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Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors
BIOCHIMIE, 1978, 60, 315-320.
Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors. G. R. BF_~LL *, J i n - P o L E E * , H. D. P E G K Jr. * a n d J. L E GA,LI, "*.
" D e p a r t m e n t of B i o c h e m i s t r y , University of Georgia, Athens, Georgia 30602 (U.S.A.). *" Laboratoire de Chimie Bact~rienne, C.N.R.S., 1327~ Marseille Cedex 2 (France).
R6sum6.
Summary.
Une pr6paration d'hydrog6nase de Desuliovibrio qiqas ne peut pas r6duire la ferr6doxine, la flavodoxine ou la rubr6doxine en l'absence de cytochrome c3 (P.M. 13000), sous atmosph6re d'hydroqbne. Seuls le benzy1 viologbne ou le FMH sont directement r6duits. La production d'hydrogbne mol6culaire & partir du dithionite est possible avec cette pr6paration, en pr6sence de m6thyl violoq~ne comme m6diateur. La m6me r6action a aussi lieu en pr6sence de cytochrome c3 (P. M. 13 000) ou de cytochrome c3 (P. M. 26 000). L'addition de ferr&doxine ou de flavodoxine n'acc61~re pas la vitesse de production d'hydrog~ne.
A purified preparation of hydrogenase from D. qiqas was inactive toward ferredoxin, flavodoxin or rubredoxin in the absence of cytochrome c3 (M. W. 13,000), in an atmosphere of hydrogen, although direct reduction of benzy1 viologen or FMN w a s possible. The hydrogen evolution reaction from dithionite w a s possible with methyl viologen. The same reaction also occured with cytochrome c3 (M. W. 13,000) or cytochrome c3 (M.W. 26,000). Addition of either ferredoxin or flavodoxin did not accelerate the reaction.
Introduction.
Chemical and reagents. Trizma-base, DNase a n d bovine serum a l b u m i n were p u r c h a s e d from Sigma Chemical Company. Sodium d i t h i o n i t e was o b t a i n e d from F i s h e r Chemical Company. Benzyl viologen was supplied b y S c h w a r t z - M a n n ; m e t h y l - v i o l o g e n by Brit i s h Drug Houses Ldt. ; s p e c t r o p h o t o m e t r i c a l l y pure FMN by F l u k a AG, Chemische Fabrik. All o t h e r chemicals were of the highest p u r i t y available.
Yagi et al. [1] h a v e p r o p o s e d a d i f f e r e n t i a t i o n of the hydrogenases from clostridia and the sulfater e d u c i n g b a c t e r i a b e l o n g i n g to t h e g e n u s Desul[ovibrio o n t h e b a s i s of e l e c t r o n c a r r i e r s p e c i f i c i t y . A c c o r d i n g to t h e i r s t u d y , d e s u l f o v i b r i o n e s h y d r o g e n a s e is s p e c i f i c f o r c y t o c h r o m e c a a n d n o t f e r r e d o x i n w h i l e c l o s t r i d i a l h y d r o g e n a s e is s p e c i f i c f o r ferrodoxin. Other investigations have supported this view that, in desulfovibriones, either ferred o x i n o r f l a v o d o x i n a r e c o u p l e d to h y d r o g e n a s e v i a c y t o c h r o m e c a [2, 3, 4]. W e t h o u g h t it w o u l d b e of i n t e r e s t to i n v e s t i g a t e t h e r e l a t i o n s h i p s b e t ween hydrogenase and other redox proteins in a d e s u l f o v i b r i o n e , Desul[ovibrio gigas, f r o m w h i c h several electron carrying proteins have been characterized.
Materials
and
Methods.
Growth conditions. D. 9iyas was cultivated in a lact a t e - s u l f a t e m e d i u m a n d h a r v e s t e d as previously described [5].
Chromatographic materials. Calcinated a l u m i n a was from the L a b o r a t o i r e du Bois de Boulogne ; DEAEcellulose was p u r c h a s e d f r o m Schleieher and Sehnell, Inc. and Sephadex from P h a r m a e i a Fine Chemicals. Assays [or hydrogenase. 1) The reduction of benzyl viologen by h y d r o g e n a s e w i t h H2 was followed m a n o metrically by m e a s u r i n g the rate of H2 c o n s u m p t i o n . The center well of the W a r b u r g vessel contained 0.1 ml 10 per cent CdClo ; m a i n c o m p a r t m e n t , 0.6 ml of a benzyl viologen solution, r00 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.6) a n d distilled w a t e r to 2.3 ml. E n z y m e was added from the s i d e a r m a f t e r gassing the vessel w i t h H~ and s h a k i n g for 30 m i n u t e s at 3'0°C. Best results were obtained with reagents previously v a c u u m degassed. The u n i t of activity was defined as t~moles H~ oxidized per minute. 2~ The oxidation b y h y d r o g e n a s e of d i t h i o n i t e - r e d u cod m e t h y l viologen (or a n o t h e r electron carrier to be tested) was followed m a n o m e t r i c a l l y at 30°C by m e a s u ring the rate of H2 evolution. The m a i n c o m p a r t m e n t of each W a r b u r g vessel c o n t a i n e d h y d r o g e n a s e prepa-
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G. R . B e l l a n d coll.
r a t i o n a n d 5. X 10-4 M m e t h y l viologen (or a n o t h e r electron carrier) in 10~0 mM p o t a s s i u m p h o s p h a t e buffer (at the pH o p t i m u m for each reaction) to a final volume of 2.3 ml. The center well c o n t a i n e d 0.2 ml a l k a l i n e pyrogallol. The gas p h a s e was argon. A n h y drous sodium d i t h i o n i t e (2.4 mg) was added from t h e s i d e a r m to i n i t i a t e the reaction. The u n i t of activity was defined as lxmoles H2 evolved per m i n u t e .
Purification of electron carriers. The electron carriers were o b t a i n e d f r o m D. gigas according to m e t h o d s already p u b l i s h e d ; a review of these m e t h o d s h a s been p u b l i s h e d r e c e n t l y [6]. Enzymatic reduction of pure carriers. The r e d u c t i o n of p u r e electron t r a n s f e r p r o t e i n s f r o m D. 9igas was tested u s i n g purified h y d r o g e n a s e from the same organism. Reactions were followed s p e e t r o p h o t o m e t r i e a l l y in either B e c k m a n Aeta GV or Gilford 2000 recording s p e e t r o p h o t o m e t e r s u s i n g T h u n h e r g euvettes w i t h a 1 - em l i g h t p a t h . Solutions of eytoehromes e~, ferred o x i n a n d r n b r e d o x i n were dialyzed a g a i n s t several changes of 50 mM Tris-ttC1 buffer, pH 7.6. The same buffer was used to dissolve lyophilized flavodoxin a n d to p r e p a r e a s o l u t i o n of FMN. In each e x p e r i m e n t the volume of electron carrier solution added to t h e euvette was b r o u g h t up to 1.2 ml w i t h the same buffer. The s i d e a r m c o n t a i n e d 0.4 ml of the buffer a n d hydrogenase (0.1)63 rag). Cytoehrome e8 (M.W. 13,1)00) was added to the euvette in those e x p e r i m e n t s in w h i c h it was mixed w i t h a n o t h e r electron carrier. W i t h eytoehrome e3 (M.W. 26t,001)), 0.068 mg h y d r o g e n a s e i n 0.1 ml of 101) mM Tris-HG1, pH 7.6, was placed in t h e sidearm a n d 1.5 m l of the eytoehrome (in 1 M Tris-HC1) in the euvette. This a l t e r a t i o n was necessitated b y the insol u b i l i t y of eytochrome e~ (M.W. 26;000) in more dilute buffer. Each cuvette was p r e p a r e d b y connecting it to a closed system w h i c h p e r m i t t e d a l t e r n a t e flushing w i t h h y d r o g e n a n d v a c u u m pumping. Hydrogen was passed over copper filings at 5,00°C to remove traces of oxygen. A m i n i m u m of 15 g a s - v a c u u m cycles were done before e q u i l i b r a t i n g the system for 30 m i n u t e s at 25°C u n d e r 1 a t m o s p h r e of hydrogen, a f t e r w h i e h the cuvette stopcock was closed. To i n i t i a t e the r e a e t i o n h y d r o g e n a s e was tipped in a n d the following a b s o r b a n e e changes were m o n i t o r e d : for cytochromes c3 (M.W. 13,000) a n d ca (M.W. 26,000) the a p p e a r a n c e of the a b a n d of the redueed eytoehrome at 553 n m ; decreases i n a b s o r b a n c e for f e r r e d o x i n (at 450 nm), flavodoxin (at 456 nm), r u b r e d o x i n (at 490 nm) a n d FMN (at 450 nm). A b s o r p t i o n spectra of the elect r o n carriers were reeorded before, d u r i n g a n d a f t e r reduction. In all cases the v a c u u m - - gassing procedure caused some e v a p o r a t i o n of the s t a r t i n g solutions, so t h a t the e o n e e n t r a t i o n of electron carriers was calculated on t h e b a s i s of t h e m e a s u r e d v o l u m e of s o l u t i o n in t h e cuvette a f t e r the experiment.
Protein determination. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y the Biuret m e t h o d [7] using b o v i n e s e r u m a l b u m i n as standard.
Results.
H!tdrogenase preparation. T h e . a i m of t h e p u r i f i c a t i o n w a s to o b t a i n a h y d r o g e n a s e p r e p a r a t i o n BIOCHIMIE, 1978, 60, n ° 3.
f r e e of a n y t r a c e of h e m o p r o t e i n . T h i s w a s a c h i e ved using the following procedure. Although this procedure does not yield hydrogenase with a high s p e c i f i c a c t i v i t y , it is fast a n d l e a d s to a f r a c t i o n c o m p l e t e l y d e v o i d of c y t o c h r o m e , as e v i d e n c e d b y t h e a b s e n c e of a p e a k o r s h o u l d e r a r o u n d 420 n m a f t e r r e d u c t i o n b y d i t h i o n i t e . B e c a u s e of t h e e x t r e m e s e n s i t i v i t y of h y d r o g e nase to oxygen, the following procedure was devel o p e d w h i c h w o u l d a l l o w t h e e n z y m e to b e p u r i fied u n d e r a i r - f r e e c o n d i t i o n s as r a p i d l y as p o s s i b l e . All c h r o m a t o g r a p h i c c o l u m n s w e r e e x h a u s t i vely equilibrated with buffers which had been b o i l e d t h e n c o o l e d i n ice u n d e r a s t r e a m of a r g o n , a n d w h i c h c o n t a i n e d 2. m M 2 - m e r c a p t o e t h a n o l . All b u f f e r s u s e d i n t h e p u r i f i c a t i o n w e r e k e p t at p H 7.6 a n d f r a c t i o n s w e r e c o l l e c t e d u n d e r a s t r e a m of a r g o n . All s t e p s w e r e c a r r i e d o u t at 0 t o 4°C and fractions were stored under hydrogen.
Step I. W a s h i n g of intact cells. I F r e s h l y t h a w e d c e l l s of D. gigas (150 g w e t w e i g h t ) w e r e c a r e f u l l y m i x e d w i t h 1'5~0' m l of 50 mM Tris-HC1 b u f f e r , 2 m M i n BME. T h e s u s p e n s i o n w a s c e n t r i f u g e d at 37,00,0 × g f o r 2,0 m i n u t e s , t h e s u p e r n a rant wash fluid retained, and the cells suspended a g a i n i n 1,50 m l of b u f f e r . A f t e r a s e c o n d c e n t r i f u g a t i o n at 37,1~0,0 × g t h e t w o s u p e r n a t a n t f r a c t i o n s w e r e c o m b i n e d a n d c e n t r i f u g e d at 120,000 X g f o r 9. h o u r s i n s c r e w - c a p p e d t u b e s u n d e r h y d r o gen. T h e w a s h i n g p r o c e d u r e i t s e l f c o n s t i t u t e d a 3-fold p u r i f i c a t i o n as c o m p a r e d to c e l l - f r e e e x t r a c t s . A p p r o x i m a t e l y 99 p e r c e n t of t h e h y d r o g e n a s e of t h e cell c o u l d b e e x t r a c t e d i n t h i s m a n ner. Step II. Calcinated alumina chromatography. - T h e cell w a s h f r a c t i o n w a s p a s s e d t h r o u g h a c o l u m n of c a l c i n a t e d a l u m i n a (4 × 1~5 c m ) e q u i l i b r a t e d w i t h 50 m M Tris-HC1 b u f f e r . Most of t h e h y d r o g e n a s e a c t i v i t y is r e t a i n e d o n t h e a l u m i n a c o l u m n a l o n g w i t h c y t o c h r o m e s c z. T h e c o l u m n w a s w a s h e d w i t h 50 mM Tris-H'C1 b u f f e r u n t i l all t h e g r e e n p r o t e i n w a s e l u t e d ( u s u a l l y a b o u t 50 m l w a s r e q u i r e d ) , t h e n w i t h 50 m l of 10 m M p o t a s s i u m p h o s p h a t e b u f f e r (KPi). T h e c o l u m n w a s e l u t e d w i t h ~0~), m M K P i b u f f e r a n d at t h i s i o n i c s t r e n g t h h y d r o g e n a s e w a s e l u t e d s l i g h t l y a h e a d of t h e m a j o r i t y of t h e a d s o r b e d c y t o c h r o m e . T w o hydrogenase-containing fractions were collected (a a n d b i n t a b l e IV) ; t h e first ( a b o u t 7'5 m l ) w a s g o l d e n y e l l o w a n d c o n t a i n e d s o m e c y t o c h r o m e as w a s e v i d e n t f r o m its v i s i b l e a b s o r p t i o n s p e c t r u m , w h i l e t h e s e c o n d ( a b o u t 150 m l ) w a s r e d d i s h g o l d a n d c o n t a i n e d m u c h m o r e c y t o c h r o m e . Most of t h e c y t o c h r o m e r e m a i n e d a b s o r b e d to t h e alu-
Reactivity of D. g i g a s hydrogenase. m i n a c o l u m n and could be eluted w i t h 1.0 M KPi buffer. Because the most difficult c o n t a m i n a t i n g p r o t e i n to separate from h y d r o g e n a s e had been f o u n d i n i n i t i a l h y d r o g e n a s e purifications to be a c-type c y t o c h r o m e with sinfilar ionic and hydrod y n a m i c properties, fractions a and b from the a l u m i n a c o l u m n were not c o m b i n e d in the subseq u e n t step.
Step III. Chromatography on Sephadex G-lO0. - F r a c t i o n a from the previous step was c o n c e n t r a ted to 5 nfl (Diaflo UM-10 ultrafilter, Amicon Corp.) and applied to a 2.5 × 100 cm c o l u m n of Sephadex G-100 p r e p a r e d for a s c e n d i n g flow at a rate of 10 ml p e r hour, a n d fractions of 10 ml were collected and stored u n d e r h y d r o g e n . In the same way, f r a c t i o n b Yeas c o n c e n t r a t e d to 25 ml, applied
317
(5.0 ml) were collected d u r i n g elution and tubes with high hydrogenase specific activity w e r e pooled. I n most cases a second DEAE step was needed to effects complete s e p a r a t i o n of hydrogenase from cytochrome. W h e n this was necessary the pooled fraction from the first DEAE c o l u m n was diluted 5-fold with distilled water, adsorbed on a 1.6 × 16 cm DEAE c o l u m n e q u i l i b r a t e d as before, and eluted w i t h a l i n e a r Tris-HC1 gradient from 30 to 10# mM (20'0 ml each). Only those fractions h a v i n g no 418 n m peak (c-type c y t o c h r o m e Sorer p e a k ) in t h e i r d i t h i o n i t e - r e d u c e d a b s o r p t i o n spectra were combined. The p u r i f i c a t i o n of D. gigas h y d r o g e n a s e is summ a r i z e d in table I. The activity of the enzyme was followed d u r i n g the purifi'cation p r o c e d u r e by the
TABLE I.
Purification of the hydrogenase from Desulfovibrio gigas. Step I. Wash fraction II. After caleinated (a) alumina (b) III. After Sephadex G°100 IV. After D E A E
Volume (ml)
Protein (rag/
units ('}
320 75 150
12.3 4.86 7.15
23,700 3,150 7,730
100 24
1.3 1.65
3,260 2,400
ml}
Total
Specific
Recovery
activity
(per cent)
6.0 8.65 7.2
100 13.4 32.6
(units/rag)
25.0 50.5
14 10
(*) One unit = 1 ~,mole hydrogen oxidized per minute per mg protein. Benzyl viologen was used as the electron aeceptor.
to a 5 × 10@, cm a s c e n d i n g flow G-190. c o l u m n and 10 ml fractions were collected at a flow rate of 15 ml per hour. The tubes c o n t a i n i n g high specific activities of h y d r o g e n a s e from both c o l u m n s w e r e pooled into a single fraction of about 100 ml. By r u n n i n g these two G-IO0 c o l u m n s sinmltaneously, r a t h e r t h a n c o m b i n i n g fractions a a n d b a n d u s i n g a single column, better s e p a r a t i o n of h y d r o g e n a s e from c y t o c h r o m e was achieved and the d u r a t i o n of the step was not lengthned.
Step IV. DEAE-column chromatography. - - The pooled f r a c t i o n from the G-10,0 c o l u m n s was diluted to 150 ml w i t h cold H_o-saturated distilled water. This m a t e r i a l was absorbed on a c o l u m n of D,I~AE-cellulose (1.8 X 27 cm) e q u i l i b r a t e d w i t h 50 mM Tris-HC1 buffer. The c o l u m n was w a s h e d with 1,0@ ml of the same buffer and the enzyme was eluted by m e a n s of a l i n e a r Tris-HCl g r a d i e n t from 5'0 to 5,00 mM (250 ml each). F r a c t i o n s BIOCHIMIE, 1978, 60, n ° 3.
h y d r o g e n uptake assaye described in the <> section. The p r o c e d u r e produced a 1~0 per cent yield of h y d r o g e n a s e purified $0-fold as c o m p a r e d to cell-free extracts.
Effects of pH on the rate of hydrogenase reactions. The pH optima of the following hydrogenase-catalyzed reactions were studied : (1.) h y d r o gen oxidation w i t h benzyl viologen as electron acceptor, h y d r o g e n evolution from (2:) dithioniter e d u c e d m e t h y l viologen and from (3) dithioniter e d u c e d D. gigas c y t o c h r o m e c a. The following s e r i e s of buffers w a s used : s o d i u m citrate-Na 2 H P04, pH 4.5 to 5.5 ; K2HPO4-KH2P04, pH 5.4): to 8.0 ; Tris-HC1, pH 7.1 to 9.t) ; glycine-NaOH, pH 9.@,to l(k0~. All buffers were at a final c o n c e n t r a t i o n of 125 raM. The o p t i m u m pH of the c y t o c h r o m e ca-dependent H.2 e v o l u t i o n was 5.5, that of the m e t h y l violo-
G. R. Bell and coll.
318
g e n - d e p e n d e n t H 2 evolution 6.(}-6.'5 and that of the benzyl viologen r e d u c t i o n by H 2 7.0-8.2.
Apparent Km values for carrier-dependent hydrogen evol'ution. All reactions c o n t a i n e d 0.0.63 mg D. gigas h y d r o g e n a s e a n d were r u n in an argon atmosphere. Double r e c i p r o c a l plots were used to calculate the k i n e t i c constants. The apparent Km value for m e t h y l viologen is 2:5 × 10 -5 M (at pH 6.5), for c y t o c h r o m e Ca (M.W. 26,0(}(}), 1.0 × 10' - 6 ~ (at pH 5.5) a n d for c y t o c h r o m e c a (M.W. 13,,0~00'), 4.0 × 1,0-7 M (at pH 5.5). The maxim u m velocities o b t a i n e d in these e x p e r i m e n t s were 9.5, 6.0, a n d 2.~0 l~moles per m i n u t e per mg p r o t e i n for m e t h y l viologen, c y t o c h r o m e % (M.W. 26,0(}(}~), and c y t o c h r o m e c 3 (M.W. 13,04)0) respectively. These rates of h y d r o g e n evolution were 6, 1~0, and ~0-fold less t h a n the rate of h y d r o g e n uptake i n the p r e s e n c e of benzyl-viologen.
Hydrogen evo?ution [rom reduced natural electron carriers. H y d r o g e n evolution from pure, d i t h i o n i t e - r e d u c e d electron carriers of D. gigas was tested i n the presence of D. gigas hydrogenase. The m a n o m e t r i c p r o c e d u r e described i n ¢ Materials and Methods )> was used. Each r e a c t i o n was started by a d d i n g solid d i t h i o n i t e from the s i d e a r m to the m i x t u r e of electron c a r r i e r a n d hydrogenase. Assays i n v o l v i n g f e r r e d o x i n of flav o d o x i n (also c y t o c h r o m e % (M.W. 13,0~0) for
TABLE II.
Hydrogen evolution from reduced natural electron carriers o[ Desulfovibrio gigas Electron carrier added
None Ferredoxin I Flavodoxin Cytochrome % (M.W. 2 6 , 0 0 0 )
Amount o[ carrier
Rate oI HI evolution
(~moles per minute)
0 0.01 ~mole 0.01 ~
0.00 0.00 0.00
0.16
0.033(*)
~
Cytoehrome ca (M.W. 1 3 , 0 0 0 ) Ferredoxtn -~- c 3 F l a v o d o x i n -Jr- e 3
0.005 , 0 . 0 1 --[- 0 . 0 0 5 0 . 0 1 -~- 0 . 0 0 5
0.013 0.012 0.012
The reaction mixture in the Warburg vessel containcd in a total volume of 2.3 ml : D. gigas hydrogenasc (0.063 my) ; pure electron carriers, ferredoxin (0.01 tanole) or flaxodoxin (0.01~i~mole) or cytochromc ca (M.W. 2~,000) (0.16 p2aole) or cytochrome cs (M.W. 13,000) (0.005 tgmole) ; 23() l~moles potassium phosphate buffer (pI~ 7.0 or 5.5) (*) ; 0.2 ml alkaline pyrogallol (center well). The gas phase was argon and the temperature 30°C. Reactions were started by adding solid sodium dithionite from the sidearm.
BIOCHIMIE, 1978, 60, n ° 3.
comparison) w e r e done at pH 7.,0 to avoid possible d e n a t u r a t i o n w h i c h could occur below this pH. Cytochrome c 3 (M.W. 26,0(}(}) was assayed at pH 5.5. As i n d i c a t e d i n table II, no h y d r o g e n was evolved in the p r e s e n c e of f e r r e d o x i n or flavodoxin as well as i n the absence of electron carriers. H y d r o g e n was p r o d u c e d in the presence of either c y t o c h r o m e ca (M.W. 13,,0(}~) or c a (M.W. 26,()00), i n d i c a t i n g that both c y t o c h r o m e s can t r a n s f e r electrons directly to hydrogenase. The rate of h y d r o g e n evolution from c y t o c h r o m e c a (M.W. 13;004)) was not affected a p p r e c i a b l y b y the addition of f e r r e d o x i n or flavodoxin. However, w h e n the r e a c t i o n was i n i t i a t e d by t i p p i n g c y t o c h r o m e c a (M.W. 13,'0~),) into the m i x t u r e of hydrogenase, d i t h i o n i t e and buffer, no h y d r o g e n was evolved. This a p p a r e n t i n a c t i v a t i o n of h y d r o g e n a s e by d i t h i o n i t e r e d u c t i o n is not understood. N~either the relatively high redox potential cytochrome c55a from Desulfovibrio vulgaris n o r FMN coupled the evolution of h y d r o g e n from d i t h i o n i t e by D. gigas h y d r o g e n a s e .
Reduction of natural electron carriers with hydrogenase. Cytochrome c3 (M.W. 13,(},0.0) or cytoc h r o m e c a (M.W. 26,(}0~}) from D gigas was fully r e d u c e d b y h y d r o g e n a s e from the same o r g a n i s m u n d e r a h y d r o g e n atmosphere, resulting in the a p p e a r a n c e of the a b a n d of the r e d u c e d cytochrome at 553 nm. T h e r e was a short lag p e r i o d p r e c e d i n g r e d u c t i o n . T h e p r e s e n c e of a 1-5 m i n u t e lag period was c o m m o n in c y t o c h r o m e c 3 (M.W. 13,60~) r e d u c t i o n s but it was not correlated w i t h the rate once the r e a c t i o n began. Slight v a r i a t i o n s from l i n e a r i t y w e r e seen w h e n the c y t o c h r o m e was about half-reduced. This r e d u c e d s p e c t r u m of h y d r o g e n a s e r e d u c e d c y t o c h r o m e c 3 is superimposable on the s p e c t r u m of d i t h i o n i t e - r e d u c e d c y t o c h r o m e c aRates of c y t o c h r o m e c a (M.W. 13,~0~(}) r e d u c t i o n , d e t e r m i n e d from the steepest p o r t i o n of s i m i l a r t r a c i n g s of A 553 versus time, were plotted against c y t o c h r o m e c o n c e n t r a t i o n . The c o n c e n t r a t i o n s of this electron c a r r i e r w h i c h w e r e used (from 2 to 9.10~-6M) w e r e not sufficient to saturate h y d r o genase. I n contrast to the results w i t h c y t o c h r o m e c 3 (M.W. 1~,~)(}#) a n d ca (M.W. 26;0#0), ferredoxin, flavodoxin a n d r u b r e d o x i n w e r e not r e d u c e d by h y d r o g e n a s e from D. gigas. However, the r e d u c t i o n of each of these electron c a r r i e r s could be effected b y the a d d i t i o n of a small a m o u n t of c y t o c h r o m e c a (M.W. 13,,0~0,) to the r e a c t i o n mixtures. Ferre-
Reactivity of D. g i g a s hydrogenase. doxin was fully r e d u c e d (compared to d i t h i o n i t e r e d u c t i o n ) in a r e a c t i o n m i x t u r e c o n t a i n i n g a m o l a r ratio of 20 f e r r e d o x i n : 0.3 c a : 0.0~63. mg hydrogenase. The results were s i m i l a r in an e x p e r i m e n t w i t h r u b r e d o x i n , in w h i c h the m o l a r ratio w a s 268 r u b r e d o x i n : 1.3, c 3 ; c 3 :,0:063 mg hydrogenase. The ~, ~ and Soret b a n d s of the c y t o c h r o m e were evident i n the r e d u c e d spectrum. F l a v o d o x i n was r e d u c e d i n a r e a c t i o n m i x t u r e of 73 flavodoxin :,0.3 c 3 : 0.0'63 mg hydrogenase. At e q u i l i b r i u m , a m a j o r a b s o r p t i o n above 550 n m was seen that was due to the f o r m a t i o n of a flavin s e m i q u i n o n e i n t e r m e d i a t e w h i l e a peak at 418 n m a n d the small b u m p at ~53 n m were due to the Soret and (x b a n d s of the r e d u c e d cytochrome. Although D. gigas h y d r o g e n a s e c a n n o t r e d u c e flav o d o x i n ( w h i c h c o n t a i n s 1 FMN/mole) w i t h o u t the i n t e r v e n t i o n of c y t o c h r o m e ca, it can reduce free FM~N i n the absence of cytochrome. These results suggest that 19. gigas h y d r o g e n a s e is coupled directly w i t h c y t o c h r o m e c a (M.W. 13,0,0,0,) or c y t o c h r o m e cz (M.W. 26,00,0). F e r r e d o xin, flavodoxin a n d r u b r e d o x i n do not serve as direct substrates for this h y d r o g e n a s e but are r e d u c e d i n d i r e c t l y via c y t o c h r o m e c a (M.W. 13,,(~0,0) and possible also c y t o c h r o m e c a (M.W. 26;0,0~0,) although this w a s not tested. These results are in agreement w i t h the studies on the c a r r i e r d e p e n d e n t evolution of h y d r o g e n by D. glgas hydrogenase.
Discussion.
The different reactivities of D. gigas a n d C. pastorianum h y d r o g e n a s e s t o w a r d n a t u r a l electron acceptors c a n n o t be i m m e d i a t e l y u n d e r s t o o d since both enzymes, in highly purified p r e p a r a t i o n s [8, 9] c o n t a i n the same a m o u n t of i r o n atoms a r r a n ged in three F%S 4 clusters. Since oxidative p h o s p h o r y l a t i o n s are present in
D. gigas [10], it is possible to postulate that the electrons have to go t h r o u g h a c y t o c h r o m i c c h a i n i n v o l v i n g c y t o c h r o m e c a. This is not the case as far as clostridia such as C. pastorianum are concerned, since this type of o r g a n i s m derive t h e i r energy entirely from substrate-level p h o s p h o r y l a tions. If this hypothesis is correct, direct c o u p l i n g b e t w e e n h y d r o g e n a s e and n o n - c y t o c h r o m e elect r o n c a r r i e r s has to be avoided in desulfovibriones
BIOCHIMIE, 1978,
60, n ° 3.
319
in order to p r e v e n t a loss of energy by ¢ short c i r c u i t i n g >> of redox carriers. Although c y t o c h r o m e ca is necessary for the t r a n s f e r of electrons b e t w e e n h y d r o g e n a s e and other redox proteins, this is not the case as far as smaller molecules such as viologens or FMN are c o n c e r n e d ; the smaller size of these molecules is not sufficient to e x p l a i n t h e i r direct c o u p l i n g w i t h h y d r o g e n a s e since ferric ions are not reduced by D. gigas h y d r o g e n a s e in the absence of c y t o c h r o m e c 3 [11]. However, the lack of reactivity b e t w e e n D. gigas h y d r o g e n a s e and the n o n c y t o c h r o m i c electron carriers could be e x p l a i n e d by the existence of specific p r o t e i n interactions. F l a v o d o x i n can be taken as an example : the t e r t i a r y s t r u c t u r e of both Clostridium MtP [12] and D. vtdyaris flaxodoxin is k n o w n [13]. A c o m p a r a t i v e study of the two structures has been p u b l i s h e d r e c e n t l y [14]. The authors p o i n t out the quite s u r p r i s i n g differences that exist in the b i n d i n g of the FMN moiety w i t h the p r o t e i n and conclude that <>. In this view, it is t e m p t i n g to speculate that in clostridial flavodoxins the FMN is m u c h less ~ protected >> t h a n in Desat[ovibrio flavodoxins, allowing r e d u c t i o n of flavodoxin by h y d r o g e n a s e in the first type of bacteria. Since FMN can react directly w i t h D. 919as hydrogenase, it is possible that direct electron t r a n s f e r from h y d r o g e n a s e to flavodoxin can only be p r e v e n t e d by a m a x i m a l shielding of the proslhetic group by the apo-protein. A specific interaction b e t w e e n hydrogenase, c y t o c h r o m e c 3 and flavodoxin w o u l d restore the electron transfer. It is to be noted that a p r o t e i n - p r o t e i n i n t e r a c t i o n has been d e m o n s t r a t e d between f e r r e d o x i n and c y t o c h r o m e c 3 from D. gigas [15]. Differences can be noted in the reactivity of cytochrome % : c y t o c h r o m e c a from D. vttlgaris (Miyazaki strain) stimulates h y d r o g e n evolution from d i t h i o n i t e in the presence of methyl viologen as electron c a r r i e r [163 w h e r e a s c y t o c h r o m e c a from D. gigas has no action on D. gigas h y d r o genase in the same r e a c t i o n [9]. The drastic changes in the p r i m a r y s t r u c t u r e of cytochromes % from different strains of bacteria is a well k n o w n fact [4]. These changes lead to modifi'cations of the t e r t i a r y s t r u c t u r e and redox properties of the molecule : they are so great bet-
320
G. R . B e l l a n d c o l l .
w e e n D. g i g a s a n d D. d e s u l [ u r i c a n s ( N o r w a y 4) c y t o c h r o m e s c 3 t h a t a s h i f t of t h e f u n c t i o n of t h e s e t w o m o l e c u l e s h a s b e e n p r o p o s e d [17]. T h e e s t a b l i s h m e n t of t h e t e r t i a r y s t r u c t u r e of t h i s p r o t e i n , w h i c h is n o w u n d e r w a y [18], t o g e t h e r w i t h a d e t a i l e d s t u d y of its m a g n e t i c a n d r e d o x p r o p e r t i e s [19, 20], w i l l b e e s s e n t i a l i n o r d e r to u n d e r s t a n d its i n t r i c a t e r e l a t i o n s h i p s w i t h h y d r o genase and the other redox carriers. This work is part of G. R. Bell's dissertation (Studies on electron transfer s y s t e m s in Desulfovibrio : p u r i f i cation and characterization of hydrogenase, catalase and superoxide d i s m u l a s e ; P h . D . Thesis, The Uniuersitg of Georgia, Athens, Georgia, U.S.A., 1973).
Acknowledgements. This work was supported by Research Grant GB-2961 X awarded by the National Science Foundalion to J. Le Gall, and research grant PCM 762.2~10 to H. Peck.
REFERENCES. 1. Yagi, T., Honya, M. & Tamiya, N. (1968) Biochim. Biophys. Acta, 153. 692-698. 2. Akagi, J. M. (1964) J. Bacleriol., 88, 813-814.
BIOCHIMIE, 1978, 60, n ° 3.
3. Irie, K., Kobayashi, K., Kobayashi, M. ~ Ishimoto, M. (1973) J. Biochem. (Tokyo) 73, 353-3~6. 4. D e r V a r t a n i a n , D. V. a Le Gall. J. (1974) Biochim. Biophys. Acta, 346, 79-99. 5. Le Gall, J., Mazza, G. ~ Dragoni, N. (1965) Biochim. Biophys. Acta, 90, 385-387. 6. Le Gall, J. ~ Forget, N. Methods in E n z y m o l o g y : Biomembranes, vol. 53 (part D) : S. Fleischer, L. Packer Eds., Acad. Press, Inc. (in press). 7. Gornall, A. G., Bardawill, C. J. a David M. M. (1949) J. Biol. Chem., 177, 751-766. 8. Gillum, W. D., Mortenson, L. E., Chen, J. S. ,~ Holm, R. M. (1977) J. Am. Chem. Soe., 99, 584-595. 9. Hatchikian, E. C., Bruschi, M. ~ Le Gall, J., to be published. 10. Peck, H. D. Jr. (196.6) Bioehem. Biophys. Res. Comm., 22, 112-114. l t . Hatchikian, E. C., p e r s o n n a l c o m m u n i c a t i o n . 12. Andersen, R. D., Apgar, P. A., Bornett, R. M., Darling, G. D., Le Quesne, M. E., Mayhew, S. G. Ludwig, M. L. (1971) Proc. Nat. Aead. Sciences U~. (1972), 69, 3189-3192. 13. W a t e n p a u g h , K. D., Sieker, L. C., Jensen, L. H., Le Gall, J. ,~ Dubourdieu, M. (1972) Proc. Natl. Acad. Sc. U~., 69, 3185-3188. 14. Mayhew, S. G. ~ Ludwig, M. L. (19759 in The E n z y m e s , Boyer, p. D. Ed., Acad. Press, New York, vol. XII, pp. 57-118. 15. Moura, J. J. G., Xavier, A. V., Cookson, D. J., Moore, G. R., Williams, R. J. P., Bruschi, M. ~ Le Gall, J. (1977) FEBS Letters, 81, 275-260. 16. Yagi, T. (1970) J. Biochem. (Tokyo), 68, 649-657. I7. Bruschi, M., Hatchikian, E. C., Golovleva, L. A. Le Gall, J. (1977) J. Bacteriol., 129, 30-38. 18. Frey, M., Haser, R., Pierrot, M., Brusehi, M. ,~ Le Gall, J. (1976) J. Mol. Biol., 104. 741-743. !9. DerVartanian, D. V., Xavier, A. V. ~ Le Gall, J., this issue. 20. Xavier, A. V. ~ Moura, J. J. G., t h i s issue.
Reactivity of Desulfovibrio gigas hydrogenase toward artificial and natural electron donors or acceptors. G. R. BF_~LL *, J i n - P o L E E * , H. D. P E G K Jr. * a n d J. L E GA,LI, "*.
" D e p a r t m e n t of B i o c h e m i s t r y , University of Georgia, Athens, Georgia 30602 (U.S.A.). *" Laboratoire de Chimie Bact~rienne, C.N.R.S., 1327~ Marseille Cedex 2 (France).
R6sum6.
Summary.
Une pr6paration d'hydrog6nase de Desuliovibrio qiqas ne peut pas r6duire la ferr6doxine, la flavodoxine ou la rubr6doxine en l'absence de cytochrome c3 (P.M. 13000), sous atmosph6re d'hydroqbne. Seuls le benzy1 viologbne ou le FMH sont directement r6duits. La production d'hydrogbne mol6culaire & partir du dithionite est possible avec cette pr6paration, en pr6sence de m6thyl violoq~ne comme m6diateur. La m6me r6action a aussi lieu en pr6sence de cytochrome c3 (P. M. 13 000) ou de cytochrome c3 (P. M. 26 000). L'addition de ferr&doxine ou de flavodoxine n'acc61~re pas la vitesse de production d'hydrog~ne.
A purified preparation of hydrogenase from D. qiqas was inactive toward ferredoxin, flavodoxin or rubredoxin in the absence of cytochrome c3 (M. W. 13,000), in an atmosphere of hydrogen, although direct reduction of benzy1 viologen or FMN w a s possible. The hydrogen evolution reaction from dithionite w a s possible with methyl viologen. The same reaction also occured with cytochrome c3 (M. W. 13,000) or cytochrome c3 (M.W. 26,000). Addition of either ferredoxin or flavodoxin did not accelerate the reaction.
Introduction.
Chemical and reagents. Trizma-base, DNase a n d bovine serum a l b u m i n were p u r c h a s e d from Sigma Chemical Company. Sodium d i t h i o n i t e was o b t a i n e d from F i s h e r Chemical Company. Benzyl viologen was supplied b y S c h w a r t z - M a n n ; m e t h y l - v i o l o g e n by Brit i s h Drug Houses Ldt. ; s p e c t r o p h o t o m e t r i c a l l y pure FMN by F l u k a AG, Chemische Fabrik. All o t h e r chemicals were of the highest p u r i t y available.
Yagi et al. [1] h a v e p r o p o s e d a d i f f e r e n t i a t i o n of the hydrogenases from clostridia and the sulfater e d u c i n g b a c t e r i a b e l o n g i n g to t h e g e n u s Desul[ovibrio o n t h e b a s i s of e l e c t r o n c a r r i e r s p e c i f i c i t y . A c c o r d i n g to t h e i r s t u d y , d e s u l f o v i b r i o n e s h y d r o g e n a s e is s p e c i f i c f o r c y t o c h r o m e c a a n d n o t f e r r e d o x i n w h i l e c l o s t r i d i a l h y d r o g e n a s e is s p e c i f i c f o r ferrodoxin. Other investigations have supported this view that, in desulfovibriones, either ferred o x i n o r f l a v o d o x i n a r e c o u p l e d to h y d r o g e n a s e v i a c y t o c h r o m e c a [2, 3, 4]. W e t h o u g h t it w o u l d b e of i n t e r e s t to i n v e s t i g a t e t h e r e l a t i o n s h i p s b e t ween hydrogenase and other redox proteins in a d e s u l f o v i b r i o n e , Desul[ovibrio gigas, f r o m w h i c h several electron carrying proteins have been characterized.
Materials
and
Methods.
Growth conditions. D. 9iyas was cultivated in a lact a t e - s u l f a t e m e d i u m a n d h a r v e s t e d as previously described [5].
Chromatographic materials. Calcinated a l u m i n a was from the L a b o r a t o i r e du Bois de Boulogne ; DEAEcellulose was p u r c h a s e d f r o m Schleieher and Sehnell, Inc. and Sephadex from P h a r m a e i a Fine Chemicals. Assays [or hydrogenase. 1) The reduction of benzyl viologen by h y d r o g e n a s e w i t h H2 was followed m a n o metrically by m e a s u r i n g the rate of H2 c o n s u m p t i o n . The center well of the W a r b u r g vessel contained 0.1 ml 10 per cent CdClo ; m a i n c o m p a r t m e n t , 0.6 ml of a benzyl viologen solution, r00 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.6) a n d distilled w a t e r to 2.3 ml. E n z y m e was added from the s i d e a r m a f t e r gassing the vessel w i t h H~ and s h a k i n g for 30 m i n u t e s at 3'0°C. Best results were obtained with reagents previously v a c u u m degassed. The u n i t of activity was defined as t~moles H~ oxidized per minute. 2~ The oxidation b y h y d r o g e n a s e of d i t h i o n i t e - r e d u cod m e t h y l viologen (or a n o t h e r electron carrier to be tested) was followed m a n o m e t r i c a l l y at 30°C by m e a s u ring the rate of H2 evolution. The m a i n c o m p a r t m e n t of each W a r b u r g vessel c o n t a i n e d h y d r o g e n a s e prepa-
316
G. R . B e l l a n d coll.
r a t i o n a n d 5. X 10-4 M m e t h y l viologen (or a n o t h e r electron carrier) in 10~0 mM p o t a s s i u m p h o s p h a t e buffer (at the pH o p t i m u m for each reaction) to a final volume of 2.3 ml. The center well c o n t a i n e d 0.2 ml a l k a l i n e pyrogallol. The gas p h a s e was argon. A n h y drous sodium d i t h i o n i t e (2.4 mg) was added from t h e s i d e a r m to i n i t i a t e the reaction. The u n i t of activity was defined as lxmoles H2 evolved per m i n u t e .
Purification of electron carriers. The electron carriers were o b t a i n e d f r o m D. gigas according to m e t h o d s already p u b l i s h e d ; a review of these m e t h o d s h a s been p u b l i s h e d r e c e n t l y [6]. Enzymatic reduction of pure carriers. The r e d u c t i o n of p u r e electron t r a n s f e r p r o t e i n s f r o m D. 9igas was tested u s i n g purified h y d r o g e n a s e from the same organism. Reactions were followed s p e e t r o p h o t o m e t r i e a l l y in either B e c k m a n Aeta GV or Gilford 2000 recording s p e e t r o p h o t o m e t e r s u s i n g T h u n h e r g euvettes w i t h a 1 - em l i g h t p a t h . Solutions of eytoehromes e~, ferred o x i n a n d r n b r e d o x i n were dialyzed a g a i n s t several changes of 50 mM Tris-ttC1 buffer, pH 7.6. The same buffer was used to dissolve lyophilized flavodoxin a n d to p r e p a r e a s o l u t i o n of FMN. In each e x p e r i m e n t the volume of electron carrier solution added to t h e euvette was b r o u g h t up to 1.2 ml w i t h the same buffer. The s i d e a r m c o n t a i n e d 0.4 ml of the buffer a n d hydrogenase (0.1)63 rag). Cytoehrome e8 (M.W. 13,1)00) was added to the euvette in those e x p e r i m e n t s in w h i c h it was mixed w i t h a n o t h e r electron carrier. W i t h eytoehrome e3 (M.W. 26t,001)), 0.068 mg h y d r o g e n a s e i n 0.1 ml of 101) mM Tris-HG1, pH 7.6, was placed in t h e sidearm a n d 1.5 m l of the eytoehrome (in 1 M Tris-HC1) in the euvette. This a l t e r a t i o n was necessitated b y the insol u b i l i t y of eytochrome e~ (M.W. 26;000) in more dilute buffer. Each cuvette was p r e p a r e d b y connecting it to a closed system w h i c h p e r m i t t e d a l t e r n a t e flushing w i t h h y d r o g e n a n d v a c u u m pumping. Hydrogen was passed over copper filings at 5,00°C to remove traces of oxygen. A m i n i m u m of 15 g a s - v a c u u m cycles were done before e q u i l i b r a t i n g the system for 30 m i n u t e s at 25°C u n d e r 1 a t m o s p h r e of hydrogen, a f t e r w h i e h the cuvette stopcock was closed. To i n i t i a t e the r e a e t i o n h y d r o g e n a s e was tipped in a n d the following a b s o r b a n e e changes were m o n i t o r e d : for cytochromes c3 (M.W. 13,000) a n d ca (M.W. 26,000) the a p p e a r a n c e of the a b a n d of the redueed eytoehrome at 553 n m ; decreases i n a b s o r b a n c e for f e r r e d o x i n (at 450 nm), flavodoxin (at 456 nm), r u b r e d o x i n (at 490 nm) a n d FMN (at 450 nm). A b s o r p t i o n spectra of the elect r o n carriers were reeorded before, d u r i n g a n d a f t e r reduction. In all cases the v a c u u m - - gassing procedure caused some e v a p o r a t i o n of the s t a r t i n g solutions, so t h a t the e o n e e n t r a t i o n of electron carriers was calculated on t h e b a s i s of t h e m e a s u r e d v o l u m e of s o l u t i o n in t h e cuvette a f t e r the experiment.
Protein determination. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y the Biuret m e t h o d [7] using b o v i n e s e r u m a l b u m i n as standard.
Results.
H!tdrogenase preparation. T h e . a i m of t h e p u r i f i c a t i o n w a s to o b t a i n a h y d r o g e n a s e p r e p a r a t i o n BIOCHIMIE, 1978, 60, n ° 3.
f r e e of a n y t r a c e of h e m o p r o t e i n . T h i s w a s a c h i e ved using the following procedure. Although this procedure does not yield hydrogenase with a high s p e c i f i c a c t i v i t y , it is fast a n d l e a d s to a f r a c t i o n c o m p l e t e l y d e v o i d of c y t o c h r o m e , as e v i d e n c e d b y t h e a b s e n c e of a p e a k o r s h o u l d e r a r o u n d 420 n m a f t e r r e d u c t i o n b y d i t h i o n i t e . B e c a u s e of t h e e x t r e m e s e n s i t i v i t y of h y d r o g e nase to oxygen, the following procedure was devel o p e d w h i c h w o u l d a l l o w t h e e n z y m e to b e p u r i fied u n d e r a i r - f r e e c o n d i t i o n s as r a p i d l y as p o s s i b l e . All c h r o m a t o g r a p h i c c o l u m n s w e r e e x h a u s t i vely equilibrated with buffers which had been b o i l e d t h e n c o o l e d i n ice u n d e r a s t r e a m of a r g o n , a n d w h i c h c o n t a i n e d 2. m M 2 - m e r c a p t o e t h a n o l . All b u f f e r s u s e d i n t h e p u r i f i c a t i o n w e r e k e p t at p H 7.6 a n d f r a c t i o n s w e r e c o l l e c t e d u n d e r a s t r e a m of a r g o n . All s t e p s w e r e c a r r i e d o u t at 0 t o 4°C and fractions were stored under hydrogen.
Step I. W a s h i n g of intact cells. I F r e s h l y t h a w e d c e l l s of D. gigas (150 g w e t w e i g h t ) w e r e c a r e f u l l y m i x e d w i t h 1'5~0' m l of 50 mM Tris-HC1 b u f f e r , 2 m M i n BME. T h e s u s p e n s i o n w a s c e n t r i f u g e d at 37,00,0 × g f o r 2,0 m i n u t e s , t h e s u p e r n a rant wash fluid retained, and the cells suspended a g a i n i n 1,50 m l of b u f f e r . A f t e r a s e c o n d c e n t r i f u g a t i o n at 37,1~0,0 × g t h e t w o s u p e r n a t a n t f r a c t i o n s w e r e c o m b i n e d a n d c e n t r i f u g e d at 120,000 X g f o r 9. h o u r s i n s c r e w - c a p p e d t u b e s u n d e r h y d r o gen. T h e w a s h i n g p r o c e d u r e i t s e l f c o n s t i t u t e d a 3-fold p u r i f i c a t i o n as c o m p a r e d to c e l l - f r e e e x t r a c t s . A p p r o x i m a t e l y 99 p e r c e n t of t h e h y d r o g e n a s e of t h e cell c o u l d b e e x t r a c t e d i n t h i s m a n ner. Step II. Calcinated alumina chromatography. - T h e cell w a s h f r a c t i o n w a s p a s s e d t h r o u g h a c o l u m n of c a l c i n a t e d a l u m i n a (4 × 1~5 c m ) e q u i l i b r a t e d w i t h 50 m M Tris-HC1 b u f f e r . Most of t h e h y d r o g e n a s e a c t i v i t y is r e t a i n e d o n t h e a l u m i n a c o l u m n a l o n g w i t h c y t o c h r o m e s c z. T h e c o l u m n w a s w a s h e d w i t h 50 mM Tris-H'C1 b u f f e r u n t i l all t h e g r e e n p r o t e i n w a s e l u t e d ( u s u a l l y a b o u t 50 m l w a s r e q u i r e d ) , t h e n w i t h 50 m l of 10 m M p o t a s s i u m p h o s p h a t e b u f f e r (KPi). T h e c o l u m n w a s e l u t e d w i t h ~0~), m M K P i b u f f e r a n d at t h i s i o n i c s t r e n g t h h y d r o g e n a s e w a s e l u t e d s l i g h t l y a h e a d of t h e m a j o r i t y of t h e a d s o r b e d c y t o c h r o m e . T w o hydrogenase-containing fractions were collected (a a n d b i n t a b l e IV) ; t h e first ( a b o u t 7'5 m l ) w a s g o l d e n y e l l o w a n d c o n t a i n e d s o m e c y t o c h r o m e as w a s e v i d e n t f r o m its v i s i b l e a b s o r p t i o n s p e c t r u m , w h i l e t h e s e c o n d ( a b o u t 150 m l ) w a s r e d d i s h g o l d a n d c o n t a i n e d m u c h m o r e c y t o c h r o m e . Most of t h e c y t o c h r o m e r e m a i n e d a b s o r b e d to t h e alu-
Reactivity of D. g i g a s hydrogenase. m i n a c o l u m n and could be eluted w i t h 1.0 M KPi buffer. Because the most difficult c o n t a m i n a t i n g p r o t e i n to separate from h y d r o g e n a s e had been f o u n d i n i n i t i a l h y d r o g e n a s e purifications to be a c-type c y t o c h r o m e with sinfilar ionic and hydrod y n a m i c properties, fractions a and b from the a l u m i n a c o l u m n were not c o m b i n e d in the subseq u e n t step.
Step III. Chromatography on Sephadex G-lO0. - F r a c t i o n a from the previous step was c o n c e n t r a ted to 5 nfl (Diaflo UM-10 ultrafilter, Amicon Corp.) and applied to a 2.5 × 100 cm c o l u m n of Sephadex G-100 p r e p a r e d for a s c e n d i n g flow at a rate of 10 ml p e r hour, a n d fractions of 10 ml were collected and stored u n d e r h y d r o g e n . In the same way, f r a c t i o n b Yeas c o n c e n t r a t e d to 25 ml, applied
317
(5.0 ml) were collected d u r i n g elution and tubes with high hydrogenase specific activity w e r e pooled. I n most cases a second DEAE step was needed to effects complete s e p a r a t i o n of hydrogenase from cytochrome. W h e n this was necessary the pooled fraction from the first DEAE c o l u m n was diluted 5-fold with distilled water, adsorbed on a 1.6 × 16 cm DEAE c o l u m n e q u i l i b r a t e d as before, and eluted w i t h a l i n e a r Tris-HC1 gradient from 30 to 10# mM (20'0 ml each). Only those fractions h a v i n g no 418 n m peak (c-type c y t o c h r o m e Sorer p e a k ) in t h e i r d i t h i o n i t e - r e d u c e d a b s o r p t i o n spectra were combined. The p u r i f i c a t i o n of D. gigas h y d r o g e n a s e is summ a r i z e d in table I. The activity of the enzyme was followed d u r i n g the purifi'cation p r o c e d u r e by the
TABLE I.
Purification of the hydrogenase from Desulfovibrio gigas. Step I. Wash fraction II. After caleinated (a) alumina (b) III. After Sephadex G°100 IV. After D E A E
Volume (ml)
Protein (rag/
units ('}
320 75 150
12.3 4.86 7.15
23,700 3,150 7,730
100 24
1.3 1.65
3,260 2,400
ml}
Total
Specific
Recovery
activity
(per cent)
6.0 8.65 7.2
100 13.4 32.6
(units/rag)
25.0 50.5
14 10
(*) One unit = 1 ~,mole hydrogen oxidized per minute per mg protein. Benzyl viologen was used as the electron aeceptor.
to a 5 × 10@, cm a s c e n d i n g flow G-190. c o l u m n and 10 ml fractions were collected at a flow rate of 15 ml per hour. The tubes c o n t a i n i n g high specific activities of h y d r o g e n a s e from both c o l u m n s w e r e pooled into a single fraction of about 100 ml. By r u n n i n g these two G-IO0 c o l u m n s sinmltaneously, r a t h e r t h a n c o m b i n i n g fractions a a n d b a n d u s i n g a single column, better s e p a r a t i o n of h y d r o g e n a s e from c y t o c h r o m e was achieved and the d u r a t i o n of the step was not lengthned.
Step IV. DEAE-column chromatography. - - The pooled f r a c t i o n from the G-10,0 c o l u m n s was diluted to 150 ml w i t h cold H_o-saturated distilled water. This m a t e r i a l was absorbed on a c o l u m n of D,I~AE-cellulose (1.8 X 27 cm) e q u i l i b r a t e d w i t h 50 mM Tris-HC1 buffer. The c o l u m n was w a s h e d with 1,0@ ml of the same buffer and the enzyme was eluted by m e a n s of a l i n e a r Tris-HCl g r a d i e n t from 5'0 to 5,00 mM (250 ml each). F r a c t i o n s BIOCHIMIE, 1978, 60, n ° 3.
h y d r o g e n uptake assaye described in the <
Effects of pH on the rate of hydrogenase reactions. The pH optima of the following hydrogenase-catalyzed reactions were studied : (1.) h y d r o gen oxidation w i t h benzyl viologen as electron acceptor, h y d r o g e n evolution from (2:) dithioniter e d u c e d m e t h y l viologen and from (3) dithioniter e d u c e d D. gigas c y t o c h r o m e c a. The following s e r i e s of buffers w a s used : s o d i u m citrate-Na 2 H P04, pH 4.5 to 5.5 ; K2HPO4-KH2P04, pH 5.4): to 8.0 ; Tris-HC1, pH 7.1 to 9.t) ; glycine-NaOH, pH 9.@,to l(k0~. All buffers were at a final c o n c e n t r a t i o n of 125 raM. The o p t i m u m pH of the c y t o c h r o m e ca-dependent H.2 e v o l u t i o n was 5.5, that of the m e t h y l violo-
G. R. Bell and coll.
318
g e n - d e p e n d e n t H 2 evolution 6.(}-6.'5 and that of the benzyl viologen r e d u c t i o n by H 2 7.0-8.2.
Apparent Km values for carrier-dependent hydrogen evol'ution. All reactions c o n t a i n e d 0.0.63 mg D. gigas h y d r o g e n a s e a n d were r u n in an argon atmosphere. Double r e c i p r o c a l plots were used to calculate the k i n e t i c constants. The apparent Km value for m e t h y l viologen is 2:5 × 10 -5 M (at pH 6.5), for c y t o c h r o m e Ca (M.W. 26,0(}(}), 1.0 × 10' - 6 ~ (at pH 5.5) a n d for c y t o c h r o m e c a (M.W. 13,,0~00'), 4.0 × 1,0-7 M (at pH 5.5). The maxim u m velocities o b t a i n e d in these e x p e r i m e n t s were 9.5, 6.0, a n d 2.~0 l~moles per m i n u t e per mg p r o t e i n for m e t h y l viologen, c y t o c h r o m e % (M.W. 26,0(}(}~), and c y t o c h r o m e c 3 (M.W. 13,04)0) respectively. These rates of h y d r o g e n evolution were 6, 1~0, and ~0-fold less t h a n the rate of h y d r o g e n uptake i n the p r e s e n c e of benzyl-viologen.
Hydrogen evo?ution [rom reduced natural electron carriers. H y d r o g e n evolution from pure, d i t h i o n i t e - r e d u c e d electron carriers of D. gigas was tested i n the presence of D. gigas hydrogenase. The m a n o m e t r i c p r o c e d u r e described i n ¢ Materials and Methods )> was used. Each r e a c t i o n was started by a d d i n g solid d i t h i o n i t e from the s i d e a r m to the m i x t u r e of electron c a r r i e r a n d hydrogenase. Assays i n v o l v i n g f e r r e d o x i n of flav o d o x i n (also c y t o c h r o m e % (M.W. 13,0~0) for
TABLE II.
Hydrogen evolution from reduced natural electron carriers o[ Desulfovibrio gigas Electron carrier added
None Ferredoxin I Flavodoxin Cytochrome % (M.W. 2 6 , 0 0 0 )
Amount o[ carrier
Rate oI HI evolution
(~moles per minute)
0 0.01 ~mole 0.01 ~
0.00 0.00 0.00
0.16
0.033(*)
~
Cytoehrome ca (M.W. 1 3 , 0 0 0 ) Ferredoxtn -~- c 3 F l a v o d o x i n -Jr- e 3
0.005 , 0 . 0 1 --[- 0 . 0 0 5 0 . 0 1 -~- 0 . 0 0 5
0.013 0.012 0.012
The reaction mixture in the Warburg vessel containcd in a total volume of 2.3 ml : D. gigas hydrogenasc (0.063 my) ; pure electron carriers, ferredoxin (0.01 tanole) or flaxodoxin (0.01~i~mole) or cytochromc ca (M.W. 2~,000) (0.16 p2aole) or cytochrome cs (M.W. 13,000) (0.005 tgmole) ; 23() l~moles potassium phosphate buffer (pI~ 7.0 or 5.5) (*) ; 0.2 ml alkaline pyrogallol (center well). The gas phase was argon and the temperature 30°C. Reactions were started by adding solid sodium dithionite from the sidearm.
BIOCHIMIE, 1978, 60, n ° 3.
comparison) w e r e done at pH 7.,0 to avoid possible d e n a t u r a t i o n w h i c h could occur below this pH. Cytochrome c 3 (M.W. 26,0(}(}) was assayed at pH 5.5. As i n d i c a t e d i n table II, no h y d r o g e n was evolved in the p r e s e n c e of f e r r e d o x i n or flavodoxin as well as i n the absence of electron carriers. H y d r o g e n was p r o d u c e d in the presence of either c y t o c h r o m e ca (M.W. 13,,0(}~) or c a (M.W. 26,()00), i n d i c a t i n g that both c y t o c h r o m e s can t r a n s f e r electrons directly to hydrogenase. The rate of h y d r o g e n evolution from c y t o c h r o m e c a (M.W. 13;004)) was not affected a p p r e c i a b l y b y the addition of f e r r e d o x i n or flavodoxin. However, w h e n the r e a c t i o n was i n i t i a t e d by t i p p i n g c y t o c h r o m e c a (M.W. 13,'0~),) into the m i x t u r e of hydrogenase, d i t h i o n i t e and buffer, no h y d r o g e n was evolved. This a p p a r e n t i n a c t i v a t i o n of h y d r o g e n a s e by d i t h i o n i t e r e d u c t i o n is not understood. N~either the relatively high redox potential cytochrome c55a from Desulfovibrio vulgaris n o r FMN coupled the evolution of h y d r o g e n from d i t h i o n i t e by D. gigas h y d r o g e n a s e .
Reduction of natural electron carriers with hydrogenase. Cytochrome c3 (M.W. 13,(},0.0) or cytoc h r o m e c a (M.W. 26,(}0~}) from D gigas was fully r e d u c e d b y h y d r o g e n a s e from the same o r g a n i s m u n d e r a h y d r o g e n atmosphere, resulting in the a p p e a r a n c e of the a b a n d of the r e d u c e d cytochrome at 553 nm. T h e r e was a short lag p e r i o d p r e c e d i n g r e d u c t i o n . T h e p r e s e n c e of a 1-5 m i n u t e lag period was c o m m o n in c y t o c h r o m e c 3 (M.W. 13,60~) r e d u c t i o n s but it was not correlated w i t h the rate once the r e a c t i o n began. Slight v a r i a t i o n s from l i n e a r i t y w e r e seen w h e n the c y t o c h r o m e was about half-reduced. This r e d u c e d s p e c t r u m of h y d r o g e n a s e r e d u c e d c y t o c h r o m e c 3 is superimposable on the s p e c t r u m of d i t h i o n i t e - r e d u c e d c y t o c h r o m e c aRates of c y t o c h r o m e c a (M.W. 13,~0~(}) r e d u c t i o n , d e t e r m i n e d from the steepest p o r t i o n of s i m i l a r t r a c i n g s of A 553 versus time, were plotted against c y t o c h r o m e c o n c e n t r a t i o n . The c o n c e n t r a t i o n s of this electron c a r r i e r w h i c h w e r e used (from 2 to 9.10~-6M) w e r e not sufficient to saturate h y d r o genase. I n contrast to the results w i t h c y t o c h r o m e c 3 (M.W. 1~,~)(}#) a n d ca (M.W. 26;0#0), ferredoxin, flavodoxin a n d r u b r e d o x i n w e r e not r e d u c e d by h y d r o g e n a s e from D. gigas. However, the r e d u c t i o n of each of these electron c a r r i e r s could be effected b y the a d d i t i o n of a small a m o u n t of c y t o c h r o m e c a (M.W. 13,,0~0,) to the r e a c t i o n mixtures. Ferre-
Reactivity of D. g i g a s hydrogenase. doxin was fully r e d u c e d (compared to d i t h i o n i t e r e d u c t i o n ) in a r e a c t i o n m i x t u r e c o n t a i n i n g a m o l a r ratio of 20 f e r r e d o x i n : 0.3 c a : 0.0~63. mg hydrogenase. The results were s i m i l a r in an e x p e r i m e n t w i t h r u b r e d o x i n , in w h i c h the m o l a r ratio w a s 268 r u b r e d o x i n : 1.3, c 3 ; c 3 :,0:063 mg hydrogenase. The ~, ~ and Soret b a n d s of the c y t o c h r o m e were evident i n the r e d u c e d spectrum. F l a v o d o x i n was r e d u c e d i n a r e a c t i o n m i x t u r e of 73 flavodoxin :,0.3 c 3 : 0.0'63 mg hydrogenase. At e q u i l i b r i u m , a m a j o r a b s o r p t i o n above 550 n m was seen that was due to the f o r m a t i o n of a flavin s e m i q u i n o n e i n t e r m e d i a t e w h i l e a peak at 418 n m a n d the small b u m p at ~53 n m were due to the Soret and (x b a n d s of the r e d u c e d cytochrome. Although D. gigas h y d r o g e n a s e c a n n o t r e d u c e flav o d o x i n ( w h i c h c o n t a i n s 1 FMN/mole) w i t h o u t the i n t e r v e n t i o n of c y t o c h r o m e ca, it can reduce free FM~N i n the absence of cytochrome. These results suggest that 19. gigas h y d r o g e n a s e is coupled directly w i t h c y t o c h r o m e c a (M.W. 13,0,0,0,) or c y t o c h r o m e cz (M.W. 26,00,0). F e r r e d o xin, flavodoxin a n d r u b r e d o x i n do not serve as direct substrates for this h y d r o g e n a s e but are r e d u c e d i n d i r e c t l y via c y t o c h r o m e c a (M.W. 13,,(~0,0) and possible also c y t o c h r o m e c a (M.W. 26;0,0~0,) although this w a s not tested. These results are in agreement w i t h the studies on the c a r r i e r d e p e n d e n t evolution of h y d r o g e n by D. glgas hydrogenase.
Discussion.
The different reactivities of D. gigas a n d C. pastorianum h y d r o g e n a s e s t o w a r d n a t u r a l electron acceptors c a n n o t be i m m e d i a t e l y u n d e r s t o o d since both enzymes, in highly purified p r e p a r a t i o n s [8, 9] c o n t a i n the same a m o u n t of i r o n atoms a r r a n ged in three F%S 4 clusters. Since oxidative p h o s p h o r y l a t i o n s are present in
D. gigas [10], it is possible to postulate that the electrons have to go t h r o u g h a c y t o c h r o m i c c h a i n i n v o l v i n g c y t o c h r o m e c a. This is not the case as far as clostridia such as C. pastorianum are concerned, since this type of o r g a n i s m derive t h e i r energy entirely from substrate-level p h o s p h o r y l a tions. If this hypothesis is correct, direct c o u p l i n g b e t w e e n h y d r o g e n a s e and n o n - c y t o c h r o m e elect r o n c a r r i e r s has to be avoided in desulfovibriones
BIOCHIMIE, 1978,
60, n ° 3.
319
in order to p r e v e n t a loss of energy by ¢ short c i r c u i t i n g >> of redox carriers. Although c y t o c h r o m e ca is necessary for the t r a n s f e r of electrons b e t w e e n h y d r o g e n a s e and other redox proteins, this is not the case as far as smaller molecules such as viologens or FMN are c o n c e r n e d ; the smaller size of these molecules is not sufficient to e x p l a i n t h e i r direct c o u p l i n g w i t h h y d r o g e n a s e since ferric ions are not reduced by D. gigas h y d r o g e n a s e in the absence of c y t o c h r o m e c 3 [11]. However, the lack of reactivity b e t w e e n D. gigas h y d r o g e n a s e and the n o n c y t o c h r o m i c electron carriers could be e x p l a i n e d by the existence of specific p r o t e i n interactions. F l a v o d o x i n can be taken as an example : the t e r t i a r y s t r u c t u r e of both Clostridium MtP [12] and D. vtdyaris flaxodoxin is k n o w n [13]. A c o m p a r a t i v e study of the two structures has been p u b l i s h e d r e c e n t l y [14]. The authors p o i n t out the quite s u r p r i s i n g differences that exist in the b i n d i n g of the FMN moiety w i t h the p r o t e i n and conclude that <
320
G. R . B e l l a n d c o l l .
w e e n D. g i g a s a n d D. d e s u l [ u r i c a n s ( N o r w a y 4) c y t o c h r o m e s c 3 t h a t a s h i f t of t h e f u n c t i o n of t h e s e t w o m o l e c u l e s h a s b e e n p r o p o s e d [17]. T h e e s t a b l i s h m e n t of t h e t e r t i a r y s t r u c t u r e of t h i s p r o t e i n , w h i c h is n o w u n d e r w a y [18], t o g e t h e r w i t h a d e t a i l e d s t u d y of its m a g n e t i c a n d r e d o x p r o p e r t i e s [19, 20], w i l l b e e s s e n t i a l i n o r d e r to u n d e r s t a n d its i n t r i c a t e r e l a t i o n s h i p s w i t h h y d r o genase and the other redox carriers. This work is part of G. R. Bell's dissertation (Studies on electron transfer s y s t e m s in Desulfovibrio : p u r i f i cation and characterization of hydrogenase, catalase and superoxide d i s m u l a s e ; P h . D . Thesis, The Uniuersitg of Georgia, Athens, Georgia, U.S.A., 1973).
Acknowledgements. This work was supported by Research Grant GB-2961 X awarded by the National Science Foundalion to J. Le Gall, and research grant PCM 762.2~10 to H. Peck.
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