Vol. 108, No. 3, 1982 October 15, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1002-1009
ISOLATION OF P590 FROM ME’ilHAlDSARCINA BARKERI: EVIDENCE FORTHE PRESENCE OFSULFITE REWCTASE ACTIVITY a,b , Isabel
JosgJ.G.Moura
Moura a& , Helena Santos',
Monique ScandellariC,
Ant&io
V.Xavier a,b ,
and Jean LeGalld
a Centro de Q&mica
Estrutural, Complexo I, IST, Av.Rovisco Pais,. 1000 Lisboa (Portugal) b Gray Freshwater Biological Institute, University of Minnesota, P.O.Box 100, Navarre, Minnesota 55392 (U.S.A.) ' Laboratoire de Chimie Bacteri&ne, CNRS, 13274 Marseille, Cedex 2 (France). d Department of Biochemistry, University of Georgia, Athens, Georgia 30602(USA). Received
August
2, 1982
A protein (P5gD) which catalyses the 6-electron reduction step from to sulfide was isolated from the methanogenic bacterium, Methanosarcina barkeri. The enzyme has a specific bisulfite reductase activity of 90 mU/mg and its spectralcharacteristics are similar to the siroheme containing sulfite reductases. sulfite
INTRODUCTION The methanogenic unique group of bacteria and H2. different
Additionally substrates:
methanol,
acetate,
formaldehyde
importance
amino-acids,
M, which contains
that can produce methane following is a versatile
The metabolism
containing
of sulfur
of methyl-cobalamin,
and for-mate (1). it
to the usual needs, such as sulfur
they are known to synthetize sulfur
large amounts of coenzyme (2).
In addition
it has been
is necessary for maximal growth and methane
(3) and that
sulfide
0006-291X/82/191002-08$01.00/0 Copyrighf 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
of CO2
organism since it can reduce
methyl moiety
both reduced and oxidized
in M.barkeri
the reduction
in those organisms remains obscure although
since further
known for some time that sulfide production
to which belongs Methanosarcina barkeri are a
M.barkeri
methyltetrahydrofolate,
is of special
bacteria
1002
is not merely active
as a reducing
Vol. 108, No. 3, 1982
BIOCHEMICAL
agent since addition sulfite
to sulfide
reductase. It is a central
is a 6-electron reaction
thiosulfate
bacteria,
to sulfite.
Assimilatory
catalysed by the dissimilatory
The active
of their
as in the of
have been suggested to occur during the reactions enzymes. The mechanismof the dissimilatory
from different
active
(5).
Assimilatory
sources and their
sulfite
physiological
centers have been studied in detail
role
(6,7,8).
center was found to contain a 14Fe-4SI core and a siroheme. Recent
siroheme and the iron sulfur
center are linked as a functional
Dissimilatory
sulfite
reductases have been isolated
coZi indicate unit
from sulfate
(10) , D.gigas (11) and D.saZetigens from D.desuZ,Furicans
substrate is protonated.
Norway 4 (12) and D.desu2furican.s
strain
spp. (13,14).
reductases is not well defined.
from
of the active
when the
centers of dissimilatory
Approximately
per enzyme molecule which also contains iron-sulfur
9974 (our
These enzymes
reductases since they have higher activity The structure
(9).
(our unpublished data) ; desulfo-
unpublished data) ,and P-582 from Desdfotomaculum were denominated bisulfite
that the
reducing
and used as taxonomic markers of the genus: desulfoviridin
D. vuZgaris
sulfite
which are
but the presence of intermediates such
reductase from Escherichia
rubidin
in the
in the oxidation
Wssbauer studies of sulfite
bacteria
There are
reductases can carry out the
is, however, quite controversial
reductases have been purified and the structure
which participate
reduction of sulfite,
sulfite
to sulfide,
and thiosulfate
reduction of sulfate
biocycle.
or primary oxidases participating
complete reduction of sulfite as trithionate
instead of
that is catalysed
biomolecules, and the dissimilatory,
terminal reductases in the respiratory
sulfate-reducing
reaction
in the sulfur
reductases: the assimilatory,
synthesis of sulfur-containing either
citrate,
for maximal growth (4).
Reduction of sulfite
two types of sulfite
RESEARCH COMMUNICATIONS
into the growth mediumof titanium(II1)
is not sufficient
by sulfite
AND BIOPHYSICAL
two sirohemes are bound
centers.
Under reducing
conditions
these enzymes show an EPR signal at g=1.94, but in general no precise
definition
of the type of center was yet made, although two (4Fe-4SI centers
were indicated
to be present in D.gigas
desulfoviridin
(15) y The spectroscopic
BIOCHEMICAL
Vol. 108, No. 3, 1982
data here reported strong
similarities
AND BIOPHYSICAL
for the enzyme isolated
RESEARCH COMMUNICATIONS
from Methanosarcina
with the siroheme containing
sulfite
barkeri
shows
reductases.
MATERIALSANDMETHODS Organism and growth conditions grown on methanol as previously utilization.
barkeri strain DSM 800, was (1) and stored at -800C until
- Methanosarcina
described
Acrylamide gel electrophoresis and molecular weight determination - Analytical gel electrophoresis was performed according to the method of Davis (16) on a 7% (v/v) gels at pH=8.0. The molecular weight determinations were done using electrophoresis SDS-polyacrylamide gel (17) using the following protein standark: D.gigas Mo(Fe-S) protein (Mr= 120,000) (18), egg albumin (Mr= 68,000) hman albumin (Mr= 45,000) and chymotrypsin (Mr= 25,000). Protein determination - Protein was determined Bradford (19) using C.omassie brilliant blue. - Ultraviolet
and visible
using the method described
by
spectra were recorded on a Beckman
-photometer. Extraction of the active center - The active center was extracted with 9 volumes of- acidified acetone (5% v/v) per volume of protein solution at 4OC (20). 120 $I protein solutions were used in the extraction procedure. The precipitated apoprotein was removed by centrifugation and the extracted solution was concentrated by evaporation under vacuum. Enzymatic assays - The sulfite reductase activity was measured by a manometric assay, as described by Schedel et aZ. (21). It requires the generation of reduced methylviologen by an excess of hydrogenase activity under hydrogen atmosphere. The reduced dye then serves as electron donor to the reductase. A freshly prepared sodium sulfite solution (0.1 ml of 0.01 M) was added from the sidearm to the main compartment of each manometric vessel containing 0.1 ml of 1 M phosphate buffer (pH as required): 0.1 FI methylviologen 2% (w/v); hydrogenase (approx. 40 ug of protein) and 0.75 ml of enzyme solution (2.2 m&al); water was added to a final volume of 2.8 ml. The center well containing 0.05 ml of 10 N Nash and 0.05 ml of 10% (w/v) Cd(CH3COO)2.Hydrogenase, purified either from D.gigas as described by Bell (33) or from M.barkeri (our unpublished work), was added (in all cases) to the system to ensure an excess of this activity. Before addition of sodium sulfite the flask was incubated under hydrogen atmosphere for 30 min. at 370C. In typical experiments, the blue color of reduced methylviologen appears 3-5 mins. after incubation under H2. -90 - purification scheme - All operations were carried out at +4oC, unless otherwise stated. Trls-HCl and phosphate buffers, pH = 7.6 of the appropriate molarity were used. Gradients were accomplished with Tris-HCl or NaCl in 10 mM Tris-HCl buffer medium. A frozen past of (700 g) of cells was suspended in 500 ml of 10 mM Tris-HCl, CNA'se was added and the mixture was passed twice through a French pressure cell. The disrupted cell suspention was centrifuged at 13,200 g for two hours. The supernatant (1,000 ml) was applied to a DEAE 52 column (2.5 x 26 cm) equilibrated with 10 &I Tris-HCl. A fraction containing cytochrome b and hydrogenase in a particulate form was not adsorbed. The adsorbed proteins were washed with 200 ml of 10 a&l Tris-HCl and eluted with a discontinuous gradient of 200 ml of 0.05, 0.15, 0.20 up to 0.60 M Tris-HCl. Several fractions were collected according to their spectral characteristics. The position of the pigment during the chromate graphic steps was located by following the absorption at 590 nm. One of the less acidic fractions (150 ml) contained a band around 590 nm and was subsequently 1004
BIOCHEMICAL
Vol. 108, No. 3, 1982
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
purified. It was diluted twice with distilled water and readsorbed on a second DEAE-52 column (2.5 x 15 cm). A gradient was performed using 100 ml of the following NaCl concentrations in 10 mM Tris-HCl: 0.050, 0.075, 0.100, 0.125, up to 0.250 M. Four main fractions were obtained: the first fraction (350 ml) contained a protein with an absorption band at 350 nm; the second fraction (100 ml) contained a protein with an absorption band at 590 nm; the thirdfraction (50 ml) contained a non-heme iron protein, with an absorption band at 425 nm and a shoulder at 445 nm. The first, third and fourth fractions have been subsequen tly purified. The second fraction (containing P590) was adsorbed in a hydroxylapatite column (3 x 5 cm) equilibrated with 0.15 M NaCl (the same ionic strength of elution of the fraction). Before starting the phosphate gradient the column was washed with 30 ml of the following decreasing NaCl molarities, in 10 mM Tris -HCl: 0.15, 0.10,. 0.05, and finally with 30 ml of 0.010 M Tris-HCl. The column was washed with 30 ml of each of the following phosphate molarities: 0.01, 0.05, 0.10, 0.15, 0.20, up to 0.50 M. The fraction containing the PsgO was eluted at 0.25 Mphosphate buffer and collected in a volume of 50 ml. It had an absorption ratio A28O/AsgO = 20.7. This fraction was concentrated in a Diaflo Amicon Membrane PMlO, and adsorbed again in a hydroxylapatite coltmm (2.5 x 5 cm ). A phosphate gradient was performed again using the following molarities: 0.100, 0.125, 0.150, up to 0.300 M. The P5go was eluted at 0.15 - 0.20 M, in a volume of 150 ml. This fraction was concentrated down to 10 ml in a Diaflo system and applied to a G-50 Sephadex column (2 x 100 cm) equilibrated with 10 mM Tris-HCl buffer. The absorption ratio of the main band (25 ml) was A28O/AsgO = 5.3. The protein was adsorbed on a DEAE column (2.5 x 5 cm) and a Tris-HCl gradient was performed using 25 ml of the following molarities: 0.1, 0.12, 0.14, 0.16, 0.18, and 0.20 M. The protein eluted at 0.16-0.18 M. Tris was obtained in a final volume of 30 ml and had an absorption ratio of A280/A5go = 4.3.
RESULTS Molecular
Weight and Purity
SDS polyacrylamide /A275 ratio
gel electrophoresis
of the preparation
with a ASgo/
of 4.3 showed two strong bands at Mr. = 56,000 and 31,000 and two
very weak bands at Mr. = 37,000 and 27,000 after protein
staining.
However, in
the absence of SDS, only the strong band was observed before staining additional
minor bands were detected
suggest that the enzyme may exist Electronic
Spectral
The absorption the native (0.23),
395 (0.63)
bands at the following and 275 (1.0)
sulfite
isolated
other siroheme containing E.coli,
Those results state.
spectra of the Psgo is shown in Figure 1. The spectra
It is very similar
spinach,
staining.
as a dimer in the native
in parenthesis). reductase
protein
Data
form shows absorption
545 (0.17),
after
and two
to the absorption
from D.~utgutis
intensities spectra
from different Treatment
1005
(nm): 590 are indicated
of "assimilatory"
(2) 'and does not differ
enzymes isolated
and D.chitrifieans).
(relative
wavelengths
of
markedly
organisms
(e.g.,
with acetone/He1
yields
from
a
BIOCHEMICAL
Vol. 108, No. 3, 1982
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fj.gure 1. Ultraviolet and visible absorption spectra of the native M.barkeri P5go (sulfite reductase) (-) and the acidic acetone extract (----) . Figure 2. Profile of hydrogen consum@iondeterminedas described in Materials andMethodsmeasuredin the presenceof D.gigas hydrogenaseat pH = 6.0 (o), 6.5 (o), 7.0 (e), and 7.5 ( CJ) ; in the presence of M.barkeri hydrogenaseat pH = 6.5 (A ) , and in the absenceof sulfite reductase (endogenous) (0).
heme-containing extract
similar
to those obtained with P5g2 and B.coti
sulfite
reductase (20). Enzymatic Activity Sulfite
can be used as a terminal electron
acceptor with the P590 pre-
parations with consequent consumption of hydrogen in the enzymatic reaction described before (32) as: Hase--aMV-
H2 -
= SO3
Reductase (P590) _l_g
Figure 2 shows the rate of hydrogen consumption together with its pH activity
profile
indicates
(pH = 6.0 to 7.5) . The pH dependence of the enzymatic reaction
that bisulfite
the slightly
is the substrate since maximal activity
acidic region.
by the yellow precipitate
CdSwas formed during the reaction as was attested formed in the center well.
tested with hydrogenase from two different (our unpublished data). The specific
sulfite
was observed in
No difference
reductase activity
The enzymatic reaction was
sources: D.gigas
in activity
(23)
and M.barkeri
could be detected (see Fig.2).
measured at pH = 6.5 was 90 mU/mg 1006
Vol. 108, No. 3, 1982
BIOCHEMICAL
AND BIOPHYSICAL TABLE
CO?&‘ARISON Sulfite
reductase
BEl?TEEN
SULFITE
Absorption
mulfotomcuLm
700w,582,392,280
denitrificans ('582)
A3g2/A582
ORIGINS
w.
Activity huh%)
1o-3
Active
145
center
SIR
+ Fe-S
= 3.8
D.desulfuricms
720w,580,545,392,28Ll A3921545
D.gigas
628,580,408,390,357,280
225
420
SIR
+ Fe-S(a2B2)
632
632
SIR
+ Fe-S(a2B2)
220
260
SIR
+ Fe-S(a2B2)
27
900
SIR
+ Fe-S
(a)
30
90
SIR
+ Fe-S
(LX,)
136
SIR
+ Fe-S
(2.3W
SIR
= 3*3
A408/A628
D.vuZga&
FROM DIFWRFXT
(nm)
Norway 4 desulforubidin
desulfoviridin
I
RELXJCTASE
bands
RESEARCH COMMUNICATIONS
= 3.0
628,580,408,(309)
,(375),220,279
desulfoviridin
A408'%28 D. vulgaris
= 3*o
590,545,405,275 A275/A5go”.0
M.barkeri
A405/A5g0=2.7
7oow,590,s45,395,275 A275/A5go=4.3
T.denitrificans
A3g5/A5g0=2.
595,(450)
,390,280
A3g0/A3g5
= 3.0
E.COli
7 160
705,587,(445),386,280 A3@j/A587
= 4.5
589,404,(385)
Spinach
A27g/A58g=6.0 w - weak
( ) - shoulder
of protein
(1 unit
SIR
685
85
,279 A404/A58g=5.1
- sirohem
Fe-S
- iron-sulfur
center
catalysing
the consumption of
(u) is the enzyme activity
1 pmole H2 per min. at 3O'C). DISCUSSION Siroheme is an universal sulfite
to sulfide
compares sulfite molecular weight,
and is also involved in the reduction of nitrite reductases from different specific
Although the constitution
activities
reductases exhibit
(20). Table 1
origins in terms of absorption bands,
and constitution
of the active
centers.
of the active centers is not well defined in terms of
type and number of iron-sulfur sulfite
choice as active center for the reduction of
centers present,
characteristic
g=6 (18,19). 1007
assimilatory
and dissimilatory
ferric-heme type of EPR signals around
BIOCHEMICAL
Vol. 108, No. 3, 1982
The dissimilatory
sulfite
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
reductases are complex structures.
Desulfoviridin
has a 220,000 Dalton molecular weight and possesses a tetramer ~1282structure. and P582 were reported to have molecular weights of approx.225,000
Desulforubidin
Dalton and 145,000 Dalton respectively. have optical
spectra typical
but desulfoviridin
denitrificans
with a 01 $ structure 22 Visible
trophic
bands at larger wavelenghts (628 nm), also contains a sulfite
characteristi‘cs
are typical
enzymes. However, since T.denittificans
centers.
is an obligate
chemolitho-
organism, whose growth depends on the presence of reduced sulfur
reduced sulfur
the sulfite
isolated
from M.barkeri
reductase isolated
reductase in M.barkeri
of sulfur
of sulfate
Bisulfite
reductase would
compoundsin a way very similar
(18). The fact that sulfide has been found
There also remains the possibility
reductase is used at an assimilatory genie bacteria
sulfite
for maximal growth and methane production
of this hypothesis.
utilization
with our present knowledge of M.barketi
would be that !!.barkeri
to that suggested for T. denitrificans
reductases have up to
However, a respiratory
to gain energy from reduced sulfur
to be essential
with
type of metabolism such as that
reducing bacteria.
metabolism. Another possibility
level.
(3) could be in favour
that M.barkeri
The redox potential
sulfite
at which methano -
grow makes it most probable that they do have large amounts of
at their
disposal. However, since coenzyrre EIcontains a sulfonate group
can be speculated that the sulfite
to provide sulfur
reductase functions
at the proper oxidation
that M.barkeri needs to reduce nitrite the isolated
shows spectroscopic similarities
raises many questions.
compoundsdoes not correlate
be utilized
of those
in other systems. The presence of a bisulfite
now been associated with a dissimilatory characteristic
oxidation
compounds
compounds.
The protein
it
reductase (Mr=16C),000)
of those from the
the enzyme was proposed to have a role on dissimilatory
sulfide
reductases
(15,18) with two siroheme units and iron-sulfur
and EPR spectra
assimilatory
sulfite
of siroheme containing proteins with bands at 590nm
shows additional
ThiObmillus
The dissimilatory
enzyme could function
level.
Although there is no indication
ions, the possibility
as a nitrite 1008
in a reversed manner
reductase.
still
remains that
Vol. 108, No. 3, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICA;TIONS
ACKNCWLEDGEMENTS We are indebted to Mrs. I.Carvalho for her skilful technical help. This work was supported by NIH Grant Nq25879, and the INIC, JNICT, Calouste Gulbenkian Foundation of Portugal and grants from SERI (Contract NO XK-1-1182-1) and from COMES. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Blaylock,B.A., and Stadtman,T.C. (1966) Arch.Biochem.Biophys., 116, 138 Taylor,C.D., and Wolfe,R.S. (1974) J.Biol.Chem. 249, 4879-4885. Ebonfort,D.O., and Asher,R.A. (1979) Appl.and EnZ-?on.Microbiol. 37, 670-675. Scherer,J., and Sahm,M. (1981) European J.Appl.Microbiol.Biotechn~. -12, 28-35. DerVartanian,D.V. and Peck,H.D. (1979) Current Topics in BioLeGall,J., energetic5 9, 237 Siege1,L.M. (19757 in "Metabolic Pathways", 3rd ed., vol.7 (ed.D.M.Greeberg) Academic Press, N.Y., p.217. and Kamin,H. (1978) Methods in Enzymol. 51, 436 Siegel,L.M., Murphy,M.J., Lancaster,L.R., Vega,J.M., Kamin,H., Orme-Johnson,N.R., Orme-Johnsz,W.H., Krueger,R.J., and Siege1,L.M. (1979) J.Biol.Chem. 254, 1268 Christner,J.A., M&rck,E., Janick,P.A. and Siege1,L.N. (lm) J.Biol.Chem. 256, 2098 mayashi,K., Takahashi,E., and Ishimoto,M. (1974) J.Biochem.(Tokio) 75,519 Lee,J.P. andPe&H D. (1971( Biochem.Biophys.Res.Conunun. 45, 583 and Peck,H.D. (1973) J.BactGiol. Lee,J.P., Yi,C.S., LeGall,J., -115, 453-455. Trudinger,P.A. (1970) J.Bacteriol. 104, 158 Akagi,J.M. and Adams,V. (1973) J.Bazriol. 116, 392 Hall,D.O., Prince,R.H. and Cammack,R. (1979)-Biochim.Biophys.Acta 581, 27 Davis,B.J. (1964) Ann.N.Y.Acad.Sci. 121, 404 Wctber,K. and Osborn,M. (1969) J.BiolT&em. 244, 4406 Moura,J.J.G., Xavier,A.V., Bruschi,M., LeGarJ., Hal1,D.O. and Cammack,R. (1976) Biochem.Biophys.Res.Commun. 12, 782 Bradford,M. (1976) Anal.Biochem. 72, 248 and Siege1,L.M. (197v J.Biol.Chem. 248, 6911 Murphy,M.J., Schedel,M. , LeGall,J. and Baldensperger,J. (1975)xch.Microbiol. 105,339 and Peck,H.D. (1973) J.Bacteriol. 115, szi5 Lee,J.P., Yi,C., LeGall,J. Bel1,G.R. (1973) Studies on electron transfer systems in Desu~~ibrio: purification and characterization of hydrogenase, catalase and superoxide dismutase. Ph.D.Thesis,p.206, Univ.of Georgia,Athens (U.S.A.).
1009