The presence of multiple intrinsic membrane nickel-containing hydrogenases in Desulfovibrio vulgaris (hildenborough)

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 16, 1986

Pages 70]-708

THE PRESENCE OF MULTIPLE I N T R I N S I C MEMBRANE NICKEL-CONTAINING HYDROGENASES IN DESULFOVIBRIO VULGARIS (HILDENBOROUGH)

Thierry

Lissolo,

Eui

S.

Choi,

Jean

LeGall

and

Harry

D.

Peck,

Jr.

School of Chemical Sciences Department of Biochemistry University of Georgia Athens, Georgia 30602

Received July 30, 1986

Three intrinsic membrane proteins exhibiting oxygen stable hydrogenase activity have been isolated f r o m 2- 2 ~ ! g a r i s . In contrast to the periplasmtc exclusively non-heme iron hydrogenase, all three hydrogenases contain Ni i n a d d i t i o n to non-heme iron, have low s p e c i f i c activities and a r e i n s e n s i t i v e to inhibition by CO. None o f t h e t h r e e hydrogenases cross react w i t h IgA a g a i n s t the periplasmic hydrogenase o f D. v u l g a r i s b u t t w o o f t h e new hydrogenases cross react w i t h IgA a g a i n s t the periplasmic nickel containing hydrogenase o f D. g ! g a s a n d t h e o t h e r new h y d r o g e n a s e cross reacts w i t h IgA a g a i n s t the periplasmic nickel and s e l e n i u m

hydrogenase

Three been

of D. d e s u l f u r i c a n s

soluble

isolated

reducing

and

belonging

exclusively

from

D.

iron

hydrogenase

~!g~E!~

baculatus hydrogenase

Desulfovibrio established

the

[(NiFe)

D.

the

iron

activities

have

(12,13);

been

however,

these

D.

gigas

and

the

not

within been

represent

Abbreviations: BV, B e n z y l v i o l o g e n ; SDS, s o d i u m a n d PAGE, p o l y a c r y l a m i d e gel electrophoresis.

701

sulfate

hydrogenase] non-heme

(3,4,5);

and

the

[(NiFeSe)

closely and

have

the

[(Fe)

hydrogenase

reported

activities

of

containing

(8,9,10,11)

has

hydrogenase

Desulfovibrio:

from

(6),

it

of

species

nickel

selenium

associated

forms

hydrogenase

periplasmic

iron

© 1986AcademicPress, Inco

different

genus

desulfuricans

Membrane

whether

from

hydrogenase]

non-heme

from (7).

to

--4).

molecular

non-heme

(1,2);

containing

hydrogenase]

stable

characterized

bacteria

periplasmlc

nickel

oxygen

(Norway

related

multiple

a single

D.

soluble

species

of

conclusively distinct dodecyl

molecular sulfate;

0006-291X/86 $1.50 Copyr~ht © 1986 ~ Academ~ Pre~, Inc. A# r~h~ ~ r~roduction in a ~ ~ r m reservel

VoI. 139, No. 2,1986

forms

of

hydrogenase

hydrogenase In

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

this

other

in

are

artifacts

we

report

hydrogenases

addition

due

to

the

association

of

proteins.

communication,

nickel-containing vu!garis

or

to

the

in well

the

the

presence

membrane

characterized

of

three

fraction periplasmtc

of

D. (Fe)

hydrogenase. METHODS Assays

and

PreRarations

D. v u l g a K ~ s (NCIB 8303) was grown on lactate-sulfate medium and extracts prepared as previously described (14). Hydrogen uptake and evolution activities were assayed spectrophotometrically (15) and protein was determined by a modified Lowry method (16). Native polyacrylamide gel electrophorests employed 5~ slab gels containing 0.1~ Triton X-100 (17) and hydrogenase activities were visualized by staining with BV ( 0 . 5 mM) a n d 2 , 3 , 5 - t r l p h e n y l tetrazoltum chloride (1 mM). S l a b g e l SDS e l e c t r o p h o r e s i s was carried out and the reactivities of hydrogenases wi£h polyclonal antibodies raised against purified hydrogenases f r o m D. f f ~ f f a s ( 3 ) , D. v u l g a E ! s (2) and D. d e s u l f u r t c a n s (Norway 4) (6) were analyzed by the enzyme-linked immuno electrotransfer blot technique (18) using horse radish peroxtdase-conjugated second antibody (19). Fe, Nt and Se were determined by plasma emission spectroscopy using a Jarrell Ash model 750 atomcomp. For the inhibition o f H2 u p t a k e activity by C0, the concentration o f CO w a s v a r i e d by the addition o f CO a n d H 2 saturated buffer solutions. Purification

of

membrane-bound

h~drogenases

The crude extract was centrifuged at 100,000 xg to obtain the membrane fraction which was extracted with 1~ Triton (X-114) containing 1 5 0 mM NaC1 a n d 10 mM t r i s - H C 1 , pH 7 . 4 (20,21). After incubation a t O°C f o r 2 h o u r s , the suspension was centrifuged for 20 m i n . a t 1 2 , 0 0 0 xg to remove cell debris and the clear supernate was incubated at 30°C for 5 min. to allow for precipitation of the detergent which was removed by centrifugation at 12,000 xg for 20 min. 28°C. The lower detergent phase was collected and redlssolved in the same buffer without Triton (X-114) by incubation at 0°C. The phase separation was repeated and the washed detergent phase was diluted 10x with a solution of Triton (X-100) plus 5 mM t r i s - H C l pH7.8, to give a final concentration o f 1~ T r i t o n (X-100). The protein was loaded onto a DEAE-Bio-Gel column (4.5 x 29 cm), the column washed with a liter of 0.01M Tris - HC1, and the hydrogenases eluted with a tris-HCl linear gradient (one liter each of 0.01M and 0.40 M tris-HC1). The elution profile is shown in Figure 1, u p p e r graph. The hydrogenase fractions were pooled, and applied to a hydroxyapatite (Bio-Rad) column ( 3 x 15 c m ) , a n d t h e proteins eluted by a linear phosphate gradient ( 5 0 0 ml e a c h o f 0 . 0 2 M and 0.30 M potassium phosphate buffer). As s h o w n i n F i g u r e 1, lower graph, the first peak of hydrogenase activity contained two different hydrogenases when analyzed by gel electrophoresis and activity staining. The third hydrogenase was well separated from the other two hydrogenase activities. All procedures were carried out in air at 4°C and all buffers were adjusted t o pH 7 . 8 a n d c o n t a i n e d 0.1~ Triton X-IO0. 702

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2 , 1 9 8 6

£ 2 -4 ;O

Z 0.4

O m

I--

"4

Iu

0.2

/" 100

.

' 150 FRACTION

2

i

c v

>" 1

t,,m

20 22 24 26 28 30 0.4

pig

t~ 0.2

Fig.

1.

z

,.(

i

!

25

O

50 75 FRACTION

Chromatography of m e m b r a n e ( U p p e r ) and h y d r o x y a p a t i t e

100

bound hydrogenases (Lower)

on

DEAE-BioGel

The d e t e r g e n t - r i c h phase containing membrane bound hydrogenases was l o a d e d o n t o a D E A - B i o G e l c o l u m n a f t e r 10X d i l u t i o n w i t h T r i t o n X - 1 0 0 ( f i n a l conc. 1~) and the p r o t e i n s were eluted with a linear 0.01-0.40 M tris-HCl gradient (Upper) . The h y d r o g e n a s e fractions from DEA-BioGel a p p l i e d to a h y d r o x y a p a t i t e c o l u m n and the eluted with a linear 0.02-0.30M potassium (Lower). The f r a c t i o n s of the f i r s t p e a k gel e l e c t r o p h o r e s i s and a c t i v i t y s t a i n i n g

were pooled, proteins were phosphate gradient w e r e a n a l y z e d by (inset).

RESULTS AND D I S C U S S I O N

Cell-free the to

high

preparations

specific

contaminate

activities.

all

was

(Fe)

and

(Fe)

hydrogenase

by

activity

Our

hydrogenase

(NiFe)

CO w h i l e

the

of

cell first

perlplasmic fractions

evidence

obtained

as

for

As

mask

the CO a s shown

anticipated,

membrane-bound

(Fe)

and

employing

hydrogenases. is,

vu!g~[!~ contain

D.

in

highly

hydrogenase 703

amounts

hydrogenase other

presence a

large

probe Figure

tends

hydrogenase of

multiple

to

differentiate

2,

sensitive activity

which

of

the to was

periplasmic inhibition relatively

Vol. 139, No, 2,1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,°° k 60 ko 40

20

I

I

Io

20

m

Inhibition

2.

o f D. ~ ! g ~ E ~

hydrogenases

The h y d r o g e n a s e a c t i v i t y (-0-0-) and t h e p u r i f i e d measured in the presence extent of inhibition was

insensitive

to

inhibition

hydrogenase(s).

This

solubilization

and

hydrogenase

activities.

with

X-114

Triton

hydrogenase the

(21).

has

properties

After

forms

hydrogenase-l, hydrogenase hydroxyapatite, the

first

in

at the

of

30°C

is

hydrogenase

which

remains

the on

periplasmic

DEAE-Biogel

hydrogenase

activity (NiFe)

in

eluted

and

were the by

early 30

and

an

(NiFe) 704

extended

membrane a

the

contained

of

hydrogenase-3

that

hydrophilic (Table

phase I).

three

follows: the

buffer in

the

membrane

hydroxyapatite,

fractlons

of

indicating

intrinsic

as

fractlon

portion

hydrogenase

identified

by

membrane-bound

phase

in

mM p h o s p h a t e

hydrogenase-2, peak

(Fe)

three

the

hydrophobic

(NiFe)

been

(20),

activity

contained

activity

extraction

of

has of

for

of

of

presence

observation

partitioning remains

chromatography

molecular

of

The

the

characterization

responsible

protein the

and

activity

enzyme(s)

preliminary

After

by CO

of t h e c r u d e membrane f r a c t i o n periplasmic hydrogenase ( - e - O - ) was of different a m o u n t s o f CO. The expressed as % remaining activity.

suggesting

partial

50

(pL)

CO Fig.

40

30

(NiFe)

first

peak

of

from

the

late

which

fractions was

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table i.

Comparison of the Hydrogenase

from D. vulgaris

Specific Activities (kat/@) H 2 Uptake H 2 Evolution

Subunit Structure (KDa)

Effect of CO

Hydrogenase

Electrophoresis (Rf)

Hase l q A Dv. Dg. Db.

Periplasmic (Fe) hydrogenase

S

800x103

80x103

0.49

43,110

I

5.3

3.9

0.ii

85,45

+ +

Membrane(NiFe) hydrogenase (NiFe) hydrogenase

(2)

I

328

112

0.14

85,45

(NiFe) hydrogenase

(3)

I

560

1850

0.06

86,45

i.

S, sensitive,

contained buffer.

in The

hydrogenases gel

(i)

containing

activity

stain

the

+

I, insensitive second

activity

electrophoretic and

the

0.i~ is

(Fe) Triton

shown

in

peak

eluting

Soluble periplasmic

with

behavior

of

the

hydrogenase

on

a native

(X-IO0) Figure

as 3.

detected In

the

123

200

three

by

mM p h o s p h a t e

(NiFe) (5~) the

absence

polyacrylamide hydrogenase

of

the

) Membrane t b°und

&

D. vulgaris Hydrogenases Fig.

3.

-

Native

gel

electrophoresis

o f D.

vu~gar~s

hydrogenases.

Three membrane hound hydrogenases separated on hydroxyapatite c o l u m n w e r e a n a l y z e d by n a t i v e g e l electrophoresis (5~ a c r y l a m i d e and 0 . 1 ~ T r i t o n X-IO0) and compared with the periplasmic h y d r o g e n a s e by a c t i v i t y staining w i t h BV (0.SmM) a n d 2 , 3 , 5 - t r i p h e n y l tetrazolium chloride (1.OmM). 705

Vol. 139, No. 2,1986

detergents, stacking

the

(NiFe)

gel.

Each

protein

and their

(12.5~

acrylamide

into

two

also

were

minor were

not

of

giving

are

and

0.1~

SDS)

(~6

and

dissociated

completely

nickel

to it

completely

the

of

be

(Fe)

hydrogenase

of

D.

gigas,

Western

blot

technique.

that

vulgaris

bydrogenases

of

D.

D.

the

but

each

of

(NiFe)

2

4 to

5

of

the of

0.3

g.

bydrogenase

selenium

(NiFe)

and

hydregenases

presence

molecule

the

the

in

an

amount

hydrogenase-1

hydrogenase-1

and

is

3;

not

hydrogenase-1

-2

of

D.

against

~g~!~,

the

cytoplasmic

each

results

of

antibody provided

least

three

the

purified

periplasmic

(NiFeSe)

reactivities

the

(Fe)

the

(NiFe)

hydrogenase

of

different

determined definitive

antigenically

hydrogenase

against

but

with

-2

not

the

fact

the

does

not

(NiFe)

by

the

evidence distinct

other

two

of

desulfuricans

D.

subunit large

with

706

subunits

with of

of

the

subunits

antibody

(NiFe)

against mean

and

(Norway

of

that

the D.

~igas

(NiFe)

hydrogenases;

of

necessarily

only

bydrogenase

large

the

react

reacted

the

large

that

cross

gigas

the

against

hydrogenase

The and

raised

1).

(NiFeSe)

vulgaris.

and

at

reactivity

exclusively

D.

with

antibody

and

were

the

The

against

hydrogenase; weak

4)

contains

antibody

hydrogenase

major

dissociates

these of

the

On SDS-PAGE

Kda)

analyses

per

in

the

of

vulgaris

(Table

hydrogenase-1

reacts

that

45

that

addition,

found

and

(Norway of

against

In

antibodies

hydrogenases

exhibited

+ 1.

was

and

indicate

iron

1.

on

hydrogenases-1

indicating

of

one

hydrogenase-3

(85

emission

hydrogenase

desulfuricans

(Fe)

13

Table

(NiFe)

Metal

atom

noted

rabbit

periplasmlc

Thus,

(NiFe) the

remained

purified.

Polyclonal

D.

g.

in

subunits

detected

nickel

should

the

and

contained

presented

two

plasma

4.0

ratio

preparation

purified.

by

and

a Fe/Ni

however,

were

precipitated

45 K D a ) ;

into

bands

yet

equivalent

D.

values

hydrogenases

atom

hydrogenases

hydrogenase

Rf

subunits

protein

three

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

antibody 4)

hydrogenase-3

the

(NiFe)

the

(NiFe)

the

two

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VoI. 139, No. 2 , 1 9 8 6

hydrogenases

are

forms

results

these

two

from

D.

and

results

vulgaris

kay

of play

hydrogen

hydrogen

E.

coll

for

of

to

a

model specific

one

be

of

of

the

the The by

D.

nickel

the

electrophoretlc relationship

of

further

not

hydrogenase

been

reductions

kay or

of

D.

be

g~gas

and

however,

postulated

involved

oxidations

of

to

physiological

established; as

fraction

hydrogenases

The

hydrogenase

they

membrane

containing

desulfuricans.

cytoplasmic or

that

(NiFe)

have

(23)

of

established

establish

hydrogenases role

that

characterization.

types

hydrogenase

cycling

of

two

related

these the

only

biochemical

contains

(NiFeSe)

roles

can

or

modification.

conclusively

immunologically the

(22)

proteolytic

hydrogenases

purification Our

identical

in as

in

the the

in

one the

activation case

of

(24,25).

ACKNOWLEDGEMENTS T h e s e s t u d i e s w e r e s u p p o r t e d in part u n d e r c o n t r a c t no. D E A - 5 0 9 - 7 9 ER I 0 4 9 9 - A 0 0 2 f r o m the U.S. D e p a r t m e n t of E n e r g y to H . D . P . , and g r a n t s from the N a t i o n a l S c i e n c e F o u n d a t i o n no. DMB 8 4 1 9 6 3 2 to J,L. and H . D . P . and f r o m the N a t i o n a l I n s t i t u t e s of H e a l t h no. I Rol GM 3 4 9 0 3 to J.L. and H . D . P . The a u t h o r s w i s h to t h a n k the p e r s o n n e l of the U n i v e r s i t y of Georgia fermentation p l a n t for the g r o w t h of D. vulgari_ss, B e n e t C. P r i c k r i l and N a n c y B o n d for a s s i s t a n c e in the p r e p a r a t i o n of h y d r o g e n a s e antibodies, Dr. D a l u l a t S. P a t i l for the p r e p a r a t i o n of a n t i b o d i e s to D. b a c u l a t u s hydrogenase, and Dr. N a n d a K. M e n o n for a s s i s t i n g the W e s t e r n i m m u n o b l o t s .

REFERENCES 1. 2,

3.

4. 5.

6. 7. 8. 9.

Van der Westen, H., Mayhew, S.G. and Veeger, C. 1978. FEBS Lett. 86: 122-26. Huynh, B.H., Czechowskt, M.H., Kruger, H.J., DerVartanian, D.V., Peck, H.D., Jr., and LeGall, J. 1984. Proc. Natl. Acad. Sci. (US) 8~: 3728-3732. LeGall, J., Ljungdahl, P.O., Noura, I., Peck, H.D., Jr., Xavier, A.V., Moura, J.J.6., Teixeira, M., Huynh, B.H., and DerVartanian, D.V. 1982. Biochem. Biophys. Res. Commun. ~06: 610-816. Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E.C., 1982. FEBS L e t t . ~42: 289-292. Teixeira, M., Moura, I., Xavier, A.V., Ruynh, B.H., DerVartanian, D.V., Peck, H.D., Jr., LeGall, J., and J.J.6. Noura. 1985. 3. Biol. Chem. 280: 8942-8950. Rieder, R., Cammack, R., and Hall, D.O., 1984. Eur. J. Biochem. ~45: 637-643. Teixeira, N., Moura, I., Xavier, A.V., Moura, J.J.6., Fauque, G., Pickril, B., and LeGall, J. (1985) Rev. Port. Quim. 27: 194-195 Martin, S.N., Glick, 8.R., and Martin, W.G., 1980. Can. J. Biochem. 26: 1204-1213. Lalla-Naharajh, W.V., Hall, D.O., Cammack, R., Rao, K.K., and LeGall, J., 1983. Blochem. J. 209: 445-454. 707

Vol. 139, No. 2,1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10. Yagi, T., Kimura, K., Daldoji, H., Sakai, F., Tamura, S., and Inokuchi, H., 1978. J. B i o c h e m (Tokyo) 79: 661-671. 11. Yagi, T., Kimura, g., and Inokuchi, H., 1985. J. Biochem. (Tokyo) 97: 181-187. 12. Ackrell, B.A.C., Asato, R.M., and Mower, H.F. (1966) J. B a c t e r l o l 92: 8 2 8 - 8 3 8 . 13. Kruger, H.J., Huynh, B.H., Ljungdahl, P.O., Xavier, A.V., D e r V a r t a n l a n , D.V., Houra, J., Peck, H.D., Jr., Telxeira, M., Moura, J.J.G., and LeGall, J., (1982) J. Biol. Chem. ~ Z : 14620-14623. 14. L e G a l l , J . , M a z z a , G . , and D r a g o n i , N . , ( 1 9 6 5 ) B i o c h e m . B i o p h y s . Acts 99: 385-387. 15. L i s s o l o , T., Poluin, S . , and Thomas, D . , ( 1 9 8 4 ) J. B i o l . Chem. 259: 11725-11729. 16. M a r k w e l ] , H . A . K . , Haas, S.M., B i e b e r , L . L . , and T o l b e r t , N.E., (1978) A n a l . Btochem. 87: 2 0 6 - 2 1 0 . 17. L a e m m l i , U . K . , ( 1 9 7 0 ) N a t u r e 2 2 7 : 6 8 0 - 6 8 5 . 18. H a t d , A. and S u t s s a , M ., ( 1 9 8 3 ) M e t h o d s E n z y m o l . 9 6 : 1 9 2 - 2 0 5 19. Hawkes, R . , N i d a y , E. and G o r d o n , J . , (1982) Anal. Btochem. 119: 142-147 20. B o r d i e r , C . , ( 1 9 8 1 ) J. B i o l . Chem. 2 5 6 : 1 6 0 4 - 1 6 0 7 . 21. B r i c k e r , T . M . , and S h e r m a n , L . A . , (!984) Arch. Biochem. Biophys. 235: 2 0 4 - 2 1 1 . 22. B a l l a n t i n e , S.P., and Boxer, D.H., (1985) J. Bacteriol. !~: 454-459. 2 3 . Odom, J . H . , and Peck, H.D., Jr., ( 1 9 8 1 ) FEMS L e t t . !~: 47-50. 24. S a w e r s , R . G . , B a l l a n t i n e , S.P., and Boxer, D.H., (1985) J. Bacteriol. !~: 1324-1331. 25. Harker, A . R . , Z u b e r , M., a n d E v a n s , H . J . (1986) J. Bactertol. 165: 579-584. m

m

708