H2 Metabolism in Photosynthetic Organisms: I. Dark H2 Evolution and ...

Plant Physiol. (1975) 56, 72-77

112

I.

Metabolism in Photosynthetic Organisms

DARK H2 EVOLUTION AND UPTAKE BY ALGAE AND MOSSES'

Received for publication January 31, 1975 and in revised form March 27, 1975

AMI BEN-AMOTZ, DAVID L. ERBES,2 MARY ANN RIEDERER-HENDERSON,3 DWIGHT G. PEAVEY, AND MARTIN GIBBS Department of Biology, Brandeis University, Waltham, Massachusetts 02154 and Marine Biological Laboratories, Woods Hole, Massachusetts 02543 In most studies (2, 13), hydrogenase has been detected by measuring H2 uptake, H2 evolution, and related reactions in intact algae. Recently, there have been reports of H2 metabolism with hydrogenase isolated from anaerobically adapted green and blue-green algae. Success has been limited because of the extreme sensitivity of the enzyme to 02. Abeles (1) first successfully isolated a cell-free preparation of hydrogenase from Chlamydomonas eugametos. The preparation was particulate, catalyzed the reduction of methylene blue and pyridine nucleotides, and also evolved H2 from reduced methyl viologen. Ward (25, 26) obtained a hydrogenase preparation from Chlamydomonas similar to that of Abeles and showed reactivation of an oxidized cell-free hydrogenase with H2. Lee and Stiller (15) reported hydrogenase activity in a cell-free preparation of Chlorella pyrenoidosa. Their enzyme preparation catalyzed the reduction of phenazine methosulfate, methylene blue, and ferricyanide but not of p-quinone. Fujita and Myers (6) prepared a cell-free preparation of hydrogenase from Anabaena cylindrica and concluded a poor coupling between hydrogenase and ferredoxin. The purpose of this paper was multifold. We surveyed the distribution of hydrogenase in marine and fresh water red algae by measuring H2 uptake and evolution under darkness. Similar studies were undertaken with five species of moss. The properties of adaptation were studied in these organisms with emphasis on the red alga, Chondrus crispus. Finally, characteristics of cell-free preparations of hydrogenase from two algae, Chlamydomonas reinhardii and Chondrus crispus, and the moss, Leptobryum pyriforme, are described.

ABSTRACT Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to 112 evolution. H2 evolution was sensitive to disalicylidenepropanediamine. An immediate H2 uptake was observed in these algae. Immediate dark H2 uptake but no evolution was observed in the mosses. A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p-benzoquinone, NAD, NADP, but not spinach ferredoxin. H2 evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H2 evolution and for H2 uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve 112. Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H2 by a different pathway than Chlamydomonas.

The investigation of H2 metabolism in algae was initiated by Gaffron (8) in 1940 when he discovered that certain unicellular green algae utilized hydrogen in the dark after a period of anaerobic incubation. Subsequently, Gaffron and Rubin (9) found that algae which were capable of consuming H2 were also able to evolve H2 in the dark. The capacity to include H2 in the metabolism of these algae appeared after an obligatory variable period of anaerobic adaptation and was lost in the presence of small quantities of 02. In Chl-containing organisms, except for the unconfirmed report of Boichenko (4), studies on H2 metabolism have been limited to algae.

MATERIALS AND METHODS

Algae. The filamentous marine algae Codium fragilis, Ulva lactuca, Entermorpha linza, Cladophora gracilis, Fucius vesiculosus, Chondrus crispus, Corallina officinalis, Grinnellia sp., Ceramium rubrum, Porphyra umbilicalis, Polysiphonia urecolata, Rhodymenia palmata, Gigartina stellata, Champia paravula, Chordaria flagelliformis, and Dictyosiphon foeniculaceus were collected on the beaches in the Cape Cod area of Massachusetts. Porphyridium aerugineuim, a gift from Dr. J. Ramus, was grown on fresh water medium as previously described (21). Chlorella autotrophica, Chlamydomonas sp. (marine), Pyramimonas sp., Platymonas sp., Cyclotella cryptica, Oscillatoria sp., Synechococcus sp., and Porphyridum sp. (marine) were obtained from Dr. R. R. L. Guillard and were grown on "f/ 2" medium (16) at 20 C in the light. Suspensions of the unicellular algae concentrated to give 5 to 10 mg dry weight/ml of growth medium were used for assays. The filamentous marine algae were washed a few times in seawater,

'This research was generously supported by Atomic Energy Commission Grant AT-i1-1 3231 and National Science Foundation Grants GB29126X2 and BMS 73-00978. 2Present address: Department of Biochemistry, University of Wisconsin, Madison, Wis. 53706. 'Present address: Department of Biology, Rollins College, Winter Park, Fla. 32789. 72

Plant Physiol. Vol. 56, 1975

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HYDROGEN METABOLISM

cut into pieces, and resuspended in seawater filtered through a Millipore filter. H, metabolism in the filamentous marine algae was not sensitive to UV radiation for 1 min nor to washing in 3% Clorox for 30 sec, and not to gentamycin (100 ,ug/3 ml). Cultures of bacteria isolated from these algae showed no H. uptake or evolution. It is clear, therefore, that the H, metabolism was not due to bacterial contamination. Chlamydomonas reinhardii was obtained from the Indiana Culture Collection. Algae were grown in a medium which contained per liter: 14.3 mg KXHPO4; 7.2 mg KH,,PO; 2.4 g tris; 1 ml glacial acetic acid; 0.4 mg NH,Cl; 50 mg CaCl1, 2 H,O; 100 mg MgSO0 7 H,O; 1 ml of a trace element solution; made up to 1 liter with distilled H,O. Trace elements contained the following: 50 g Na,EDTA; 22 g ZnSO4' 7 H,O; 11.4 g H,BO,; 5 g MnCI,A, H,0; 4.9 g FeSO4 7 H,O; 1.6 g CaCl, 6 H,0; 1.57 g CuSO4 5 H_O; 1.1 g (NH,)Mo,O 4- 4 H,O in a total volume of 1 liter. The final pH after autoclaving was 7.3. Cultures were grown with slow shaking at 22 C under white fluorescent lamps with an intensity of about 200 ft-c. Cells were harvested by centrifugation and were resuspended in growth medium or in 25 mm potassium phosphate, pH 7.3, to give a suspension of cells containing about 0.4 mg Chl/ml. Mosses. Bryum argenteum, Physcomitrium turbinatum (wild type), Polytrichum formosum (Hedw), Homomallium adnatum

(Hedw) Brothers, and Leptobryum pyriforme (L) Schimper were gifts from Dr. H. Dyer. They were grown on Knudson's medium which contained the following in 1 liter: 1 g Ca(NO,), 4 H,O; 0.5 g (NHJ,S04; 0.25 g K,HPO,; 0.25 g MgSO, 7 H,O; 1 ml ferric nitrate (200 mg/ 100 ml); 1 ml Nitsch minor element solution (19); 1 ml HCI; 17.5 g agar; and 2.5 g sucrose. Mosses were grown at 20 C under white fluorescent lights with an intensity of about 200 ft-c. Whole plants containing 30 to 50 mg dry weight were suspended in 3 ml of 20 mm potassium phosphate, pH 7.3, and used for the

Table I. H2 Evolution and Uptake by Algae and an Extract of Chondrus crispus The reaction mixture for marine algae and Choondrus extract contained 10 ,M DCMU and about 0.3 g dry weight in 3 ml of filtered seawater. The reaction mixture for fresh water algae contained 10 Mm DCMU, 5 mg dry weight Porphyridium, or 15 mg dry weight Chiamydomonas in 3 ml of 25 mm potassium phosphate, pH 7.3. Choondrus extract was prepared by grinding in liquid N2 anaerobically adapted algae. Aliquots of the ground mixture were placed in Warburg vessels which were sitting on Dry Ice. The vessels were attached to their manometers, placed in the water bath at 20 C, and quickly flushed with gas to keep the content anaerobic as it thawed. H2 evolution with Choondrus extract was assayed with reduced methyl viologen as described in Table IV. The gas phases for H2 evolution and H2 uptake were No and H2, respectively. A 10:1 (v/v) mixture of 25%7 pyrogallol and 40% KOH was present in the center well. Preparation of Algae

H2Uptake AdPtation H, Evolution Adaptation

dry mulg wt hr

hr

tnl/gwt hrdry

0.1 0.1 0.25 0.22 0.15 0.35

0.2

0.2 0 0.12 0.20 0.05 0.3

-

Choondrus Chonzdrus extract Corallina Ceramium

Porphyridium Chlamydomonas

0.2 0.2 0.2 0.2

hr

20 20 16 3.0 0. 2

Hydrogenase Assays in Extracts and Whole Cells. H, uptake and evolution were measured manometrically at 20 C in the dark room. Conical Warburg flasks with center wells and one or two side arms were used. Alkaline pyrogallol was put in the center well. A mixture of methylene blue and 5% palladium on aluminum oxide in the side arm was used for identification of H, released. Intact algae and mosses were transferred into flasks containing 3 ml of reaction mixture and were flushed with prepurified N, or with H, purified by passing through a Deoxo cartridge (Englehard) for 1 hr before gas uptake or evolution was monitored. Cell-free preparations were injected through rubber stoppers during gas flushing. For H, uptake with methylene blue, the conditions were the same, but with 0.2 ml of 20 mm methylene blue tipped into the reaction mixture from the side arm. H2 evolution with reduced methyl viologen was measured in the presence of excess sodium dithionite in a Warburg vessel under an atmosphere of N,. Spinach ferredoxin was prepared according to Boger et al. (3) with modification of Nelson and Neumann (18).

experiments. Adaptation. Whole cells were incubated in darkness in Warburg flasks under H. or N2 until H, consumption or evolution was initiated. To prepare cell-free extracts containing hydrogenase, whole cells were first adapted under similar conditions. Chondrus, Moss and Chlamydomonas Extracts. Threemonth-old cultures of Leptobryum pyriforme were put into a 500 ml-suction flask with 50 ml of 20 mm potassium phosphate, pH 7.3, and 0.5 ml of 1 mM DCMU. Chondrus crispus washed a few times in seawater was placed in a similar flask with 50 ml of seawater passed through a Millipore filter and 0.5 ml of 1 mm DCMU. The flask was filled with H2, covered with aluminum foil, and left at room temperature. Six hr later, the moss was covered with liquid N2. Similarly, liquid N, was poured onto the algae when a control batch of the same cells evolved gas in the Warburg respirometer. The frozen suspensions were then ground with a mortar and pestle. RESULTS Aliquots of the ground mixture were placed in Warburg vessels, attached to their manometers, placed in the water bath, and H2 Metabolism in Whole Algae and Mosses. Among the quickly flushed with gas to keep the content anaerobic as it algae listed under "Materials and Methods," dark H2 evoluthawed. tion and uptake were demonstrated consistently only in the Chlamydomonas suspensions containing about 0.4 mg of marine red algae, Chondrus crispus, Corallina officinalis, and Chl in 25 mm potassium phosphate, pH 7.3, were flushed with Ceramium rubrum, in the fresh water red alga, Porphyridium H2 or N. and permitted to adapt for 1 hr in the dark at room aerugineum, and in the green alga, Chlamydomonas reinhardii temperature in the presence of 10 [tM DCMU. After adaptation (Table I). On a dry weight basis, uptake and evolution were the cells were sonicated with a Branson sonic power sonifier roughly similar. A period of anaerobic incubation was deterfor 30 sec at 6 amp under H2 or N2. The sonicated suspensions mined for hydrogenase prior to H, evolution and uptake. were transferred by syringe into anaerobic plastic tubes and Adaptation time for H, evolution varied with the strain of the centrifuged for 60 min at 30,000g to remove whole and alga and ranged from minutes in Chlamydomonas to a few broken cells. Unless otherwise indicated 2.8 ml of the 30,000g hours in Chondrus and 1 to 2 days in Corallina. In contrast, H, supernatant fluid were used for hydrogenase assays. uptake in all of these algae was apparently initiated within

74


BEN-AMOTZ ET AL.

tion by 0.4 mm DSPD. H2 evolution by Chondrus was not sensitive to 10 ,M FCCP (data not shown). All of the mosses took up H2 (Table III). The average specific activity varied from 1.3 ml H2/g dry weight hr for P. formosum to 8.3 for P. turbinatum. H2 uptake was immediate, following the customary 15-min flushing of the Warburg flasks with H2. Attempts to obtain H2 evolution by adapted mosses were unsuccesful.

4(

0

XADAPTATION TIME

LJ 25

25-

160 -6

a o -o I

. 15

H2 METABOLISM IN CELL-FREE PREPARATIONS FROM CHLA MYDOMONAS

7"

-80

15

48 >48 >48 34 36 27 20 15 12

0 0 0 18 75 117 80 165 239

22

the 15 min of H2 flushing of the Warburg flasks. H2 evolution and consumption continued at a linear rate for 72 hr. Figure 1 illustrates the effects of temperature on the adaptation period and, in turn, on H2 evolution by Chondrus. This alga became adapted over 1 day at 20 C and within a few hours at 30 C. H2 was evolved with a rate of 0.03 ml/g dry weightvhr at 20 C and 0.24 at 30 C. The Q1o for H, evolution was 6. The adaptation period and the rates also varied with the season, in that Chondrus collected in June was characterized by an anaerobic incubation at 20 C of roughly 1 to 2 days, while the period was shortened to less than 1 day for algae removed from the same ocean location in the latter part of August (Table II). In order to determine whether ferredoxin or ATP were involved in the process of H2 evolution by Chondrus, the effect of DSPD,4 an inhibitor of ferredoxin and an uncoupler (FCCP), were studied. Figure 2 illustrates 50% inhibition of H2 evolu-

Abbreviations: DSPD: disalicylidenepropanediamine; FCCP: carboxycyanide p-trifluoromethoxyphenylhydrazone. I

2

I:

vJo

1.8 1.2 0.6 DSPD CONCENTRATION (mM)

FIG. 2. Effect of DSPD on H2 evolution by Chondrus crispus. The reaction mixture contained 3 ml of filtered seawater, 10 AM DCMU, and algae containing about 0.3 g dry weight. DSPD was tipped into the algal suspension after an adaptation period of 33 hr. An activity of 100% is 0.22 ml H2/g dry weight hr.

Table III. Hydrogeni Uptake by Mosses anid ant Extr-act of Leptobryuim pyriforme The reaction mixtures contained 3 ml of 20 mm potassium phosphate, pH 7.3, 10,Mm DCMU, and whole plant materials containing about 50 mg dry weight or extracts of L. pyriforme containing about 30 mg dry weight. The extract was prepared by grinding L. pyriforme adapted for 6 hr under anaerobic conditions in liquid N2. Aliquots of the ground mixture were placed in Warburg vessels which were sitting on Dry Ice. The vessels were attached to their manometers, placed in the water bath at 20 C, and quickly flushed with H2 to keep the content anaerobic as it thawed. Alkaline pyrogallol was present in the center well.

AMoss

H2 Uptake

inl/g dry wI hr

Homomallium adlnatum Polytrichum formosuni Bryum argeniteum Physcomitrium turbiniatum

Leptobryum pyriforme L. pyriforme extract L. pyriforme extract + 2 mM methylene blue

4.1 1.3 6.7 8.3 6.4 7.4 14.1

Plant Physiol. Vol.

Table IV. Assays for H2 Evolution anzd H2 Uptake by Cell-free Preparation of Chlamydomonas An algal suspension containing about 0.4 mg Chl/ml in 25 mM potassium phosphate, pH 7.3, was flushed with H2 or N2 and adapted for 1 hr at room temperature in the dark in the presence of 10 ,M DCMU. After adaptation the cells were sonicated for 30 sec under H2 or N2. Sonicated suspensions were then transferred by syringe into anaerobic plastic tubes and were centrifuged for 60 min at 30,000g. Of the 30,000g supernatant, 2.8 ml, containing about 20 mg of protein, were injected through a serum stopper into the Warburg vessel. H2 evolution with reduced methyl viologen was measured under N2 in the following manner. Fifty mg of sodium dithionite and a few ml of 100 mm tris-HCl, pH 8, were flushed with N2 in separate serum bottles. One ml of the anaerobic buffer was then injected into the bottle containing dithionite. Two hundred ,Al of the buffered dithionite and 7.5 ul of 200 mm methyl viologen were injected separately through a serum stopper into the side arm of the Warburg vessel. The reaction was initiated by tipping the reduced methyl viologen into the Chlamydomontas extract. H2 uptake with methylene blue was measured under H2 in the following manner. Twenty mm methylene blue were flushed with N2 and 0.2 ml was injected into the side arm of the Warburg vessel. The oxidized methylene blue was then tipped into the vessel and H2 uptake was monitored. H2 evolution and uptake were assayed at 20 C. H2 Evolved

Reaction Mixture

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HYDROGEN METABOLISM

56, 1975

H2 Uptake

Reaction Mixture

pIdmg

pI/mg

protein * hr

Complete No dithionite No methyl viologen No dithionite, no methyll viologen

5.3 0

Complete No methylene blue

1.8 0

No enzyme

No enzyme

0

prolein hr 3.8

0.5 0

Table V. Relative Rates of Hydrogen Uptake with Differenlt Acceptors by a Cell-free Preparationz of Chlamydomonzas The reaction mixture (3 ml) contained 25 mm potassium phosphate, pH 7.3, the indicated acceptors, and cell-free preparation of Chlamydomonzas containing 22 mg of protein. Spinach ferredoxin saturating for NADP photoreduction with spinach chloroplasts was added. Other conditions were as described under Table IV. Acceptor

ferredoxin were unsuccesful. Control experiments indicated NADP photoreduction at a rate of 40 ptmoles/mg Chl-hr by sonicated Chlamydomonas supplemented with saturated amount of spinach ferredoxin. Concentrations of ferricyanide, up to 5 mM, did not stimulate H2 uptake. H2 was not evolved with other reductants including NADH, NADPH, dithiothreitol, ascorbate, and ascorbate with dichlorophenolindophenol. The question of the role of ferredoxin in the process of H2 evolution was approached by the use of purified spinach ferredoxin at saturated amounts for NADP photoreduction and by the inhibitor sulfo-DSPD. Spinach ferredoxin did not stimulate hydrogenase activity (data not shown). Sulfo-DSPD, up to 1.5 mm, had no effect on H2 evolution from reduced methyl viologen and only slightly inhibited H2 evolution from dithionite. Effect of Centrifugation on Cell-free Preparation. Sonicated suspensions of Chlamydomonas were subjected to centrifugal fields up to 100,000g for various periods of time to determine whether hydrogenase was a particulate enzyme. Centrifugation at 500g for 10 min removed whole cells. Centrifugation at 30,000g for 1 hr removed chloroplasts and broken cells. Finally, centrifugation at 100,000g for 1 hr yielded a clear yellowish-greenish solution which contained the same amount of hydrogenase as in the original cell-free preparation. pH Effect. The dependence of hydrogenase activity on pH is shown in Figure 3. H2 evolution with reduced methyl viologen proceeded best at pH 7.2. Dithionite was not stable at low pH values, and measurements below pH 6.5 were not reliable. H2 uptake with methylene blue had a broad pH optimum between 7.4 and 9.5. Stability. Enzyme preparations were stored in liquid N2 for at least 2 months without loss of activity. Lyophilized preparations of the enzyme retained maximal activity for a few weeks. Small quantities of 02 irreversibly inactivated the hydrogenase activity and attempts to reactivate by NADH, dithiothreitol, sodium ascorbate, or cysteine under H2 or N2 were unsuccessful. H2 Uptake in Extracts from Chondrusand Leptobryum. Ex:2 0

z

0

j;000-

XH2 UPTAKE

H2 Uptake

pt/ yng protein-hr Methylene blue, 1.3 mM p-Benzoquinone, 1 mM NADP, 1.6 mM

5.1 5.1 1.3

NAD,1.6mM NADP, 1.6 mM + ferredoxin NAD, 1.6 mm + ferredoxin Ferricyanide, 3.3 mm None

1.3 1.0 0.77 0.77

1.0

assayed routinely by H2 evolution with dithionite and methyl viologen. Acceptors and Donors. Table V shows H2 consumption rates with different acceptors. The highest activity was obtained with methylene blue and p-benzoquinone. Uptake of H2 with pyridine nucleotide varied widely from no uptake to a 2-fold stimulation. Attempts to stimulate this reaction with spinach

o I0

0

50 0~~~~~~

>-

i0 4

6

8

tO0

FIG. 3. Effect of pH on hydrogenase dependent Hi uptake and H2 evolution by cell-free preparation of Chlamydomonas. The following buffers were used at 25 mm over the following pH ranges: Tricine-maleate, pH 5 to 8; Tricine-NaOH, pH 6 to 8.5; and Tricine-glycine, pH 7 to 9. The reaction mixture for H2 evolution and uptake were as described under Table IV. An activity of 100% refers to 4.2 Al H- evolved/mg protein*hr or to 4.7 p1 H2 uptake/mg protein *hr.

76

BEN-AMOTZ ET AL.

tracts prepared from anaerobically adapted Chondrus consumed H, (Table I). The appearance of H2 uptake was immediate and proceeded at a rate of 0.1 ml H./g dry weight hr. H2 evolution was not detected upon addition of dithionite and methyl viologen. In contrast to intact algae, which consumed and released H2, the extracts were capable of consuming gas. An extract prepared from anaerobically adapted L. pyriforme consumed H2 at a rate of 7.4 ml H,/g dry weight hr (Table III). Uptake was stimulated 1.3- and 1.9-fold by methylene blue. H2 evolution was not detected in the presence of dithionite and methyl viologen.

DISCUSSION Since the early paper of Gaffron and Rubin (9), research on the dark reactions of chlorophyllous organisms with H2 and in the absence of externally supplied electron acceptors has been scanty. This fact is perhaps due to the absence of added CO2 or dyes, such as methylene blue and methyl viologen, H2 uptake is limited to reduction of cellular material (9), and evolution in the dark is meager compared to photoinduced gas release. Nonetheless, it seemed important to investigate the properties of endogenous dark H, metabolism in a number of photosynthetic cells to determine if the pathways were similar and also for comparison with the heterotrophic, anaerobic, and aerobic hydrogen-metabolizing bacteria. Similar to published reports (13), hydrogen metabolism was not found in all the intact algae tested and not until the organism was put under anaerobic conditions. The reaction time varied with the reaction and the organism (Table I). In Chlamydomonas, uptake and evolution had an identical adaptation period. On the other hand, H2 uptake by the red algae was apparently immediate when contrasted to gas evolution (Table I). Finally, hydrogen metabolism was found in all the mosses tested (Table II). In these bryophytes, only uptake of H2 was detected. Immediate H2 metabolism in these differing organisms is in agreement with the studies of Stiller and Lee (22) that activation of hydrogenase was the result of a modification of a constitutive enzyme. Our results with Chondrus (Fig. 1) demonstrate that temperature and possibly other environmental factors (Table II) influence the adaptation period and also support evidence that hydrogenase activation is an enzyme-catalyzed process. With respect to the difference in the onset of H2 uptake and evolution in the red algae, we could envisage two unidirectional functioning hydrogenases or, more likely, the slow buildup of reduced carbon compounds which were the sources of H2 since the final rates of both processes were essentially identical. If the latter is correct, then the failure to detect H2 metabolism in photosynthetic cells may not necessarily reside with the presence or absence of a hydrogenase but with an inherent initial slow rate of fermentation of cellular substances (11). Three types of bacterial hydrogenases have been recognized (20). The most widely distributed is a particulate enzyme characterized by considerable variation with respect to artificial and physiological electron acceptors and donors but does not react directly with pyridine nucleotides. The second type, also particulate, is found in N2-fixing microorganisms and catalyzes an ATP-dependent evolution of H2 from reduced viologen or ferredoxin. The third type described in Hydrogenomonas is soluble and catalyzes the direct reduction of NAD and has been designated as hydrogenase dehydrogenase. The algal enzymes described by Abeles (1), Ward (24, 25), Fujita et al. (6, 7), and in this study would suggest comparison with the


particulate bacterial enzyme with diverse specificity in accepting and donating electrons. Similar to the hydrogenase preparation of Abeles (1), the C. reinhardii enzyme catalyzed H, uptake with methylene blue (Table IV), pyridine nucleotides (Table V), and evolution with dithionite (Table IV). The pH profiles (Fig. 3) for the two Chlamnydomonas preparations were essentially similar for H2 uptake brought about the reduction of methylene blue and of a quinone, already reported in intact cells (12, 14) but, unlike whole cells of green alga (14), did not reduce ferricyanide. The enzyme was extremely sensitive to 02 and contrary to the report of Ward (26), attempts to reactivate hydrogenase under an atmosphere of H, up to 24 hr were without success. If our enzyme was associated with a particle, its size is much smaller than that of other algal hydrogenases. Unlike the bacterial enzyme (17, 24) and the Anabaena preparation of Fujita and Myers (6), soluble spinach ferredoxin stimulated neither H2 uptake nor evolution by the C. reinhardii enzyme with the appropriate substrates (Table V). Furthermore, sulfo-DSPD, an inhibitor of ferredoxin (23), did not impair H, evolution with dithionite or H, uptake with the pyridine nucleotides. Low concentrations of DSPD did inhibit H, evolution by intact Chondrus (Fig. 2). These reactions involving H, are the result of multicomponent systems and not until studies with purified proteins are completed can definitive conclusions concerning the role of ferredoxin and other factors be presented. Finally, it is noteworthy to compare H, metabolism in the mosses and green and red algae. In contrast to Chlamydomonas extracts, reduced methyl viologen did not support H, evolution in Chondrus and moss preparations (Table I and Table IV). In addition, H, evolution of Chondrus was not sensitive to an uncoupler, and the opposite has been reported for Chlamydoinonas (10) and Scenedesmus (9). Most important, preliminary results of this laboratory show that in contrast to the green algae (2) and the report of Frenkel and Rieger (5) with Porphyridiunz cruentum the metabolism of H, in the mosses and the red algae, Chondrus crispus and Porphyridium aerugineum, was not light stimulated and was not coupled to CO2 reduction in the light. From the information now available, we propose that there may be variations in the H2metabolizing pathways in Chl-containing cells. Clearly, an understanding of the relationship among the pathways will not be elucidated until information concerning the cellular location and structural studies on the purified hydrogenases becomes known. LITERATURE CITED

1. ABELES, F. B. 1964. Cell-free hydrogenase from Chlamydomanas. Plant Physiol. 39: 169-176. 2. BISHOP, N. I. 1966. Partial reactions of photosynthesis and photoreduction. Annu. Rev. Plant Physiol. 17: 185-208. 3. BOGER, P., C. C. BLACK, AND A. SAN PIETRO. 1966. Photosynthetic reactions with pyridine nucleotide analogs. II. 3-Pyridinealdehyde-diphosphoridine nucleotide and 3-pyridinealdehydedeamino-diphosphopyridine nucleotide. Arch. Biochem. Biophys. 115: 35-43. 4. BOICHENKO, E. A. 1946. Evolution of hydrogen by isolated chloroplasts. Compt. Rend. Acad. Sci. USSR 52: 521-24. 5. FRENTEEL, A. W. AND C. RIEGER. 1951. Photoreduction in algae. Nature 167: 1030. 6. FUJITA, V. AND J. MYERS. 1965. Hydrogenase and NADP-reduction reactions by cell-free preparations of Anabaena cylindrica. Arch. Biochem. Biophys. 111: 619-625. 7. FUJITA, V., H. OHANIA, AND A. HATTORI. 1964. Hydrogenase activity of cell-free preparations obtained from the blue-green alga Anabaena cylindrica. Plant Cell Physiol. 5: 305-314. 8. GAFFRON, H. 1940. Carbon dioxide reduction with molecular hydrogen in green algae. Am. J. Bot. 27: 273-83. 9. GAFFRON, H. AND J. RUBIN. 1942. Fermentative and photochemical production of hydrogen in algae. J. Gen. Physiol. 26: 219-240.


HYDROGEN METABOLISM

10. HEALEY, F. P. 1970. The mechanism of hydrogen evolution by Chlamydomonas moewusii. Plant Physiol. 45: 153-159. 11. GIBBS, M. 1962. Fermentation. In: R. A. Lewin, ed., Physiology and Biochemistry of Algae. Academic Press, New York. pp. 91-97. 12. KESSLER, E. 1957. Stoffwechselphysiologische Untersuchungen an hydrogenase enthaltenden Grunalgen II. Dunkel-Reduction von Nitrate und Nitrit mit molekularem Wasserstoff. Arch. Mikrobiol. 27: 166-181. 13. KESSLER, E. 1974. Hydrogenase, photoreduction and anaerobic growth. In: W. D. P. Stewart, ed., Algal Physiology and Biochemistry. Blackwell, Oxford. pp. 456473. 14. KESSLER, E. AND H. MAiFARTH. 1960. Vorkommen und Leistungsfahigkeit von Hydrogenase bei einigen Griinalgen. Arch. Mikrobiol. 37: 215-225. 15. LEE, J. K. H. AND M. STnLER. 1967. Hydrogenase activity in cell-free preparation of Chlorella. Biochim. Biophys. Acta 132: 503-505. 16. McLcmN, J. 1973. Growth media-marine. In: J. R. Stein, ed., Handbook of Phycological Methods, Culture Methods, and Growth Measurements. Cambridge University Press, New York. pp. 25-51. 17. MORTENSON, L. E., R. C. VAmzNTu, AND J. E. CAsNuN. 1963. Ferredoxin in the phosphoroclastic reaction of pyruvic acid and its relation to nitrogen fixation in Clostidium pa8teurianum. J. Biol. Chem. 238: 794-800. 18. NELSON, N. AND J. NEumANN. 1969. Interaction between ferredoxin and ferre-

19.

20. 21.

22. 23. 24.

25. 26.

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