(Received for publication, June 5, 1995; and in revised form, November 9, 1995)
From the
Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as
the primary dehydrogenase of the ethanol oxidase respiratory chain,
where it donates electrons to ubiquinone. ADH is a membrane-bound
quinohemoprotein-cytochrome c complex which consists of
subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several
hemes c as well as pyrroloquinoline quinone as prosthetic
groups. To understand the role of the heme c moieties in the
intramolecular electron transport and the ubiquinone reduction, the ADH
complex of Gluconobacter suboxydans was separated into a
subunit I/III complex and subunit II, then reconstituted into the
complex. The subunit I/III complex, probably subunit I, contained 1 mol
each of pyrroloquinoline quinone and heme c and exhibited
significant ferricyanide reductase, but no Q reductase
activities. Subunit II was a triheme cytochrome c and had no
enzyme activity, but it enabled the subunit I/III complex to reproduce
the Q
and ferricyanide reductase activities. Hybrid ADH
consisting of the subunit I/III complex of G. suboxydans ADH
and subunit II of Acetobacter aceti ADH was constructed and it
had showed a significant Q
reductase activity, indicating
that subunit II has a ubiquinone-binding site. Inactive ADH from G.
suboxydans exhibiting only 10% of the Q
and
ferricyanide reductase activities of the active enzyme has been
isolated separately from active ADH (Matsushita, K., Yakushi, T.,
Takaki, Y., Toyama, H., and Adachi, O(1995) J. Bacteriol. 177,
6552-6559). Using these active and inactive ADHs and also
isolated subunit I/III complex, we performed kinetic studies which
suggested that ADH contains four ferricyanide-reacting sites, one of
which was detected in subunit I and the others in subunit II. One of
the three ferricyanide-reacting sites in subunit II was defective in
inactive ADH. The ferricyanide-reacting site remained inactive even
after alkali treatment of inactive ADH and also after reconstituting
the ADH complex from the subunits, in contrast to the restoration of
Q
reductase activity and the other ferricyanide reductase
activities. Thus, the data suggested that the heme c in
subunit I and two of the three heme c moieties in subunit II
are involved in the intramolecular electron transport of ADH into
ubiquinone, where one of the two heme c sites may work at, or
close to, the ubiquinone-reacting site and another between that and the
heme c site in subunit I. The remaining heme c moiety
in subunit II may have a function other than the electron transfer from
ethanol to ubiquinone in ADH.
Alcohol dehydrogenase (ADH) ()of acetic acid
bacteria, consisting of the genera Acetobacter and Gluconobacter, catalyzes the first step of acetic acid
production, oxidation of ethanol to acetaldehyde. ADH is a
quinohemoprotein-cytochrome c complex bound to the periplasmic
side of the cytoplasmic membrane and functions as the primary
dehydrogenase in the ethanol oxidase respiratory chain, where ADH
oxidizes ethanol by transferring electrons to ubiquinone embedded in
the membrane phospholipids. The resulting ubiquinol is oxidized by
terminal ubiquinol oxidase, cytochrome o or a
(1) . ADH has been purified from five
strains and it consists of subunits I, II, and
III(1, 2, 3, 4) , except for one ADH
purified from Acetobacter polyoxogenes which consists only of
subunits I and II(5) . ADH contains pyrroloquinoline quinone
(PQQ) (6) and several heme c moieties in subunits I
and II(1) . The genes encoding subunits I and II have been
cloned and sequenced from several sources including Acetobacter
aceti(7, 8) , A.
polyoxogenes(9) , and Acetobacter pasteurianus(10) . Takeda et al.(11) have also
cloned the gene encoding the CO-binding cytochrome c from Gluconobacter suboxydans, which is identical to subunit II of
ADH. These genetic data suggest that subunit I is a typical secretory
protein with a cleavable signal sequence which has significant homology
to the putative PQQ-binding motif found in the methanol dehydrogenase
subunit, and a heme c binding motif, and that subunit II
is also a secretory protein with three heme c binding motives.
Coupled with ethanol oxidation, ADH reduces phenazine methosulfate, dichlorophenolindophenol, or ferricyanide as an artificial electron acceptor in vitro(12) . Since ferricyanide reacts with heme components having a high redox potential, the heme c sites in the ADH complex should reduce ferricyanide. Furthermore, ADH reacts with several ubiquinone homologues and also with native ubiquinone in proteoliposomes(1) . To couple with the reduction of ubiquinone, an electron from ethanol must be transferred inside the ADH complex, where PQQ and several heme c moieties may be involved in the electron transfer and thus in the reduction of ubiquinone. Furthermore, ADH is involved in the CN-insensitive by-pass oxidase system of the G. suboxydans respiratory chain (13, 14) and may mediate electron transfer from another primary dehydrogenase, glucose dehydrogenase, to ferricyanide(15) . Thus, ADH appears to have several additional functions in vivo, besides the oxidation of ethanol to acetaldehyde.
To understand why there are so many prosthetic groups and how the intramolecular electron is transported in the ADH complex, we separated and reconstituted individual subunits from the ADH complex. In addition, during the course of the investigation, inactive ADH was isolated from G. suboxydans(16) , which has at least 10 times lower activity, although there are no differences in the subunit composition or prosthetic groups. Thus, we also studied the kinetic properties of active and inactive ADH and the reactivation of inactive ADH. The results indicated that subunit I of ADH is a quinohemoprotein which contains one molecule each of PQQ and heme c, that subunit II contains three heme c moieties which are responsible for ubiquinone reduction and that the four heme c sites of ADH are separately involved in the various ferricyanide reductase activities of the ADH complex. Furthermore, based on the results obtained in this study, the intramolecular electron transport of the ADH complex to ubiquinone is discussed.
Escherichia coli and Pseudomonas aeruginosa were grown in LB medium. P.
aeruginosa was also grown on a minimal medium composed of 4.52 g
of KHPO
, 11.76 g of
K
HPO
, 3.0 g of
(NH
)
SO
, 0.5 g of
MgCl
6H
O, 15 mg of CaCl
, 15 mg
of FeSO
7H
O, 7.8 µg of
CuSO
5H
O, 10 µg of
H
BO
, 10 µg of
MnSO
4H
O, 125 µg of
ZnSO
7H
O, and 10 µg of MnO
in 1 liter of distilled water, supplemented with 0.5% (v/v)
ethanol or 0.5% (w/v) sodium gluconate as the sole carbon source.
Antibiotics when added, were routinely used at the final concentrations
as follows: 100 µg/ml kanamycin and 25 µg/ml tetracycline for
acetic acid bacteria and P. aeruginosa; and 50 µg/ml
kanamycin and 12.5 µg/ml tetracycline for E. coli.
Figure 1:
Dissociation of ADH complex into
subunit II and subunit I/III complex in CM-Toyopearl column
chromatography. The supernatant (245 mg of protein) solubilized with
1.0% Triton X-100 (TX) was applied to a 50-ml of
DEAE-Toyopearl column and ADH was eluted with a linear gradient as
described under ``Experimental Procedures.'' Fractions
exhibiting ADH activity (27 mg of protein) were pooled and dialyzed as
described under ``Experimental Procedures,'' then applied to
a 7-ml CM-Toyopearl column. The enzymes were eluted in 40 mM acetate buffer (pH 5.0) containing 0.1% Triton X-100, then by a
linear gradient from 40 to 100 mM acetate buffer (pH 5.0)
containing 0.1% Triton X-100 and by another linear gradient from 100 to
200 mM acetate buffer (pH 5.0) without the detergent. ADH
activity () was measured by ferricyanide reductase assay (pH 5.0).
Elution of the protein (
) and cytochrome (
) was measured at
290 and 420 nm, respectively. At the point indicated as Tx-free, the buffer system was exchanged to that excluding the
detergent.
Figure 2:
Protein and heme staining as well as
immunoblotting of G. suboxydans ADH, the subunit II, and the
subunit I/III complex in SDS-PAGE. ADHs were heated in SDS sample
buffer with dithiothreitol (for protein staining and for
immunoblotting) or without dithiothreitol (for heme staining) for 30
min at 60 °C, then applied to a SDS gel containing 12.5%
acrylamide. The gels were stained for protein and heme, and also
immunoblotted as described under ``Experimental Procedures.''
Protein staining; 12, 4, and 8 µg of protein were applied on the
lanes for ADH (lane 1), subunit II (lane 2), and
subunit I/III complex (lane 3), respectively. M shows
protein staining markers as described under ``Experimental
Procedures.'' Heme staining; lanes 1, 2, and 3 contained 140, 100, and 40 pmol of heme c of ADH, subunit
II, and subunit I/III complex, respectively. Lane M contained
pre-stained markers. Immunoblotting with anti-CO-binding cytochrome c (A) and with anti-subunit I (B); 0.38, 0.19, and 0.59 µg of protein of subunit I/III
complex, subunit II, and ADH were applied to lanes 1, 2, and 3, respectively, in both A and B. Prestained
markers are in lane M.
CO-binding cytochrome c has
been purified from G. suboxydans where the cytochrome can be
solubilized from the membrane with 0.2% Triton X-100 and separated from
ADH by CM-cellulose column chromatography(24) . Since the first
cytochrome c fraction exhibited CO-binding ability and almost
the same molecular weight and heme c contents as the
cytochrome (see below), it seems to be similar to the CO-binding
cytochrome c
. Therefore, the
immunocross-reactivity of the first cytochrome c fraction with
the antibody raised against the CO-binding cytochrome c
was examined by immunoblotting (Fig. 2). The antibody cross-reacted at the same intensity with
the cytochrome of the first fraction and also with the second subunit
in ADH complex of the second fraction but not with the third fraction.
Immunoblotting confirmed that the third fraction contained the subunit
I present in the ADH complex (Fig. 2). Thus, it was shown that
the ADH complex can be separated into subunit II, which is identical to
the CO-binding cytochrome c
, ADH complex, and
subunit I/III complex by CM-Toyopearl column chromatography. The pI
values of the ADH complex, subunit I/III complex, and subunits I and II
were also determined by isoelectrofocusing to be 5.1, 5.3 (5.5 in the
apo-form), 6.4, and 4.7, respectively.
Figure 3: Absorption spectra of subunit II, subunit I/III complex, and ADH purified from G. suboxydans. Triton X-100 included in ADH and subunit II was depleted as described (1) . First, each spectrum (broken lines) was taken with subunit II (0.3 mg/ml), subunit I/III complex (0.58 mg/ml), and ADH (0.24 mg/ml), then taken again after adding a few grains of borohydride (solid lines).
Although the N-terminal amino acids of subunits I and II were blocked by some modifications and thus could not be determined, the N-terminal amino acid sequence of subunit III was determined without deblocking, to be Gln-Asp-Gln-Leu-Gly-Ala-Pro-Val-Gly.
Figure 4: The pH profiles for the ferricyanide reductase activities of ADH, subunit I/III complex, and the reconstituted ADH. Ferricyanide reductase activity was measured in McIlvaine buffer at pH 3.5 to 8.0 using active ADH (A) subunit I/III and reconstituted ADH complexes (B) as described under ``Experimental Procedures.'' The reconstituted ADH was prepared by mixing subunit II and subunit I/III complex at a heme c ratio of 3 (mol/mol) as described under ``Experimental Procedures.'' In panel A, the thin lines indicate the ideal values of four ferricyanide-reacting sites, and the respective numbers correspond to those mentioned under ``Discussion.'' In panel B, the ferricyanide reductase activity of subunit I/III complex (triangles) and the reconstituted ADH complex (circles) is indicated.
ADH complex was reconstituted from the
isolated subunits by mixing subunit I/III complex and subunit II in 10
mM KPB (pH 6.0) containing 0.1% Triton X-100 and incubating it
at 25 °C for 20 min. ADH activities, ferricyanide reductase
activities at pH 5.0 and pH 7.0, and Q reductase activity
at pH 5.0, of subunit I/III complex were titrated with subunit II, in
which the enzyme activities were measured following holoenzyme
formation with both PQQ and Ca
. As the added subunit
II was increased, ferricyanide reductase activity increased slightly at
pH 5.0 and drastically at pH 7.0 and most importantly, ubiquinone
reductase activity was recovered to almost the same level as that of
the native ADH complex (Fig. 5). In the reconstitution
experiments, the enzyme activity of the reconstituted ADH seemed to
reflect that of the subunit I/III complex used. Since subunit I/III
complex was so unstable that the activity was difficult to maintain
constantly during storage, the activity of the reconstituted ADH varied
largely among experiments even if the holoenzyme was formed with PQQ
(see Fig. 4and Fig. 5). Nonetheless, the ratio between
Q
reductase activity and ferricyanide reductase activity at
pH 7.0 of the reconstituted enzyme was constant through the study. When
the molar ratio was calculated based on the heme contents of the
subunits where subunit I/III complex and subunit II were estimated to
contain 1 and 3 mol of heme c, respectively, the activities
were saturated with 0.5-1.0 mol of subunit II per mol of subunit
I/III complex. In high performance liquid chromatography gel filtration
(data not shown), the reconstituted enzyme was eluted at the same
position as the native ADH. This was faster than subunit I/III complex,
suggesting that subunit II binds with subunit I/III complex at an
equimolar ratio to form the ADH complex. Considering that the
reconstituted enzyme consisted of a one to one ratio of both subunits,
it seems that the reconstituted activity can also be saturated at a
ratio of roughly 1 mol of subunit II per 1 mol of subunit I/III
complex. One specific ferricyanide reductase activity of the native
ADH, which functions at acidic to neutral pH regions, was not
functional in the reconstituted ADH (Fig. 4). This also shows
the pH profiles of the ferricyanide reductase activities of the
reconstituted ADH.
Figure 5:
Reconstitution of ferricyanide and
ubiquinone reductase activities of subunit I/III complex with various
amounts of subunit II. The holo-enzyme was initially formed by
incubating subunit I/III with 4 µM PQQ and 2 mM CaCl in 10 mM KPB (pH 6.0) for 10 min at 25
°C, then the subunit was reconstituted with various amounts of
subunit II in the presence of 0.1% Triton X-100 for 20 min at 25
°C. Using the reconstituted ADH, ferricyanide reductase (A) and Q
reductase (B) activities were
measured and are expressed as units/mg of protein for subunit I/III
complex. Ferricyanide reductase activity was measured at pH 5.0
(
) and 7.0 (
). The molar ratio was estimated from the heme c contents as subunit I/III containing one heme c and
subunit II containing three heme c molecules.
Figure 6:
Immunoblots of the ADH produced in A.
pasteurianus 2503C and P. aeruginosa 3445A. A and B, immunoblots of the membranes of A.
pasteurianus NP2503 (parent strain, lane 1), A.
pasteurianus 2503B (lane 2, not related in this
experiment), and A. pasteurianus 2503C (lane 3) with
anti-A. aceti ADH were performed using 10 (A) or 60 (B) µg of membrane protein. Prestained markers were also
run in lane M. C, immunoblots with anti-CO-binding cytochrome c were performed with the soluble and membrane
fractions of P. aeruginosa IFO 3445 grown on ethanol-minimal
medium (lanes 1 and 2), of P. aeruginosa 3445A grown on ethanol-minimal medium (lanes 3 and 4), and of the same strain on LB medium (lanes 5 and 6). Lanes 1, 3, and 5 contain membrane
fractions (50 µg of protein each) and lanes 2, 4, and 6 contain soluble fractions (50 µg of protein each). Lane A contains purified G. suboxydans ADH (1.5
µg of protein).
To construct a hybrid ADH, we attempted to
reconstitute ADH from subunit I/III complex of G. suboxydans ADH with subunit II of A. aceti ADH, and the kinetics for
Q reductase activity were compared with those of whole ADH
complexes of A. aceti K6033 and G. suboxydans. When
the subunit I/III complex was titrated with the subunit II, ADH
activity was gradually increased but not saturated, even when excess
subunit II was added to the subunit I/III complex (data not shown).
This implies that affinity of the interaction between subunit I/III
complex and subunit II from different origins is not so high.
Importantly, however, Q
reductase activity could also be
reproduced in the ``hybrid ADH'' as well as the ferricyanide
reductase activities at pH 5.0 and 7.0. Thus, kinetics of Q
reductase activity can be compared between native complex and
hybrid ADH complex (Table 3). Affinity for Q
of ADH
from G. suboxydans was high (K
;
32-40 µM) while that of native ADH from A. aceti was relatively low (K
; 204 µM).
The K
value for Q
of the hybrid ADH
(205 µM) was comparable to that of A. aceti native ADH. Thus, the results suggested that the
ubiquinone-binding site of ADH is present in subunit II of ADH.
Figure 7:
Effect of alkali-treatment on inactive
ADH. ADH was diluted in 50 mM Tris (pH 8.0) then left at 25
°C for 60 min. Left panel, ferricyanide reductase
activities were measured with active () and inactive (
) ADHs
and the alkali-treated inactive ADH (
), as described under
``Experimental Procedures.'' Right panel,
ferricyanide (ferri at pH 5 and 7) and Q
reductase
activities were also measured with active and inactive ADHs and the
alkali-treated inactive ADH (alkali inactive), as described under
``Experimental Procedures.''
Incubation of enzyme with alkali
conditions causes a conformational change in inactive ADH(16) .
As shown in Fig. 7, the alkali treatment restored several enzyme
activities of inactive ADH. Ferricyanide reductase activity at neutral
pH regions was restored to about 80% of the activity of the active ADH,
while only 50% of the ferricyanide reductase activity was restored
around pH 5. Thus, inactive ADH could not restore one of the
ferricyanide reductase activities detected in active ADH even after
exposure to alkali. On the other hand, the Q reductase
activity of inactive ADH was almost completely restored to
90% of
the activity of the active ADH by the same procedure.
ADH of acetic acid bacteria is a highly sophisticated enzyme
complex composed of subunits I (78 kDa), II (48 kDa), and III (14 kDa).
In this study, from the ADH of G. suboxydans, subunit I was
isolated as a complex with subunit III, and subunit II was isolated as
a free form. The subunit I/III complex exhibited ferricyanide reductase
activity only at acidic pH but not Q reductase activity,
whereas subunit II had no activity. The electron flow of ADH from
ethanol to ubiquinone was reproduced by reconstituting subunit I/III
complex with subunit II, indicating that subunit I/III complex,
probably subunit I, is responsible for the dehydrogenation of ethanol.
By sequence homology with the methanol dehydrogenase of methylotrophs (29, 30) and the alcohol dehydrogenase of Comamonas testosteroni, (
)as well as by the
presence of a heme c-binding motif in their amino acid
sequences,
subunit I of ADH complex should have PQQ and
heme c as the prosthetic group. Actually, this study showed
that subunit I/III complex contained 1 mol each of PQQ and heme c and functioned as the dehydrogenase. Thus, subunit I can be
classified as a quinohemoprotein ADH termed type II ADH(31) ,
which includes ADHs from C. testosteroni(32) , Pseudomonas putida (ADHs IIB and IIG; 26), and Rhodopseudomonas acidophila(33) , as well as polyvinyl
alcohol dehydrogenase from Pseudomonas sp. VM15C, (
)all of which have 1 mol each of PQQ and heme c and a relative molecular mass of around 70 kDa.
Subunit II of
ADH was shown to be identical to cytochrome c isolated from the membranes of G.
suboxydans(24) , which had been thought to contain 2 mol
of heme. However, the amino acid sequence of the cytochrome c deduced from the DNA sequence has suggested that there are three
heme c-binding motives(11) . The heme determination of
the purified subunit II or ADH in this study actually showed that
subunit II contained three heme c moieties. This notion has
also been confirmed by redox titration with subunit II, which shows the
cytochrome c behaving as three one-electron carriers. (
)Thus it can be concluded that the ADH complex contains a
total of four heme c moieties, one in subunit I and three in
subunit II.
Data obtained using active and inactive ADHs and the
isolated subunit I/III complex in this study indicate that these four
heme c moieties in the ADH complex can be distinguished by
their kinetic differences with ferricyanide, since four specific
ferricyanide-reacting sites were detected. The first site functions
with high affinity at acidic pH, the second with low affinity at
neutral pH, the third with extremely high affinity at acidic pH, and
the fourth with middle affinity over a range of pH. Since the first
ferricyanide-reacting site (high affinity at acidic pH) was detected
even in subunit I/III complex ( Fig. 4and Table 4), it may
be located at the heme c site in subunit I and termed heme c site I (see Fig. 4). Thus, the other three
ferricyanide-reacting sites should locate at or near one of the three
heme c moieties in subunit II, in which the second, third, and
fourth ferricyanide-reacting sites are tentatively termed heme c sites II, II
, and II
,
respectively (see Fig. 4).
Inactive ADH and also the
reconstituted ADH complex may lack one of the ferricyanide-reacting
sites, namely the fourth site with middle affinity working at broad pH
regions, the II site. One of the heme c moieties
in inactive ADH remains oxidized and is not reduced with ethanol,
although the individual subunits seemingly remain intact(16) .
Inactive ADH can be activated by alkali treatment, where, despite the
Q
reductase activity being almost completely recovered, the
fourth ferricyanide-reacting site, II
, remained
unrecovered. This is consistent with the notion that the oxidized heme c moiety of inactive ADH remains oxidized after exposure to
alkali(16) . Thus, these data suggested that the ubiquinone
reductase activity of ADH can function properly irrespective of whether
the fourth ferricyanide site works or not. Therefore, the heme c site II
would not be functioning in the pathway of
electron transport from ethanol to ubiquinone within the ADH complex.
Thus other heme c moieties (I, II
, and
II
) should function for intra- and inter-subunit electron
transport within subunits I or II. Furthermore, this study showed that
inactive ADH, except for missing one ferricyanide-reacting site, kept
the same K
values for ferricyanide and also for
Q
as active ADH. Although inactive ADH has an electron
transfer rate of only 10% of active ADH(16) , the electrons
from ethanol at the PQQ site in subunit I should be effectively
extracted in inactive ADH and thus even in subunit I/III complex alone,
like active ADH. Thus, we speculate that in inactive ADH, an improper
interaction between subunit II and subunit I/III complex impairs
efficient intersubunit electron transport in the ADH complex.
This
study also showed that Q reductase activity can be
reproduced by reconstituting subunit II to the subunit I/III complex
and furthermore, its kinetics for Q
in a hybrid
reconstituted ADH complex reflected the feature of the original ADH
from which subunit II was derived. These results indicated that the
ubiquinone-reacting site of ADH is located in subunit II. The
ubiquinone site would be very close to either the second or third
ferricyanide-reacting sites (II
or II
site)
since three heme c sites (I, II
, and
II
) may be involved in the electron transport to ubiquinone
in ADH as described above and sites II
and II
are present in subunit II. It cannot be determined at this
moment, which should be the actual site or close to the
ubiquinone-reacting site, because we could not obtain any evidence
indicating a relationship between the ubiquinone-reacting site and the
ferricyanide-reacting sites in the ADH of G. suboxydans. Thus,
to understand whether the II
or the II
site is
related to the ubiquinone-reacting site, we are searching for some
specific inhibitors of Q
reductase activity and also the
ferricyanide reductase activity of G. suboxydans ADH.
Thus,
we speculate that electrons extracted from ethanol at the PQQ site may
be transferred via heme c site I in subunit I to either heme c site II or II
in subunit II, then to
the ubiquinone site, which may also be at or near either of heme c sites II
or II
. If so, the physiological
function of the heme c site II
in subunit II
remains to be elucidated. The respiratory chain of G. suboxydans branches at the site of ubiquinone, with CN-sensitive terminal
oxidase and -insensitive by-pass oxidase, of which the former is
cytochrome o(19) and the latter may be constituted at
least partly with subunit II of ADH (13, 14, 34) which may connect the quinone
pool to the by-pass oxidase(15) . We found that ADH can oxidize
ubiquinol and the ubiquinol-ferricyanide oxidoreductase activity works
at somewhere other than the ubiquinone-reacting site, but which has
similar affinity to ferricyanide as the II
site.
Thus, the II
site may be involved in the electron
transport from ubiquinol to the CN-insensitive by-pass oxidase
independent of the intramolecular electron transport from ethanol to
ubiquinone.