A tightly bound quinone functions in the ubiquinone reaction sites of quinoprotein alcohol dehydrogenase of an acetic acid bacterium, Gluconobacter suboxydans

Article abstract of DOI:10.1271/bbb.80363

Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH₂) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q₂H₂) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q₂ reductase and Q₂H₂ oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH₂ oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH₂ in the low-affinity sites.

Full text of DOI:10.1271/bbb.80363

Biosci. Biotechnol. Biochem., 72 (10), 2723–2731, 2008
A Tightly Bound Quinone Functions in the Ubiquinone Reaction Sites
of Quinoprotein Alcohol Dehydrogenase of an Acetic Acid Bacterium,
Gluconobacter suboxydans
y
Kazunobu MATSUSHITA,1; Yoshiki KOBAYASHI,1 Mitsuhiro MIZUGUCHI,1
1
2
3
Hirohide TOYAMA,1; Osao ADACHI, Kimitoshi SAKAMOTO, and Hideto MIYOSHI
*
1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi, Yamaguchi 753-8515, Japan
2Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo,
Tokyo 113-0033, Japan
3Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
Received May 28, 2008; Accepted July 5, 2008; Online Publication, October 7, 2008
[doi:10.1271/bbb.80363]
Quinoprotein alcohol dehydrogenase (ADH) of acetic
acid bacteria is a membrane-bound enzyme that
functions as the primary dehydrogenase in the ethanol
oxidase respiratory chain. It consists of three subunits
and has a pyrroloquinoline quinone (PQQ) in the active
site and four heme c moieties as electron transfer
mediators. Of these, three heme c sites and a further site
have been found to be involved in ubiquinone (Q)
reduction and ubiquinol (QH2) oxidation respectively
(Matsushita et al., Biochim. Biophys. Acta, 1409, 154–
164 (1999)). In this study, it was found that ADH
solubilized and purified with dodecyl maltoside, but not
with Triton X-100, had a tightly bound Q, and thus
two different ADHs, one having the tightly bound Q
(Q-bound ADH) and Q-free ADH, could be obtained.
The Q-binding sites of both the ADHs were character-
ized using specific inhibitors, a substituted phenol PC16
(a Q analog inhibitor) and antimycin A. Based on the
inhibition kinetics of Q2 reductase and ubiquinol-2
(Q2H2) oxidase activities, it was suggested that there are
one and two PC16-binding sites in Q-bound ADH and
Q-free ADH respectively. On the other hand, with
antimycin A, only one binding site was found for Q2
reductase and Q2H2 oxidase activities, irrespective of
the presence of bound Q. These results suggest that
ADH has a high-affinity Q binding site (QH) besides low-
affinity Q reduction and QH2 oxidation sites, and that
the bound Q in the QH site is involved in the electron
transfer between heme c moieties and bulk Q or QH2 in
the low-affinity sites.
Key words: ubiquinone; quinoprotein; alcohol dehydro-
genase; acetic acid bacteria; electron trans-
fer in bacterial respiratory chain
Acetic acid bacteria are obligate aerobes that have
a characteristically active respiratory chain oxidizing
ethanol, several sugars, and sugar alcohols, that
branches into cytochrome bo3 ubiquinol oxidase and
cyanide-insensitive bypass oxidase at the ubiquinone
site.1–3) Acetic acid bacteria produce acetic acid from
ethanol by two sequential catalytic reactions of mem-
brane-bound alcohol dehydrogenase (ADH) and alde-
hyde dehydrogenase, and thus are well known as vinegar
producers. ADH, catalyzing the first step of acetic acid
production, is a quinohemoprotein-cytochrome c com-
plex bound to the periplasmic side of the cytoplasmic
membrane, and it functions as the primary dehydrogen-
ase in the ethanol oxidation respiratory chain, where it
oxidizes ethanol by transferring electrons to ubiquinone
(Q) embedded in the membrane phospholipids. ADH has
been purified from several strains of acetic acid bacteria
(see Ref. 4 for a review) and has been shown to consist
of subunits I, II, and III, as in case of the enzyme
of Gluconobacter suboxydans,5) or of only subunits I
and II, as ADH purified from Gluconoacetobacter
europeaus.6) Subunit I contains pyrroloquinoline qui-
none (PQQ) and a heme c moiety as the prosthetic
groups, and functions as the catalytic site for ethanol.5)
Subunit II contains three heme c moieties, and works as
the electron transfer mediator from subunit I to ubiq-
y
To whom correspondence should be addressed. Fax: +81-839-33-5859; E-mail: kazunobu@yamaguchi-u.ac.jp
Present address: Department of Bioscience and Biotechnology of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa
903-0123, Japan
*
Abbreviations: ADH, alcohol dehydrogenase; DM, n-dodecyl-ꢀ-D-maltoside; FR, ferricyanide; KPB, potassium phosphate buffer; OG, ꢀ-octyl-D-
glucopyranoside; PC-16, 2,6-dichloro-4-dicyanovinylphenol; PQQ, pyrroloquinoline quinone; Q, ubiquinone; QH2, ubiquinol; Qn, ubiquinone-n;
Q2H2, ubiquinol-2; QH, high affinity Q binding site; QL, low affinity quinol oxidation site; QN, low affinity quinone oxidation site

2724
K. MATSUSHITA et al.
Acetaldehyde
Ethanol
A
B
PQQ
H
Q
-
e
Q10
II
3
II 2
QN
QL
-
e
I
II1
FR
-
e
Q2
Q2H2
Q2
II2
QN
II3
-
e
-
e
QL
Q2H2
Q2
Fig. 1. Hypothetical Mode of Electron Transfer from PQQ to Ubiquinone and Q-Binding Sites in ADH Complex.
A, In intramolecular electron transfer model of ADH, ADH has PQQ in the catalytic site of subunit I for ethanol oxidation and four heme c
sites in subunit I (I) and subunit II (II1, II2, II3). In Q reduction site (QN, see ‘‘Discussion’’), electrons were transferred to bulk Q (Q2 in this case)
from PQQ via three heme c sites (I, II1, and II2) to generate QH2, whereas in QH2 oxidation site (QL), electrons were transferred to ferricyanide
(FR) from QH2 (Q2H2 in this case) via one heme c site (II3). B, In the model of Q-binding sites, heme c II2 site is located near QH site, where Q10
is present as a bound-Q. Bulk Q (Q2) or bulk QH2 (Q2H2) freely enters QN and QL sites respectively, which are located adjacent to each other,
and also near QH site. Heme c II3 site might be located near QL site.
uinone5) (see Fig. 1A). Subunit III exists freely in the
periplasmic space besides in ADH complex, and is
thought to work as a molecular chaperone.7)
oxidase,11) DsbB12) and a quinoprotein glucose dehy-
drogenase13) from Escherichia coli, have been studied
in terms of the tightly bound Q. Although, like these
enzymes, ADH of acetic acid bacteria is also a Q-
reacting enzyme, it is more complicated and interesting,
since it is thought to have multiple electron transfer
reactions from a two-electron donor (PQQ) to a one-
electron redox center (heme), from heme to heme, and
also from a one-electron redox center (heme) to a two-
electron acceptor (Q), and also has an additional Q-
reacting site (QH2 oxidation) besides usual Q reduction
site (see Fig. 1A).
In this study, ADH was purified with n-dodecyl-ꢀ-D-
maltoside (DM) instead of Triton X-100 which is known
usually to deplete bound-Q, and was found to have a
tightly bound Q. In order to determine whether the
bound-Q is involved in the intramolecular electron
transfer, the effects of bound-Q on Q reduction and QH2
oxidation activities of ADH were also investigated by
kinetic analysis of inhibition by specific inhibitors.
In G. suboxydans, ADH is constitutively expressed as
a major protein even if ethanol is not present in the
growth medium, and is thought to be required for the
non-energy generating cyanide-insensitive bypass oxi-
dase activity.8) ADH is present in two different forms,
active and inactive, and generation of the inactive form
depends on growth conditions.8) Inactive ADH, which
is convertible to active ADH in vivo and in vitro, has
10-fold lower Q reductase activity, although there is no
difference in subunit composition or prosthetic groups.
By kinetic study with native, reconstituted, and hybrid
ADHs, four heme c sites of ADH could be distinguished
as ferricyanide (FR)-reacting sites,5) and also as the sites
of different redox potentials.9) The data also suggest that
heme c in subunit I and two of the three heme c moieties
in subunit II are involved in intramolecular electron
transport from PQQ to Q5,9) (Fig. 1A). Furthermore,
ADH of acetic acid bacteria has been found to have novel
ubiquinol (QH2) oxidation activity, the site of which is
different from the Q reduction site10) (Fig. 1A). Thus
ADH appears to have two separate Q reaction sites: one
for Q reduction and another for QH2 oxidation.
The coupling mechanism of one-electron redox
centers (such as heme) to two-electron donor/acceptor
systems (such as Q) in the respiratory chain is a
challenging subject in electron transport systems. The
electron transfer reaction to Q appears to proceed via
two discrete univalent steps. In most Q-reacting redox
proteins, there appear to be two Q/QH2 binding sites,
one site stabilizing a ubisemiquinone radical, and the
other site in dynamic equilibrium with the Q pool. Thus
a number of Q-reacting proteins, such a cytochrome bo3
Materials and Methods
Materials. Ubiquinone-2 (Q2) and Ubiquinone-6, 8 or
10 (Q6, Q8, or Q10) were kindly supplied by Eizai
(Tokyo). Ubiquinol-2 (Q2H2) was prepared by the
method of Rieske.14) Substituted phenol 2,6-dichloro-4-
dicyanovinylphenol (PC-16) was the same inhibitor as
that used previously.15) DM and ꢀ-octyl-D-glucopyrano-
side (OG) were obtained from Calbiochem (San Diego,
CA). All other materials were of reagent grade and were
obtained from commercial sources.
Bacterial strains and growth conditions. G. suboxy-
dans IFO 12528 was maintained and cultivated as

Bound Quinone in Quinoprotein Alcohol Dehydrogenase
2725
described previously,5) and was used throughout the
experiment.
after addition of ethanol (final 10 mM) to a reaction
mixture containing McIlvaine buffer (pH 3.5, 5.0, or 7.0,
a mixture of 100 mM Na2HPO4 and 50 mM citrate), 1 mM
potassium FR, and enzyme solution at 25 ꢂC. One unit of
enzyme activity was defined as the oxidizing ability of
1 mmol of substrate per min. Q2 reductase activity was
also measured by following the decrease in absorbance
at 275 nm (12.25 mMꢁ1 cmꢁ1) in a reaction mixture
consisting of appropriate amounts of enzyme, McIlvaine
buffer (pH 5.0), 10 mM ethanol, 0.25% OG, and 3–30 mM
Q2 dissolved in dimethyl sulfoxide (final dimethyl
sulfide concentration, 0.2% v/v) at 25 ꢂC. One unit of
enzyme activity was defined as the amount of enzyme
oxidizing 1 mmol of ethanol per min. Ubiquinol:FR
oxidoreductase activity (Q2H2 oxidation activity) was
measured with the wavelength pair 273–298 nm, as
described previously.10) Reaction mixture contained
McIlvaine buffer (pH 5.0), 0.15% (w/v) OG, 5–15 mM
Q2H2, 250 mM potassium FR, and appropriate amounts of
enzyme. In this case, the buffer solution was prepared
with special care to remove traces of ethanol contami-
nated by the chemicals. One enzyme unit (oxidation of
Preparation of membrane fraction. Cells were har-
vested by centrifugation at 8;800 ꢀ g for 10 min and
washed twice with 10 mM potassium phosphate buffer
(KPB, pH 6.0). The washed cells were resuspended at
about 1 g of cells (wet weight) per 4 ml in 10 mM KPB
(pH 6.0), and passed twice through a French pressure cell
press (Spectronic Instrument, Rochester, NY) at 10,000
psi. After centrifugation at 8;800 ꢀ g for 10 min to re-
move intact cells, the supernatants were ultracentrifuged
at 86;000 ꢀ g for 90 min to obtain the membrane fraction.
Purification of Q-free and Q-bound ADHs. ADH was
purified by two different methods to obtain enzymes
having a tightly bound Q (Q-bound ADH) and, one not
having it (Q-free ADH). Q-free ADH was purified in the
presence of Triton X-100 by essentially the same method
as described previously.5) In order to obtain Q-bound
enzyme, ADH was purified in the presence of DM,
as described below. The membranes were suspended
to a final protein concentration of 15 mg/ml in 10 mM
KPB (pH 6.0), and DM was added to a final concen-
tration of 0.75%. The suspension was incubated on
ice for 90 min and ultra-centrifuged at 86;000 ꢀ g for
60 min. The supernatant obtained was dialyzed against
10-fold volumes of 5 mM KPB (pH 6.0) containing
0.015% DM 3 times overnight. The supernatant fluid
was applied onto a DEAE-Toyopearl column (about 1 ml
of bed volume per 5 mg of protein) which had been
equilibrated with the same buffer. After the column was
washed with 3 bed volumes of the same buffer containing
0.015% DM, the enzyme was eluted by a concave-type
gradient system. The gradient was made up of two-buffer
systems consisting of 5 bed volumes of 5 mM KPB
(pH 6.0) and 3 bed volumes of 50 mM KPB (pH 6.0), in
which both buffers contained 0.015% DM. The peak
having ADH activity appeared at the midpoint of the
gradient. This column chromatography was repeated
as necessary. Thus the peak fractions from DEAE-
Toyopearl column were collected and then dialyzed
against 5 mM acetate buffer (pH 5.0) containing 0.015%
DM. The dialyzed sample was applied onto a CM-
Toyopearl column (about 1 ml of bed volume per 5 mg of
protein applied) which had been equilibrated with the
same buffer. After it was washed with the same buffer,
subunit II was eluted with 100 mM acetate buffer (pH
5.0) containing 0.015% DM. Then ADH complex was
eluted at the mid-point of a linear gradient consisting of
4-bed volumes each of 100 and 400 mM acetate buffer
(pH 5.0), both containing 0.015% DM. At almost the end
of the gradient, subunit I/III complex was also eluted.
1 mmol of Q2H2 per min) was defined by a calculation
ꢁ1
using a millimolar extinction coefficient of 16.84 mM
.
An inhibition experiment with PC16 and antimycin A
was performed in the presence of PC16 or antimycin A
dissolved in DMSO in the assay mixture (final dimethyl
sulfoxide concentrations, 0.2% v/v and 0.6% v/v
respectively).
Reversed phase-HPLC analysis. Q was extracted from
enzyme with Q8 as the internal standard with 10-fold
volumes of ethanol/hexane (2:5). After 30 min of
incubation at 50 ꢂC, the mixture was centrifuged at
12,000 rpm for 10 min and the upper layer was collected.
Extraction was repeated 3 times. These extracted upper
layers were combined and dried, and dissolved in a
small volume of HPLC solvent. The extracted Q was
analyzed in a reversed phase-HPLC column by mon-
itoring it at 278 nm at the flow rate of 0.5 ml/min. The
HPLC solvent was ethanol/methanol/70% HClO4
(700:300:1, v/v/v) containing 0.05 M NaClO4. Q was
identified by comparing the retention times and the
absorption spectra measured simultaneously using a
photodiode array detector with standard Q, Q8, and Q10.
Protein content. Protein contents were determined by
amido black method.16) Bovine serum albumin was used
as the standard protein. The absorption at 630 nm was
taken with a Hitachi Model U-2000 spectrophotometer
(Tokyo).
Results
Enzyme assay. ADH activity was determined by three
different methods. Ferricyanide (FR) reductase activity
was measured spectrophotometrically by following the
Detection of bound-Q in ADHs purified in different
ways
To obtain insight into the Q reaction sites of ADH, we
first determined Q content of ADHs purified under
decrease in absorbance at 420 nm (1.01 mMꢁ1 cmꢁ1
)

2726
K. MATSUSHITA et al.
Retention time (min)
Fig. 2. HPLC Elution Profiles of Ubiquinone Extracted from ADHs.
Q was extracted from 2 nmol of DM-purified ADH (A) or Triton X-100-purified ADH (B), which was mixed with an internal standard Q8
(2 nmol), and then analyzed as described in ‘‘Materials and Methods.’’ The standard Q mixture (C), 2 nmol each of Q2, Q6, Q8, and Q10, was also
analyzed. Insert shows the absorption spectrum of the peak at a retention time of 26.2 (A) as observed by photodiode array detector.
buffer prepared with de-ionized water contained about
0.7 mol of Q10/mol of the enzyme. Thus the DM-
purified and Triton X-100-purified enzymes are regarded
as Q-bound and Q-free ADHs respectively.
Table 1. Ubiquinone Contents of Several Preparations of ADHs in
the Presence of DM
FR reductase
activity
(units/mg)
Q10 content
(mol of Q10/mol
of ADH)
Enzymea
Comparison of Q2 and FR reduction kinetics between
Q-free and Q-bound ADHs
b
b
ADH (DM)
ADH (DM/ultra-pure water)b
ADH (Triton)
238 ꢃ 7:4ð3Þ
0:69 ꢃ 0:05ð3Þ
248
300
0.94
0
In order to determine the effects of bound-Q on Q
reduction, the kinetics of Q2 reductase activity were
compared as between Q-free and Q-bound ADHs
(Table 2). Triton-purified ADH (leaving to contain
Triton X-100) exhibited somewhat different Km value
for Q2 from the enzyme the detergent included in the
original Triton-purified ADH of which was exchanged
with DM. Thus using the Q-free ADH dissolved in DM,
the Q2 reductase activity was compared with Q-bound
enzyme purified with DM. As for the results, bound-Q
itself appeared not to affect the affinity of Q2 (Km ¼ 3:5
vs. 6.4 mM) very much.
The effect of bound-Q on FR reductase activity was
also examined in this study. Four heme c sites of ADH
can be distinguished based on its FR reductase activity
using ethanol as an electron donor, which has different
optimum pHs depending on the site.5) The effect of
bound-Q on each heme c site was examined by kinetic
analysis of FR reductase activity. Only one heme c site,
II2 site (see Fig. 1), exhibited different kinetics; the
other sites, I, II1, and II3, did not (data not shown). The
Km value of II2 site for FR was found to be 0.022 and
0.12 mM in Q-free and Q-bound ADHs respectively
(Table 2), suggesting that FR reduction at the II2 site is
affected by the presence of bound-Q.
aADH (DM) and ADH (Triton) were purified in the presence of
dodecylmaltoside (DM) and Triton X-100 (Triton) respectively. ADH
(DM/ultra-pure water) was purified in the presence of DM using buffer
prepared with ultra-pure water.
bData are shown as standard deviations (ꢃ) and the experimental numbers in
parentheses. FR reductase activity was measured at pH 5.0.
different detergent and buffer conditions. Q was ex-
tracted with ethanol/hexane from several preparations
of purified ADHs, and was analyzed by reversed-phase
HPLC. As shown in Fig. 2, a compound from the extract
of DM-purified ADH detected at a retention time of
around 26 min was determined to be Q10 based on a
comparison of the retention times with standard Qs and
also with the absorption spectrum, while no Q10 was
detected in ADH purified with Triton X-100. The
presence of Q10 in ADH is consistent with the fact that
Q10 is the predominant Q species in Gluconobacter
sp.17) The Q10 content was calculated from the peak area
after correction based on the extraction yield of the
internal standard, Q8. As summarized in Table 1, DM-
purified ADH contained nearly 1.0 mol of Q10/mol of
the enzyme, when purification was done with a buffer
prepared with ultra-pure water, while one purified with a

Bound Quinone in Quinoprotein Alcohol Dehydrogenase
2727
Table 2. Kinetic Parameters of Q2 Reductase and FR Reductase (II2 site) Activities of Various ADHs
Q2 reductase
Vmax (U/mg)
FR reductase (II2 site)b
Enzymesa
Km (mM)
11.7
Km (mM)
n.d.c
Vmax (U/mg)
Triton-purified ADH
(ꢁBound Q, +Triton X-100)
Q-bound ADH
64.5
41.2
64.5
n.d.c
294
109
3.5
0.12
(+Bound Q, +DM)
Q-free ADH
6.4
0.022
(ꢁBound Q, +DM)
aTriton-purified ADH and Q-bound ADH were purified in the presence of Trition X-100 and DM respectively. Q-free ADH was prepared from the Triton-purified
enzyme by changing the detergent to DM, as described in ‘‘Materials and Methods.’’ The parentheses show the detergent and bound Q included in the enzymes.
bFR reductase activity was measured at pH 3.5, which is specific to II2 site activity (9).
cn.d., not determined.
Q reduction and QH2 oxidation activities of ADH and
their relationship
Q-bound ADH, while at higher concentrations, the
inhibition rate appeared to be not very different between
these enzymes (data not shown). Thus, bound-Q might
affect inhibitor binding, and so inhibition mode of PC16
was analyzed by Dixon plot (Figs. 4, 5). The modes of
inhibition are summarized in Table 3. As shown, in
PC16 titration of Q2 reductase activity, two competitive
inhibition curves were observed in Q-free ADH
(Fig. 4A), while only a single competitive inhibition
curve was observed in Q-bound ADH (Fig. 4C). In
PC16 titration of Q2H2 oxidase activity, two different
curves, representing competitive and non-competitive
inhibition, were observed in Q-free ADH (Fig. 4B), but
only a single line that could be regarded as non-
competitive inhibition was observed in Q-bound ADH
(Fig. 4D). Thus, it is suggested that a high-affinity
Q-binding site (QH, see Fig. 1B) overlaps with the high-
affinity PC16-binding site, and that the presence of
bound-Q prevents the binding of PC16 to the high
affinity site, so that one of two competitive inhibitions
with PC16 is missing. As can be seen in the Ki values,
PC16 appears to disturb Q2H2 binding more severely
than Q2 binding to QH site. The remaining inhibition of
PC16 might occur at a low-affinity Q reduction (QN)
site, but not at a low-affinity QH2 oxidation (QL) site,
because, in Q-bound ADH, PC16 inhibited Q2 reductase
competitively, and Q2H2 oxidase activity non-compet-
itively. On the other hand, inhibitor sensitivity of the Q2
reductase and Q2H2 oxidase activities against antimycin
A was not much different as between Q-free and
Q-bound ADHs, and the same inhibition mode, com-
petitive inhibition, was observed in the two enzymes
(Fig. 5). Thus antimycin A appears to bind to a site near
QN and also near QL, which are adjacent to each other,
and the binding appears to be affected a little by the
presence of bound-Q.
Studies of the structure-activity relationship of short-
chain Q and QH2 and of inhibitor sensitivity have
indicated that ADH of acetic acid bacteria has an
additional electron transfer ability, QH2 oxidation, as
well as Q reduction, and that the two reactions occur at
different sites in the enzyme.10) During enzyme activity
measurement, it was found that the reaction rates of
Q2 reductase and Q2H2 oxidase activities gradually
decreased in a time-dependent manner. Therefore, since
the Q reductase and QH2 oxidase activities can be
inhibited by the reaction products, Q2H2 and Q2
respectively, the inhibition kinetics of Q2 reductase
and Q2H2 oxidase activities were determined with
Triton-purified (Q-free) ADH. As shown in Fig. 3, Q2
reductase activity was inhibited non-competitively with
Q2H2 (Ki value, 27.5 mM), and the Q2H2 oxidase activity
was also done with Q2 in a non-competitive fashion
(Ki value, 10 mM). The Ki values were similar to the Km
values (8.4 mM for Q2 and 36 mM for Q2H2) of Q2
reductase and Q2H2 oxidase activities of ADH.10) Thus,
the inhibition might not be due to the product inhibi-
tion, but rather to the binding of Q2 and Q2H2 to
their respective Q reduction and QH2 oxidation sites,
which disturbs the Q2H2 oxidation and Q2 reduction
respectively.
Effect of inhibitors on Q-free and Q-bound ADHs
In order to determine the effects of bound-Q on Q2
reductase and Q2H2 oxidase activities of ADH, inhibitor
sensitivities of the activities were compared as between
Q-free and Q-bound ADHs. PC16 and antimycin A
were used as specific inhibitors because they have the
ability to inhibit Q reduction and QH2 oxidation
activities of Triton-purified ADH.10) It is noteworthy
that PC16 was originally found to be a competitive
inhibitor against short-chain quinol with cytochrome
bo3 enzyme.15) In those experiments, Q-free ADH was
prepared from Triton-purified ADH by changing the
detergent to DM to eliminate the effect of Triton on
kinetics as described above. At lower concentrations of
PC16, both Q2 reductase and Q2H2 oxidase activities
appeared to be more sensitive in Q-free ADH than in
Discussion
Quinoprotein ADH of acetic acid bacteria is a primary
dehydrogenase of the ethanol oxidase respiratory chain,
that transfers electrons from ethanol to membranous
bulk Q. Electron transfer perhaps occurs from PQQ at
the catalytic site to heme c in subunit I, and then to a

2728
K. MATSUSHITA et al.
A
100
80
60
40
20
Concentration (µM)
0
5
10
15
B
C
7.5 µM
7.5 µM
1/V
1/V
0.04
0.04
15 µM Q2
0.02
0.02
15 µM Q2H2
-30
-20
-10
0
10
-15
-10
-5
0
5
10
15
Q2H2 (µM)
Q2 (µM)
Fig. 3. Inhibition Mode of Q2 Reductase and Q2H2 Oxidase Activities by the Opposite Redox Q Species, Q2H2 and Q2 Respectively.
A, Q2 reductase activity ( ) was measured at pH 5.0, with 15 mM Q2 and several concentrations of Q2H2. Q2H2 oxidase activity ( ) was
measured at pH 5.0, with 15 mM Q2H2 and several concentrations of Q2. B, Q2 reductase activity was measured at pH 5.0, with 7.5 ( ) or 15 mM
(
) Q2 and several concentrations of Q2H2. Then specific activities obtained were plotted by Dixon plot. C, Q2H2 oxidase activity was measured
at pH 5.0, with 7.5 ( ) or 15 mM ( ) Q2H2 and several concentrations of Q2. Then specific activities obtained were plotted by Dixon plot.
heme c site in subunit II, then finally to bulk Q via
additional heme c within subunit II (Fig. 1). Besides Q
reduction, ADH also has a QH2 oxidation reaction that
occurs at a different site from Q reduction site. Thus, one
goal of this study was to elucidate the unique Q redox
reaction of ADH.
Q-binding site (QH) close to both the QL site and heme b
has been established by biochemical and spectroscopic
studies,19,20) and it has been shown that the QH site is
present in the periplasmic regions of helices I and II of
subunit I.21,22) In ADH, although the detailed localiza-
tion is not yet clear, the Q reacting sites are most
probably located at subunit II, because Q reduction
activity has been shown to require subunit II of ADH in
a reconstitution study of the activity of the subunits.5) In
addition, it was found that subunit II of ADH has a
tightly bound Q10 and is also cross-reacted with azido-
Q2 in a photo-affinity labeling experiment (data not
shown). It has also been speculated that the Q reduction
and QH2 oxidation sites are very close to either the third
or forth heme c site of subunit II (II2 or II3 site in
Fig. 1), since electrons extracted from ethanol at the
PQQ site can be transferred via heme c site I in subunit I
to heme c sites II1 and II2 in subunit II, and then to the Q
reduction site (QN), and that QH2 is oxidized at the QL
site by transferring electrons to FR via one heme c site
(II3 site) (see Fig. 1).
It was found that native ADH purified with DM, but
not with Triton X-100, had a tightly bound Q10, and this
suggests that ADH has two Q-binding sites, high- and
low-affinity ones; bound Q10 is located at the high-
affinity QH site and bulk Q2 is reduced at the low-affinity
site. Since ADH has been shown to have QH2 oxidation
activity at the site different from Q reduction site,10) it
should have two low affinity Q-reacting sites, QN and
QL, in which bulk Q and QH2 respectively react, in
addition to a single high affinity QH site (Fig. 1). The
reason that Q-free ADH, which lacks bound Q10, retains
sufficient electron transfer ability to bulk Q2 is most
likely that the reduction in bulk Q2 at QN site is
mediated by a tightly bound Q2 that occupies QH site
instead of the bound Q10. In E. coli cytochrome bo3
oxidase, in addition to the low-affinity QH2 oxidation
site (QL) in subunit II,18) the presence of a high-affinity
In addition, in this study, inhibition kinetics with
PC16 clearly indicated that there are two PC16-binding

Bound Quinone in Quinoprotein Alcohol Dehydrogenase
2729
1/V
B
1/V
A
3 µM
5 µM Q2H2
0.15
0.06
15 µM
0.10
0.04
10 µM Q2
0.06
0.02
0.04
0.02
1
-3
3
5
-5
-1
0
-60
-40
-20
0
20
40
60
80
100
-30
-20
-10
0
10
20
30
40
50
C
1/V
0.05
1/V
0.3
D
5 µM
5 µM
0.2
0.1
0.03
0.01
10 µM Q2
15 µM Q2H2
-20
-10
0
10
20
30
40
50
-50 -40 -30 -20 -10
0
10 20 30 40 50
PC16 (µM)
Fig. 4. Dixon Plots of Q2 Reductase and Q2H2 Oxidase Activities Inhibited by PC16.
Q2 reductase activities (A, C) and Q2H2 oxidase activities (B, D) were measured with Q-free ADH (A and B), Q-bound ADH (C and D), and
in the presence of several concentrations of PC16, as described in ‘‘Materials and Methods’’. Q2 reductase activities were measured with 3 mM (A)
or 5 mM (B) Q2
( ) and with 10 mM (A, B) Q2 ( ), and Q2H2 oxidase activities with 5 mM ( ) and 15 mM ( ) Q2H2 as the substrates.
sites in Q-free ADH, and further suggested that one of
the two PC16-binding sites overlaps or is identical with
the high-affinity site, QH, because competitive inhibition
at the high-affinity site by PC16 is missing in Q-bound
ADH. This effect of bound Q10 on PC16 inhibition
kinetics was observed in both Q2 reductase and Q2H2
oxidase activities. Therefore, together with the finding
that ADH has only one mol of bound Q10, the results
strongly suggest that one common bound Q10 at QH site
is involved in both Q reduction and QH2 oxidation
reactions (Fig. 1B). Additionally, since further compet-
itive inhibition with PC16 was observed in both Q-free
and Q-bound ADHs, PC16 appears also to bind to the
low-affinity QN and/or QL sites. In Q-bound ADH,
PC16 simply inhibits Q2 reduction competitively, while
Q2H2 oxidation probably does by non-competitive
fashion. Therefore, PC16 might bind to QN site only,
and might disturb Q2H2 oxidation reaction at QL site by
generating ADH-Q2H2-PC16 complex (ESI complex).
Similar inhibition with two Q-binding sites has also been
observed in succinate dehydrogenase (SDH) from
Saccharomyces cerevisiae, in which SDH is inhibited
by s-BDNP (2-sec-butyl-4,6-dinitrophenol).23) In this
case, s-BDNP inhibits SDH in two non-competitive
ways, where the two inhibitor-binding sites correspond
to QP (QH) site and QD (QN) site, and the higher Ki
value was remarkably increased by a mutation around
the QD site. Unlike PC16, antimycin A showed a single
competitive inhibition in both Q2 reductase and Q2H2
oxidase activities of Q-free and Q-bound ADHs,
suggesting that antimycin A binds at a single site. Such
a bulky compound might sterically hinder QN and QL
sites, which are perhaps adjacent to each other, as in
Fig. 1B. Such close proximity of QN and QL sites may
also be verified by the observation that Q2 reductase
activity is inhibited non-competitively by Q2H2, and
Q2H2 oxidase activity by Q2 (Fig. 3). Since the Ki
values for antimycin A were somewhat different
between Q-free ADH and Q-bound ADH, the binding
of antimycin A at the site might be affected by bound-Q.
FR reduction kinetics at II2 site were largely affected by
Q-binding, and so the results also suggest that QH site is
located near the heme c II2 site and is involved in the
intramolecular electron transfer at least between the
heme site and bulk Q at QN site (Fig. 1B).
Thus, in ADH, a tightly bound-Q in QH site may work
as an electron mediator between a one-electron redox
center (probably heme c II2 site) and a two-electron
acceptor (bulk Q) at the QN site, and also between a two-
electron donor (bulk QH2) and a one-electron redox
center (probably heme cII3 site) at the QL site (Fig. 1).
Although the physiological meaning of the tightly bound
Q in ADH is not clear, such a tightly bound Q has been
observed to protect the production of H2O2 during the
redox reaction in NADH-Q oxidoreductase (Ndi1) of
S. cerevisiae.24) Thus, in ADH, functioning in a vast,
rapid electron transfer for oxidative fermentation to
produce acetic acid, the tightly bound Q may also be

2730
K. MATSUSHITA et al.
1/V
B
1/V
A
0.12
5 µM
0.10
5 µM
0.08
0.08
0.04
10 µM Q2
10 µM Q2
0.04
0.02
-30
-20
-10
0
10
20
30
40
50
-50 -40 -30 -20 -10
0
10
20
30
40 50
1/V
D
1/V
C
5 µM
5 µM
0.12
0.10
0.06
0.02
0.08
0.04
15 µM Q2H2
15 µM Q2H2
-10
0
10
20
30
40
50
-20
-10
0
10
20
30
40
50
Antimycin A (
)
M
µ
Fig. 5. Dixon Plots of Q2 Reductase and Q2H2 Oxidase Activities Inhibited with Antimycin A.
Q2 reductase (A and B) and Q2H2 oxidase (C and D) activities were measured with Q-free ADH (A and C) and Q-bound ADH (B and D) in the
presence of several concentrations of antimycin A at pH 5.0 as described in ‘‘Materials and Methods.’’ Q2 reductase activities were measured
with 5 mM ( ) and 10 mM ( ) Q2, and Q2H2 oxidase activities with 5 mM ( ) and 15 mM Q2H2
( ) as the substrates.
Table 3. Modes of Inhibition and Apparent Ki Values of Several ADHs with PC16 and Antimycin A
PC16
Activities
Enzymes
Antimycin A
High affinity
Low affinity
Ki values (mM)a
Q2 reduction
Q-free ADH
Q-bound ADH
Comb (ꢄ7:5)
Comb (ꢄ34)
Comb (ꢄ25)
Comb (ꢄ15)
Comb (ꢄ16)
n.d.c
Q2H2 oxidation
Q-free ADH
Q-bound ADH
Comb (ꢄ1:5)
Nond (ꢄ30)
Comb (ꢄ34)
n.d.c
Nond (ꢄ25)
Comb (ꢄ4)
aApparent Ki values are shown in parentheses.
bCom, competitive inhibition
cn.d., not detected
dNon, non-competitive inhibition
important to keep the electron transfer safe by prevent-
ing the leakage of electrons to O2.
Ministry of Education, Culture, Sports, Science, and
Technology of Japan (to KM). We thank Dr. Mamoru
Yamada and Dr. Toshiharu Yakushi for helpful dis-
cussion.
To elucidate such structural and functional features,
and also to understand the physiological meaning of the
high- and low-affinity sites for Q in the ADH complex in
more detail, identification of ubisemiquinone radical and
structural data by X-ray diffraction are required. This is
now under investigation.
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This study was supported by a Grant-in-Acid for
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