CHAPTER THREE

Thiosulfate-Oxidizing Multi-component System

in the Green Sulfur Bacterium Chlorobaculum tepidum
Takuro Ogawa1,4, Daisuke Seo2, Hidehiro Sakurai1,3, and Kazuhito Inoue1,4

Abstract Green sulfur bacteria grow phototrophically using sulfur compounds such as sulfide,
sulfur, or thiosulfate as electron donors. The components of the thiosulfate oxidoreductase system,
and the functions of each component are controversial. The thiosulfate-dependent mammalian
cytochrome c reducing activity of the cell extract
from Chlorobaculum tepidum was resolved into
four fractions (Fraction I, II, III and IV) by ammonium sulfate fractionation, anion-exchange chromatography and cation-exchange chromatography.
Fraction I is a heterodimer of SoxY and SoxZ,
Fraction II SoxB, Fraction III is composed of SoxA
and SoxX, and Fraction IV is SoxF2. For reduction of mammalian cytochrome c by thiosulfate,
all of Fraction I-III is indispensable. The optical

Department of Biological Sciences, Faculty of Science,

Kanagawa University, Hiratsuka, Kanagawa 259-1293,
Japan
2
Department of Chemistry, Faculty of Science, Kanazawa
University, Kanazawa, Ishikawa 920-1192, Japan
3
Department of Biology, School of Education, Waseda
University, Nishiwaseda, Shinjuku, Tokyo 169-8050, Japan
4
Department of Biological Sciences, Graduate School of
Science, University of Tokyo, Bunkyo, Tokyo 113-0033,
Japan

J.F. Allen, E. Gantt, J.H. Golbeck, and B. Osmond (eds.),

Photosynthesis. Energy from the Sun:
14th International Congress on Photosynthesis,
1114. 2008 Springer.

spectrum of dithionite-reduced SoxAX showed

characteristic of cyt c with an peak at 551 nm.
SoxYZ and SoxB are colorless. SoxF2 is yellow
and binds flavin. We have also purified a soluble
cyt c-554 (about 10 kDa). Addition of SoxF2 and
cyt c-554 to Fraction I-III enhanced the thiosulfate
oxidation.
Keywords Electron transfer, green sulfur bacteria, inorganic sulfur, thiosulfate oxidation

Introduction
Green sulfur bacteria grow phototrophically using
sulfur compounds such as sulfide, sulfur, or thiosulfate as electron donors. They are strict anaerobes and obligate phototrophs. In green sulfur
bacteria, the thiosulfate oxidizing system had been
most intensively studied with Chlorobium limicola
f. thiosulfatophilum: thiosulfate is oxidized by colorless thiosulfate-cytochrome (cyt) c oxidoreductase (Kusai and Yamanaka 1973) and it donates
electrons to cyt c-551 complex, which in turn transfers
electrons to soluble small molecular weight cyt
c-555. The flavocytochrome c which catalyzes

12

Thiosulfate-Oxidizing Multi-component System in the Green Sulfur Bacterium Chlorobaculum tepidum

sulfide-dependent reduction of cyt c-555 can also

reduce horse heart cyt c in in vitro. In Chlorobaculum
tepidum, soluble cyt c-554 (a counterpart of C.
limicola f. thiosulfatophilum cyt c-555) donates
electrons to cyt cZ bound to the photosynthetic
reaction center (Itoh et al. 2002). There seems to
be another pathway of inorganic sulfide metabolism that by-pass cyt c-554 (Tsukatani et al. 2006).
Sulfide is also oxidized by membrane-bound sulfide
quinone oxidoreductase (SQR), and the reduced
quinone in the quinone pool reduces cyt b/c complex
(Shahak et al. 1992).
The thiosulfate oxidation system is most intensively
studied in the facultatively lithoautotrophic bacterium Paracoccus pantotrophus, and the oxidation is
catalyzed by multiple proteins encoded by the sox gene
cluster (Friedrich et al. 2001): all of SoxAX, SoxB, and
SoxYZ have been shown to be essential for thiosulfate
oxidation with cyt c as the electron acceptor. SoxYZ
has been proposed to act as a scaffold for covalently
binding sulfur compounds. SoxB contains two manganese atoms, and proposed to catalyze hydrolysis of thiosulfate. The SoxAX protein covalently binds C heme.
A complex reaction scheme of sequential oxidation of
thiosulfate involving all the above factors is proposed,
but many of the biochemical reaction steps remain to
be established.
The sox gene cluster of C. limicola and
C. tepidum encodes ten periplasmic proteins which
constitute the sulfur-oxidizing enzyme system. In
C. tepidum, the sox genes constitute clusters that
code for SoxF2, X, Y, Z, A, CT1020, B, and W
and, apart about 1 Mb from these gene cluster,
soxE and soxF1 (Vert et al. 2002). To understand biochemical reactions of thisulfate oxidation
in green sulfur bacteria, we purified thiosulfate
oxidizing proteins from C. tepidum.

Materials and methods

Bacterial strains and growth conditions.
Chlorobaculum tepidum strain TLS (ATCC49652)
was photoautotrophically grown in Pf-7 medium
(pH6.5) at 40 C by illuminating with light from
incandescent lumps at 30 E m2 s1.

Purification of SoxYZ, SoxB, SoxAX and SoxF2.

The cells harvested by centrifugation (8,000 g)
were washed twice with 50 mM Tris-HCl (pH 7.8)
containing 100 mM NaCl and 5 mM sodium ascorbate. The precipitated cells were resuspended in
50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 10 mM
EDTA, 5 mM sodium ascorbate, 5 mM dithiothreithol, 1 mM 6-amino-n-caproic acid, 1 mM phenylmethylsulfonyl fluoride and p-aminobenzamidine
2HCl, and homogenized by sonication followed by
disruption with a French pressure cell at 140 MPa.
The suspension was centrifuged at 20,000 g for
20 min, and unbroken cells were removed as a precipitate. The supernatant was further centrifuged at
160,000 g, and the fraction of the resultant supernatant precipitated between 40% and 80% ammonium sulfate saturation was saved. Precipitated
proteins were collected by centrifugation, and the
pellet was resuspended in 20 mM Tris-HCl (pH 7.8)
and dialyzed against the same buffer. The dialyzed
preparation was applied to a DEAE-Toyopearl
650 M column (TOSOH) equilibrated with 20 mM
Tris-HCl (pH 7.8). The thiosulfate-cyt c redyction
activity was obtained in flow-though fractions
unbound to the column. Proteins which were
bound to the column were eluted with 0.3 M NaCl
in the same buffer, yielding fractions containing
SoxF2. The buffer of the flow-though fraction was
changed to 10 mM MES-NaOH buffer (pH 6.0)
by ultrafiltration (YM-3, Millipore) and applied
to a Hitrap SP column (GE Healthcare) equilibrated with 10 mM MES-NaOH buffer (pH 6.0).
The column was washed with a linear gradient of
NaCl (0300 mM), and the fractions that contain a
thiosulfate-dependent cyt c reduction activity were
saved. The combined solution was concentrated
and washed with 10 mM Tris-HCl (pH8.7) by
ultrafiltration (YM-3, Millipore), and applied to a
Hitrap Q column (GE healthcare) equilibrated with
the same buffer. Protein was eluted with a linear
gradient of NaCl (0300 mM) (Fig. 1). SoxYZ
containing fraction was desalted by ultrafiltration
(YM-3, Millipore) and applied to a MonoS column
(GE Healthcare) equilibrated with 10 mM MESNaOH (pH 6.0). Proteins were eluted with a linear
gradient of NaCl (0300 mM) yielding purified

Thiosulfate-Oxidizing Multi-component System in the Green Sulfur Bacterium Chlorobaculum tepidum

Fig. 1 Separation of thiosulfate-oxidizing factors by Hitrap

Q column chromatography. Fractionation was performed
using the FPLC system (GE-healthcare)

SoxYZ. SoxB containing fraction was desalted by

ultrafiltration and applied to a MonoQ column (GE
Healthcare) equilibrated with 10 mM Tris-HCl (pH
8.7). Proteins were eluted with a linear gradient of
NaCl (0300 mM) yielding purified SoxB. SoxAX
containing fraction was applied to a MonoQ column (GE Healthcare) equilibrated with 10 mM
Tris-HCl (pH8.7). Protein was eluted with a linear
gradient of NaCl yielding purified SoxAX. For
SoxF2 purification, the fraction eluted by 0.3 M
NaCl from a DEAE-Toyopearl 650 M column,
was dialyzed against 10 mM MES-NaOH (pH 6.0),
and the dialyzed preparation was applied to a SP
Sepharose FF column (GE Healthcare) equilibrated
with 10 mM MES-NaOH (pH 6.0). After washing
the column with 10 mM MES-NaOH (pH 6.0),
protein fractions finally eluted by 400 mM NaCl
in the same buffer were saved. The fractions were
desalted by ultrafiltration (PM-30, Millipore) and
applied to a Hitrap SP column (GE Healthcare)
equilibrated with 10 mM MES-NaOH (pH 6.0).
Proteins were eluted with a linear gradient of NaCl
(0300 mM), each fractions were assayed for facil-

13

itation of thiosulfate-dependent cyt c reduction,

and active fractions were saved. The buffer of the
active fractions was changed to 50 mM Tris-HCl
(pH7.8) by ultrafiltration and applied to a Hitrap
Q column equilibrated with 50 mM Tris-HCl (pH
7.8). Proteins were eluted with a linear gradient of
NaCl (0300 mM), yielding purified SoxF2.
Purification of cytochrome c-554. Cytochrome
c-554 was purified to homogeneity from cell
extracts as described previously (Itoh et al. 2002).
Enzyme assays. Thiosulfate-dependent cytochrome c reduction assays were carried out at 25 C
in a volume of 0.1 ml reaction mixture. The reaction
mixture contained 10 mM Tris-HCl (pH 7.8), 20 M
cytochrome c-554 or horse heart cyt c, 2 mM sodium
thiosulfate and 0.5 M each of purified thiosulfateoxidizing factors (SoxYZ, SoxB, SoxAX) unless otherwise stated. The time course of the cyt c reduction
was monitored by spectrophotometer (Shimadzu,
UV2500PC). Reduction rate of cytochrome c-554
from C. tepidum and cyt c (horse heart, Wako) were
calculated from 554 = 23.8 mM1 cm1 and 550 =
20.0 mM1 cm1, respectively.

Results
The cell-free extract contained thiosulfate-dependent
cytochrome c-554 reduction activity, which disappeared in any of the single fractions separated
by Hitrap Q column chromatography. However,
a mixture of all of the three fractions I, II, and
III (Fig. 1) restored the activity. The factors in
the above three fractions essential to thiosulfate
oxidation were purified to apparent homogeneity,
yielding SoxYZ, SoxB and SoxAX (see below).
In addition to these three factors, another factor
(SoxF2) is not essential to, but facilitates thiosulfate-dependent cytochrome c-554 reduction was
purified to homogeneity from the fraction bound
to the DEAE column.
SoxYZ is a color-less heterodimer with masses
of SoxY and SoxZ of 13 and 9 kDa, respectively,
as estimated by SDS-PAGE. SoxB was a color-less
monomer with a molecular mass of was 62 kDa.
SoxAX binds a total of two heme c and proposed

14

Thiosulfate-Oxidizing Multi-component System in the Green Sulfur Bacterium Chlorobaculum tepidum

to act as an electron acceptor to link the -S adduct

of thiosulfate to SoxY. The molecular mass of
SoxF2 was 43 kDa. SoxF2 shows an absorption
spectrum typical of a flavoprotein, with peaks at
about 278, 359, 451 and 476 nm. As with SoxF of
P. pantotrophus, CtSoxF2 was purified as a monomeric protein and does not bind heme c. From
comparison of N-terminal amino acid sequence and
the sequence deduced from the gene, it was found
that all of the purified Sox proteins except for
SoxZ had experienced N-terminus signal peptide
cleavage indicating localization of these proteins
in periplasm.
A combined system of purified SoxAX, SoxB
and SoxYZ reconstituted thiosulfate dependent cyt
c-554 reduction activity without participation of
SoxF2. When SoxF2 was added the reconstituted
system, the reduction rate was increased further.
The stoichiometry of horse heart cyt c reduced
was 2.1 0.02 mole per mole of thiosulfate oxidized. Addition of SoxF2 accelerated the rate, but
did not affect the stoichiometry of cyt c reduction
(2.1 0.01 mole/mole) nor Km for thiosulfate.
These results indicate that SoxF2 is not involved in

binding of thiosulfate, but accelerate some step(s)

after initial thiosulfate oxidation takes place.

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