First publ. in: Biochimie 60 (1978), pp.

297-305

Hydrogen metabolism in aerobic hydrogen-oxidizing bacteria.

Bernhard SCHINK and Hans-Giinter SCH~LEGEL.

Institut [i~r Mikrobiologie der Gesellscha[t [iir Strahlen- und Umwelt[orschung mbH Miinchen, und der Universitiit G6ttingen, 3400 G6ttingen, Grisebachstr. 8, Federal Republic o[ Gerlnany.

R~sum~.
O n rencontre d e u x t y p e s d ' h y d r o q 6 n a s e s d a n s les bact6ries a 6 r o b i e s c a p a b l e s d ' o x y d e r l ' h y d r o q ~ n e m o l 6 c u l a i r e : les u n e s sont solubles et r6duisent le NAD (H~: NAD oxydor~ducrases), les a u t r e s sont m e m b r a n a i r e s et ne r6duisent p a s les pyridine-nucl6otides. Les bact6ries qui o x y d e n i l ' h y d r o q ~ n e p e u v e n t ~tre divis6es e n trois q r o u p e s s u i v a n t q u ' e l l e s contiennent (i) les d e u x t y p e s d ' e n z y m e s (A/caliqenes eutrophus), (ii) settlement u n e e n z y m e soluble (Nocardia o p a c a lb), (iii) s e u l e m e n t u n e e n z y m e m e m b r a n a i r e (la majorit~ des q e n r e s et des esp~ces). Nous r 6 s u m o n s les 6tudes faites sur l ' e n z y m e & NAD de A. eutrop h u s . Les r6sultats c o n c e r n a n t la solubilisation et l a purification d e l ' h y d r o q 6 n a s e m e m b r a n a i r e d e A. e u t r o p h u s sont pr6sent6s e n d6tail. L ' e n z y m e a 6t6 solubilis6e & partir de m e m b r a n e s purifi6es & r a i d e de Triton X-100 et de d 6 s o x y c h o l a t e ou de p h e s p h o l i p a s e D. L'extrait m e m b r a n a i r e brut a 6t6 fractionn6 p a r pr6cipitation a u sulfate d ' a m m o n i u m et chromatoq r a p h i e sur c a r b o x y m 6 t h y l c e l l u l o s e & p H 5,5. L ' e n z y m e est s t a b l e en t a m p o n p h o s p h a t e ; s a stabilit6 en milieu o x y d a n t est c o m p a r a b l e & celle de l ' e n z y m e soluble. Des donn6es biochimiques et i m m u n o l o q i q u e s indiquent cepend a n t q u e les d e u x e n z y m e s p o s s ~ d e n t d e s structures diff6rentes.

Summary. A s u r v e y on o r q a n i s m s a b l e to u s e molec u l a r h y d r o q e n a s electron donor in the e n e r q y yieldinq p r o c e s s is p r e s e n t e d . In the cyroup of the a e r o b i c hydroqen-oxidizing b a c t e r i a so far two t y p e s of h y d r o q e n a s e s h a v e b e e n encountered, a NAD-reducinq, soluble e n z y m e (H2 : NAD oxidoreductase) a n d a m e m b r a n e b o u n d e n z y m e u n a b l e to r e d u c e p y r i d i n e nucleotides. With respect to the distribution of both t y p e s of h y d r o q e n a s e s three q r o u p s of hydroqen-oxidizinq b a c t e r i a c a n b e diffentiated containinq (i) both t y p e s (Alcaliqenes eutrophus), (ii) a soluble e n z y m e o n l y (Nocardia o p a c a lb), a n d (iii) a m e m b r a n e - b o u n d h y d r o q e n a s e o n l y (majority of g e n e r a a n d species). The results of studies on the NADspecific h y d r o q e n a s e of A. e u t r o p h u s a r e s u m m a r i z e d . Results on the solubilization a n d purification of the m e m b r a n e - b o u n d hydroq e n a s e of A. e u t r o p h u s a r e p r e s e n t e d in detail. The e n z y m e w a s solubilized from purified m e m b r a n e s b y Triton X-100 a n d s o d i u m desoxycholate or p h o s p h o l i p a s e D. The c r u d e m e m b r a n e extract w a s f r a c t i o n a t e d b y a m m o n i u m sulfate precipitation a n d c h r o m a t o g r a p h y on c a r b o x y m e t h y l c e l l u l o s e at pH 5.5. The e n z y m e w a s s t a b l e in p o t a s s i u m p h o s p h a t e buffer ; it r e s e m b l e s the soluble e n z y m e with respect to stability u n d e r oxidizinq conditions. Further b i o c h e m i c a l a n d i m m u n o l o q i c a l d a t a indicate, h o w e v e r , that b o t h e n z y m e s a r e different with r e s p e c t to their n a t i v e structure.

Several metabolic types of organisms are able to use molecular hydrogen (dihydrogen or H 2) as an electron donor in the energy-yielding process. Two groups have to be considered. The first group comprises bacteria utilizing hydrogen under aerobic conditions. These are the cheumlithoautotrophic, aerobic hydrogen-oxidizing bacteria (e.g. Alcaligenes eutrophus); they are able to

use hydrogen for aerobic respiration and the reduction of carbon dioxide. The second group, which is able to utilize hydrogen under anaerobic conditions only, comprises various metabolic types, chemotrophic as well as phototrophic bacteria. The chemotrophic hydrogen-utilizing bacteria are the methanogenic bacteria (e.g., Methanosarcina barkeri), the acetogenic bacteria (e.g., 21

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/6288/ URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-62888

298

Ci'ostridium aceticum and Acetobaclerium woodii), the sulfate-reducing bacteria (e.g., Desul[ovibrio vulgaris) , the n i t r a t e - r e d u c i n g , d e n i t r i f y i n g bacteria (e.g. Paracoccus denitri[icans) and the fumar a t e - r e d u c i n g bacteria (e.g., Escherichia colt).
Among the p h o t o t r o p h i c b a c t e r i a there are at least some species able to grow w i t h h y d r o g e n as an electron d o n o r in each of the lnajor families, Chromatiaceae (e.g., Chromatium vinosam, Thiocapsa roseopersicina), Rhodospirillaceae (e.g.,

Rhodospirillum rubrum, Rhodopseudomonas capsulata) and Chlorobiaceae (e.g., Chlorobium limicola var. thiosalphatophilum). Some c y a n o b a c t e r i a
and a few green algae can be adapted to use h y d r o g e n for a n o x y g e n i c photosynthesis. The c a r b o n source used together w i t h h y d r o g e n is c a r b o n dioxide, at least in the m a j o r i t y of the b a c t e r i a using H2 as source of r e d u c i n g p o w e r a n d e n e r g y ; only the sulfate- and the fumarater e d u c i n g bacteria lack the ability to fix CO 2 autotrophically. There are m a n y b a c t e r i a that are able to activate h y d r o g e n in a d d i t i o n to those m e n t i o n e d . They share the presence of hydrogenases, as the enzymes activating o r / a n d releasing h y d r o g e n gas are collectively called. For example, all nitrogenfixing bacteria, the rhizobia included, possess hydrogenases ; it is assumed that in these bacteria h y d r o g e n a s e recycles the h y d r o g e n evolved by nitrogenase thus p r e v e n t i n g waste of energy and p r o t e c t i n g the nitrogenase from i n a c t i v a t i o n by oxygen [1]. The obvious questions related to h y d r o g e n utilization for energy g e n e r a t i o n by n o n - a u t o t r o p h i c bacteria have not been definitely resolved. This report will deal w i t h the first group only, the aerobic c h e m o a u t o t r o p h i c bacteria, hereafter p l a i n l y called the h y d r o g e n - o x i d i z i n g bacteria. These b a c t e r i a comprise a physiological g r o u p ; they are facultatively a u t o t r o p h i c b a c t e r i a and share the ability to grow with h y d r o g e n as electron donor and with c a r b o n dioxide as the sole c a r b o n source. The s t o i c h i o m e t r y of the c o n s u m p t i o n of the gaseous substrates by these bacteria is d e p e n d e n t on the growth c o n d i t i o n s and growth rate and varies from strain to strain. Hydrogen is used in the o x y g e n - h y d r o g e n reaction serving the gener a t i o n of e n e r ~ (equation 1) and in the fixation of CO 2 to the level of cellular material (equation 2). The ratio of h y d r o g e n uptake over the uptake of c a r b o n dioxide (H2/CO 2) by a s u s p e n s i o n of g r o w i n g cells is d e p e n d e n t on the efficiency of coupling, on the e x p e n d i t u r e for m a i n t e n a n c e

energy a n d on the level, at w h i c h the electrons enter the r e s p i r a t o r y c h a i n ; H J C O 2 ratios of 4 to 19 have been measured. The average ratio measured w i t h Alcaligenes eatrophas is represented in equation 3. 2 H 2 + 02 )'2 H20 (1) 2 ~ + C09 ~-<CH20 > + H20 (2) fi H 2 + 2 02 + CO 2 > < C H 2 0 > +~5 H20 (3) Carbon dioxide tixation occurs via the ribulosebisphosphate cycle. So far, a n m n g the aerobic autotrophs no m e m b e r has been f o u n d w h i c h assimilates CO 2 via a n o t h e r pathway. Thus, the spect a c u l a r ability of the aerobic h y d r o g e n - o x i d i z i n g b a c t e r i a to grow w i t h H 2 + CO 2 as the sole energy and c a r b o n substrates is due to the c o m b i n a t i o n of CO~ fixation and h y d r o g e n oxidation, w i t h r i b u l o s e b i s p h o s p h a t e carboxylase, p h o s p h o r i b u l o Ikinase and hydrogenase(s) as the m a i n key enzymes. Otherwise, the h y d r o g e n bacteria are not different from n o r m a l h e t e r o t r o p h i c b a c t e r i a ; they are able to grow on o r g a n i c substrates as well. T a x o n o m i c a l l y the aerobic h y d r o g e n - o x i d i z i n g b a c t e r i a are a heterogeneous group c o n t a i n i n g Gram-negative genera, such as Alcaligenes, Pseu-

domonas, Paracoccus, Aquaspiril?um, Flavobacterium, Corynebacterium as well as Gram-positive genera such as Nocardia, Mycobacterium and Bacillus ([2] ; Aragno and Schlegel, in preparation).

Hydrogenases, their distribulion and regulation.

Hydrogenase is the collective name of a multitude of enzymes either activating or releasing hydrogen. In most cases n e i t h e r the physiological electron acceptor nor the properties of the enzymes are k n o w n [3]. Some h y d r o g e n a s e s reduce N~AD, others ferredoxin, m e n a q u i n o n e or cytochrome c ; the ability to reduce some redox dyes is c o m m o n to all hydrogenases. So far two types of h y d r o g e n a s e have been encountered in the aerobic h y d r o g e n - o x i d i z i n g bacteria. The one is a NAD-reducing, soluble enzyme (H~ : N,~D oxidoreductase) and the other is a memb r a n e - b o u n d enzyme u n a b l e to reduce p y r i d i n e nucleotides. W i t h respect to the d i s t r i b u t i o n of both types of hydrogenases three groups of h y d r o gen-oxidizing b a c t e r i a can be differentiated (table I) : (i) Alcaligenes eutrophus [4], A. ruhlandii, [5] and Pseudomonas saccharophila [6] c o n t a i n both types, the soluble and the m e m b r a n e - h o u n d h y d r o genase ; (it) Nocardia opaca lb (7) and some further species of the genus Nocardia c o n t a i n a soluble, NAD-reducing enzyme o n l y ; (iii) the majority of the h y d r o g e n - o x i d i z i n g bacteria, such

299 as Alcaligenes latus, Aquaspirillum aulolrophicum, Paracoccus den itri[icans, several p s e u d o m o n a d s and the c o r y n e f o r m nitrogen-fixing strains, cont a i n only a m e m b r a n e - b o u n d h y d r o g e n a s e [8]. (Entner-Doudoroff) enzymes is repressed [11]. In contrast, in Paracoccus denitri[icans glucose is the d o m i n a n t substrate, and h y d r o g e n a s e form a t i o n is repressed E12~. The latter type of regu-

TABLE I.

Hydrogen-oxidizing bacteria and the localization o[ hydrogenases.

Hydrogenase Species
soluble membranebound

nitrogen
fixation

Gram
stain

Alcaligenes eutrophus Alcaligenes paradoxus Alcaligenes ruhlandii Alealigenes latus Pseudomonas [acilis Pseudomonas saccharophila Pseudomonas carboxydooorans Pseudomonas pseudoflava Pseudomonas palleronii Pseudomonas hydrogenooora Pseudomonas hgdrogenothermophila Flavobacterium autothermophilum Hydrogenomonas thermophilus Aquaspirillum autotrophieum Paracoccus denitrificans < < Cor!lnebacterium > > autotrophicum Nocardia opaca lb Nocardia aulotrophica Mgcobacterium 9ordonae Arthrobacter sp. (llX, Pd-I 12) Seliberia carboxgdohgdrogena

dt71_ __ w

.~.ql-A I-qq.-q.-q"JI----------------

d[_

-31-

--

--

-q71-

---

__ __

+
-__

-q.qt_

-~-

__ __

AV
m

+ +

---

+ +

The regulation of h y d r o g e n a s e f o r m a t i o n in these b a c t e r i a is not well k n o w n [9]. All aerobic h y d r o g e n bacteria are facultatively a u t o t r o p h i c b a c t e r i a and are able to grow u n d e r a u t o t r o p h i c c o n d i t i o n s w i t h CO2 + H 2 as well as u n d e r heterot r o p h i c c o n d i t i o n s on a n u m b e r of organic comp o u n d s as substrates, such as sugars, o r g a n i c acids, and a m i n o acids. Maximum h y d r o g e n a s e activity is found in a u t o t r o p h i c a l l y g r o w n ceils. In h e t e r o t r o p h i c a l l y g r o w n cells h y d r o g e n a s e is either absent or present in m i n o r specific activities ; in Alcaligenes eutrophus the specific total h y d r o g e n a s e activity d e p e n d s on the k i n d of organic substrate (table Ii) a n d the growth rate [101. W h e n the cells are exposed to both energy donors, h y d r o g e n and an organic substrate, the response is different in different strains ; a k i n d of catabolite repression nlay be operative. In Alcaligenes eutrophus h y d r o g e n is the d o m i n a n t substrate ; in the presence of h y d r o g e n and fructose the formation of the fructose d e g r a d i n g

lation seems to p r e d o m i n a t e among the h y d r o g e n bacteria. In addition, h y d r o g e n can exert an i n h i bitory effect on the utilization of the organic substrate. In Alcaligenes eutrophus H16 h y d r o g e n i n h i b i t s the utilization of f r u c t o s e ; this effect t u r n e d out to be due to the i n h i b i t i o n of glucose-6phosphate d e h y d r o g e n a s e [13] ; in A. eutrophus this enzyme is sensitive to ATP and NAD'H2. F r o m following the pool sizes of glucose-6-phosphate, 6-phosphogluconate, ATP and NA,DH2 d u r i n g t r a n s i t i o n of the cells from air to a h y d r o g e n oxygen m i x t u r e it was c o n c l u d e d that glucose-6phosphate d e h y d r o g e n a s e is the regulatory enzyme and that in vivo the i n h i b i t i o n is caused by the increase in the NADH 2 c o n c e n t r a t i o n [141. The presence of two h y d r o g e n a s e s has been described for Pseudomonas saccharophila [6~, Alcaligenes ruhlandii [15; 5] and Alcaligenes eutrophus [4]. The soluble enzyme has been recently purified and characterized. The results

300 have been p u b l i s h e d [16; 81 and w i l l only be s u m m a r i z e d here. of a flavin c o m p o n e n t . It was identified as being FMN by t h i n layer c h r o m a t o g r a p h y , fluorescence s p e c t r u m and fluorescence q u e n c h i n g by the addition of apoflavodoxin. The molecular weight is 2,0.5t)00.. The isoelectric p o i n t was found to be 4.85. The pH o p t i n m m was 8.0, the optimal t e m p e r a t u r e 33C, and the Mi-

The soluble hlldrogenase o[ Alcaligenes eutrophus.

Eleven strains of Alcaligenes eutrophus were used to e x a m i n e the stability of the soluble hydrogenase, and s t r a i n HI6 was selected as the enzyme

TABLE II.

Rate o[ hydrogen oxidation by intact cells o[ Alcaligenes eutrophus H 16 after growth on glutamate, lactate or fructose compared to autotrophically grown cells.
Rate of hydrogen oxidation (l~mol H~/min.mg protein)
after g r o w t h on

Multiplication
factor of cell protein during growth

Glutamate
8 5 p e r cent N~ + 5 par cent O~ + 10 per cent C0~

Lactate
85 per centN~ + 5 per cent O~ + 5 per cent CO~

Fructose Air

on organic substrate

Autotrophic culture ca. 10 ~ ca. 104 ca. 1 0 6 10 8

1.36 0.454 -0.160 --

1.45 0.056 -0.056 0

1.26 0.651 0.484 i 0.424

Rates of total gas uptake were measured in an atmosphere of 85 per cent H~2+ 5 per cent O2 + 10 per cent COs (10). The rate of hydrogen oxidation was calculated assuming ratio. H~ :O~. :CO~= 6 : 2 : 1 .

source. The enzyme was purified 68-fold w i t h a yield of 20 p e r c e n t and a specific activity (NADr e d u c t i o n ) of 54 !~mol H 2 oxidized per m i n and mg protein. The enzyme is not only i n s e n s i t i v e to oxygen, its stability can even be increased by oxygen t r e a t m e n t or the a d d i t i o n of oxidizing agents like f e r r i c y a n i d e (,0.5 raM). The enzyme, w h i c h was homqgeneous by the c r i t e r i u m of polya c r y l a m i d e gel electrophoresis, is not strictly NAD-specific. In its reactive state it has the ability to react in the absence of I'AI)(H) w i t h a large n u m b e r of arti,ficial and physiological h y d r o g e n acceptors such as m e t h y l e n e blue, f e r r i c y a n i d e , dichlorophenol indophenol, phenazinemethosulfate, viologen dyes, FMtN, FAD, u b i q u i n o n e , cytochrome c a n d oxygen. In some cases the r e d u c t i o n rates were even up to 5-fold higher t h a n w i t h NAD. The enzyme catalyzed the evolution of h y d r o g e n from NADH, d i t h i o n i t e - r e d u c e d methylviologen and benzylviologen. It exhibited diaphorase and b~AD ( P ) H oxidase activity. In a c c o r d a n c e w i t h the low acceptor specificity and with diaphorase activity was the presence

chaelis constants for NAD and H 2 were '0.56 and 0:037 mM, respectively.

The membrane-bound hydrogenase

of A. eutrophus. The existence of two enzymes for the activation of h y d r o g e n in A. eatrophus, a soluble and a m e m b r a n e - b o u n d one, raised the question, w h e t h e r these two h y d r o g e n a s e s were two i n d e p e n d e n t isozymes or one enzyme protein e x h i b i t i n g different properties due to its localization in different sites in the cell. P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d almost c o o r d i n a t e f o r m a t i o n of both enzymes [17]. However, these observations contradicted the differences in their b i o c h e m i c a l behaviour. F o r f u r t h e r differentiation of both hydrogenase activities, the inembrane-bound h y d r o g e n a s e had to be purified and compared to the soluble enzyme. Since the results have not yet been published, the solubilization, purification, and some properties of the m e m b r a n e - b o u n d h y d r o g e n a s e will be described in some detail.

301

TABLE III.

S'olubilization of the membrane-botmd hydrogenase of A. e u t r o I ) h u s by di[ferent agents ( u s u a l l y i n

K P b u f f e r 50' mM, p H 7..0).

Agent

Incubation time (hours)

Incubation temperature / "C)

Per cent protein solnbilizeO

Per cent activity solubilized

EDTA 10 mM KC1 3 M KC1 3 M -~- EDTA 10 mM KCi 3 M -1(- MgC1._, 10 mM Glyeine 2 M KCI 3 M in K a c e t a t e buffer pH 4.0 KCI 3 M in Na citrate buffer p H 5.0 KC1 3 M in Tris-HCl buffer pH 9.0 n-pentanol j iso-pentanol ( 50 per cent t e r t pentanol ~ n-butanol ~, NaSCN ! NaCI04 i 0.5 M in Tris-HCl buffer i KNO:, pH 8.0 -~ 0.25 M sucrose LiBr Guanidine HCI ; Phospholipase D (3 U / g wet weight) in K acetate buffer p H 5.5 Brij 35 2 per cent Lubrol W X 2 per cent Digitonin 0.1 per cent Na deoxycholate 0.05 per cent Na deoxycholate 0.1 per cent Na deoxyeholate 0.2 per cent Triton X-100 0.2 per cent Triton X-100 0.5 per cent Triton X-100 1.0 per cent Triton X-100 2.0 per cent Triton X-100 0.5 p. 100 -1- Na deoxychol. 0.1 per cent -~- sucrose 10 per cent -J- EDTA 10 mM

2 2 2 2 22
2

4 4 4 4 4
20

3.0 12.0 12.3 99 13.0

1 6

2.0 0.4 2.2 0.3 1.1

--

2 2 0.5 0 5 o 5 0.5 0 5 o 5 0.5 0.5 o 5 0 5 2 2 2 2 2 2 2 2 2 2 0.5

20 2o 4 4 4 4 4 4 4 4 4 30 20 20 20 20 2o 20 20 20 20 20 20

4.8 13.0 14.5 15.8 19.0 36.5 22 6 18.4 20.0 14.8 16.3 8.2 11 6 12.5 12.0 9 0 13.5 22.0 20.0 30.0 36.0 300 23 8

4.0 5.0 3 5 3.7 35 ---3.5 1.3 4.8 14.1 0.2 1.3 3.0 4.8 11.0 9.0 9.7 11.8 9.2 4 2 22.0

When the investigation on the membrane-bound hydrogenase was started only a few data were known; these concerned the hydrogen oxidase system in intact membranes. T h e a c t i v i t y ol hydrogen oxidase was stable in frozen membrane p r e p a r a t i o n s u n d e r air, a n d it w a s i n h i b i t e d b y o x y g e n at c o n c e n t r a t i o n s o v e r 70 ~ [18, 19]. Q u i n o n e s a n d c y t o c h r o m e s of t h e b, c, a, a3, a n d o type were involved in the electron transfer s y s t e m f r o m h y d r o g e n to o x y g e n [203. In silu t h e hydrogenase reacted only with artificial electron acceptors like methylene blue, phenazinemethosulfate, and dichlorophenolindophenol ; it d i d n o t react with pyridine nucleotides. The physiological electron acceptors were unknown. Hydrogenase a c t i v i t y of m e m b r a n e p r e p a r a t i o n s w a s u s u a l l y

determined manometrically under hydrogen atmos p h e r e w i t h m e t h y l e n e b l u e as e l e c t r o n a c c e p t o r . F o r r a p i d d e t e r n f i n a t i o n of h y d r o g e n a s e a c t i vity in membranes and in solubilized preparations t h e f o l l o w i n g t e s t s y s t e m w a s u s e d . A s o l u t i o n of 200' ~M m e t h y l e n e b l u e i n '50, 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 , p H 7.0, w a s k e p t u n d e r h y d r o g e n gas a n d p i p e t t e d i n t o 3 nil c u v e t t e s . T h e s e w e r e flushed with hydrogen before and after adding t h e b u f f e r . G l u c o s e (0.2 !mml), g l u c o s e o x i d a s e (1 u n i t ) a n d c a t a l a s e (1 u n i t ) w e r e a d d e d as a n o x y gen trap. The reaction was started by adding the membrane suspension or solution containing the hydrogenase, and the absorption decrease was f o l l o w e d p h o t o m e t r i c a l l y at 570 n m w a v e - l e n g t h .

302 The results were i n a c c o r d a n c e with those derived from m a n o m e t r i c tests. The cytoplasmic m e m b r a n e s were p r e p a r e d by s o n i c a t i o n , t r e a t m e n t by lysozyme followed by osmotic shoctk, and in the F r e n c h pressure cell. In these p r e p a r a t i o n s the h y d r o g e n a s e activity was stable w h e n frozen u n d e r air. Due to their relatively high specific h y d r o g e n a s e activities, sonicared m e m b r a n e s were used for solubilization. The m e m b r a n e s were washed w i t h phosphate buffer and r e s u s p e n d e d in the same buffer to a protein c o n t e n t of about 3-4 m g / m l . After a d d i n g the solubilizing agent, the m e m b r a n e s u s p e n s i o n was s t i r r e d for ,0.5 to 2;2 h. The insoluble debris was r e m o v e d by c e n t r i f u g a t i o n at 1,0~)~00~)g. Hydrogenase activity a n d p r o t e i n c o n t e n t were d e t e r m i n e d in the s u p e r n a t a n t . The results of various attempts to solubilize the enzyme are s u m m a r i z e d i n table III. The following p r o c e d u r e for solubilizing hydrogenase i n a p r e p a r a t i v e scale has been finally used (after Yu a n d W o l i n , [21~ ; modified) : m e m b r a n e s p r e p a r e d b y s o n i c a t i o n w e r e washed w i t h potassium phosphate buffer and again w i t h this buffer s u p p l e m e n t e d by 0..1;5 M NaC1 and '0.26 M sucrose for r e m o v i n g p r o t e i n s loosely b o u n d to the memb r a n e surface. The m e m b r a n e s w e r e r e s u s p e n d e d i n phosphate buffer to a p r o t e i n c o n t e n t of 4 to 5 m g / m l . 10, per cent sucrose, 110 mM EDTA, 0.1 per cent deoxycholate, a n d ,0.5 per cent T r i t o n X-IO0 (final c o n c e n t r a t i o n s ) were added, a n d after stirr i n g for 30, rain at room temperature, m e m b r a n e particles not solubilized were r e m o v e d by centrifugation. A m a x i m a l yield of 22 p e r cent of the h y d r o g e n a s e activity of intact m e m b r a n e s was solubilized. As e x p l a i n e d later, this c o r r e s p o n d s to ahnost the total a m o u n t of h y d r o g e n a s e present in the m e m b r a n e s . The a p p a r e n t loss of activity t u r n e d out to be due to a change in the pH optim u m of the h y d r o g e n a s e after solubilization. The pH o p t i m u m change made it possible to follow the solubilization process by m e a s u r i n g the decrease of enzyme activity in the m e m b r a n e sus-

100
>_ < ~> 50

~5
E E
0.4

rr

%--0
0 0 I 10 I 20 ~ 30 ~ 40 L 50 I 150 Time (min) ~,

LIJ

O,3

<

0.2

FIG. 1. Kinetics of the solubilization of the membrane-bound hydrogenase of A. eutrophus H 16 by the combination of Triton X-IO0 and Na-deoxgcholate. Protein content : 2.4 mg/ml. The decrease of hydrogenase activity is the result of the shift of the pH optimum during solubilization.
-

30

6O

Time (days)

Decrease of activity of the solubilized membrane-bound hydrogenase during storage at --18C under air.
Fro. 2. --

Only low h y d r o g e n a s e activities w e r e released by the i n c u b a t i o n of the m e m b r a n e s in the presence of potassium chloride, m a g n e s i u m chloride or E~)TA, in acidic or aIkaline buffers. Alcohols as well as c h a o t r o p i c agents decreased the hydrogenase activity. Satisfactory results were o b t a i n e d w i t h T r i t o n X-I'&0, and sodium deoxycholate as well as w i t h phospholipase I). These results i n d i cated the h y d r o g e n a s e enzyme b e i n g tightly b o u n d to the m e m b r a n e . The enzyme p r o t e i n is a p p a r e n tly i n close contact to the lipid m e m b r a n e layer a n d has to be regarded as an integral, not a peripheral m e m b r a n e protein.

p e n s i o n after the a d d i t i o n of detergents (fig. 1). Already after 29 m i n more t h a n 9,0 per cent of the h y d r o g e n a s e was solubilized. The velocity of this process was not influenced b y the i n c u b a t i o n t e m p e r a t u r e b e t w e e n 4 a n d 3'0C. The yellow s u p e r n a t a n t tolerated storage u n d e r air at - - 1 8 C for two m o n t h s almost w i t h o u t loss of enzyme activity (~i'g. 2). D u r i n g storage at 4C u n d e r air for two days the loss was 10 per cent, w h e r e a s u n d e r r e d u c i n g c o n d i t i o n s (addition of

303 m e r c a p t o e t h a n o l or hydrogen) the loss was 70 per cent. The m e m b r a n e - b o u n d h y d r o g e n a s e t u r n e d out to be stable u n d e r oxidizing and unstable u n d e r r e d u c i n g c o n d i t i o n s ; i n this respect it resembles the soluble h y d r o g e n dehydrogenase. The yellow s u p e r n a t a n t , w h i c h c o n t a i n e d proteins, lipids, detergents, sucrose, a n d EDTA, was f r a c t i o n a t e d by a m m o n i u m sulfate p r e c i p i t a t i o n . I n the first step a m m o n i u m sulfate was added to give a 25 per cent saturated solution. After centrifugation the emulsion was separated into an oily, red coloured s u p e r n a t a n t layer and a clear aqueous solution c o n t a i n i n g the hydrogenase. The s u p e r n a t a n t layer could be fixed to the surface by a d d i n g light petroleum or h e x a n e before centrifuphate buffer. After dialysis and centrifugation, the s u p e r n a t a n t was c h r o m a t o g r a p h e d on CM~cellulose ( W h a t m a n CM 52) e q u i l i b r a t e d w i t h 25 mM potassium acetate buffer, pH '5.5, in a 1.5 30 cm column using a 2{)0 ml l i n e a r g r a d i e n t from 0 to 0.5 M KCl. F r a c t i o n s w i t h a volume of 2 ml were collected. Profiles of h y d r o g e n a s e and p r o t e i n c o n t e n t are presented in figure 3. A high a m o u n t of p r o t e i n w i t h low h y d r o g e n a s e activity passed the column. The h y d r o g e n a s e was eluted in two adjacent peaks. The fractions of both activity peaks were c o n c e n t r a t e d separately by ultrafiltration to a protein c o n t e n t of 1.0 m g / m l and dialyzed against potassium phosphate buffer, pH 7.0.

21
Og 0.4 / 02
ol : .../

..t-

'":
/ / / 7 f J J

05

/t~~ m ~ / /
n ""'~".'~-'o"'~'er
Number of

5 ,

c LU

/~
'e' n

0
0

~~,

t4t I

5o
fraction

-o-4-.6.~. . . . - ~I I -o--Q4 ,O-,I~,D100

Fro. 3. - - Elution profile of the membrane-bound hgdrogenase during chromatography on CM-cellalose. 9 mg of protein were applied to the column equilibrated with potassium acetate buffer 25. m M p H 5.5 and eluted by a linear gradient of 0 to 0.5 M KC1. Fractions of 2 ml were collected. Symbols : @--@ protein content, measured as extinction at 280 n m ; /X--A hydrogenase activity, measured as the rate of
m e t h y l e n e b l u e reduction.

gation ; the aqueous solution was easily sucked off w i t h a pipette. This p r o c e d u r e was repeated after i n c r e a s i n g the a m m o n i u m sulfate c o n c e n t r a tions to 3.0 per cent saturation. The h y d r o g e n a s e r e m a i n e d in the aqueous solution. Hydrogenase was p r e c i p i t a t e d by a m m o n i u m sulfate b e t w e e n 4,0 and 5'5 per cent saturation. The precipitate was dissolved in phosphate buffer a n d again p r e c i p i tated by a m m o n i u m sulfate b e t w e e n 4~-6() per cent saturation. The precipitate was dissolved in phos-

The total p u r i f i c a t i o n p r o c e d u r e is s u m m a r i z e d in table IV. F o r c o m p r e h e n s i o n of the low values of activity recovered after e n z y m e solubilization it has to be m e n t i o n e d that activity was consistently measured at pH 7.0, w h i c h is about the optim u m pH for the h y d r o g e n a s e b o u n d to the membrane. The solubilized enzyme has a pH o p t i m u m at pH 5:5. At pH 7.'0,, the solubilized enzyme has only 24 per cent of the activity m e a s u r e d at pH 5.5. For m a k i n g out a balance-sheet of the

304

TABLE IV.

Solubilization and purification of the membrane-bound hydrogenase

of A. eutrophus. Total protein (mg) 1 890 1 620 360 37 9 4.2 Total activity (units) 1 870 1 810 430 (1 770) 286 (1 180 240 (990) 165 (685) Specific activity (units/g protein) 990 120 200 950) 700 700) 700 000) 300 000) Purilication {--fold) 1.0 1.13 1.21 (5.0) 7.8 (32.0) 27.0 (111.0) 41.7 (172) Yield (per cent) 100 96 23 (95) 15.3 (63 0) 12.8 (55.0) 8.8 (35.8)

Step

Washed membranes Washing by NaCl/sucrose Solubilization 1. Precipitation with ammonium sulfate (40-55 per cent) 2. Precipitation with ammonium sulfate (45-60 per cent) CM-cellulose chromatography

1 1 (4 7 (31 26 (110 41 (170

whole p u r i f i c a t i o n procedure, the activities have to be m u l t i p l i e d by the factor 4.1 (values in brackets). C o m p a r i n g the values o b t a i n e d after the first w a s h i n g of the m e m b r a n e s w i t h those of the two purified enzyme fractions o b t a i n e d after CM-cellulose c h r o m a t o g r a p h y , one calculates a yield of 35.8 per cent, an e n r i c h m e n t factor of 172 a n d a specitff~c activity of 170 i~mol H 2 oxidized per rain a n d mg protein. Both fractions of purified enzyme separated by c a r b o x y m e t h y l cellulose c h r o m a t o g r a p h y were homogeneous by m e a n s of p o l y a c r y l a m i d e gel electrophoresis at different p o l y a c r y l a m i d e concentrations, only t h e i r R~-values differed slightly. However, these ~ e r e the only differences b e t w e e n the two fractions we found. A m i x t u r e of both t u r n e d out to be homogeneous in isoelectric focusing, in gel filtration on Sephadex G 210,0., and in sucrose g r a d i e n t centrifugation. Sodium dodecylsulfate gel electrophoresis revealed no differences in s u b u n i t composition. The greater > > or (( less polar )> molecule, however, was not stable, but g r a d u a l l y c h a n g e d to the other one d u r i n g storage for several days. I n fresh crude solubilized prepar a t i o n s almost onIy the (~ greater > > molecule was f o u n d as d e m o n s t r a t e d by activity s t a i n i n g the h y d r o g e n a s e in p o l y a c r y l a m i d e gel slabs by phenazinemethosulfate and n i t r o b l u e tetrazolium, chloride u n d e r h y d r o g e n (method a c c o r d i n g to 16). On the basis of these data it m a y be assumed that both types of hydrogenuse molecules differ from each other b y the a m o u n t of a d h e r e n t phosp h o l i p i d or detergent attached to u n p o l a r regions of the enzyme.

Concluding remarks.
The m a j o r i t y of species of the h y d r o g e n bacteria have a m e m b r a n e - b o u n d h y d r o g e n a s e only [8]. The possession of a single enzyme is shared w i t h Azotobacter vinelandii [22], Rhizobium bacteroids [1] a n d m a n y facultatively a n d strictly a n a e r o b i c b a c t e r i a as well as p h o t o t r o p h i c bacteria [23, 24]. The study of these enzymes lends itself as an a p p r o a c h to reveal r e l a t i o n s h i p s among the h y d r o g e n - o x i d i z i n g bacteria as ~vell as b e t w e e n this group, the p h o t o t r o p h i c a n d the nona u t o t r o p h i c h y d r o g e n a s e c o n t a i n i n g bacteria. The progress in the studies on the h y d r o g e n a s e s of Alcaligenes eutrophus so far revealed similarities and differences. Both the NAD-reducing, soluble and the m e m b r a n e - b o u n d h y d r o g e n a s e are r e m a r k a b l y stable to oxygen, they are inactivated w h e n stored u n d e r r e d u c i n g conditions. However, the enzymes are different in several basic p r o p e r t i e s ; these differences c o n c e r n the electron acceptor specificities, the isoelectric points, the molecular weights and the pH and t e m p e r a t u r e optima. I m m u n o d i f f u s i o n experiments done w i t h antisera p r e p a r e d against both purified e n z y m e s did not show any cross reactions b e t w e e n both enzyme proteins. The native enzymes are different proteins.

REFERENCES. 1. Dixon, R. O. D. (1972) Arch. Mikrobiol., 85, 193-201. 2. Schlegel, H. G. (1975) in < (Marine Ecology > >(Kinne, O. ed.), vol. II, part I, pp. 9-60, John Wiley ,~ Sons, London.

305
3. Mortenson, L. E. ~ Chen, J.-S. (1974) in (( Microbial Iron Metabolism > > (Neilands, J. B. ed.), pp. 231282, Academic Press, New York and London. 4. Eberhardt, U. (1966) Arch. Mikrobiol., 53, 288-3(12. 5. Bone, D. H., Bernstein, S. ~ Vishniac, W. (1963) Biochim. Biophys. Acta, 67, 581-588. 6. Bone, D. H. (19'60) Biochem. Biophys. Res. Commnn, 3, 211-214. 7. Aggag, M. ~ Schlegel, H. G. (1974) Arch. MicrobioL, 100, 25-39. 8. Schneider, K. & Schlegel, H. G. (1977) Arch. Microbiol., 112, 229-238. 9. Schlegel, H. G. & Eberhardt, U. (1972) Adv. Microb. Physiol., 7, 205-242. 10. Eberhardt, U. (1966) Arch. Mikrobiol., 54, 115-124. 11. Gottschalk, G. (1965) Biochem. Z., 341, 249-259. 12. Vogt, M. (1965) Arch. Mikrobiol., 50, 256-281. 13. Black,kolb, F. a Schlegel, H. G. (1968) Arch. Mikrobiol., 63, 177-196. 14. Bowien, B., Cook, A. M. & Schlegel, H. G. (19'74) Arch. Microbiol., 97, 273-281. 15. Vishniac, W. ~ Trudinger, Ph. A. (1962) Bacteriol. Rev., 26, 168-169. 16. Schneider, K. ~ Schlegel, H. G. (1976) Biochim. Biophys. Acta, 452, 66-80. 17. Schink, B. (1977) P h . D . Thesis, University of GiSttingen. 18. Probst, I. (1975) P h . D . Thesis, University of GSttingen. 19. Probst, I. ~ Sehlegel, H. G. (1976) Biochim. Biophys. Acta, 440, 412-428. 20. Pfitzner, J. (1972) Zentralbl. Bakteriol. Parasitenkd. I n f e k t i o n s k r . Hyg. Abt. 1, Orig. R e i h e A, 220, 396-401. 21. Yu, L. ~ Wolin, M. J. (1972) J. Bacteriol., 109, 59-68. 22. H y n d m a n , L. A., Bnrris, R. H. Wilson, P. W. (1953) J. Bacteriol., 65, 52~-531. 23. Gitlitz, P. H. ~ Krasna, A. I. (1975) Biochemistry, 14, 2561-2567. 24. Adams, M. W. W. ~ Hall, D. O. (1977) Biochem. Biophys. Res. Commun., 77, 730-737.