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(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
Alcohol dehydrogenase (ADH) ( 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 KH
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 (
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
CO-binding cytochrome c
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
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
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
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
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 (
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 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 Subunit II of
ADH was shown to be identical to cytochrome c 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 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 This
study also showed that Q 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
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4850-4857
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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.
Materials
Monoclonal antibodies against the subunit I of ADH of G.
suboxydans were prepared as described(17) . Ubiquinone
homologues (Q) were supplied by Eizai Co., Tokyo. DEAE- or
CM-Toyopearl, which was used as a medium performance ion-exchanger, was
from Tosoh Co. (Tokyo). Phenyl-Sepharose and Ampholine (pH
3.5-10.0 for IEF) were purchased from Pharmacia LKB. PQQ was from
Wako Chemical Co. (Osaka). An immunoblotting kit and prestained marker
proteins were obtained from Bio-Rad. The polyvinylidene difluoride
microporous membrane (PVDF) was obtained from Millipore. High
performance liquid chromatography marker proteins and pI marker
proteins were supplied by Oriental Yeast Co. Ltd. (Osaka). All other
materials were of reagent grade and obtained from commercial sources.Bacterial Strains, Plasmids, and Growth Conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. These organisms were cultivated at 30 °C
with rotary shaking (200 rpm). Acetic acid bacteria were maintained on
agar slants containing 1.5% agar, 0.5% CaCO
, and potato
medium(18) . Cells maintained on the agar slant were inoculated
into 5 ml of the potato medium and shaken for 24 h as seed cultures. G. suboxydans was grown on sugar-rich (19) or sorbitol
medium(20) . Acetobacter species including A.
aceti and A. pasteurianus were grown on glycerol
medium(18) . When necessary, 50 mM potassium phosphate
buffer (KPB) was added to adjust the pH of the medium. The seed culture
was inoculated into 100 ml of the respective medium in a 500-ml
Erlenmeyer flask, which was then shaken for 16-24 h. For large
scale cultivation, 100 ml of the culture was transferred into 1.5
liters of the same medium in a 3-liter Erlenmeyer flask and when
necessary, further transferred into 20 liters of the same medium in a
50-liter jar fermentor.
PO
, 11.76 g of
KHPO, 3.0 g of
(NH)SO, 0.5 g of
MgCl
6HO, 15 mg of CaCl, 15 mg
of FeSO7HO, 7.8 µg of
CuSO5HO, 10 µg of
HBO, 10 µg of
MnSO4HO, 125 µg of
ZnSO7HO, 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.Preparation of A. pasteurianus and P. aeruginosa Strains
Harboring the Plasmid Containing adh Gene
Plasmids, pAA025 (21) and pRK2013(22) , and A. pasteurianus NP2503 (21) (Table 1) were
supplied by Dr. Masao Fukuda (Department of Bioengineering, Nagaoka
University of Techology). E. coli HB101 was transformed with
the plasmids by a standard CaCl procedure(23) . The
transformants were screened on an LB plate containing tetracycline or
kanamycin. The plasmid pAA025 was transferred from E. coli to A. pasteurianus NP2503 or P. aeruginosa IFO 3445 by
the triparental mating method using pRK2013 as a helper
plasmid(7) . The resulting transconjugants were isolated on
plates of glycerol medium containing 1% acetic acid or of the minimal
medium supplemented with gluconate, respectively, both of which
contained tetracycline. The transconjugated strains were termed A.
pasteurianus 2503C or P. aeruginosa 3445A. These strains
were cultivated in glycerol medium or the minimal medium supplemented
with 0.5% ethanol, respectively, and both contained tetracycline.Preparation of the Membrane Fraction
Cells were harvested by centrifugation at 9,000 
g for 10 min, and washed twice with 50 mM KPB (pH 6.0). The
washed cells were resuspended at about 1 g of wet cells per 5 ml of 50
mM KPB (pH 6.0), and passed twice through a French press
(American Instrument Co.) at 16,000 psi. After centrifugation at 9,000
g for 10 min to remove intact cells, the supernatant
were ultracentrifuged at 86,000 g for 90 min to obtain
the membrane fraction.Purification of ADH, Subunit I/III Complex, and Subunit
II from G. suboxydans
ADH was purified essentially as described (1, 2) with some modifications as follows. The
membrane fraction was suspended in 10 mM KPB (pH 6.0) at a
protein concentration of 20 mg/ml, and Triton X-100 was added to the
suspension at a final concentration of 1.0% (w/v). After an incubation
at 4 °C for 60 min, solubilized ADH was recovered by
ultracentrifugation and dialyzed against 5 mM KPB (pH 6.0)
containing 0.1% Triton X-100. The dialyzate was applied to a
DEAE-Toyopearl column (about 5 mg of protein per 1-ml of bed volume)
equilibrated with the same buffer. ADH was eluted with a linear
gradient consisting of 5-bed volumes each of 5 and 50 mM KPB
(pH 6.0), both of which contained 0.1% Triton X-100. Rose red fractions
having ADH activity were eluted at around 20-30 mM buffer. The active fractions were collected and dialyzed against 5
mM acetate buffer (pH 5.0) containing 0.1% Triton X-100. The
dialyzate was applied to a CM-Toyopearl column (about 5 mg of protein
per 1-ml bed volume) equilibrated with the same buffer. After washing
with 5 mM buffer, the column was further washed with 5-bed
volumes of 40 mM acetate buffer (pH 5.0) where a cytochrome c was eluted as a purified subunit II. ADH was eluted with a
linear gradient consisting of 5-bed volumes each of 40 and 100 mM acetate buffer (pH 5.0) and an enzyme fraction having relatively
high ADH activity was eluted as a purified ADH complex, at the midpoint
of the gradient. All buffer systems used until this step contained 0.1%
Triton X-100. The detergent was omitted from the system at this point.
Another ADH fraction having relatively low enzyme activity was eluted
with a linear gradient consisting of 5-bed volumes each of 100 and 200
mM acetate buffer (pH 5.0) followed by 5-bed volumes of 200
mM buffer. The enzyme, subunit I/III complex, was eluted
almost at the end of the gradient. When necessary, the latter ADH
fraction was further purified as follows. Solid ammonium sulfate was
added to the fraction at a final concentration of 30% saturation, and
the suspension was applied to a Phenyl-Sepharose column (about 5 mg of
protein per 1-ml bed volume) equilibrated with 50 mM KPB (pH
6.0) containing 30% saturated ammonium sulfate and 2 mM CaCl. An orange-colored active fraction was eluted
with the same buffer without ammonium sulfate.Purification of the Inactive ADH of G. suboxydans and A.
aceti ADH from A. pasteurianus 2503C Strain
Inactive ADH of G. suboxydans was purified from the
membranes of G. suboxydans grown on sugar-rich medium as
described(16) . ADH of A. aceti was purified from the
membranes of A. pasteurianus 2503C strain, harboring pAA025
including the gene encoding ADH subunits I and II of A. aceti,
as described(1) .Purification of Subunit II of A. aceti ADH
P. aeruginosa 3445A strain harboring pAA025 was
grown to the late-logarithmic phase on minimal medium containing
ethanol. The membranes were prepared from about 20 g of wet cells, and
suspended in 5 mM KPB (pH 6.0) at a protein concentration of
around 10 mg/ml. Triton X-100 was added to the membrane suspension at a
final concentration of 1.0% (w/v), followed by standing with stirring
at 4 °C for 30 min. The solubilized supernatant was recovered by
ultracentrifugation at 86,000 g for 90 min, and
dialyzed overnight against 50-fold volumes of 5 mM KPB (pH
6.0) containing 0.1% Triton X-100. The dialyzate was applied to a
DEAE-Toyopearl column (about 5 mg of protein per 1-ml of bed volume)
which had been equilibrated with the same buffer. The enzyme was eluted
with a linear gradient of 3-bed volumes each of 5 and 50 mM KPB (pH 6.0) containing 0.1% Triton X-100, after washing the
column with 3-bed volumes of 5 mM buffer containing the
detergent. Orange-colored cytochrome was eluted around 40 mM buffer. The fractions containing cytochrome c were
collected, concentrated by ultrafiltration using a UP-20 membrane
(Advantec Toyo), and dialyzed overnight against 50 volumes of 5 mM acetate buffer (pH 5.0) containing 0.1% Triton X-100. After
dialysis and centrifugation (10,000 g for 10 min), the
sample was applied to a CM-Toyopearl column (2.4 mg of protein per 1-ml
of bed volume) equilibrated with the same buffer. After washing the
column with 5-bed volumes of the buffer containing 0.1% Triton X-100,
the cytochrome c was eluted with a linear gradient of 10-bed
volumes each of 5 and 100 mM acetate buffer (pH 5.0)
containing detergent. Cytochrome c eluted around 20 mM buffer concentrations was collected separately from another
cytochrome c (20-kDa protein) successively eluted, and
concentrated by ultrafiltration. The sample was dialyzed against 10
mM KPB (pH 6.0) containing 0.1% Triton X-100, and used as
subunit II of A. aceti ADH. It contained 3.2 µM heme c with about 30% impurities.Preparation of Polyclonal Antibodies Raised against
CO-binding Cytochrome c of G. suboxydans and ADH of A. aceti
CO-binding cytochrome c from G.
suboxydans and ADH of A. aceti were purified as
described(1, 24) . Antibodies against both proteins
were prepared as follows. About 1 mg of the cytochrome or ADH was
injected subcutaneously into a rabbit after emulsifying with an equal
volume of complete adjuvant. One month later, about 0.5 mg of the
cytochrome or ADH was mixed with an equal volume of incomplete adjuvant
and the rabbit was given a booster injection. Another 10 days later,
about 30 ml of blood was collected from the rabbit, left at room
temperature for 4 h and centrifuged at 3,000
g for 15
min to remove red cells. The supernatant containing the antiserum was
used as a polyclonal antibody for CO-binding cytochrome c or
the ADH of A. aceti.Enzyme Assays
Ferricyanide reductase activity of ADH was measured
colorimetrically using potassium ferricyanide as an electron acceptor
as described(12, 16) . Ferricyanide reductase activity
was also measured spectrophotometrically in the reaction mixture (1 ml)
containing buffer, potassium ferricyanide, and enzyme solution. The
reaction was started by adding 10 mM ethanol and the
absorbance at 417 nm was followed. Enzyme activity was defined as the
amount of enzyme oxidizing 1 µmol of substrate per min, calculated
from a millimolar extinction coefficient of potassium ferricyanide of
1.0 mM. Q
reductase activity of
ADH was measured spectrophotometrically by following the decrease of
absorbance at 275 nm at 25 °C in a reaction mixture (1 ml)
consisting of appropriate amounts of enzyme, 10 mM ethanol, 50
µM Q, and McIlvaine buffer (pH 4.5), as
described(1) . One unit of these activities was defined as the
amount of enzyme oxidizing 1 µmol of ethanol per min.Analytical Procedures
Electrophoresis
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on
12.5% acrylamide slab gels. The standard marker proteins were a mixture
of phosphorylase b (92 kDa), bovine serum albumin (68 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and lysozyme (14 kDa)
or prestained molecular weight markers (low molecular weight range,
Bio-Rad) for protein staining or heme staining and immunoblotting,
respectively. The gel was stained for protein or heme using 0.1%
Coomassie Brilliant Blue R-250 or heme-catalyzed peroxidase
activity(25) , respectively. Immunoblotting was performed as
described (26) after the samples treated with 2.0% SDS were
applied to SDS-PAGE and resolved as described above. Isoelectrofocusing
was performed on 7% (w/v) polyacrylamide gels containing 5.0% (w/v)
Ampholine (pH 3.5-10.0) as described(26) .Analysis of N-terminal Sequence
Purified ADH was
applied to SDS-PAGE using glycine-free buffers 200 mM Tris-HCl
(pH 8.5) and 100 mM Tris-Tricine containing 0.1% SDS (pH 8.25)
as the anode and cathode buffers, respectively. The proteins in the gel
were transferred electrophoretically onto a PVDF membrane in 10 mM CAPS-NaOH buffer (pH 11.0) containing 10% methanol for 9 h at 25
to 50 mA. The transferred membrane was stained with Coomassie Brilliant
Blue R-250 and destained with 50% methanol. The visible bands were
excised and applied to a peptide sequence analyzer (Shimadzu).Measurement of PQQ Content
Purified enzyme
solution was mixed with 9 volumes of methanol. After incubation at 25
°C for 30 min, this solution was centrifuged at 3,000 g for 10 min. The supernatant was evaporated and used as PQQ
extract. The PQQ content was measured enzymatically using the membrane
fraction containing apo-glucose dehydrogenase from E. coli K12
strain as described(27) .Heme c Contents
Heme was measured from
dithionite-reduced minus ferricyanide-oxidized difference spectrum of
its pyridine hemochrome with a dual-wavelength spectrophotometer. The
pyridine hemochrome was prepared by mixing the sample with a final
concentrations of 20% (v/v) pyridine and 0.2 N NaOH. The heme
content was calculated by a millimolar extinction coefficient of 24.3
(549-535 nm).Protein Content
The protein content was determined
by the modified Lowry method(28) . Bovine serum albumin was
used as the standard protein.
Isolation of Subunit I/III Complex and Subunit II of
ADH from G. suboxydans
ADH purified by DEAE-Toyopearl column was
dialyzed against pH 5 buffer, then applied to a CM-Toyopearl column to
separate three cytochrome c fractions (Fig. 1). The
first cytochrome fraction that eluted at 40 mM buffer had
CO-binding ability, according to the CO-reduced minus reduced
difference spectrum (data not shown), but no ADH activity. The second
fraction that eluted at 70-80 mM buffer showed a
relatively high ADH activity. The third fraction that eluted at 200
mM buffer had lower ADH activity. As shown in SDS-PAGE (Fig. 2), these fractions consisted of a single peptide of 48
kDa, three bands of 78, 48, and 14 kDa, and two peptides of 78 and 14
kDa. Thus, although the principle of this separation was unclear, the
ADH complex was separated into three fractions in the CM-Toyopearl
column chromatography. The second fraction was the same as the reported
native ADH complex(2) , and the first and third fractions
seemed to correspond to subunit II and subunit I/III complex of ADH,
respectively.
) 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.
(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.
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.
Characterization of Subunit I/III Complex and Subunit II
of G. suboxydans ADH
The contents of the prosthetic groups, PQQ
and heme c, in ADH complex, subunit I/III complex, and subunit
II were determined (Table 2), and their contents were estimated
based on their relative molecular masses of 140, 92, and 48 kDa with
the ADH complex, subunit I/III complex, and subunit II, respectively.
The ADH and subunit I/III complexes contained about 0.6 mol of PQQ per
mol. Heme c was present at 3.5, 0.74 and 2.5 mol/mol of the
ADH complex, subunit I/III complex, and subunit II, respectively. In
addition to subunit II, as shown by heme-stained SDS-PAGE (Fig. 2), the heme c moiety was detected in subunit I
but not in subunit III. The absorption spectra of these fractions are
shown in Fig. 3. The ADH and subunit I/III complexes were
completely reduced whereas subunit II was oxidized. ADH complex
exhibited the same absorption spectrum as the purified ADH(2) ,
having , 
, and peaks of 553, 522, and 417 nm. The
absorption spectra of subunit II was similar to that of CO-binding
cytochrome c
(24) , which exhibited
, , and peaks of 553, 522, and 418 nm, respectively, in
the reduced state and a
peak of 410 nm in the oxidized form. The
subunit I/III complex exhibited absorption peaks at 551, 522, and 416
nm.
Reconstitution of ADH Activity from the Separated
Subunits
The first fraction, subunit II, did not exhibit any ADH
activity, while the third fraction, subunit I/III complex, showed a
relatively weak ADH activity of around 100 units/mg at pH 5.0. In
contrast to the ADH complex acting at a broad pH range from acidic to
neutral pH, the subunit I/III complex exhibited ADH activity only at
acidic pH (Fig. 4). Notably, the subunit I/III complex showed no
Q reductase activity although it had ferricyanide reductase
activity (Table 2).
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.
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.
Construction of Hybrid ADH from Subunit I/III Complex of
G. suboxydans ADH and Subunit II of A. aceti ADH
The affinity
for Q between ADH from G. suboxydans and that from A. aceti IFO 3284 largely differs(1) . Therefore, if a
hybrid ADH can be prepared from the subunits of both strains, the
subunit containing Q-site could be identified. Since plasmid pAA025
encodes the genes for subunits I and II, but not subunit III, of ADH of A. aceti K6033(21) , transformants harboring this
plasmid may produce whole ADH complex or part of the subunits and thus
may be useful for the purpose described above. As shown in Fig. 6, when this plasmid was transconjugated into the
ADH-deficient strain, A. pasteurianus NP2503, the
transconjugant A. pasteurianus 2503C, produced whole ADH
complex, probably because the mutant strain retains the ability to
produce subunit III, but not subunits I and II. On the other hand, when
the transconjugant, P. aeruginosa 3445A, was prepared with the
same plasmid, the strain produced only subunit II of A. aceti ADH. Although the reason for this is not yet clear, the host
strain, P. aeruginosa, may not have any genes for the ADH of
acetic acid bacteria and thus subunit I of ADH encoded in the plasmid
might not be produced properly without subunit III, which is not
present in the plasmid. Thus, the ADH complex and subunit II of A.
aceti K6033 were purified from the membranes of these
transconjugants, A. pasteurianus 2503C and P. aeruginosa 3445A, respectively.
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).
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.
Kinetic Characterization in the Subunits of Active and
Inactive ADHs
An inactive ADH has been detected and purified,
separate from the active (native) enzyme, in the membranes of the cells
grown on acidic pH, and it has enzyme activities that are 10 times
lower than those of active ADH(16) . Like active ADH as
described above, inactive ADH was also partially dissociated into the
subunit I/III complex and subunit II. Although the subunit I/III
complex from inactive ADH exhibited less ferricyanide reductase
activity, it was re-activated by holoenzyme formation with PQQ and
Ca to the level with the subunits obtained from
active ADH. Thus, the K
values for electron
acceptors, ferricyanide and Q, were determined and compared
with active and inactive ADHs, and also with the subunit I/III complex
derived from inactive ADH (Table 4). When ferricyanide reductase
activity was measured at pH 5.0 and 7.0, active ADH exhibited two
significantly distinct K values for ferricyanide
at either pH, 0.09 and 0.40 mM at pH 5, and 0.47 and 4.5
mM at pH 7. Two of these K values (0.40
and 0.47 mM) seemed to be identical. On the other hand,
inactive ADH exhibited only one K value at both pH
values which were almost the same as one of two of the K values for active ADH: only the low value (0.09 mM) at
pH 5.0 and only the high value (more than 2 mM) at pH 7.0, in
which the saturation curve became sigmoidal against ferricyanide
concentrations so that V
could not be obtained.
Furthermore, although subunit I/III complex exhibits ferricyanide
reductase activity only at pH 5, the K value for
ferricyanide was also the same as that of ADH complex. In addition to
these activities, an additional K value for
ferricyanide was detected with ADH complex at pH 3.5, in which the K value with active ADH was below 20
µM. The value is so high that a real K value could not be determined from the usual steady state
kinetics. Although the K value at pH 3.5 was not
determined with inactive ADH, the enzyme also had this high affinity
site that reacted with ferricyanide, judging from the pH profile (see Fig. 7). On the other hand, both active and inactive ADHs
exhibited the same K value for Q.
Thus, it was suggested that inactive ADH was defective in one of the
ferricyanide-reacting sites, which is a middle affinity (K; 0.4 mM) site working at acidic
to neutral pH regions with the active ADH, although it has a normal
ubiquinone-reacting site.
) 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.''
reductase
activity of inactive ADH was almost completely restored to 90% of
the activity of the active ADH by the same procedure.
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. 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., II, and II,
respectively (see Fig. 4). 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. 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. 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.
))))
We are indebted to Dr. Masao Fukuda for providing
bacterial strains and plasmids. We also thank Keiko Kimura and Fumiyo
Itoh for their technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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