Relationships in hydrogen metabolism between hydrogenase and nitrogenase in phototrophic bacteria

BIOCHIMIE, 1978, 60, 267-275.

Relationships in hydrogen metabolism between hydrogenase and nitrogenase in phototrophic bacteria. Ivan N. GOGOTOV.

Institute o[ Photosynthesis, USSR Academy o[ Sciences, Pushchino, Moscow Region, 142292 (USSR).

R~sum~.

Summary.

Les bact6ries p o u r p r e s Rhodospirillum rubruin et Thiocapsa r o s e o p e r s i c i n a poss~dent

Purple b a c t e r i a Rhodospirillum r u b r u m a n d Thiocapsa r o s e o p e r s i c i n a f o r m two e n z y m e s ,

d e u x e n z y m e s : l ' h y d r o q 6 n a s e et la nitroq6nase qui participent a u m 6 t a b o l i s m e de l'hydroq~ne. Chez ces bact6ries, la photoproduction d'H~ est associ6e en m a j e u r e p a t t i e ou m ~ m e totalement & l'action de la nitroq6nase.

h y d r o q e n a s e a n d n i t r o q e n a s e , w h i c h particip a t e in h y d r o q e n m e t a b o l i s m . H2 photoproduction in these b a c t e r i a is a s s o c i a t e d m a i n l y or c o m p l e t e l y with the action of nitrocjenase. The soluble a n d m e m b r a n e - b o u n d h y d r o q e n a s e s of 7". r o s e o p e r s i c i n a h a v e similar physicoc h e m i c a l properties (tool. weiqht, subunit composition, N-terminal a m i n o acids, Fe 2÷ a n d S 2content, pl. Eo'). In c o m p a r i s o n with other hyd r o q e n a s e s the e n z y m e from R. r u b r u m a n d 1". r o s e o p e r s i c i n a e v o l v e H~ with hiqh r a t e from r e d u c e d c y t o c h r o m e c3, but not from ferredoxins. H2 production a n d N~ fixation t a k e p l a c e in the p r e s e n c e of NAD(P)H. NADP-reductase, ferredoxin a n d c y t o c h r o m e c3 p a r t i c i p a t e in this reaction. Possible relationships b e t w e e n hydroq e n a s e - n i t r o g e n a s e in the metabolism of mole-

Les h y d r o q 6 n a s e s soluble et m e m b r a n a i r e de T. r o s e o p e r s i c i n a p o s s ~ d e n t d e s propri6t6s p h y s i c o - c h i m i q u e s similaires (poids mol6culaire, composition en sous-unit6s, a c i d e s a m i n 6 s N-terminaux, teneur en Fe 2+ et $2-, pI, E°'). Compar6e & d'autres hydrog6nases, l'enzyme de R. r u b r u m et de T. r o s e o p e r s i c i n a d 6 q a q e de I'H~ en quantit6 i m p o r t a n t e & partir du cytoc h r o m e c3 r6duit, m a i s non & partir d e s ferredoxines. La production d'H2 et la fixation de N2 ont lieu en p r 6 s e n c e de NAD(P)H. La NADP-r6ductase, la ferredoxine et le c y t o c h r o m e c3 sont impliqu6s d a n s cette r6action. Les relations p o s s i b l e s entre l ' h y d r o q 6 n a s e et la nitroq6nase d a n s le m 6 t a b o l i s m e de l ' h y d r o q ~ n e m o l 6 c u l a i r e sont discut6es.

According to some production by purple completely associated nase. This concept is facts.

data [1-7], hydrogen photobacteria may be largely or with the action of nitrogeconfirmed by the following

Hydrogen is evolved only by cells capable of nitrogen fix,ation in conditions of N~_ deficiency. Ammonia inhibits H 2 photoevolution as well as nitrogen fixation. Abbreviations : MV, meth!tl viologen ; Fd, [erredoxin.

cular hydroqen are discussed.

CO, a.n inhibitor of hydrogenase, does not inhibit H 2 photoevolntion or its effect is negligible [8]. Mutants of the purple bacterium Rhodopseudomohas capsnlata lacking nil genes do not evolve H e in the light but such ability is restored in reverrants and transferants which can fix N 2 [9]. The repression of nitrogenase synthesis during growth of purple bacteria in a medium containing ammonium salts results in the loss of their ability to photoevolve H 2 [2, 10]. It has recently been found that in addition to the effect of NH4 ÷, the synthesis of nitrogenase in Rhodospirillum rubrnm Ell] as well as in Klebsiella pneumoniae [12, 133 is also repressed when

I. N. Gogotov.

268 glutamine Sou,r c e ,

and

asparagine

are used

as n i t r o g e n

T h e e l e c t r o n t r a n s f e r c h a i n f r o m h y d r o g e n to terminal reductase in the pho~otrophic bacteria is n o t k n o w n . H y d r o g e n a s e f r o m T. roseopersieina [11, 12], C h r o m a t i u m sp. [13] a n d R. r u b r u m [14] h a v e b e e n p u r i f i e d . C e l l - f r e e e x t r a c t s of R. rubr u m r e d u c e N A D a n d N A D P w i t h H 2. H o w e v e r , t h e p u r e h y d r o g e n a s e of T. roseopersicina [15] a n d R. r u b r a m [16] c a n n o t r e d u c e t h e s e n u c l e o t i d e s dir e c t l y i n d i c a t i n g t h e p r e s e n c e of i n t e r m e d i a t e elect r o n c a r r i e r s i n t h e s e b a c t e r i a . R. r u b r u m h y d r o g e n a s e i n t e r a c t s w i t h t h e s o l u b l e . R. r u b r u m f e r r e d o x i n s I a n d H. R e d u c e d f e r r e d o x i n s f r o m R. r u b r u m c a n r e p l a c e r e d u c e d m e t h y l v i o l o g e n as a s u b s t r a t e f o r H e e v o l u t i o n (16]. H o w e v e r , o t h e r b a c t e r i a l ferr e d o x i n s w e r e u n a b l e to m e d i a t e e l e c t r o n t r a n s f e r t o R. r u b r u m h y ( i r o g e n a s e a n d s i m i l a r l y , Chromatium h y d r o g e n a s e w o u l d n o t c a t a l y s e t h e e v o l u t i o n o r u p t a k e of H 2 w i t h f e r r e d o x i n as t h e elect r o n c a r r i e r [13, 17]. T h i s is i n c o n t r a s t to t h e h y d r o g e n a s e of C. p a s t e u r i a n u m [18]. W i t h R. r u bruin, t h e s o l u b l e f e r r e d o x i n s I a n d II also r e s t o r e phosphorylating a c t i v i t y ¢o f,e r r e d o x i n - d e p l e t e d c h r o m a t o p h o r e s [19] a n d m e d i a t e e l e c t r o n t r a n s f e r to t h e n i t r o g e n a s e [20]. H e n c e t h e i r p h y s i o l o g i c a l r o l e is u n c l e a r . Further understanding of t h e m e c h a n i s m of electron transfer in H2 metabolism ~nd N 2 fixation c a n b e o b t a i n e d b y t h e s t u d y , in vitro, of i n t e r a c t i o n s b e t w e e n t h e c o m p o n e n t s of t h e p a t h w a y s . In this paper we report on the hyd:rogenase-niirogenase relationship in phototrophic bacteria and p o s s i b l e f u n c t i o n of t h e f e r r e d o x i n s a n d c y t o c h r o m e <> i n t h i s p r o c e s s .

Materials and Methods. Chemicals. All chemicals a n d reagents were of the highest a v a i l a b l e purity. Sephadex G-25, DEAE-sephaeel were obtained f r o m P h a r m a c i a ; W h a t m a n DEAEcellulose (DE~) f r o m W h a t m a n Bioehemicals, U.K. ; A l u m i n i u m Hydroxide Gel from Research P r o d u c t s Division, Miles Laboratories, U.K. ; glucose-6-phosphate, p h o s p h o t r a n s a c e t y l a s e , NAD, NADP, NADH a n d ATP from Boehringer Corp., U.K. ; Ultrogel AcA 54 f r o m I2KB (Sweden). All other chemicals were o b t a i n e d f r o m Sigma (London) Ltd, U.K. a n d R e a c h i m (USSR). Organisms. The n o n - s u l p h u r purple b a c t e r i u m Rhodospirillum rubrum s t r a i n 1 a n d p u r p l e s u l p h u r bact e r i u m Thioeapsa roseopersicina s t r a i n BBS were t a k e n for i n v e s t i g a t i o n from the collection of the Microbiology D e p a r t m e n t of Moscow State University. The BIOCHIMIE, 1978, 60, n ° 3.

Ormerod m e d i u m [2] w i t h 0.3 per cent m a l a t e was used for c u l t i v a t i o n of R. rubrum a n d P f e n n i g med i u m [21] w i t h 0.1 per cent t h i o s u l p h a t e a n d 0.2 p e r cent acetate for T. roseopersicina. NH~C1, L-arginine, L-glutamate, L-glutamine, L-asparagine, urea or KNO3 (0.1-0.05 per cent) were added to t h e media as a source of nitrogen. Both b a c t e r i a were also cultivated u n d e r conditions of n i t r o g e n fixation (5 per cent CO2 d- 95 per cent N:). The b a c t e r i a were grown in the light (30.103 erg/cm2.see) u n d e r a n a e r o b i c conditions [22]. The cells f r o m the cultures in exponential g r o w t h phase were used for experiments. After c e n t r i f u g a t i o n (20.103 g ; 20 rain ; 4°C) a n d w a s h i n g in p h o s p h a t e buffer the cells were stored in argon.

The cell-free extracts. To o b t a i n cell-free p r e p a r a tions t h e b a c t e r i a were d i s r u p t e d w i t h a sonic disinteg r a t o r (20 kHz for 1 min) or b y pressure [23]. The i n t a c t cells a n d cell debris were removed b y e e n t r i f n gation (20.1{)3 g ; 30 m i n ; 4 ° 0 a n d the s u p e r n a t a n t was used for experiments. Purification of hydrogenase. The hydrogenases were purified f r o m the cells of T. roseopersicina, R. rubrum, Chromatium vinosum and C. pasteurianum b y the methods described earlier [11, 243. Purification of ferredoxins. The f e r r e d o x i n s from Pisum safivum, C. pasleurianum, Spirulina platensis a n d Chromatium vinosum s t r a i n D were isolated by the m e t h o d of Rao et al. [253. R. rubrum a n d T. roseopersicina f e r r e d o x i n s purified according to the m e t h o d of Yoch et al. [26]. For the final stage of the purificat i o n for all ferredoxins, c h r o m a t o g r a p h y on a Ultrogel AcA 54 c o l u m n was used. Purification of cylochrome e~. Bacterial sonicate prepared from 100 g wet ceils T. roseopersicina was centrifuged at 100,000 g for 60 min. The s u p e r n a t a n t fluid (800 ml) was passed t h r o u g h a c o l u m n (3 × 50, cm) of DEAE-cellulose DE~o ( W h a t m a n ) w h i c h h a d been equil i b r a t e d w i t h 0.02 M p o t a s s i u m p h o s p h a t e buffer (pH 7.0) a n d w a s h e d w i t h 10 vol. of distilled water. Cytochromes were elntcd by 0.02 M p o t a s s i u m phosp h a t e buffer (pH 7.0) c o n t a i n i n g 0.2 M NAG1. The r e s u l t i n g cytochrome f r a c t i o n was desalted on a Sephadex G-25 column a n d concentrated. The cytoehrome f r a c t i o n was f u r t h e r purified b y c h r o m a t o g r a p h y on a DEAE-sephacel c o l u m n (1.5 × 2D em) e q u i l i b r a t e d w i t h 10 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.0). E l u t i o n of cytoehromes is accomplished w i t h 0.15-0.8 M NaC1 g r a d i e n t in the buffer. The f r a c t i o n s (from the DEAE cephacel column) c o n t a i n i n g cytochrome <> were concentrated, desalted and separated from h y d r o g e n a s e by a l u m i n a gel c h r o m a t o g r a p h y or p r e p a r a t i v e elcct r o p h o r e s i s in 7.5 per cent p o l y a e r y l a m i d e gel. Purification of NAD(P)~-reduetase. The e n z y m e s were purified from the cells of T. roseopersieina by the m e t h o d of Gogotov a n d L a u r i n a v i t e h e n e [27]. Enzyme assays. H~ evolution f r o m s u b s t r a t e s or diff e r e n t reduced H~-carriers was m e a s u r e d by gas chrom a t o g r a p h y [10]. Hydrogen u p t a k e in t h e presence of m e t h y l viologen (MV) (10 raM) or f e r r e d o x i n s was d e t e r m i n e d m a n o m e t r i c a l l y or spectrophotometrieally. Hydrogenase activity was also d e t e r m i n e d b y the gas c h r o m a t o g r a p h y m e t h o d using the D.~-H_oO exchange reaction [28]. Nitrogen fixation by whole cells and cell-free prepar a t i o n s was d e t e r m i n e d b y 15N_~or acetylene reduction m e t h o d s [29].

H y d r o g e n m e t a b o l i s m in p h o t o t r o p h i c bacteria. Ferredoxin or cytoehrome c~-NADP + oxido-reductase activity was measured by a previously described method [3]. Cytochrome c3-NADP * oxido-reductase activity was followed by the reduction of cytoehromc c:, at 553nm using 2 nmoles of cyt. c3 and 50 ixmoles of NADPH. Fcrredoxin-NADP+ oxido-reductase activity was m e a s u r e d spectrophotomctrieally at 390 nm using 50 nmoles of Spirulina platensis ferredoxin and 0.2 ilxmoles of NADPH. W h e n d e t e r m i n i n g H2 evolution and N: fixing activity inalatc, pyruvate, formate (10 mM) and MV, ferredoxins or cytochrome <, reduced w i t h dithionite (l mM), were used as substrates. ATP-dependent evolution of HL, in cell free extracts was measured, in a final volume of 2 ml, in the presence of Na2S.~O4 (5 raM), ATP (2.5 mM), MgCI.o (10 mM), creatine phosphate (2,5 raM) and creatine p h o s p h o k i n a s e (50 :lxg).

Protein determination. Protein was determined by the method of Lowry et al. [311.

269

a b i l i t y w a s s h o w n in R. rubrum c e l l s g r o w n in t h e m e d i u m w i t h glul.anlale o r in c o n d i t i o n s of No fixation. If a m m o n i u m salts, u r e a , g l u t a m i n e , a s p a r a g i n e (or NO a- f o r T. roseopersicina) s e r v e d as s o u r c e s of n i t r o g e n t h e n H e p h o t o e v o ' l u t i o n a n d a c e t y l e n e r e d u c t i o n s t a r t e d a f t e r a lag p e r i o d (30-180 rain) w i t h l o w r a t e f o r b o t h p r o c e s s e s . Cell f r e e e x t r a c t s d i d not c a t a l y s e A T P - d e p e n d e n t e v o l u t i o n of H,, f r o m d i t h i o n i t e w h i c h is c o n s i d e r e d to b e a c h a r a c t e r i s t i c t e s t f o r t h e p r e s e n c e of n i t r o g e n a s e (table l). In c o n t r a s t t h e u p t a k e a n d e v o l u t i o n of H..~ b y

T. roseopersicina i n t h e p r e s e n c e of r e d u c e d a n d o x i d i z e d MV, r e s p e c t i v e l y as w e l l as t h e D.2-H,,O

TABLE I.

Metabolism of H.e and nitrogen fixation in T. r o s e o p e r s i c i n a depending on nitrogen source in growlh medium. Nitrogen source Reaction

NH~+

Photoevolution of H~ in the presence of pyruvate Photoreduetion of C2H~. in the presence of pyruvate Consumption of H~ in the presenee of MV D~-H~O exchange Evolution of H~ in the presence of MV ~- NauS204 ATP-dependent evolution at H~. from dithionite

Glutamine

Asp.aragme

Nu

0.03 a

0

0

1.84

trace a

0

0

26.5

Arginine

1.50 100.8

14.57 19.6

3.63 12.5

0.88 126.6

3.87 11.3

4.08 24.2

5.82

9.84

6.10

6.73

4.50

0

0

0

0.38

0.72

Rates of H~ evolution and uptake are expressed as Ixmolcs h.-1. mg-1 protein. For the D_~-H~_O exchange reaction they are expressed as umoles D2 consumed h.-1.mg-1 protein. C..,H., reduction is m e a s u r e d as nmoles C.,H~ formed h-l.mg I protein ATP-dependent evolution of H: f r o m dithionite was d e t e r m i n e d using cell-free extracts. All other activities were measured using intact cells. (a) Photoevolution of H~ and Co_H4 started after a lag-period (30-180 rain).

Results. ROLE

OF N I T R O G E N A S E IN P H O T O E V O L U T I O N OF H 2.

E F F E C T OF SOME C O M P O U N D S ON H 2 METABOLISM AND NITROGEN FIXATION.

T h e c e l l s of T. roseopersicina g r o w n i n a r g i n i n e m e d i u m o r u n d e r N2-fixation c o n d i t i o n s r e d u c e d a c e t y l e n e a n d e v o l v e d I4_2 u n d e r i l l u m i n a t i o n (20.10:3 e r g / c m 2 . s e c ) i n t h e p r e s e n c e of p y r u v a t e o r m a l a t e ' w i t h o u t a lag p e r i o d (table I). T h e s a m e

BIOCHIMIE, 1978, 60, n ° 3.

e x c h a n g e reaction p r o c e e d e d w i t h o u t a lag-time. T h e r a t e s of s u c h p r o c e s s e s in t h e cells g r o w n in m e d i u m w i t h a m u l o n i u m salts, g l u t a m i n e o r a s p a r a g i n e w e r e n o t l o w e r a n d in s o m e c a s e s w e r e e v e n h i g h e r , t h a n in cell's g r o w n in t h e p r e s e n c e of a r g i n i n e o r t i n d e r n i t r o g e n f i x a t i o n c o n d i t i o n s (table I). T h e a d d i t i o n of NHa +, g l u t a m i n e , a s p a r a g i n e o r KNO a to cell s u s p e n s i o n s of T. roseopersicina 30 r a i n b e f o r e t h e e x p e r i m e n t s d i d not a f f e c t t h e i r

I. N. Gogotov.

270

h y d r o g e n a s e a c t i v i t y e s t i m a t e d b y D,~-H~O e x c h a n ge r e a c t i o n a n d H 2 p r o d u c t i o n f r o m r e d u c e d MV ( t a b l e II). I n c o n t r a s t , H,, a n d C2H 4 p b o t o f o r m a t i o n

b y t h e c e l l s f r o m r e d u c e d MV a n d also r e d u c t i o n of C~H.~ b u t it d i d n o t a f f e c t H 2 p h o t o e v o l u t i o n i n t h e p r e s e n c e of p y r u v a t e ( t a b l e II) a n d A T P -

TABLE II.

E[fect of some compoands on hydrogenase and nitrogenase activities of T. r o s e o p e r s i c i n a cells.

Compound

D.rH~0 exchange

Hu Iormation in the presence of MV + Na~S~04

C.~H4 photoproduction in presence of pyruvate

H.2 photoevolution in presence of pyruvate

117 97 137 87 93 0 n.d.

98 97 126 96 95 4 92

2a 25 a 50 a 25 a 30 a 0 n.d.

12a 0 0 15 a 32 a 100 0

NH4 + (10 -3 M) Glutamine (10-a M) Asparagine (10 -3 M) KNO a (10-3 M) N~ (20 per cent) CO (20 per cent) C~H~ (10 per cent)

Bacteria were g r o w n in P f e n n i g ' s m e d i u m (21) w i t h a r g i n i n e as nitrogen source, as described in Methods. The celts were h a r v e s t e d at exponential phase, w a s h e d a n d r e s u s p e n d e d in p h o s p h a t e buffer u n d e r argon. Cell suspensions were exposed to the test compounds for 30 m i n before h y d r o g e n a s e and nitrogenase activities were d e t e r m i n e d . Control samples were stored u n d e r argon and results are expressed as percentage of activity of control. (a) C~H4 a n d H~ p h o t o p r o d u e t i o n started a f t e r a t i m e lag (30-60 min). n.d. = not determined.

i n t h e p r e s e n c e of t h e s e c o m p o u n d s e i t h e r d i d n o t t a k e p l a c e o r s t a r t e d a f t e r a l a g - p e r i o d (30-60 m i n ) a n d p r o c e e d e d a t a l o w r a t e ( t a b l e II). C a r b o n monoxide completely inhibited both H 2 formation

d e p e n d e n t f o r m a t i o n of H 2 b y t h e cell e x t r a c t s from ditbionite (results not shown).

PROPERTIES

OF

HYDROGENASE

FROM

THE

CELLS

OF

T. roseopersicina. TABLE III.

Some properties o[ hydrogenase [rom T h i o c a p s a r o s e o p e r s i c i n a . Properties

Molecular weight Molecular weight of subunits Fe'~+ 168,000 daltons S~-/68,000 daltons pI Eo (pH 7.0), mV N-terminal amino acids T. opt, oC Thermostability, oC Activity, (?moles/Ho produced. min-L mg -t) (reduced MV (5raM) as substrate) The energy of activation (Kcal/mole) Half life (TI~) of enzyme inactivation in the presence of air (hr)

BIOCHIMIE, 1978, 60, n ° 3.

Soluble and chromatophore bound

68000 25000 and 47000 3.1 to 3 . 9 3 . 8 to 4 . 4 4.15 to 4.20 - - 280 Alanine, glycine 75 80

The soluble and membrane-bound hydrogenases f r o m T. roseopersicina h a v e b e e n p u r i f i e d to h o m o g e n e i t y [11, 12, 15]. B o t h h y d r o g e n a s e s h a v e a m o l e c u l a r w e i g h t of 6 8 0 0 0 a n d c o n s i s t of t w o s u b u n i t s w i t h a m o l e c u l a r w e i g h t of a b o u t 25 000 a n d 47 000 ( t a b l e III). T h e c o n t e n t of i r o n a n d a c i d - l a b i l e s u l p h u r i n t h e h y d r o g e n a s e is a p p r o x i m a t e l y e q u i m o l a r a n d c o r r e s p o n d s to 3.5 a n d 3.9 m o l e / m o l e of p r o t e i n , r e s p e c t i v e l y . Both soluble and membrane-hound hydrogenases a r e c h a r a c t e r i z e d b y t h e p r e s e n c e of a l a r g e a m o u n t of a c i d i c a m i n o a c i d s E15] w h i c h is i n a g r e e m e n t w i t h t h e i r i s o e l e c t r i c p o i n t s ( p I : 4.154.2'0).

300 C- 60.91 700 C-612.00 16.0 _+_ 2

Both soluble and membrane-bound hydrogenases h a v e t w o N - t e r m i n a l a m i n o a c i d s , a l a n i n e a n d glycine.

144

T h e t i t r a t i o n of t h e r e d u c e d a n d o x i d i z e d f o r m s of e n z y m e w i t h d y e s of l o w r e d o x p o t e n t i a l h a s

H y d r o g e n n l e t a b o l i s n l in p h o t o l r o p h i c s h o w n that the average value of r e d o x potential at pH 7.0, calculated for both the soluble and m e m b r a n e - b o u n d h y d r o g e n a s e s , is about -280 inV.

CATALYTIC

PROPERTIES

OF

HYDROGENASE

FROM

T. roseopersicina. The h m n o g e n e o u s p r e p a r a t i o n s of h y d r o g e n a s e w e r e able lo calalyse the r e d u c t i o n of MV and benzyl viologen (BV) w i t h h y d r o g e n . Unlike u n p u r i fled p r e p a r a t i o n s , the isolated e n z y m e did not reduce a z o c a r m i n e , n l e l h y l e n e blue, f e r r i c y a n i d e , NAD, NADP, FAD and FMN. The ability of hydro_ genase to catalyse De-HeO e x c h a n g e r e a c t i o n w a s lost d u r i n g the e n z y m e purificalion, although it could be r e s t o r e d by a d d i t i o n of dithionite. U n d e r such c o n d i t i o n s the h o m o g e n e o u s p r e p a r a l i o n s of soluble and m e m b r a n e - b o u n d h y d r o g e n a s e catalyse the e x c h a n g e of about 5 v,moles D,,. min-~. rag-1 protein. H o w e v e r , the r e a c t i o n rate w i t h m e m b r a n e - b o u n d h y d r o g e n a s e in the p r e s e n c e of

bacteria.

271

The rate of H 2 evolution from r e d u c e d MV by the soh~ble and m e m b r a n e - b o u n d h y d r o g e n a s e s of T. roseopersicina d e p e n d on the l e m p e r a t u r e . The m a x i m u m activity (612 .~mmles H . , . m i n -1 . m y -1 protein) has been s h o w n to take place at 70-80°C, whilst an aelivity of 60.91 i~naoles . r a i n -1 . ulg -1 p r o t e i n was o b s e r v e d at 30°C (table liD.

Natural electron carriers. Various r e d o x mediators w e r e tested to see if they w o u l d r e p l a c e methyl viologen and m e d i a t e e l e c t r o n t r a n s f e r to h y d r o g e n a s e s from sodium dithionite. The results are given in table IV. All h y d r o g e n a s e s w e r e able to catalyse H e p r o d u c t i o n in the p r e s e n c e of reduced MV. In the p r e s e n c e of r e d u c e d f e r r e d o x i n s from different sources, the h y d r o g e n a s e from C. pasteurianam was able to cat alyse H e p r o d u c tion at a h i g h e r rate w i t h C. p a s t e u r i a n u m ferred o x i n than the rate of H e p r o d u c e d f r o m r e d u c e d MV. E v o l u t i o n of H., from different r e d u c e d ferredoxins catalysed by h y d r o g e n a s e s isolated from

TABLE IV.

H.z evolution catalyzed by p u r i f i e d hydrogenases in the p r e s e n c e of reduced carriers. Source of hydrogenase Carriers

nmoles

C. pasteurianum

T. roseopersicina

R. rubrum

Chromatium vinosum

1 5 5000

0 0.26 136.08

0.03 0.06 60.91

0 0.19 13.40

0.06 0.09 0.86

C. pasteurianum Ferredoxin R. rubrum Ferredoxin Chromalium vinosum Ferredoxin

5 5

15.23 n.d.

trace 0.02

0.06 0.06

0.02 n.d.

5

2.62

n.d.

n.d.

0.02

10.16

0.72

5.72

T. roseopersicina cyt. ,c:~,,

2

T. roseopersicina cyt. ~e3~

2

15.02

n.d.

n.d.

Methyl viologen

-~ Chromalium Fd

I

5 1

n.d.

traces

The assay conditions were as described in the text. Rll electron donors were reduced

using sodium dithionite (1,0, raM). The purity index of cytochrome ¢ ~a >> from T. roseopersicina is 1.4. There was no H~ production if either hydrogenase or mediator was omitted. n.d. = not determined.

BV is about 15 times h i g h e r than that w i t h MV (1.2 and 19.0 ~moles D 2 e x c h a n g e d , rain -1 . i n g -1 p r o t e i n respectively). No A T P - d e p e n d e n t H 2 evolution could be d e m o n s t r a t e d w i t h s o d i u m dithio_ nite as the electron source w h e n tile soluble or m e m b r a n e - b o u n d e n z y m e w a s assayed.

BIOCHIMIE, 1978, 60, n ° 3.

T. roseopersicina, Chromatium v i n o s u m strain D and R. r u b r u m was only 1-30 per cent of the value o b t a i n e d in the presenc, e of r e d u c e d MV. H o w e v e r , low rates of H 2 c o n s u m p t i o n and f e r r e d o x i n red u c t i o n w e r e o b s e r v e d w i t h T. roseopersicina hydrogenase in the p r e s e n c e of H 2 and f e r r e d o x i n .

l. N. Gogotov.

272

When the reaction mixture contained both cytoc h r o m e <( c a )) f r o m T. roseopersicina a n d F d f r o m C h r o m a t i u m viuosum, a n e n h a n c e m e n t i n a c t i v i t y of T. roseopersicina h y d r o g e n a s e w a s o b s e r v e d

I n c o n t r a s t , c y t o c h r o m e ¢ c 3 >>, p u r i f i e d f r o m t h e cells of T. roseopersicina c o u l d m e d i a t e e l e c t r o n transfer with high efficiency to the hydrogenases of T. roseopersicina, Ch. v i n o s u m a n d R. r u b r u m ,

TABLE V.

H e evolution and 15N2 f i x a t i o n by extracts of T. r o s e o p e r s i c i n a cells (nmoies.h-l.mg -1 of protein). H2 evoluhon

I~Ni fixation

Assay mixture

Cell-free e x t r a c t (CE)

CE CE CE CE

+ ~~-~-

NADH-GS NADH-GS + ATP-GS NADPH N A D P H ~- ATP-GS

light

dark

43.0

25.0

190.0 n.d. 298.0 n.d.

87.2 186.0

light

dark

3.2

0

163.0 n.d.

35.0 188.0

371.5

35.5

40.0

416.0

n.d.

77.0

Reaction mixture (2 ml). Cell-free extract (1 m g / m l of protein). A~TP generating system : ATP (4 o,moles), creatine phosphate (10 v,moles), creatine kinase (~() I~g), MgCl., (10 ~moles) , NADPH-generating system : glucose-6-phosphate (10 o~moles), glucose-6-phosphate dehydrogenase (10 Ixg), N~ADP (1 l~mole) ; NADH-generating system : galactose (20 mmoles), galactose dehydrogenase (1 U), NADH (2.5 mM)' : 0.95 M potassium buffer, pH 7.0 (0.6 ml). GS = generating system. n.d. = not determined.

TABLE VL

H~ evolution in some systems. Omitted component

Hydrogenase

Methyl viologen NAD P+-reduetase --

--NADP + generating system

Methyl viologen

Additional component

Concentration of additional component

H~ evolution (~moles. h-~. mg -t protein)

---acetyl phosphate g e n e r a t i n g system FAD

---(a) I mM

2.74 2.92

FMN

1 mM

2.52

--

--

0 0 0

0

R. rubrum ferredoxin

2.5

?M

2.98

Hydrogen evolution was measured in a reaction mixture (final volume, 2 nd) containing purified hydrogenase (0,.2 nag) f r o m T. roseopersicina, m e t h y l viologen (100 ttmoles) ; NADPH generating system (see legend Table V) and NADP +reductase (0.1 mg). The rate of H~ evolution u n d e r these control conditions was 2.74 ~moles.h-l.mg-1 protein. (a) P h o s p h o t r a n s a c e t y l a s e 1U, CoA (1 mmole), a c e t y l p h o s p h a t e K, Li (25 mmoles).

b u t n o t to t h e h y d r o g e n a s e of C. p a s t e u r i a n u m ( t a b l e IV). T h e r a t e s of H 2 evolu,tion w e r e 3-90 f o l d h i g h e r t h a n t h a t f r o m r e d , u c e d MV w h e n 5 n m o l e s MV w e r e u s e d .

BIOCHIMIE, 1978, 60, n ° 3.

(table IV). U n d e r s u c h c o n d i t i o n s , t h e r a t e of H 2 p r o d u c t i o n in the t r e a t m e n t c o m b i n i n g c y t o c h r o m e <( c 3 >> + F d w a s a b o u t 66 p e r c e n t h i g h e r t h a n t h a t of c y t o c h r o m e ¢ c a )> a n d F d a l o n e .

H g d r o g e n m e t a b o l i s m in p h o t o t r o p h i c bacteria.

h y d r o g e n f o r m a t i o n in p h o t o t r o p h i c b a c t e r i a using NADH or NADPH as H+-donor. The p r o c e s s is a p p a r e n t l y o p e r a t e d by the e n e r g y p r o d u c e d by photosynthesis.

H e evolution and N~ fixation in the presence o[ NAD(P)H. The isolated h y d r o g e n a s e f r o m T. roseopersicina c a n n o t catalyse H 2 e v o l u t i o n in the p r e s e n c e of NAD(P)~H as H*-donor or r e d u c e NAD(P) + w h e n H 2 is H+-donor. H o w e v e r , cell-free extracts of T. roseopersicina and R. r u b r u m not only catalyse H 2 up,take in the p r e s e n c e of NAD(P), but t h e y are also capable of H e evolution and N e fixation in the p r e s e n c e of ATP and N & I ) f P ) H - r e g e n e r a t i n g systems (table V).

Ferredoxin and cgtochrome <> reduction in the presence of NADPH. The f e r r e d o x i n , c y t o c h r o m e c a and NADPr e d u c t a s e are i n t i m a t e l y i n v o l v e d in NADPH formation. In tile p r e s e n c e of NADP-reductase and NADPH as H÷-donor. r e d u c t i o n of f e r r e d o x i n or c y t o c h r o m e c 3 takes place (not s h o w n ) . T h e ferred o x i n - c y t o c h r o m e ¢ % >> : f l a v o p r o t e i n c o m p l e x in this case p e r h a p s serves a f u n c t i o n a l role in photosynthetic electron transport.

An a t t e m p t has thus been m a d e to r e c o n s t r u c t the system of H z f o r m a t i o n w i t h the p a r t i c i p a t i o n of the e l e c t r o n t r a n s p o r t c h a i n c o m p o n e n t s , isolated f r o m R. r u b r u m ( f e r r e d o x i n , NADP-reductase) and T. roseopersicina (hydrogenase, NADP-reductase) u s i n g NADPH or NADH as H+-donor (table VI). T h e results o b t a i n e d show that in the p r e s e n c e of m e t h y l viologen H e f o r m a t i o n takes place w i t h o u t any source of energy, i.e. a p p a r e n t l y it occurs by means of the d i a p h o r a s e r e a c t i o n of methyl viologen r e d u c t i o n by NADP-reductase, though in the case w i t h f e r r e d o x i n , H e f o r m a t i o n is c o n s i d e r a b l y stimulated by acetyl-CoA-regener a t i n g system (results not shown).

S

Fdred~

273

Discussion.

Many b a c t e r i a are c a p a b l e of using H e for red u c t i o n of m o l e c u l a r n i t r o g e n [6, 33, 34]. In this case H 2 uptake is m e d i a t e d by a h y d r o g e n a s e [9, 35]. H o w e v e r , w i t h h i g h e r c o n c e n t r a t i o n s of H 2

f

H ~

~,~

H2 : Fd. oxidoreductose

//o~~

NAD(P)H\/~"

~2H÷ NAD(P)~ \-~.

9I

,

'?/ tlkN2

~ "H2 H2:c3"~oreductase / /

Cyt 'c3'bx2

2H ÷

~

FIG. 1. - - Possible scheme o[ hgdrogenase-nitrogenase relationship in the phototrophic bacteria R. rubrum and T. roseopersieina. ( ) according to the experimental data. (. . . . . ) absence of experimental data.

Thus, the dat,a o b t a i n e d show that a p a r t of the e l e c t r o n 4 r a n s p o r t chain, consisting of f e r r e d o x i n , NADP+-reductase and h y d r o g e n a s e , takes part in

BIOCHIMIE, 1978, 60, n ° 3.

both H e p r o d u c t i o n [36] and N 2 fixation m a y be i n h i b i t e d in s y m b i o t i c systems [37] or p u r e cultures [38, 39].

274

I. N. Gogotov.

Cell-free extracts from the p h o t o t r o p h i c bacteria C h r o m a t i n m v i n o s u m and ChIorobium limicola, are capable of f e r r e d o x i n - d e p e n d e n t reduction of NAD(P) in the p r e s e n c e of H 2 as H+-donor [42, 43]. The NAD(P) ,reduction 'in the p r e s e n c e of H 2 has been c h a r a c t e r i s e d for t h e p u r p l e b a c t e r i a R. r u b r u m [40, 41, 47] and T. roseopersicina [27]. T h e r e is also some data that the p u r p l e bacter i a and c y a n o b a c t e r i a , c,apable of N 2 fixation, produce t w o e n z y m e s w h i c h p a r t i c i p a t e in the H 2 evolution. Such e n z y m e s are h y d r o g e n a s e and nit r o g e n a s e [5, 44-46]. The data r e p o r t e d h e r e c o n f i r m t h a i N~ photoe v o l u t i o n by p u r p l e b a c t e r i a is due to the action of nitrogenase, the synthesis of w h i c h is r e p r e s s e d in the p r e s e n c e of g l u t a m i n e and asp,aragine. Both c o m p o u n d s as w e l l as NH4 + and NO 3- (in the case of T. roseopersicina w h i c h is capable of assimil a t o r y n i t r a t e - r e d u c t i o n ) seem to p r o d u c e t h e inhib i t i n g action u p o n n i t r o g e n a s e activity. Carbon m o u o x i d e i n h i b i t s acetyl,ene r e d u c t i o n but not A T P - d e p e n d e n t f o r m a t i o n of H 2 by cell-free extracts. T h i s is in a g r e e m e n t w i t h the a s s u m p t i o n [32] that H e - f o r m i n g p a r t of nitrog,enase differs f r o m G2He-reducing one. The e x p e r i m e n t a l results m a y also be i n t e r p r e ted as i n d i c a t i n g that the h y d r o g e n a s e synthesis by p u r p l e b a c t e r i a is i n d e p e n d e n t of a source of n i t r o g e n in the m e d i u m . H y d r o g e n a s e a p p a r e n t l y does not take p a r t in H e f o r m a t i o n by the cells f r o m natural H+-donors, a,t least u n d e r i l l u m i n a tion. Thus H 2 p h o t o e v o l u t i o n by p u r p l e b a c t e r i a cells can be used as a test for estimation of t h e i r ability for nitrogen fixation. In oivo the h y d r o g e n a s e of R. r a b r u m and T. roseopersicina, as in other N2-fixers [45-47], are capable of r e c y c l i n g Hu, w h i c h is used by t h e nitrogenase, for N.~ fixation. This p o i n t or v i e w is supp o r t e d by the f o l l o w i n g e x p e r i m e n t a l data. Nu fixation a n d H 2 e v o l u t i o n take place in the presence of NAD(P)H (table VI). In the p r e s e n c e of NADP-reductase f e r r e d o x i n a n d c y t o c h r o m e <( % w e r e r e d u c e d . In this case the r e d u c t e d f o r m of f e r r e d o x i n in R. r u b r u m and T. roseopersicina (table IV-VI) and in C. pasteurianum [33],¢ Chrom a t i u m sp. [49], Anabaena c y l i n d r i c a and Chloropseudomonas ethylica [50] has been used as e l e c t r o n d o n o r to nitrogen,ase. H o w e v e r , the hyd r o g e n a s e of T. roseopersicina ,(E'o ~ - - 2 8 0 mV) a n d R. r u b r n m p a r t i c i p a t e d in the H 2 e v o l u t i o n only f r o m the r e d u c e d f o r m of c y t o c h r o m e ¢ c 3 >>, w h i c h p e r h a p s is the natural electron c a r r i e r for this enzyme (table IV). BIOCHIMIE, 1978, 60, n ° 3.

If the r e d u c i n g p o w e r and e n e r g y pools for N._, fixation in the cells are p r o d u c e d in excess then the n i t r o g e n a s e evolves H2, w h i c h is r e c y c l e d via the f e r r e d o x i n - c y t o c h r o m e ¢ % >>- h y d r o g e n a s e c o m p l e x to n i t r o g e n a s e (fig. 1). T h e a l t e r n a t i v e e x p l a n a t i o n is that in the p r e s e n c e of r e d u c e d f e r r e d o x i n and h y d r o g e n a s e f r o m R. rubrum, T. roseopersicina or Ch. vinosum, H 2 p r o d u c t i o n occurs at a v e r y low rate (table IV), but consumption of H 2 [24] and r e d u c t i o n of the f e r r e d o x i n takes place in the p r e s e n c e of H,, and h y d r o g e n a s e of T. roseopersicina or N&DPH and NADP-reductase [27, 47]. H o w e v e r , the f e r r e d o x i n stimulated H e p r o d u c t i o n f r o m r e d u c e d c y t o c h r o l n e <<% >> (table IV). T h e f e r r e d o x i n - l i n k e d H 2 p r o d u c t i o n f r o m redu_ ced c y t o c h r o m e (< c a >> (table IV) in the p r e s e n c e of h y d r o g e n a s e f r o m T. roseopersicina c o r r e l a t e d w i t h the a s s u m p t i o n [51] that f e r r e d o x i n m a y b i n d either to the f l a v o p r o t e i n (NA,D,P-reductase) or to c y t o c h r o m e <<% >). T h e b i n d i n g of ferred o x i n to c y t o c h r o m e <> w o u l d p r o v i d e a complex b e t w e e n the t w o p r o t e i n s w h i c h w o u l d serve as a substrate for the h y d r o g e n a s e and stimulate the e n z y m e activity. In this case NADP-reductase and h y d r o g e n a s e in the p h o t o t r o p h i c b a c t e r i a can play a c e n t r a l role in the regulation of r e d u c i n g p o w e r for the s u p p o r t of N., fixation or H e evolution. The coup l i n g b e t w e e n n i t r o g e n a s e and h y d r o g e n a s e takes place p e r h a p s t h r o u g h f e r r e d o x i n and a cytoc h r o m e ¢ c a >> : f l a v o p r o t e i n c o m p l e x .

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Hydrogen

metabolism

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BIOCHIMIE, 1978, 60, n ° 3.

in phototrophic

bacteria.

275

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