Isolation of P590 from Methanosarcina barkeri: Evidence for the presence of sulfite reductase activity

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.).

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