Biosci. Biotechnol. Biochem., 70 (7), 1584–1591, 2006

Existence of aa3 -Type Ubiquinol Oxidase as a Terminal Oxidase
in Sulﬁte Oxidation of Acidithiobacillus thiooxidans
Tsuyoshi S UGIO,1; y Tomohiro H ISAZUMI,1 Tadayoshi K ANAO,1 Kazuo K AMIMURA,1
Fumiaki T AKEUCHI,2 and Atsunori N EGISHI3
1

Division of Science and Technology for Energy Conversion, Graduate School of Natural Science and Technology,
Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan
2
Environmental Management and Safety Section, Health and Environment Center, Okayama University,
3-1-1 Tsushima-naka, Okayama 700-8530, Japan
3
Technical Research Institute, Hazama Corporation, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan
Received November 21, 2005; Accepted March 2, 2006; Online Publication, July 23, 2006
[doi:10.1271/bbb.50623]

It was found that Acidithiobacillus thiooxidans has
sulﬁte:ubiquinone oxidoreductase and ubiquinol oxidase
activities in the cells. Ubiquinol oxidase was puriﬁed
from plasma membranes of strain NB1-3 in a nearlyhomogeneous state. A puriﬁed enzyme showed absorption peaks at 419 and 595 nm in the oxidized form andat 442 and 605 nm in the reduced form. Pyridine
ferrohaemochrome prepared from the enzyme showed
an -peak characteristic of haem a at 587 nm, indicatingthat the enzyme contains haem a as a component. The
CO diﬀerence spectrum of ubiquinol oxidase showed
two peaks at 428 nm and 595 nm, and a trough at
446 nm, suggesting the existence of an aa3 -type cytochrome in the enzyme. Ubiquinol oxidase was composedof three subunits with apparent molecular masses of
57 kDa, 34 kDa, and 23 kDa. The optimum pH andtemperature for ubiquinol oxidation were pH 6.0 and
30  C. The activity was completely inhibited by sodium
cyanide at 1.0 mM. In contrast, the activity was inhibited
weakly by antimycin A1 and myxothiazol, which are
inhibitors of mitochondrial bc1 complex. Quinone
analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel
and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70%
respectively.
Key words:

Acidithiobacillus thiooxidans; ubiquinol oxidase; sulﬁte oxidation; terminal oxidase;
concrete corrosion

The sulfur-oxidizing bacterium Acidithiobacillus thiooxidans1) (formerly Thiobacillus thiooxidans) is anacidophilic chemolithotrophic bacterium that derives
energy for growth from the oxidation of inorganicyreduced sulfur compounds. A. thiooxidans and the ironoxidizing bacteria Acidithiobacillus ferrooxidans andLeptospirillum ferrooxidans are known as the mostimportant microorganisms for the bacterial leaching of
sulﬁde ores.2–5) A. thiooxidans is also known as one of
the main agents responsible for microbial concrete
corrosion.6–19) The sulfuric acid produced in sewer pipes
by A. thiooxidans-dependent hydrogen sulﬁde oxidation
reacts with the components of concrete such as calciumoxides and calcium hydroxide to form a sandy calcium
sulfate (gypsum). Therefore, it is desirable that sulfur
oxidation by A. thiooxidans cells should be activated at
the practical bacterial leaching sites, but rather inhibitedat the surface of concrete structure of sewer pipe. To
activate or repress the activity of A. thiooxidans, it isimportant to clarify the enzymes involved in sulfur
oxidation of A. thiooxidans. The oxidation of elemental
sulfur by A. thiooxidans is a complex process involving
the contact of cells with sulfur particles, the oxidation of
sulfur to sulﬁte, and the oxidation of sulﬁte to sulfate.
The ﬁrst step of sulfur oxidation from elemental sulfur
to sulﬁte is catalyzed by sulfur dioxygenase.20–22) Sulﬁteis considered to be the key intermediate in the oxidationof inorganic sulfur compounds in thiobacilli.22–27) Sulfur
is nearly quantitatively oxidized to sulﬁte by A. thiooxidans cells when the sulﬁte oxidation is inhibited by
2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO).28)
Many studies have been done on the enzymes involvedin the bacterial oxidation of inorganic sulfur compounds
since A. thiooxidans was ﬁrst isolated and characterized
by Waksman and Joﬀe.29) Sulfur dioxygenase thatoxidizes elemental sulfur to thiosulfate in the presenceof catalytic quantities of reduced glutathione was
puriﬁed approximately 12 fold from cell extract of
A. thiooxidans.20,21) The partially puriﬁed enzyme catalyzed the incorporation of 18 O into thiosulfate through a

To whom correspondence should be addressed. Tel/Fax: +81-86-251-8306; E-mail: sugio@cc.okayama-u.ac.jp

Existence of aa3 -Type Ubiquinol Oxidase in A. thiooxidans
21)

direct oxygenation reaction, but, it was found laterthat the initial product of sulfur oxidation by the
enzymes from T. thiooxidans and T. thioparus was
sulﬁte, but not thiosulfate.22) Recently we puriﬁed sulfur
dioxygenase 26.4-fold from a 105;000  g soluble
fraction of A. thiooxidans NB1-3. The enzyme puriﬁed
from NB1-3 is a monomer enzyme with a molecularmass of 19.5 kDa.30) The sulﬁte produced by sulfur
dioxygenase is further oxidized to sulfate either throughsulﬁte:cytochrome c oxidoreductase,31) or through adenosine phosphosulfate (APS) reductase.32) Sulﬁte dehydrogenase was puriﬁed from A. thiooxidans JCM
7814.33,34)
Although a great many studies have been done on the
enzymes involved in sulfur oxidation of acidophilic
chemolithotrophic sulfur-oxidizing bacteria, no information has been available on the terminal oxidase insulfur- and/or sulﬁte-oxidation of A. thiooxidans. In thisstudy we found the ﬁrst evidence that aa3 -type ubiquinoloxidase plays a role as a terminal oxidase in sulﬁteoxidation of Acidithiobacillus thiooxidans.

1585

formation of 1 mmol quinone in one min at 30  C.
Absorption spectrum was measured with a Shimazu UV1200 spectrophotometer (Kyoto, Japan).
Sulﬁte-ubiquinone oxidoreductase activity. Sulﬁteubiquinone oxidoreductase activity was assayed spectrophotometrically at 30  C by monitoring the reductionof Q2 at 275 nm. An assay mixture (980 ml) containing
50 mM MES–NaOH (pH 8.5), 0.02% Tween 20, 1 mM
KCN, 30 mM Q2 H2 , and 50 mg of cytosol or 10 mg
membranes was preincubated for 10 min at 30  C. The
reaction was started by adding 10 ml of 3 mM Q2 and
10 ml of 200 mM NaHSO3 .
Preparation of pyridine ferrohemochrome. Cytochrome aa3 was extracted from a puriﬁed ubiquinoloxidase (9 mg) with 1 ml of cold chloroform–methanol(2:1), 1 ml of cold acetone, and then tree times with
0.5 ml cold acetone–0.024 N hydrochloric acid.37) The
acetone was evaporated to near dryness. The residue wassuspended in 50 ml of pyridine and 50 ml of 0.2 N
potassium hydroxide.

Materials and Methods
Microorganisms, medium, and condition of cultivation. The sulfur-oxidizing bacterial strains used were
Acidithiobacillus thiooxidans NB1-3,14) A. thiooxidans
strains IFO 13724 and13701, and A. thiooxidans ON
106.35) The sulfur-salt medium (pH 3.0) used for thecultivation of A. thiooxidans contained elemental sulfur(1.0%), (NH4 )2 SO4 (3.0%), K2 HPO4 (0.05%), MgSO4
7H2 O (0.05%), KCl (0.01%), and Ca(NO3 )2 (0.001%).
The method for the large-scale production of cells was
described previously.25) The culture medium from six
10-liter carboys was ﬁltered with Toyo ﬁlter paper(no. 2) to remove the particles of elemental sulfur, andcentrifuged at 15;000  g to gather cells. Harvested
cells were washed three times with 50 mM MOPS–NaOH buﬀer (pH 7.0) and suspended in the same
buﬀer. The washed cells obtained was used in theexperiment.

.

Ubiquinol-2. Ubiquinone-2 (Q2 ) was kindly suppliedby Eizai (Tokyo), and ubiquinol, the reduced form ofubiquinone-2 (Q2 H2 ), was prepared as described by
Riske.36)
Ubiquinol oxidase activity. Ubiquinol oxidase activity
was measured spectrophotometrically at 30  C byfollowing the increase in absorbance at 275 nm. The
reaction mixture (1.0 ml) contained 50 mM MES–NaOH(pH 6.0), 30 mM Q2 H2 , 0.02% Tween 20, and 10 mg of
membranes. The reaction mixture without 30 mM Q2 H2
was preincubated for 1 min at 30  C. The reaction wasstarted by adding 10 ml of 3 mM Q2 H2 . A molecularextinction coeﬃcient of 12.25 mM1 cm1 at 275 nm
was used for the quinone form (Q2 ). One unit of enzyme
was deﬁned as the amount of enzyme that catalyzes the

Puriﬁcation of ubiquinol oxidase. All steps of the
puriﬁcation process were done at 4  C. Resting cells of
A. thiooxidans NB1-3 suspended in 50 mM MOPS–NaOH buﬀer (pH 7.0) were disrupted by passage threetimes through a French pressure cell at 1,500 kg/cm2 ,and centrifuged at 12;000  g for 10 min. The supernatant (cell extract) was further centrifuged at 105;000 
g for 60 min to obtain plasma membranes. The plasma
membranes (1,022 mg of protein) was suspended in
50 mM MOPS–NaOH buﬀer (pH 7.0) at concentration of
10 mg protein/ml, and incubated in buﬀer containing
0.5% n-dodecyl--D-maltopyranoside (DM) for 1 h to
extract ubiquinol oxidase from the membranes. Afterenzyme extraction, the solution was centrifuged at
105;000  g for 60 min to obtain a soluble fraction(0.5% DM extract). The 0.5% DM extract was equilibrated with 50 mM MOPS–NaOH buﬀer (pH 7.0) containing 0.1% DM, concentrated, and then applied to a
DEAE-Toyopearl 650M column (1  10 cm) equilibrated with 50 mM MOPS–NaOH buﬀer (pH 7.0) containing
0.1% DM. The enzyme, bound to a DEAE-Toyopearl
650M column, was eluted with 50 mM MOPS–NaOH
buﬀer (pH 7.0) containing 50 mM sodium sulfate. The
enzyme fraction from DEAE-Toyopearl 650M column
chromatography was concentrated and applied to a
Phenyl-Toyopearl 650M column (1  10 cm) equilibrated with 50 mM MOPS–NaOH buﬀer (pH 7.0) containing
0.1% DM and 400 mM sodium sulfate. Ubiquinoloxidase activity was eluted with 50 mM MOPS–NaOH
buﬀer (pH 7.0) containing 400 mM sodium sulfate. The
enzyme fraction after Phenyl-Toyopearl 650M column
chromatography was applied to a Butyl-Toyopearl
650M column equilibrated with a 50 mM MOPS buﬀer(pH 7.0) containing 0.1% DM and 200 mM sodium
sulfate. Ubiquinol oxidase was eluted with a buﬀer

containing 200 mM sodium sulfate. The enzyme fraction
obtained after Butyl-Toyopearl 650M column chromatography were concentrated and applied to a TSKgel
G3000SW column (7:5 mm  60 cm) equilibrated with a
50 mM MOPS–NaOH buﬀer (pH 7.0) containing 0.05%
DM and 50 mM sodium sulfate. Ubiquinol oxidase
activity was eluted with the same buﬀer at a ﬂow rateof 0.5 ml/min at room temperature. The peak having
enzyme activity was collected and concentrated andused to determinine subunit structure and enzyme
properties.
Protein measurement. Protein concentration wasmeasured by the Lowry method,38) with crystalline
bovine serum albumin as the reference protein.

(A275nm)T. S UGIO et al.
Sulfite:ubiquinone oxidoreductrase activity

1586

0

1

Results3

Fig. 1. Sulﬁte:Ubiquinone Oxidoreductase Activities of Plasma
Membranes and the Cytoplasm from A. thiooxidans NB1-3.
The enzyme activity was measured as described in ‘‘Materials andMethods.’’ Symbols: , plasma membranes (50 mg of protein); ,cytoplasm (50 mg of protein).

Oxidation of Q2H2 (A275 nm)

Existence of sulﬁte:ubiquinone oxidoreductase andubiquinol oxidase activities in A. thiooxidans cells
It is known that elemental sulfur is oxidized at ﬁrst by
sulfur dioxygenase to produce sulﬁte, and that thesulﬁde thus produced is next oxidized by the sulﬁteoxidase enzyme system to give sulfate in A. thiooxidans,
but no information has been available on the terminal
oxidase in sulfur- and/or sulﬁte-oxidation of A. thiooxidans. We ﬁrst investigated whether the cytosol orplasma membranes prepared from A. thiooxidans NB1-3
cells has sulﬁte:ubiquinone oxidoreductase activity.
Since ubiquinone (Q2 ) has a characteristic absorption
peak at 275 nm and ubiquinol does not have a peak at
275 nm, sulﬁte:ubiquinone oxidoreductase activity wasmeasured by the decrease in absorbance at 275 nm.
When plasma membranes was used as an enzyme
source, absorbance at 275 nm decreased rapidly withtime. In contrast, absorbance at 275 nm did not decreasein the reaction mixture without enzyme or with cytosol.
These results indicate that sulﬁte:ubiquinone oxidoreductase activity (58.5 mU/mg) is present in plasma
membranes of A. thiooxidans NB1-3 (Fig. 1). Since thereaction product of sulﬁte oxidation by sulﬁte:ubiquinone oxidoreductase is ubiquinol (Q2 H2 ), the results
obtained as above strongly suggest that ubiquinol is nextoxidized by ubiquinol oxidase in A. thiooxidans cells.
Hence ubiquinol oxidase activity was measured spectrophotometrically using cytosol and plasma membranesof strain NB1-3 as an enzyme source. The ubiquinoloxidase activities of the cytoplasm and plasma membranes from strain NB1-3 were 0.1 and 0.83 U/mg
respectively (Fig. 2), indicating that ubiquinol oxidase islocated in the plasma membranes of A. thiooxidans
cells. The ubiquinol oxidase activities of plasma
membranes from A. thiooxidans strains NB1-3, ON
106, IFO 13701, and IFO 13724 were 0.83, 0.07, 0.78,
and 0.36 unit/mg protein respectively, indicating that allof the A. thiooxidans strains tested have the activity.
Strain NB1-3 had the highest ubiquinol oxidase activity
among the A. thiooxidans strains tested.

2
Time (min)

0

1

2

3

4

5

6

Time (min)Fig. 2. Ubiquinol-Oxidizing Activities of Plasma Membranes and the
Cytoplasm from A. thiooxidans NB1-3.
The enzyme activity was measured as described in ‘‘Materials andMethods.’’ Symbols: , plasma membranes (30 mg of protein); ,cytoplasm (30 mg of protein).

Puriﬁcation of ubiquinol oxidase
The results of a typical puriﬁcation are summarizedin Table 1. The procedure resulted in 11.5-fold puriﬁcation over plasma membranes of A. thiooxidans NB1-3
with a yield of 0.3%. The enzyme solution obtainedby TSKgel G3000W chromatography was nearly homogeneous as estimated by native PAGE (Fig. 3A). Only
one faint band was observed under a main protein bandon native PAGE gel. The subunit structure of ubiquinoloxidase was analyzed by 12.5% SDS–PAGE (Fig. 3B).
Ubiquinol oxidase was composed of three subunits with

Existence of aa3 -Type Ubiquinol Oxidase in A. thiooxidans

1587

Table 1. Summary of Puriﬁcation Procedure of Ubiquinol Oxidase from the Plasma Membrane of A. thiooxidans NB1-3
Step
Plasma membrane
Solubilization
with 0.5% DM
DEAE-Toyopearl 650M
Phenyl-Toyopearl 650M
Butyl-Toyopearl 650M
TSKgel G3000SW

Total Protein(mg)

Total activity(U)Speciﬁc activity(U/mg)

Puriﬁcation(fold)

Recovery
(%)

1,022

848

0.83

1.0

100

578
449
433
280
2.7

2.10
4.18
6.78
6.14
9.54

2.5
5.0
8.1
7.4
11.5

68.2
53
51
33
0.3

278
108
65
46
0.3

A

B

66.3 kDa–

57 kDa–42.4 kDa
34 kDa30 kDa+23 kDa+20.1 kDa

14.4 kDa
Fig. 3. Native PAGE (A) and SDS–PAGE (B) of a Puriﬁed Ubiquinol Oxidase.
A, Disc gel electrophoresis was carried out in 5.0% polyacrylamide gel containing 0.05% DM. A ubiquinol oxidase from TSK gel G3000SW
column chromatography (30 mg protein) was applied on the column. The gel was stained with Coomassie Brilliant Blue. B, After SDS–PAGE on
a 12.5% polyacrylamide gel, subunits of ubiquinol oxidase were revealed by silver staining. Lane 1, marker proteins of Lysozyme (Mr 14,400),
trypsin inhibitor (Mr 20,100), carbonic anhydrase (Mr 30,000), aldolase (Mr 42,400), bovine serum albumin (Mr 66,267), and phosphorylase b(Mr 97,400); lane 2, 0.5% DM extract; lane 3, DEAE-Toyopearl chromatography; lane 4, Phenyl-Toyopearl chromatography; lane 5, ButylToyopearl chromatography; lane 6, TSKgel G3000SW. Each of the enzyme samples applied to a column was 2.5 mg protein.

apparent molecular masses of 57 kDa, 34 kDa, and23 kDa.
Spectral properties of ubiquinol oxidase
The puriﬁed enzyme showed absorption peaks at 419
and 595 nm in the oxidized form and at 442 and 605 nm
in the reduced form (Fig. 4). Pyridine ferrohaemochrome prepared from the enzyme showed an -peakcharacteristic of haem a at 587 nm, indicating that the
enzyme contains haem a as a component (Fig. 5). The
CO diﬀerence spectrum of ubiquinol oxidase showed
two peaks at 428 nm and 595 nm, and a trough at
446 nm, suggesting that aa3 -type cytochrome is present
in the enzyme39) (Fig. 6).
Enzymatic properties of ubiquinol oxidase
The substrate speciﬁcity of ubiquinol oxidase wasstudied. The puriﬁed enzyme used not only ubiquinol(3.13 U/mg) but also 2,3,5,6-tetramethyl-p-phenylene-

diamine (TMPD) (0.45 U/mg) as a substrate, but it did
not use reduced mammalian cytochrome c as an electron
donor (data not shown). The optimum pH for ubiquinoloxidation was pH 6.0 (Fig. 7A). Almost all of theactivity was lost when measured at pH below 5.5 andabove 8.0. The enzyme was stable between pH 6.0 and
8.0 when incubated in 0.1 M MES–NaOH buﬀer and
0.1 M sodium phosphate buﬀer for 60 min, but quite
unstable in 0.1 glycine-NaOH buﬀer with pH above 10
and in 0.1 M sodium citrate buﬀer with pH below 4.0(data not shown). The enzyme was stable whenincubated in 0.1 M MES–NaOH buﬀer (pH 6.0) and
0.1 M sodium phosphate buﬀer (pH 7.0) for 60 min, butquite unstable in 0.1 glycine–NaOH buﬀer with pH
above 10 and in 0.1 M sodium citrate buﬀer with pH
below 4.0 (data not shown). The optimum temperaturefor ubiquinol oxidation was 30  C (Fig. 7B). Almost allof the activity was lost when it was measured attemperatures above 40  C. The enzyme was quite

1588

T. S UGIO et al.

Fig. 4. Absorption Spectra of a Puriﬁed Ubiquinol Oxidase fromA. thiooxidans NB1-3.
The absorption spectra of ubiquinol oxidase were recorded with a
Beckman DU65 spectrophotometer. The sample cuvette contained
9.9 mg of a puriﬁed enzyme in 100 ml of 50 mM MOPS–NaOH buﬀer(pH 7.0) containing 0.05% DM and 50 mM sodium sulfate. Symbols:—, enzyme reduced with hydrosulﬁte; - - -, enzyme in air-oxidized
form.

Fig. 6. CO Diﬀerence Spectrum of a Puriﬁed Ubiquinol Oxidasefrom A. thiooxidans NB1-3.
CO-reduced minus reduced diﬀerence spectrum was recordedwith a Beckman DU65 spectrophotometer. The sample cuvettecontained 9.9 mg of puriﬁed enzyme in 100 ml of 50 mM MOPS–NaOH buﬀer (pH 7.0), containing 0.05% DM and 50 mM sodium
sulfate. The enzyme solution was reduced with hydrosulﬁte, andthen CO gas was bubbled in the solution for 1 min.

Table 2. Eﬀects of Metabolic Inhibitors on Ubiquinol Oxidase
Activity
Concentration

Remaining activity
(%)

None

—

100

KCN

1.0 mM
10.0 mM
1.0 mM

76
30
0

NaN3

1.0 mM
5.0 mM
10.0 mM

57
32
19

Antimycin A1

1.0 mM
10.0 mM

82
73

Myxothiazol

1.0 mM
10.0 mM

79
61

HOQNO

1.0 mM
5.0 mM
10.0 mM

43
39
30

Inhibitor

Fig. 5. Pyridine Ferrohemochrome of a Puriﬁed Ubiquinol Oxidasefrom A. thiooxidans NB1-3.
Pyridine ferrohemochrome was prepared from 9 mg of a puriﬁed
enzyme by the method described in ‘‘Materials and Methods.’’ Theabsorption spectrum of pyridine ferrohemochrome was recordedwith a Beckman DU65 spectrophotometer.

unstable when it was incubated in 0.1 M MES–NaOH
buﬀer (pH 6.0) for 30 min at temperatures above 40  C(data not shown).
The eﬀects of respiratory inhibitors on ubiquinoloxidase activity were studied. KCN, well known
inhibitor of cytochrome c oxidase activity, completely
inhibited ubiquinol oxidase activity at 1.0 mM (Table 2).
In contrast, the activity was inhibited weakly by
antimycin A1 and myxothiazol, inhibitors of the mito-

chondrial bc1 complex, but a quinone analog, 2-heptyl4-hydroxy-quinoline N-oxide (HOQNO), inhibited ubiquinol oxidase activity strongly. The eﬀects of heavy
metals on ubiquinol oxidase activity were also studied.
Agþ , Hg2þ , Cd2þ and Niþ , completely inhibited activity
at 0.1 mM (Table 3), but manganese and magnesium ionsdid not aﬀect it. Chromium ions activated the enzyme
activity 1.5-fold.

Existence of aa3 -Type Ubiquinol Oxidase in A. thiooxidans

B

Relative activity (%)

Relative activity (%)

A

1589

Temperature (°°C)

pH

Fig. 7. Eﬀects of pH (A) and Temperature (B) on the Activity of Ubiquinol Oxidase.
A, The activity was measured in 50 mM of MES–NaOH buﬀer ( ) or HEPES–NaOH buﬀer ( ) at 30  C. B, The activity was measured in
50 mM of MES–NaOH buﬀer (pH 6.0). The highest activity was designated 100%.

Table 3. Eﬀects of Heavy Metals on Ubiquinol Oxidase Activity
Heavy metal

Concentration(mM)

Remaining activity
(%)

None
AgNO3
HgCl2
CdCl2
ZnSO4
CuSO4
NiSO4
CoSO4
MnSO4
MgSO4
CrCl3
Na2 WO4Na2 MoO4

—
0.1
0.1
0.1
0.1
0.01
0.1
1.0
1.0
1.0
1.0
0.1
0.1

100
0
0
0
29
42
0
76
100
117
151
30
55

Discussion
It is known that there are two pathways for theoxidation of sulﬁte in thiobacilli. One mechanism,proposed by Peck, involves the formation of adenine
50 -sulphatophosphate (APS) from AMP and sulﬁte.40)
The APS produced by APS reductase is subsequently
converted to ATP with a concomitant regeneration of
AMP by the combined action of ADP-sulfurylase andadenylate kinase. The other mechanism, proposed by
Charles and Suzuki, does not require AMP and iscatalyzed by sulﬁte oxidase with ferricyanide or cytochrome c as an electron accepor.31,41) Nakamura et al.
reported that membranes prepared from Thiobacillus
thiooxidans JCM 7814 contain cytochromes a, b, and c
and show sulﬁte dehydrogenase activity.33,34) Most of
the activities of sulﬁte oxidation are in the membranes ofthe bacterium, and the addition of AMP to the reaction
mixture neither aﬀects the sulﬁte oxidation activity nor
produces APS.33) From these results, they concluded thatT. thiooxidans JCM 7814 has only the AMP-independent pathway. However, although a great many studies

have been done on the enzymes involved in the sulfurand sulﬁte-oxidation of A. thiooxidans, no information
is available on an actual terminal oxidase in the sulfurand sulﬁte-oxidation of A. thiooxidans. Ubiquinol oxidase was ﬁrst puriﬁed in a nearly homogeneous statefrom plasma membranes of A. thiooxidans strain NB1-3
and characterized. The results obtained from pyridine
ferrohaemochrome and the CO diﬀerence spectrum of a
puriﬁed ubiquinol oxidase strongly suggest the existenceof an aa3 -type cytochrome in the enzyme. We have
puriﬁed iron oxidase from the moderately thermophiliciron-oxidizing bacterium strain TI-1, which contains an
aa3 -type cytochrome.42) Strong inhibition of ubiquinoloxidase activity by a low concentration of sodium
cyanide (1.0 mM), but not by antimycin A1 or myxothiazol, also support the involvement of an aa3 -type
cytochrome in the enzyme activity.42) The ﬁnding that
A. thiooxidans NB1-3 has sulﬁte:ubiquinone oxidoreductase activity indicates that the bacterium uses
ubiquinol oxidase as a terminal oxidase in the oxidationof elemental sulfur as well as the oxidation of sulﬁte, anintermediate during elemental sulfur oxidation of thisbacterium.
We have shown that concrete supplemented withnickel and/or tungsten is resistant to corrosion,13,43) andthat nickel and tungsten bound to A. thiooxidans NB1-3
cells inhibit both sulfur dioxygenase and sulﬁte oxidase,
and as a result, inhibit cell growth.6,14,15,43) We prepared
portland cement bars (40  40  160 mm) supplemented with various kinds of inorganic and organic compounds, and these bars were hung from the lid of asludge tank manhole and exposed to a 5–10 ppm
hydrogen sulﬁde atmosphere.13) After 2 years of exposure, it was found that the surface of the portland
cement bar supplemented with nickel was especiallysmooth, while that without nickel was brittle andrough,13) suggesting the possibility that nickel can beused as a bacteriostatic agent for the prevention of
concrete corrosion. Recently, we showed that when

1590

T. S UGIO et al.

portland cement bars supplemented with 0.075% metal
nickel or with 0.075% metal nickel and 0.075% calcium
tungstate were exposed to the atmosphere of a sewage
treatment plant containing 28 ppm of H2 S for 2 years,
the weight loss of the portland cement bar with metal
nickel and calcium tungstate was much lower than thatof the cement bar containing 0.075% metal nickel.43)
Both nickel and tungsten are unique as bacteriostatic
agents for concrete corrosion, because the former tightly
binds to A. thiooxidans cells at neutral and slightly
alkaline pH, but the latter only at acidic pH below 3.0,
and both can inhibit growth of the bacterium markedly
at the pH at which these metal ions bind to the cells.
However, the precise site of nickel- and tungsteninhibition on the elemental sulfur oxidation enzyme
system of A. thiooxidans remains unsolved. From this,the ﬁndings that low concentrations of nickel andtungstate (0.1 mM) inhibited ubiquinol oxidase activity
100 and 70% respectively are interesting and important,because these inhibition studies indicate that one of thesites of nickel- and tungstate-inhibition in elemental
sulfur oxidation enzyme system is ubiquinol oxidase, aterminal oxidase in sulﬁte oxidation enzyme system of
A. thiooxidans.

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