figure 8
Proposed mechanism for reduction of quinone methides by NQO1 and NQO2.
24

Table Legend
Table 1
Energies of docking solutions and measured distances from FAD
Substrate
Total Energyα
NQO1
ΔGα
Distance from N-5(FAD)β
PCQM
OCQM
12.52
13.66
-5.20
-5.49
1.87 Å
2.05 Å
NQO2
PCQM
OCQM
MD
10.55
11.98
-5.48
-5.66
4.71 Å
2.42 Å
4.23 Å
Values calculated using SwissDockα and Chimeraβ for NQO1 and NQO2 with
PCQM, OCQM,or MD.
25