J. Biochem.

85, 1405-1414 (1979)

Flavocytochrome c of Chromatium vinosum

Some Enzymatic Properties and Subunit Structure1

Yoshihiro FUKUMORI and Tateo YAMANAKA

Department of Biology, Faculty of Science, Osaka University,

Toyonaka, Osaka 560

Received for publication, November 28, 1978

The function and the structural features of Chromatium vinosum cytochrome c-552 have been

investigated. Cytochrome c-552 has a sulfide-cytochrome c reductase activity and also cata

lyzes the reduction of elementary sulfur to sulfide with reduced benzylviologen as the electron

donor. In the sulfide-cytochrome reduction, horse and yeast cytochromes c act as good

electron acceptors, but cytochrome cor cytochrome c-553(550) purified from the organism

does not.

The subunit structure of cytochrome c-552 has been studied. The cytochrome is split

by 6 M urea into cytochrome and fiavoprotein moieties with molecular weights of 21,000 and

46,000, respectively. The fiavoprotein moiety is obtained by isoelectric focusing in the presence

of 6 M urea and 0.1 % -mercaptoethanol, while the hemoprotein moiety is obtained by gel

filtration with Sephacryl S-200 in the presence of 6 M urea and 0.1 M KCl. Neither subunit

has sulfide-cytochrome c reductase activity. Attempts to reconstitute the original fiavo

cytochrome c from the subunits have been unsuccessful.

Flavocytochromes c have been found in two (3-5), its function was unknown. Hind and Olson
genera of photosynthetic sulfur bacteria, Chro (30) have suggested that the cytochrome may
matium vinosum and Chlorobium limicola f. function in the non-cyclic system in photosynthesis
thiosulfatophilum (1). These organisms obtain as a link between substrates (inorganic sulfur
energy and reducing power by photosynthetic compounds) and the main photosynthetic electron
oxidation of thiosulfate and sulfide (2). Chro transport system. However, no enzymatic prop
matium vinosum cytochrome c-552 (1) possesses erties were detected with a purified preparation of
two molecules of heme c and one molecule of cytochrome c-552. In the present investigation,
covalently-bound FAD (21) per molecule. we have found that cytochrome c-552 has a sulfide
Although the physical and chemical properties of cytochrome c reductase activity and that it also
the cytochrome have been studied to some extent catalyzes the reduction of elementary sulfur with
an appropriate electron donor; e.g. with reduced
benzylviologen. It is known that sulfide is oxi
1This work has been supported in part by grants-in-aid
dized to sulfate via elementary sulfur in C. vinosum
from the Ministry of Education, Science and Culture of
(7). Some workers have also found that whole
Japan (Nos. 234041, 334038, and 312107).
cells of Chlorobium limicola f. thiosulfatophilium
Abbreviations: SDS, sodium dodecyl sulfate; PTH,
and Chromatium vinosurn catalyze the reduction of
phenylthiohydantoin derivative.

Vol. 85, No. 6, 1979 1405

1406 Y. FUKUMORI and T. YAMANAKA

elementary sulfur to sulfide under some phys Presence of Sodium Dodecyl Sulfate-Polyacryl

iological conditions (8, 9). Therefore, it seems amide gel electrophoresis in the presence of SDS

likely that cytochrome c-552 is responsible not was performed by the method of Weber and Osborn

only for the oxidation of sulfide but also for the (14) with slight modifications; a slab gel apparatus

reduction of sulfur in vivo. was used as described by Wada and Snell (15).

We have succeeded in unveiling to some The gel slab, 14.3 ~ 10 ~ 0.1 cm, was made by

extent the structural features of cytochrome c-552; polymerization of 7.5 % acrylamide and soaked

the cytochrome molecule is split into two subunits overnight in 0.1 M Tris-HCl, pH 8.5, containing

with molecular weights of 46,000 and 21,000. 0.5 % SDS, 6 M urea, and 1 % -mercaptoethanol.

The larger subunit contains flavin, and the smaller After electrophoresis had been performed for

one heme. Some properties of each subunit have 1.5 h, the gel was stained with Coomassie brilliant

been studied. blue R-250.

Isoelectric Point Determination-Isoelectric

MATERIALS AND METHODS point was determined by isoelectric focusing as
described by Vesterberg and Svensson (16), using
Cultivation of the Organism-A strain of an apparatus with a volume of 110 ml and carrier
Chromatium vinosum was kindly supplied by Drs. ampholyte (LKB, Sweden) in the pH range 3-6
R.G. Bartsch and T. Meyer (University of Cali or 3-10. The temperature of the column was
fornia, San Diego, U.S.A.), and large-scale culture maintained at 4C and the electric power at about
of the organism was performed as described by 1.8 watts. Electrophoresis was performed for
Bartsch and Kamen (10). Cells were harvested about two days.
after growth for 5 days, and stored at -20C Analysis of the Amino-Terminal Sequences
before use. The amino-terminal sequences were determined by
Reagents-Cytochrome c-552 and cytochrome the Edman degradation procedure (17). Identifica
c were purified by the methods of Bartsch and tion of the PTH-derivatives of amino acids was
Kamen (10) and of Bartsch (11), respectively, and carried out by thin-layer chromatography (18).
cytochrome c-553(550) by the method of Cusano Determination of Amino Acid Composition

vich and Bartsch (12). Cytochrome c (555, C. The sample was hydrolyzed with 6 N HCl for

limicola f. thiosulfatophilum) (26), cytochrome c 24 or 72 hat 110 in a sealed tube after evacuation.

(552, Nitrosoinonas europaea) (27), and cytochrome The hydrolysate was dried and analyzed in a

c (554, Pseudomonas aeruginosa) (28) were purified Beckman-Spinco amino acid analyzer, model

by the methods previously established in our 120B, with an acceleration system according to

laboratory, and cytochrome c2 (Rhodospirillum the method of Spackman et al. (19).

rubrum) was kindly supplied by Dr. T. Horio Analysis of Sulfide-To 3.0 ml of reaction
(Institute for Protein Research, Osaka University). mixture which contained sulfide, a mixture of
Horse cytochrome c (Type VI) was purchased 0.5 ml each of Na2CO3 (10%) and ZnSO4.7H2O
from Sigma Chemical Co., U.S.A. Elementary (20%) was added. The resulting suspension was
sulfur was prepared by the method of Roy and stirred for 1 min and centrifuged at 9,000 x g for
Trudinger (13). 10 min. To the precipitate thus collected, 1.0 ml
Spectrophotometric Determinations-Absorp of 6 N HCl was added, and H2S gas generated was
tion spectra were determined in a Cary recording trapped in 1.0 ml of 0.1 N NaOH. The alkaline
spectrophotometer, model 15 or 16. The reduction solution thus obtained was analyzed by the meth
of cytochrome with Na2S in the presence of cyto ylene blue method (20).
chrome c-552 was followed spectrophotometrically
in terms of the increase of the absorbance at the
RESULTS
a-peak of each cytochrome. The reduction of
elementary sulfur with reduced benzylviologen Oxidation of Sulfide-C. vinosum cytochrome
was followed in terms of the decrease of the c-552, purified in the present investigation by the
absorbance at 550 nm. method of Bartsch and Kamen (10), was homo
Polyacrylamide Gel Electrophoresis in the geneous as judged from the electrophoretic pattern;

J. Biochem.
FLAVOCYTOCHROME c OF Chromatium vinosum 1407

when it was subjected to polyacrylamide gel by KCN. When the cytochrome was heated at
electrophoresis in the absence of SDS a single band 80C for 2 min, its catalytic activity greatly de
was observed. Further, when the cytochrome creased. CO did not affect the catalytic activity
preparation was subjected to isoelectric focusing, of the cytochrome. Some of the catalytic prop
only a single band was detected as determined from erties of cytochrome c-552 are summarized in
the absorbances at 280 nm and 410 nm of the Table I.
eluate (Fig. 1). The highly purified preparation As shown in Table II, several C-type cyto
of the cytochrome showed sulfide-cytochrome c chromes were examined for ability to act as an elec
reductase activity. tron acceptor in place of horse cytochrome c in the
As shown in Fig. 2, the reduction of horse sulfide-cytochrome c reduction catalyzed by cyto
cytochrome c by Na2S was greatly accelerated by chrome c-552. Cytochrome C2(R. rubrum, Em,7=
the addition of a very small amount of cytochrome 0.320 volt) and cytochrome c (552, N. europaea,
c-552. Cytochrome c was scarcely reduced by Em,7=0.250 volt) acted as electron acceptors as
Na2S at the concentrations used in the present efficiently as horse cytochrome c, while cytochrome
experiment unless cytochrome c-552 was added.
The optimum pH of the reaction was 8.3 and
Km of the flavocytochrome for sulfide was 12.5 am.
The reduction of cytochrome c with Na2S in the
presence of cytochrome c-552 was strongly inhibited

Fig. 2. Time course of the reduction of horse cyto

chrome c by cytochrome c-552 with Na2S. The reaction

mixture contained 0.2 M Tris-maleate buffer, pH 8.3,

19 M horse ferricytochrome c, 40 nM cytochrome c-

552, and 10 M Na2S in a total volume of 1.0 ml. The

reaction was started by addition of Na2S, and per

formed in air at 23C. A, Complete reaction system;

B, in the absence of cytochrome c-552.

TABLE I. The sulfide-cytochrome c reductase activity

of cytochrome c-552. The experimental conditions were
as described in the legend to Fig. 2, except for the con
Fig. 1. (A) Elution pattern of cytochrome c-552 after
centration of Na2S. The molecular activity was calcu
isoelectric fractionation. Isoelectric focusing was per
lated from Vmax.
formed for 2 days with 1% carrier ampholyte of pH
range 3-10 using an apparatus with a volume of 110 ml.
Although a peak was usually seen in the elution curve
around pH 3.5 when the eluate was monitored in terms
of the absorbance at 280 nm, it appeared to be at
tributable to materials derived from the carrier am
pholyte used. (B) Polyacrylamide gel electrophoretic
patterns of the purified preparation of cytochrome
c-552. The gel was prepared from 7.5% acrylamide.
The electrophoresis was performed in 0.1 M Tris-HCl
buffer, pH 8.5, for 2 h at 20 mA and 150 volt, and at
4C. The two electrophoretic patterns were obtained
from cytochrome c-552 at different concentrations.
The cytochrome was stained with Coomassie brilliant
blue.

Vol. 85, No. 6, 1979

1408 Y. FUKUMORI and T. YAMANAKA

TABLE II. Reduction of several kinds of C-type cytochromes by cytochrome c-552 with Na2S. The reaction

mixture contained 10 mm Tris-HCl buffer, pH 8.5, 10 M Na2S, and each cytochrome with (+) or without (-)

20nM C. vinosum cytochrome c-552 in a total volume of 1.0 ml. The reactions were started by adding Na2S and

performed at 20C. The increase in the absorbance at the a peak of each cytochrome was followed spectrophoto
metrically.

c (554, P. aeruginosa, E.,7=0.225 volt) and cyto Molecular Features-Cytochrome c-552 con
chrome c (555, C. thiosulfatophilum, Em,7=0.145 tains one molecule of FAD and two molecules of
volt) were not reduced under the same experi heme c per molecule (1). We tried to split
mental conditions (Table II). Cytochrome c the cytochrome c-552 molecule into subunits to
(Em,7=0.005 volt) and cytochrome c-553(550) elucidate the relationship between its catalytic
(Em,7=0.330 volt) purified from C. vinosum did activity and structure.
not act as electron acceptors for cytochrome When cytochrome c-552 was subjected to

c-552 either. polyacrylamide gel electrophoresis in the presence

Reduction of Elementary Sulfur-Cytochrome of 0.5% SDS, 6M urea, and 1 % -mercapto

c-552 also catalyzed the anaerobic reduction of ethanol, two major bands and two minor bands

elementary sulfur with reduced benzylviologen as were observed in the gel stained with Coomassie

the electron donor (Fig. 3). The reaction was brilliant blue (Fig. 4A). The molecular weights of

inhibited by cyanide. The reaction mixture the major bands were 46,000 and 21,000 (Fig. 5).

smelled of hydrogen sulfide and changed the color The bands with molecular weights of 46,000
of lead acetate test paper to brown after the reaction and 21,000 were identified as the flavoprotein and
had proceeded to some extent. Further, the cytochrome moieties, respectively, by separate
hydrogen sulfide produced was analyzed by the electrophoresis of the preparations of these two
methylene blue formation method (20) with slight moieties obtained as described below. The other
modifications as described in " MATERIALS two minor bands were attributed to contaminants
AND METHODS " to avoid the inhibitory effect or irregularity in the staining. As described below,
of dithionite on the analysis. However, a stoi this was supported by analyses of the N-terminal
chiometric relationship between consumption of amino acid and the amino acid compositions of
the reduced benzylviologen and sulfide formation the intact cytochrome c-552 and the two moieties.
has not yet been established; as far as was tested When the cytochrome was subjected to

the molar ratio of reduced benzylviologen con isoelectric focusing for 2 days in the presence of

sumed to sulfide formed was about 10, although 6 M urea and 1 % -mercaptoethanol in addition

the ratio should theoretically be 2. to sucrose and carrier ampholyte, two peaks were

J. Biochem.
FLAVOCYTOCHROME c OF Chromatium vinosum 1409

Fig. 3. Anaerobic oxidation of reduced benzylviologen

by elementary sulfur catalyzed by cytochrome c-552.

The reaction mixture in the main chamber contained

10mm Tris-HCl, pH 8.5, 0.67mm benzylviologen, and

0.94 M cytochrome in a total volume of 3.0 ml, and

in the side chamber 1.2 mol Na2S2O4 was added as a

mixture with glucose (Na2S2O4: glucose, 1: 50). The Fig. 4. Electrophoretic profiles of cytochrome c-552

reaction was started by tipping the Na2S2O4 into the and its subunits. Electrophoresis was performed in

reaction mixture in the main chamber. Benzylviologen the presence of 0.5% SDS, 6 M urea, and 1 % -mer

was reduced instantly when Na2S2O4 was mixed with captoethanol. When the intact cytochrome c-552 was

the reaction mixture, and then the reduced pigment subjected to electrophoresis, two major and two minor

was oxidized in the presence of elementary sulfur. bands were observed in the gel. The molecular species

Although the purple color of reduced benzylviologen in the two major bands were identified as the cyto

was unchanged for more than 30 min in the absence of chrome and flavoprotein moieties, respectively, by

the cytochrome, the absorbance of the reaction mixture separate electrophoresis of these moieties (B and C).

decreased gradually as elementary sulfur precipitated The two minor bands were attributed to some con

readily. Elementary sulfur remained in the suspension taminants or irregularity in the staining (see the text).

for more than several hours in the presence of cyto A, Cytochrome c-552; B, the flavoprotein moiety; C,

chrome c-552. The reference cuvette contained all the the cytochrome moiety.

components except for Na2S2O4. When cyanide was

added to the reaction mixture, the reaction rates de

creased. (A), In the absence of cytochrome c-552;

(D), complete reaction system; (B) and (C), 12.2 and

6.1mm KCN were added, respectively.

found in the elution curve as monitored in terms

of the absorbance at 280 nm (Fig. 6). One peak
was located at pH 5.3 and the other at pH 5.6.
The eluate with pH 5.6 was yellow in color and
showed absorption peaks at 276, 355, and 453 nm
Fig. 5. Molecular weight estimations of the cyto
in the oxidized form (Fig. 7). On reduction with
chrome and flavoprotein moieties derived from cyto
dithionite, the absorption peak at 453 nm of the
chrome c-552 by polyacrylamide gel electrophoresis in
oxidized form disappeared, while no peak was the presence of 0.5% SDS, 6 M urea, and 1%
observed around 410 nm. These spectral prop mercaptoethanol. Their molecular weights were found
erties show that the yellow eluate contains flavo to be 46,000 and 21,000, respectively. The proteins

protein but not hemoprotein. The ratio of used as markers to determine the molecular weights of

A276nm/A453nm was 8.9 with this fraction. The these moieties were: horse cytochrome c (mol. wt.

yield of the flavoprotein moiety was more than 12,300), egg albumin (mol. wt. 43,000), and bovine

95 % on the basis of the absorbance at 450 nm. serum albumin (mol. wt. 68,000).

Vol. 85, No. 6, 1979

1410 Y. FUKUMORI and T. YAMANAKA

Fig. 8. Elution pattern of the cytochrome moiety

derived from cytochrome c-552. Cytochrome c-552
which had been dialyzed against 0.1 M Tris-HCl buffer,
pH 8.5, containing 0.1 M KCl and 6 M urea was chro
matographed on a Sephacryl S-200 column (2 x 62 cm)
equilibrated with the same buffer as used for the dialysis.
Fig. 6. Isoelectric focusing of cytochrome c-552 in ----, 280 nm; -, 410 run.
the presence of 6 M urea and l y, -mercaptoethanol.

The electrophoresis was performed with 1% carrier

The cytochrome moiety could not be obtained
ampholyte of pH range 3-6 for 16 h at 1.7 mA and 800
by isoelectric focusing. In the presence of
volt using the apparatus with a volume of 110 ml. The
- mercaptoethanol, the heme of the cytochrome
amount of cytochrome c-552 applied was 71 nmol.

Each fraction of the eluate was 2.0 ml. The absorbance moiety was very quickly destroyed, so that no red

was measured at 280 nm (), 450 nm (), and 410 nm band was seen, as would be expected if the cyto

(?). chrome moiety was present in the electrophoretic

column.

The cytochrome moiety was obtained by gel

filtration with Sephacryl S-200 in 0.1 M Tris-HCl
buffer, pH 8.5, containing 6 M urea and 0.1 M KCl.
The elution pattern is shown in Fig. 8. The cyto
chrome c-552 preparation was dialyzed against
0.1 M Tris-HCl buffer, pH 8.5, containing 6 M
urea and 0.1 M KCl for 12 h before being subjected
to gel filtration. As shown in Fig. 8, essentially
two peaks were found in the elution curve as
monitored in terms of the absorbance at 280 run.
The first and second peaks of the elution curve
contained the flavoprotein moiety plus unsplit
flavocytochrome c and the cytochrome moiety,
respectively, as judged from their absorption
spectra and the electrograms obtained on poly
acrylamide gel electrophoresis in the presence of
SDS. In this procedure, the cytochrome moiety
was obtained in a pure state, while the flavoprotein
moiety was eluted together with intact cytochrome
Fig. 7. Absorption spectrum of the flavoprotein
which survived the procedure. Figure 9 shows
moiety derived from cytochrome c-552. The moiety
the absorption spectrum of the cytochrome moiety.
was dissolved in 10 mm Tris-HCl buffer, pH 8.0.
There were absorption peaks at 552, 523, and 416
(A), Oxidized; (B), reduced with Na2S2O4.
nm in its reduced form. The pyridine ferro
hemochrome of the moiety showed absorption

J. Biochem.
FLAVOCYTOCHROME c OF Chromatium vinosum 1411

peaks at 550, 520, and 414 nm. The absorbance

of the peak around 280 nm of the cytochrome
moiety was rather low as compared with that of
the peak of the intact flavocytochrome; the ratio
of A275nm (oxidized)/Asoret (oxidized) was 0.18
with the cytochrome moiety, while the ratio with
the native cytochrome was 0.56. The yield of the
cytochrome moiety was about 70% on the basis of
the absorbance at 552nm.
The existence of the two subunits in the
C. vinosum cytochrome c-552 molecule was con
firmed by analysis of the N-terminal amino acid
residues. The first step of the Edman degradation
procedure gave alanine and glutamic acid and the
second step proline and glycine with the intact
flavocytochrome. N-Terminal amino acid anal
ysis of the flavoprotein moiety gave alanine followed
by glycine, while that of the cytochrome moiety
gave glutamic acid followed by proline. In these
analyses of the N-terminal residues, amino acids
Fig. 9. Absorption spectrum of the cytochrome
moiety derived from cytochrome c-552. The moiety other than those described were practically indetect
was dissolved in 0.1 M Tris-HCl buffer, pH 8.5, con able.
taining 6M urea. ----, Oxidized; , reduced with The amino acid compositions of cytochrome
Na2S2O4. c-552 and of the cytochrome and flavoprotein

TABLE V. Amino acid compositions of cytochrome c-552 and of the cytochrome and flavoprotein moieties.

Numbers of amino acid residues were calculated on the basis of the molecular weights of each component deter
mined as described in the text, and are expressed as the nearest integers. Tryptophan residues were not estimated.

Calculated on the basis of the molecular weight obtained by summation of the molecular weights of the two
moieties. b Values including heme and/or flavin.

Vol. 85, No. 6, 1979

1412 Y. FUKUMORI and T. YAMANAKA

moieties derived from it are shown in Table V. the reduction of cytochromes c by Na2S at very
The amino acid compositions shown in Table V low concentrations. The enzymatic activity of
were determined on the basis of the molecular the cytochrome could be of physiological signifi
weights of the respective proteins. The molecular cance, since the organism photosynthetically
weights of the intact cytochrome c-552 and of the oxidizes sulfide to acquire energy and reducing
two moieties were determined on the basis of the power for its growth. Although, in general,
heme content and by SDS-gel electrophoresis, cytochrome c is rapidly reduced non-enzymatically
respectively. The compositions calculated on the by sulfide, cytochrome c-552 accelerates the reduc
basis of mol of heme c and FAD present in the tion of cytochrome c even when the concentration
preparations gave molecular weights of 62,000 of sulfide is so low that cytochrome c is not spon
and 34,000 for the native cytochrome c-552 and taneously reduced. Further, as the organism has
the flavoprotein moiety, respectively. The FAD to take up electrons into the cells from sulfide
content was determined assuming the millimolar present in the medium, the biological reduction of
extinction coefficient at 453 nm of the flavoprotein the cytochromes or other redox proteins in the
moiety to be 11, and the heme content was deter cells by sulfide could be different from the chemical
mined using a coefficient at 550 nm of the pyridine reduction which occurs when cytochrome c is
hemochrome of heme c of 29.1 (29). The molec mixed with sulfide in solution.
ular weight thus obtained of the flavoprotein moiety It is of interest to clarify which protein is the

differs considerably from that determined by electron acceptor for sulfide oxidation catalyzed

SDS-gel electrophoresis. This discrepancy sug by cytochrome c-552 in vivo. Cytochrome cor

gests that the assumed value of the extinction cytochrome c-553(550) purified from C. vinosum

coefficient of the flavoprotein moiety is incorrect. does not act as the electron acceptor in the oxi

The molecular weight of the hemoprotein moiety dation of sulfide by cytochrome c-552. Although

was determined to be 13,000 on the basis of the a few kinds of C-type cytochrome exist in the

heme content, while it was determined to be 21,000 organism in addition to the above proteins, the

by SDS-gel electrophoresis. Therefore, it was quantity in the cells seems to be too small for

concluded that the hemoprotein moiety as obtained them to act as electron acceptors in the active

by the above method contained two molecules of oxidation of sulfide. Yamanaka and Kusai (24,

heme c per molecule. This is in good agreement 25) have reported that a flavocytochrome, cyto

with the results obtained by Bartsch et al. (1) that chrome c-553 of C. limicola f. thiosulfatophilum,

C. vinosum cytochrome c-552 has one molecule functions in the oxidation of Na2S, and cytochrome

of FAD and two molecules of heme c per molecule, c-555 of the organism acts as the electron acceptor

although they reported that the molecular weight in the oxidation of sulfide. C. vinosum cytochrome

is 72,000}6,000. The molecular weight of cyto c-553(550) resembles C. limicola f. thiosulfatophilum

chrome c-552 was determined to be 53,000 by gel cytochrome c-555; both cytochromes seem to be

filtration with Sephadex G-100 in the present c6-type cytochromes (12, and unpublished data).

study. In any case, the amino acid composition Therefore, it was expected that cytochrome

of the intact cytochrome c-552 was in good agree c-553(550) would act as the electron acceptor for

ment with the sum of those of the two moieties. sulfide oxidation with cytochrome c-552 in C.

After the two subunits obtained above had vinosum just as in C. limicola f. thiosulfatophilum.

been separately dialyzed against 10mm Tris-HCl, However, cytochrome c-553(550) does not act

pH 8.5, their sulfide-cytochrome c reductase activ as the electron acceptor. Morita et al. (6) have

ity was measured. Neither subunit showed activity. suggested that Chromatium cytochrome c-553(550)

Further, the activity was not restored even when may participate in a photosynthetic pathway

a mixture of the two subunits was dialyzed against different from that in which cytochrome c-552

10mm Tris-HCl, pH 8.5. functions, while it has been reported that Chro

matium cytochrome c-552 is implicated (23) in the

electron transfer between reduced sulfur substrates

DISCUSSION
and the photoreaction center . Further, it has
Chromatium cytochrome c-552 markedly accelerates been claimed that the two hemes of cytochrome

J. Biochem.
FLAVOCYTOCHROME c OF Chromatium vinosum 1413

c-552 can be distinguished by EPR (4). Thus, it obtain both subunits simultaneously have so far
seems likely that one of the two hemes in the been unsuccessful. However, it appears to be
cytochrome c-552 molecule plays the same role correct that the molecule of cytochrome c-552 is
as Chlorobium cytochrome c-555, so that the composed of two kinds of subunits, since N
electrons from sulfide may be directly transported terminal analysis of the cytochrome gave two
to the light-excited bacteriochlorophylls in the amino acid residues, alanine and glutamic acid,
active centers. It is necessary to elucidate the while the terminal residues of the cytochrome and
order in which an electron is transported between flavoprotein moieties were found to be glutamic
the prosthetic groups of cytochrome c-552 to acid and alanine, respectively. The molecular
clarify the physiological roles of the two hemes. weight of cytochrome c-552 is 53,000 as determined
C. vinosum cells accumulate granules of elementary by gel filtration on Sephadex G-100 in the present
sulfur photosynthetically in cells when sulfide or study. This value differs a little from that reported
thiosulfate is present in the medium, and the by Bartsch et al. (1). In any case, as judged from
elementary sulfur thus accumulated is finally the molecular weights of cytochrome c-552 and
oxidized to sulfate (7). On the other hand, some its two subunits, we can conclude that the molecule
workers have already found that whole cells of of the flavocytochrome is composed of one molecule
C. vinosum (9) and C. limicola f. thiosulfatophilum each of the cytochrome and flavoprotein subunits.
(8) catalyze the reduction of elementary sulfur to Yamanaka has reported that the flavocyto
sulfide under some conditions. As Chromatium chrome c of C. limicola f. thiosulfatophilum also
cytochrome c-552 catalyzes elementary sulfur to consists of two subunits, a flavoprotein subunit
sulfide with reduced benzylviologen, it seems that with a molecular weight of 47,000 and a hemo
the flavocytochromes c are responsible for the protein subunit with a molecular weight of 11,000
reduction of elementary sulfur in the cells as well (22). Flavocytochromes c such as C. limicola f.
as for the oxidation of sulfide. thiosulfatophilum cytochrome c-553 and C. vinosum
It seems clear that Chromatium cytochrome
cytochrome c-552 are isolated from very limited
c-552 consists of two subunits, a flavoprotein groups of photosynthetic bacteria which oxidize
moiety with a molecular weight of 46,000 and a
sulfide photosynthetically. Further, both cyto
chromes have sulfide-cytochrome c reductase
cytochrome moiety with a molecular weight of

21,000. The flavoprotein subunit can be obtained

activity and similar molecular features. It will be
by isoelectric focusing in the presence of 6 M urea
interesting to study the evolutionary relationship
and 1 % -mercaptoethanol, while the cytochrome
between the two flavocytochromes c, e.g. by
comparison of the amino acid sequences, and
subunit could not be obtained by the same method.
hence that between Chlorobium and Chromatium.
This may be attributable to the instability to
-mercaptoethanol of the heme of cytochrome
We thank Professor H. Matsubara for his encourage
c-552. In the absence of -mercaptoethanol,
ment and for valuable discussions during the course of
cytochrome c-552, was not completely split into the this work. We are also grateful to Dr. T. Hase for his
two moieties. Thus, even in polyacrylamide gel technical assistance in the amino acid analysis.
electrophoresis three bands were detected in the

presence of 6 M urea but absence of -mercapto REFERENCES

ethanol, while two bands were detected in the
1. Bartsch, R.G., Meyer, T.E., & Robinson, A.B.
presence of both 1 % -mercaptoethanol and 6 M
(1968) in Structure and Function of Cytochromes
urea. The excess band observed in the absence
(Okunuki, K., Kamen M.D., & Sekuzu, I., eds.)
of -mercaptoethanol seems to be due to original pp. 443-451, Univ. of Tokyo Press, Tokyo
cytochrome c-552 which survived the electro
2. van Niel, C.B. (1936) Arch. Mikrobiol. 7, 323-358
phoretic procedure. Thus, -mercaptoethanol was 3. Hendriks, R. & Cronin, J.R. (1971) Biochem.
essential to obtain pure flavoprotein subunit. Biophys. Res. Comnnaun. 44, 313-318
Accordingly, in order to obtain the cytochrome 4. Strekas, T.C. (1976) Biochim. Biophys. Acta 446,
moiety, a milder procedure is necessary where the 179-191
flavoprotein moiety is not completely separated
5. Kenney, W.C. & Singer, T.P. (1977) J. Biol. Chem.
from the intact flavocytochrome c. Attempts to
252, 4767-4772

Vol. 85, No. 6, 1979

1414
6. Morita, S., Edwards, M., & Gibson, J. (1965)
Biochim. Biophys. Acta 109, 45-58
7. Smith, A.J. & Lascelles, J. (1966) J. Gen. Microbiol.
42,357-370
8. Paschinger, H., Paschinger, J., & Gaffron, H. (1974)
Arch. Microbial. 96, 341-351
9. van Gemerden, H. (1968) Arch. Microbiol. 64,
118-124
10. Bartsch, R.G. & Kamen, M.D. (1960) J. Biol.
Chem. 235, 825-831
11. Bartsch, R.G. (1971) Methods in Enzymology 23A,
644-649
12. Cusanovich, M.A. & Bartsch, R.G. (1969) Biochim.
Biophys. Acta 189, 245-255
13. Roy, A.B. & Trudinger, P.A. (1970) The Biochem
istry of Inorganic Compounds of Sulfur pp. 43-44,
Univ. of Cambridge Press, London
14. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244,
4406-4412
15. Wada, H. & Snell, E.E. (1972) Anal. Biochem. 46,
548-556
16. Vesterberg, O. & Svenson, H. (1966) Acta Chem.
Scand. 20, 820-834
17. Blomback, B., Blomback, M., Edman, P., & Hessel,
B. (1966) Biochim. Biophys. Acta 115, 371-396
Y. FUKUMORI and T. YAMANAKA
18. Jeppson, J.-O., & Sjoquist, J. (1967) Anal. Biochem.
18,264-269
19. Spackman, D.H., Stein, W.H., & Moore, S. (1958)
Anal. Chem. 30,1190-1206
20. Fogo, J.K. & Popowsky, M. (1949) Anal. Chem. 21,
732-734
21. Walker, W.H., Kenney, W.C., Edmondson, D.E.,
& Singer, T.P. (1974) Eur. J. Biochem. 48, 439-448
22. Yamanaka, T. (1976) J. Biochem. 79, 655-660
23. Bartsch, R.G. (1968) Annu. Rev. Microbiol. 22,
181-200
24. Yamanaka, T. & Kusai, A. (1970) in Flavins and
Flavoproteins (Singer, T.P., eds.) pp. 292-301,
Elsevier Scientific Publishing Company, Amsterdam
25. Kusai, A. & Yamanaka, T. (1973) Biochim. Biophys.
Acta 325, 304-314
26. Yamanaka, T. & Okunuki, K. (1968) J. Biochem,
63,341-346
27. Yamanaka, T. & Shinra, M. (1974) J. Biochem. 75,
1265-1273
28. Horio, T. (1958) J. Biochem. 45,195-205
29. Falk, J.E. (1964) Porphyrins and Metalloporphyrins
p. 240, Elsevier, Amsterdam
30. Hind, G. & Olson, J.M. (1968) Ann. Rev. Plant
Physiol. 19, 249-282
J. Biochem.