Structural properties, functional states and

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FEMSRE 00202

Structural properties, functional states and physiological roles of hydrogenase in photosynthetic bacteria K o r n r l L. Kov~cs and C s a b a Bagyinka

Institute of Biophysics, BiologicalResearchCenter, Hunga.'!anAcademyof Sciences, Szegea[Hungary

Key words: Hydrogenase; Subunit structure; Active states; Bioenergetics; Membrane potential; Thiocapsa roseopersicina

1. S U M M A R Y The membrane-bound hydrogenase from Thiocapsa roseopersieina is composed of two subumts and contains two Fe-S centres and one Ni per molecule. The enzyme resists heat and proteolytic degradation, its activity is retained under SDSP A G E conditions. The location of the metal atoms on the subunits has been determined by proton-induced X-ray emission (PIXE). A revised hydrogenase model which concurs with the new data is suggested. The orientation of the enzyme in the photosynthetic membrane and its ability to generate membrane potential suggest that hydrogenase can play a significant role in the energetics of these bacteria. A possible link between nitrogen fixation, photosynthetic electron transport and hydrogenase is proposed. 2. I N T R O D U C T I O N Hydrogenases from photosynthetic bacteria are integral membrane proteins [1,2]. Their most im-

Correspondence to: K.L. Kov/lcs,Institute of Biophysics, Biological Research Center, Hungarian Academyof Sciences,P.O. Box 521, H-6701 Szeged,Hungary.

portant physiological function is assumed to be oxidation of hydrogen gas produced by the nitrogenase system. Metal analysis and EPR spectroscopy [3-6] show the presence of Ni and Fe in these enzymes, the Fe atoms being incocporated into Fe-S clusters. It has been recognized recently that membrane-bound Ni-Fe hydrogenases, isolated from various bacteria, are remarkably similar in their subunit composition [1,7-9]. This resemblance is also reflected in the immunological cross-reactivities [8,10]. Apparently, the membrane-bound Ni-Fe hydrogenases are composed of two subunits of approximately 60 kDa and 30 kDa, although previous data have been conflicting [11,12]. There are various calculations for the metal contents of hydrogenases, e.g., for the Thiocapsa roseopersicina enzyme the numbers range from 4 Fe and no Ni [12] to 7 Fe and 1 Ni [13]. Here we review our recent findings, leading to a more thorough understanding of the subunit composition and metal content of hydrogenas¢ from T. roseopersicina. Generation of membrane potential by hydrogcnase from molecular hydrogen is demonstrated in photosynthetic bacteria for the first time. The impfication of these observations is that hydrogenase is likely to be involved in membrane bioer, ergetic processes in addition to its role as a catalyst of disposal of molecular hydrogen.

0168-6445/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties

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FEMS MicrobiologyReviews87 (1990) 407-412 Published by Elsevier

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3. H Y D R O G E N A S E S T R U C T U R E

3.1. Gel electrophoresis Activity staining of the aerobically purified enzyme after SDS-PAGE revealed two bands which displayed hydrogenase activity and migrated to R F : 0 . 3 5 and RF =0.19, respectively. The R F : 0.19 protein was very oxygen labile and could be transformed into the R r = 0 . 3 5 form. Therefore, the two active bands represented two conformations of the same hydrogenase. Either active hydrogenase forms yielded two polypeptides when the enzyme was denatured by boiling for at least 10 rain. These proteins represent the two subunits

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Fig 1. Two-dimensionalelectrophoresisof hydrogenase from Thiocapsa roseopersicina. SDS-PAGE was performed in both dimensions(for details see [9]). The enzymewas exposed to the indicated temperatures under air or H 2 for 10 rain before electrophoresis. The proteins at apparent molecular masses of 90 and 49 kDa were active and are referred to as Rvffi0.19 and R F ffi0.35 ective forms in the text. Hydrogenase subunits at 64 and 34 kDa wereinactive.

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Fig. 2. Proposed hydrogenase model. The electron flow is directed from the Ni center on the 34-kDa subunit toward the

donor/acceptor (D/A) moleculesvia the Fe-S clusters which are embedded in the 64-kDa subunit. of the hydrogenase, their molecular masses being 64 and 34 kDa, respectively. The transition from the active to the inactive forms and vice versa can be induced by changing the preincubation conditions (e.g., temperature, H 2 treatment). A schematic summary of the results from some related experiments is shown in Fig. 1. The apparent molecular masses corresponding to the enzyme components and forms have been confirmed and corroborated by gel filtration and by equilibrium ultracentrifugation (data not shown). The homology between the active hydrogenase forms and inactive subunit polypeptides has been established by immunoelectroblotting, Cleveland mapping, N-terminal amino acid sequence and amino acid composition analyses. The results strongly suggest a native molecular mass of 98 kDa. Therefore, contrary to earlier characterizations [12,14] the polypeptide structure of the hydrogenase from T roseopersicina appears similar to the other membrane bound Ni-Fe hydrogenases.

3.2. PIXE measurements Determination of metal composition by proton-induced X-ray emission clearly indicated more than four Fe per hydrogenase molecule. The ratio of Fe to Ni of 7 : 1 ( + 1 ) was found [13] which is quite different from the 4 Fe per molecule estimated from chemical analysis [12]. Assurn-


408

4. H Y D R O G E N A S E F U N C T I O N

4.1. Intraceilular location The location of the membrane bound Ni-Fe hydrogenases has been determined for a n u m b e r of purple photosynthetic bacteria [2]. They are invariably positioned so that the hydrogen-uptake active center is at the outer surface of the photosynthetic membrane. A likely connection between the functionally related components in the photosynthetic membrane that complies with the data is depicted in Fig. 3.

4. 2. Membrane potential generation One consequence from the scheme in Fig. 3 is the spatial separation of the H2-producing nitrogenase from the H2-disposing hydrogenase. The intriguing possibility to couple H 2 oxidation

Fig. 3. Suggested location of hydrogenase in the membrane of photosynthetic bacteria and functional association among hydrogenase, photosynthetic electron transport, photophosphorylation and nitrogen fixation.

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CCCP 50 !00 Time (rain) Fig. 4. Generation of membrane potential by H 2 as measured with flow dialysis, l0 s freshly harvested cells were suspended in 20raM K-phosphate buffer containing 160 mM NaC1 (pH 7.2). "C-labeled tetraphenyl phnsphonium (TPP) was used as radioactive tracer. The effluent was collected in 8-ml fractions and the radioactivity in the fractions monitored by a liquid scintillation counting. Radioactivities in the effluent fractions are indicated by dots. The continuous line corresponds to the membrane potential of resting cells under air. An increase of membrane potential was observed in H 2 atmosphere (less TPP in the effluent, dashed line). CCCP added at the time indicated by the arrow collapsed the membrane potential. Inset: general layout of the flow dialysis cell (see also [14]).

to membrane potential generation is suggested by this arrangement. The effect of H : on membrane potential has been studied by flow dialysis as well as by ionselective electrode technique. The cell suspension has been exposed to H e at the beginning of the experiment. Replacing H z for 02 resulted in a shift to a more negative external potential (Fig. 4). The process could be repeated several times without noticeable loss of the H2-induced mere-


409

ing a single Ni per molecule, at least two Fe-S centers have to be attached to the enzyme in order to accommodate all Fe atoms (Fig. 2). When performed on dry gel samples PIXE also provides information about the location of the metal atoms [13]. The Fe atoms are bound to the large subunit, while the small subunit contains the Ni atom. The proposed new structure (Fig. 2) conforms well with the three-state functional model suggested by Cammack and coworkers [15].

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Fig. 5. Measurement of membrane potential by ion-selective electrode. A TPP-selective electrode was built according to [17,18]. The electrode and a potassium reference electrode were fitted into an anaerobic mee,suring cell. The TPP concentration changes in the extracelhilar space were followed by a pH measurement. Freshly harvested T. roseopersicinacells (a) and CCCP-treated bacteria (b) have been used. The direction of H2-indueed membrane potential coincided with the direction of fight-generated membrane potential as shown in trace (a). The same effects could not be reproduced in the CCCP-poisoned control (trace b).

b r a n e potential. A s expected, u p o n a d d i n g t h e inhibitor carbonyl cyanide m-chlorophenyl hyd r a z o n e ( C C C P ) t h e m e m b r a n e potential coll a p s e d a n d c o u l d n o t be r e g e n e r a t e d b y H e . The phenomenon has been further studied by m e a s u r i n g t h e m e m b r a n e potential with a tetrap h e n y l p h o s p h o n i u m (TPP) sensitive electrode (Fig. 5). It is n o t e w o r t h y t h a t t h e m e m b r a n e p o t e n t i a l c h a n g e associated with h y d r o g e n exp o s u r e is a n a l o g o u s to t h e m e m b r a n e potential g e n e r a t e d b y the p h o t o s y n t h e t i c a p p a r a t u s . It follows f r o m the o b s e r v a t i o n s t h a t t h e p r o t o n s are released at the extraeellular side o f t h e m e m b r a n e . T h e s a m e qualitative picture w a s o b t a i n e d w h e n t h e e x p e r i m e n t w a s repeated with Chromatium minutissimum, Ectothiorhodospira shaposhnikovii o r Rhodospirillum rubrum, indicating that t h e p h e n o m e n o n is p r e v a l e n t a m o n g p h o t o s y n t h e t i c bacteria. It s h o u l d b e n o t e d t h a t a very similar effect o f H 2 o n m e m b r a n e potential h a s been f o u n d in t h e case o f Methanobacterium thermoautotrophicum [19]. It is c o n c l u d e d that 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 m a y c o n t r i b u t e to A T P s y n t h e s i s b y g e n e r a t i n g m e m b r a n e potential a n d t h e r e f o r e p l a y a significant role in the m e m b r a n e bioenergetic processes.

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