Reduction of Nicotinamide Adenine Dinucleotide by Pyruvate:Lipoate ...

Vol. 134, No. 3

JOURNAL OF BACTERIOLOGY, June 1978, p. 830-836

0021-9193/78/0134-0830$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Reduction of Nicotinamide Adenine Dinucleotide by Pyruvate:Lipoate Oxidoreductase in Anaerobic, Dark-Grown Rhodospirillum rubrum Mutant Ct T. E. GORRELLt AND R. L. UFFEN* Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Received for publication 23 January 1978

Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, darkgrown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation. It has been demonstrated that Rhodospirillum rubrum grows under strictly anaerobic conditions in the dark and ferments sodium pyruvate (29, 30, 31). During fermentative growth, the pyruvate-formate hydrogenlyase system operates to produce acetyl phosphate (Ac-P), which is then presumably used to form ATP by a substrate phosphorylation reaction (9). Formate is produced by the same pyruvate-degrading pathway and is oxidized stoichiometrically to H2 and CO2. In experiments with cell extracts and formate, no nicotinamide adenine dinucleotide (NAD)-dependent formate dehydrogenase activity could be demonstrated (T. E. Gorrell and R. L. Uffen, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K151, p. 172). As a result, it has been suggested that reduced NAD (NADH) was formed in the cells during anaerobic dark growth by an alternate pyruvate-degrading system, such as pyruvate:lipoate oxidoreductase and/or pyruvate:ferredoxin oxidoreductase. (Pyruvate: lipoate oxidoreductase [EC 1.2.4.1] is the recommended systematic name for pyruvate dehydrogenase.) Both of these pathways have been reported in photosynthetically grown R. rubrum (2, 5, 20). Results described in the present report indicate that cell extracts from anaerobic, darkgrown R. rubrum mutant C reduce NAD via pyruvate:lipoate oxidoreductase. Since the act Journal article no. 8426 of the Michigan Agricultural Experiment Station. : Present address: Department of Microbiology, University of Iowa, Iowa City, IA 52242.

tivity of pyruvate:lipoate oxidoreductase appeared to be too low to be important in ATP production, it is believed that both this pathway and pyruvate-formate hydrogenlyase (9) operated in concert in R. rubrum mutant C to supply NADH and ATP. No pyruvate:ferredoxin oxidoreductase activity was observed with anaerobic, dark-grown C cells. In addition to pyruvate:lipoate oxidoreductase, after mutant C was grown photosynthetically, C, like wild-type cells (8, 14, 15), also exhibited succinate-linked, light (or ATP)-dependent NAD reduction. The role of both light-dependent and light-independent (i.e., pyruvate:lipoate oxidoreductase) NAD reduction in photosynthesizing C cells is discussed.

MATERIALS AND METHODS Organism, growth medium, and growth conditions. R. rubrum mutant C was grown fermentatively in the dark (9, 29), or photosynthetically in the light (T. E. Gorrell and R. L. Uffen, Photochem. Photobiol., in press), in complex medium containing sodium pyruvate (31). Cell extracts. Strictly anaerobic methods described earlier (9) were used to prepare protein extracts from R. rubrum, except that the buffered solution used to suspend cells before breakage consisted of: potassium phosphate (pH 7.5), 100 mM; dithiothreitol, 10 mM; MgCl2, 10 mM; and deoxyribonuclease, 1 jig/ml. After breakage with a French pressure cell, cellular debris and unbroken cells were removed by centrifugation (41,000 x g at 40C for 30 min). The supernatant solution was then desalted at 40C under 02-free conditions with a gel filtration chromatographic column containing coarse-grade Sephadex G25. Before use, the liquid chromatography column (2.5

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NAD REDUCTION IN FERMENTING R. RUBRUM

by 25 cm) was equilibrated with 3 volumes of the buffered solution described above, except that the potassium phosphate concentration was changed to 10 mM and deoxyribonuclease was not added. Crude, desalted cell extract was used immediately or stored at liquid N2 temperature. For use in certain studies, the crude, desalted cell extract was additionally treated. Small pieces of membrane material were removed by ultracentrifugation (144,000 x g at 4°C for 1 h), and the resulting supernatant solution was then sometimes treated with diethylaminoethyl-cellulose to extract ferredoxins (26). Determination of protein. Samples of cell extract were hydrolyzed in 1 N NaOH (9), and the amount of protein was estimated by the method of Lowry et al. (19). Bovine serum albumin was used as a standard. Enzyme assays. Measurement of NAD reduction activity was performed under O2-free argon gas. The procedures we used for preparing O2-free argon and anaerobic solutions are described elsewhere (9, 31). Reactions were incubated at room temperature (ca. 23°C) in specially constructed anaerobic cuvettes (10mm path length) that were sealed with rubber serum bottle stoppers. The change in absorbance at 340 nm was measured by using a Gilford 2400-S recording spectrophotometer, and the concentration of NADH in reactions was determined from the extinction coefficient (E:mo = 6.22 mM-' cm-l). Pyruvate-dependent NAD reduction was determined in a reaction mixture consisting of: potassium phosphate buffer (pH 7.5), 50 mM; sodium pyruvate, 6.0 mM; dithiothreitol, 2.5 mM; coenzyme A (CoA), 0.25 mM; thiamine pyrophosphate (TPP), 0.2 mM; and protein, 0.3 mg. A minimum concentration of 0.1 JM MgCl2 was also introduced with the addition of the cell extract. Reactions were started by adding sodium pyruvate, and the final volume was 1.0 ml. In one series of experiments, NAD was replaced with either NAD phosphate (NADP, 2.0 mM), methyl viologen (2.0 mM), or flavine adenine dinucleotide (0.06 mM). The ability of sodium formate (30 mM) to support the reduction of NAD and these cofactors in place of pyruvate was also tested. Light-dependent NAD reduction activity (8, 14) in cell extract from both anaerobic, dark- and light-grown mutant C was measured in reactions containing sodium succinate (6.0 mM) by the method of Keister and Yike (14). Lipoamide dehydrogenase (EC 1.6.4.3) activity in cell extracts was determined by measuring the oxidation of NADH by a modification of the procedure described by Reed and Willms (24). The reaction mixture consisted of: potassium phosphate buffer (pH 6.5), 150 mM; DL-6,8-thioctic acid amide (DL-lipoamide), 3.0 mM; and NADH, 0.1 mM. DL-Lipoamide (100 mM) was prepared by dissolving the compound in 95% ethanol. Reactions were started by adding crude, desalted cell extract (0.3 mg of protein; final volume, 1.0 ml). Formation of Ac-P during degradation of pyruvate in reactions with or without sodium hypophosphite (9 mM) was measured under conditions described elsewhere (9). The concentration of Ac-P was determined by a modification (18) of the method of Lipmann and Tuttle (17).

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Chemical compounds. Commercial, reagentgrade chemicals were used. NAD (grade III; fl-form; monosodium salt), NADP (98% purity), and other cofactor compounds were obtained from Sigma Chemical Co., St. Louis, Mo. Ferredoxin from Clostridium pasteurianum and beef heart lactate dehydrogenase (EC 1.1.1.27) were purchased from Worthington Biochemicals Corp., Freehold, N.J.

RESULTS Reduction of NAD. Crude, desalted cell extract from anaerobic, dark-grown R. rubrum mutant C catalyzed pyruvate-linked NAD reduction (Fig. 1). The reaction was specific for NAD (NADP was not used), and it occurred at a rate that was linear with time. The specific activity of the reduction reaction was 80 nmol of NADH formed per mg of protein per min. At the end of the incubation period, the formation of NADH was confirmed by adding lactate dehydrogenase, which abolished the absorb120_

o

NAD

~60-

~40z

Time (min) FIG. 1. Pyruvate-linked reduction ofpyridine adenine dinucleotides [NAD(P)] by cell extract from anaerobic, dark-grown R. rubrum mutant C. Reaction mixtures contained: sodium pyruvate, 6 mM; NAD (0) or NADP (0), 2 mM; and crude, desalted cell extract (0.3 mg of protein). Reaction mixtures also contained potassium phosphate, dithiothreitol, CoA, TPP, and MgCl2 as indicated in the text. Reduction activity was measured spectrophotometrically at 340 nm. At the end of the 5-min incubation period (vertical arrow), lactic acid dehydrogenase (1.5 IU) was added.

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GORRELL AND UFFEN

at 340 nm (Fig. 1). The amount of NADH produced was directly related to the amount of cell extract in the reaction mixture at concentrations ranging between 0.1 and 0.6 mg of protein. No reduction of NAD occurred when experiments were performed with heat-treated (100°C for 1 min) cell extracts. In a separate series of experiments, it was observed that the pyruvate-linked NAD reduction system was dependent upon catalytic amounts of CoA and TPP. (A magnesium requirement for enzyme activity was not determined.) Maximum NAD reduction occurred with 0.1 mM CoA (at a concentration of 0.04 mM, the activity decreased about 50%). Higher concentrations of CoA (up to 1 mM) did not further influence the reaction. Conversely, with excess CoA (0.25 mM), maximum NAD reduction occurred with 16 uM TPP, and enzyme activity decreased 50% by lowering the amount of TPP to 7 ,uM. (As noted above, 200 AM TPP was used in routine NAD reduction measurements. This high concentration of TPP did not adversely influence the reaction in R. rubrum and was chosen based on assay conditions reported in related studies on TPP-dependent, pyruvate-degrading reactions in other biological systems [11, 32, 33].) Under conditions with excess CoA and TPP, pyruvate-linked NAD reduction was most rapid at pH values ranging between 7.5 and 7.8 (Fig. 2). Maximum enzyme activity at pH 7.5 did not require potassium phosphate, and other buffering compounds could be used at a concentration of 50 mM (in reactions without phosphate addition) with no appreciable change in the amount of NADH formed. These buffering compounds included N-2-hydroxyethylpiperazineN'-propanesulfonate, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonate, and NY -tris(hydroxymethyl)methyl 3 amino-propanesulfonate. When reactions were performed at pH values of 6.5 and 8.3, NAD reduction activity decreased by about one-half (Fig. 2). The addition of reducing compounds stimulated pyruvate-linked NAD reduction (Table 1). (The restoration of reduction activity in sodium arsenite-treated reactions, shown also in Table 1, is discussed elsewhere.) As observed, dithiol compounds and two of the monothiol reducing agents (i.e., L-cysteine and glutathione) increased the amount of NAD formed by three- to fivefold. Other monothiol compounds examined were less effective. It was determined that none of these reducing agents supported NADH formation in the absence of pyruvate, but the reason that the compounds stimulated enzyme activity was not examined further. To determine whether the pyruvate-depend-

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ance reading

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FIG. 2. Influence of different pH values on NAD reduction with pyruvate. Potassium phosphate buffer (50 mM) (0) and N'-tris(hydroxymethyl)methyl-3amino-propanesulfonate (50 mM) (0) were used in the reactions. Other components in the reaction mixture and experimental conditions are described in the legend to Fig. 1.

ent, NAD-reducing system was soluble or associated with particulate material, the crude, desalted extract was fractionated by ultracentrifugation (144,000 x g at 40C for 1 h). By using this method, it was determined that 70% of the original activity was located in the sedirnentable fraction, whereas the remaining 30% was found in the supernatant solution. Since the reaction requirements for NAD reduction in both the sedimentable and soluble fractions, and the original crude, desalted cell extract were virtually identical, only results of experiments with the crude, desalted cell extract are presented. Pyruvate:lipoate oxidoreductase. Evidence that pyruvate:lipoate oxidoreductase occurred in anaerobic, dark-grown mutant C and acted to reduce NAD was obtained by inhibiting pyruvate-linked NAD reduction activity with sodium arsenite and by demonstrating lipoamide dehydrogenase activity in cell extracts (10, 36). In reactions with pyruvate, the addition of 0.5 mM sodium arsenite inhibited NAD reduction activity by about 90% (Fig. 3). (Sodium arsenate did not influence the reaction.) In these experiments with R. rubrum, as in related studies (10, 36) on pyruvate:lipoate oxidoreductase in other systems, the addition of dithiol compounds, such as dithiothreitol (Fig. 3, Table 1) or dithioerythritol (Table 1), relieved arsenite inhibition and allowed NAD reduction to resume almost immediately (Fig. 3). Use of monothiol compounds was ineffective in restoring activity (Table 1) or in protecting the enzyme system against arsenite inhibition when one of these reducing agents,


VOL. 134, 1978

TABLE 1. Effect of reducing agents onpyruvatelinked NAD reduction by R. rubrum cell extracts in the presence or absence of sodium arsenite Amt of NAD reduced (nmol/mg of protein per

Reducing agent added'

833

terial after fractionation of the crude, desalted extract by ultracentifugation. Like pyruvatelinked NAD reduction, lipoamide dehydrogenase was strongly inhibited with sodium arsenite (Fig. 4), but attempts to restore enzyme activity

min) -Sodium ar- +Sodium arseniteh senite

0 18 None Dithiols: 77 78 Dithiothreitol 88 98 Dithioerythritol Monothiols: 3.0 71 L-Cysteine 7.0 61 Glutathione (reduced) 0 32 2-Mercaptoethanol 0 32 3-Mercapto-1,2-propanediol 0 15 Sodium thioglycolate a The concentrations of monothiol and dithiol compounds were 5.0 and 2.5 mM, respectively. Other components in the reaction mixture and reaction conditions are described in the legend to Fig. 1. bAdded to the reaction (final concentration, 0.5 mM) to inhibit pyruvate:lipoate oxidoreductase activity. Sodium arsenite reacts chemically to form cyclic thioarsenite complexes with vicinal sulfhydryl groups in compounds such as lipoamide, dithiothreitol, and dithioerythritol (36).

such as L-cysteine, was present in the reaction mixture at a concentration of up to 10 mM before the inhibitor was added. Additional evidence that pyruvate:lipoate oxidoreductase was present in anaerobic darkgrown R. rubrum was obtained by demonstrating lipoamide dehydrogenase activity in crude, desalted cell extracts (23, 24). (Lipoamide dehydrogenase was assayed in the oxidative direction with NADH. The reductive formation of NADH with NAD and dihydrolipoamide, which is reported to occur at a faster rate [37], was not measured.) As observed in Fig. 4, NADH was oxidized at a linear rate during the first 2 min (the specific activity was about 46 nmol of NAD formed per mg of protein per min) and then the rate slowly decreased as the incubation period was continued. After 5 min, about 70 nmol of NADH was oxidized. Enzyme activity was greatest at pH 6.5 (the activity decreased by about 50% when pH values of 5.5 and 7.5 were used), and the amount of NAD produced was directly proportional to the amount of protein used at concentrations ranging between 0.1 and 0.6 mg. Heat-treated cell extract didjot catalyze NAD production. As expected from the distribution of pyruvatelinked NAD reduction activity in C cells, approximately 60% of the lipoamide dehydrogenase activity appeared in the sedimentable ma-

S -

0

+ 0

S 0

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z

Time (min) FIG. 3. Inhibition of pyruvate-linked NAD reduction with sodium arsenite (0.5 mM). Reaction mixtures were the same as described in the legend to Fig. 1, except that L-cysteine (5.0 mM) was used as a reducing agent. (0) NAD reduction in the control reaction. (0) NAD reduction in a second reaction was inhibited by addition of sodium arsenite (1) and then restored with dithiothreitol (t; 2.5 mM).

._ 0

0

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Time( min)

FIG. 4. Lipoamide dehydrogenase activity in cell extract from anaerobic, dark-grown R. rubrum mutant C in reactions with (0) or without (0) sodium arsenite (0.5 mM). Enzyme activity was measured in the oxidative direction at 340 nm. Reaction mixtures (at pH 6.5) contained: DL-lipoamide (3 mM), NADH (0.1 mM), potassium phosphate buffer (150 mM), and crude, desalted cell extract (0.3 mg of protein).

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GORRELL AND UFFEN

with dithiol compounds were not successful. In these assays with lipoamide and NADH, when dithiol-reducing compounds were added to reaction mixtures containing sodium arsenite, a precipitate of undetermined chemical composition developed. In addition to pyruvate:lipoate oxidoreductase, previously reported in photosynthesizing R. rubrum by Luderitz and Klemme (20), it has also been suggested that the light-grown cells formed pyruvate:ferredoxin oxidoreductase (2, 5). Although data presented in this report do not exclude this possibility, no evidence for a pyruvate:ferredoxin oxidoreductase pathway was obtained in these experiments with cell extract from anaerobic, dark-grown R. rubrum mutant C. When reactions were performed with sodium pyruvate and sodium hypophosphite (added to block the pyruvate formate hydrogenlyase system [10]), cell extract did not catalyze the reduction of either methyl viologen or flavine adenine dinucleotide. Reduction of these compounds would be expected, however, if pyruvate:ferredoxin oxidoreductase were functional (32). Secondly, ferredoxins did not appear to be involved in NAD reduction. No change in the specific activity of the reaction occurred when a membrane-free preparation (obtained by fractionation of crude, desalted cell extract by ultracentrifugation) was treated with diethylaminoethyl-cellulose to remove these nonheme iron proteins (data not shown). Furthermore, NAD reduction was not stimulated by adding either crude ferredoxin fractions from R. rubrum mutant C, or purified C. pasteurianum ferredoxin (50 jig of protein), to reactions with crude, desalted, or diethylaminoethyl-cellulose-treated cell extracts. From these observations, it was concluded that pyruvate:lipoate oxidoreductase represented a second important pyruvate-degrading system in fermenting R. rubrum, and it appeared to function alone to supply NADH necessary for anaerobic dark cell growth. Pyruvate-formate hydrogenlyase (the suggested ATP energyyielding pathway [9]) did not have this activity. When sodium hypophosphite (a potent inhibitor of pyruvate formate-lyase) was added to the reaction mixture, pyruvate-linked NADH formation was not affected (data not shown). NADH formation also did not occur when sodium formate was used in place of sodium pyruvate. NAD reduction in photosynthetically grown mutant C. During anaerobic light growth in medium with sodium succinate, parent strain R. rubrum Si reduced NAD by a light (or ATP)-dependent system (8, 14). Since this lightdependent reaction did not develop in mutant C

J. BACTERIOL.

during dark fermentative growth, C cells were grown photosynthetically in medium with sodium succinate, and cell extracts were examined for both succinate-linked (light-dependent) and pyruvate-linked (light-independent) NAD reduction activities. Results indicated that both reactions occurred. With pyruvate, cell extracts from light-grown mutant C produced about 52 nmol of NADH per mg of protein per min in either anaerobic, light or dark conditions. The cell extract from photosynthetically grown C also reacted with succinate, but only in the light, and formed about 2.9 nmol of NADH per mg of protein per min (or 370 nmol of NADH per mg of bacteriochlorophyll a). DISCUSSION R. rubrum grew fermentatively in the dark and formed two pyruvate-degrading pathways. Pyruvate:lipoate oxidoreductase metabolized small amounts of pyruvate and reduced NADH. Since this pathway did not operate in vitro to produce measurable quantities of Ac-P, the system did not seem to be important in ATP formation. (Formation of 1 ,umol of Ac-P could be detected by the assay system used.) Anaerobic, dark-grown R. rubrum also exhibited pyruvateformate hydrogenlyase (9), which degraded pyruvate to form Ac-P and did not participate in NAD(P) reduction. Similar pyruvate-degrading activity appeared to develop when aerobic, dark-grown R. rubrum was placed under 02-free dark conditions (25). In this study, the adaptive changes which occurred in the bacteria, however, were not shown to support dark fermentative cell growth. The presence of at least two pyruvate-degrading reactions in dark-grown R. rubrum was suggested earlier (9, 29). Since fermenting cells formed ferredoxin(s) (unpublished data), catalyzed a bicarbonate-pyruvate-exchange reaction (9), and synthesized poly-fi-hydroxybutyric acid (30) (which was suggested by Bosshard-Heer and Bachofen [2] to be produced in R. rubrum by a ferredoxin-dependent system), it was suggested in earlier reports (9, 29) that these cells contained pyruvate:ferredoxin oxidoreductase. Contrary to this notion, however, present experiments led to the identification of pyruvate:lipoate oxidoreductase and not the ferredoxin-dependent pathway. The role of pyruvate:lipoate oxidoreductase in anaerobic, dark-grown R. rubrum in synthesizing poly-fl-hydrMtybutyrate and exchanging small amounts of bicarbonate into pyruvate is consistent with the system in other microorganisms. It is clear from this comparison that pyruvate:lipoate oxidoreductase, instead of pyruvate:ferredoxin oxidoreductase, functions to

VOL. 134, 1978


form poly-/3-hydroxybutyrate in R. rubrum during dark growth as in other aerobic respiring cells (6). In addition, bicarbonate-pyruvate-exchange activity (9) in cell extracts from anaerobic, dark-grown R. rubrum was lower than could be expected when pyruvate:ferredoxin oxidoreductase occurred. For example, in C. pasteurianum and Chlorobium limicola f. thiosulfatophilum, both of which degraded pyruvate by pyruvate:ferredoxin oxidoreductase, the ratios of bicarbonate-pyruvate-exchange activity to pyruvate-dependent NAD reduction were 121 and 95, respectively. (These values were calculated from data reported by Bothe and Nolteernsting [3].) In R. rubrum mutant C, the ratio between these pyruvate-linked activities was only 0.35 and compared more favorably with the analogous activity ratios of 0.06 and 0.01 associated with pyruvate:lipoate oxidoreductase found in Azotobacter vinelandii and Escherichia coli (3). Although our attempts to detect pyruvate:ferredoxin oxidoreductase in anaerobic, dark-grown cells were not successful, it is still possible that pyruvate:ferredoxin oxidoreductase can develop in R. rubrum. The observation of Yoch and Arnon (38) that reduced ferredoxin supported the nitrogenase system strengthens this notion. On the other hand, the possibility that the pyruvate:lipoate oxidoreductase system catalyzes NAD(P)-mediated ferredoxin reduction (13, 28) should also be examined. R. rubrum is not the only microorganism which forms more than one pathway to degrade pyruvate (7, 16, 21, 27, 34). To our knowledge, however, it is the first example in which the pathways have appeared to operate in concert to satisfy the ATP and NADH requirements for cell growth. The question of how cells regulate the flow of pyruvate through the different metabolic systems has not been answered, but two factors have been suggested which could aid in this regulation. One Qf these might be the different activities of the reaction pathways. For example, the rate of ATP production (estimated from the amount of Ac-P produced from pyruvate per mg of protein per min [9]) was 3.34fold greater than the rate of NADH formation. Secondly, cellular compartmentalization may also have a regulatory influence, since the Ac-P generating system was only detected in soluble fractions from cell extract (9) and most of the NADreduction activity appeared with particulate membrane material after fractionation by ultracentrifugation. However, this postulate must be studied further. The appearance of pyruvate:lipoate oxidoreductase in membrane fractions may also have resulted solely from the high 'molecular weight of the enzyme complex (4, 23). Clearly, the regulation of the two pyru-

835

vate-degrading systems in R. rubrum during anaerobic, dark growth warrants further investigation. Pyruvate:lipoate oxidoreductase activity not only occurs in fermentatively grown R. rubrum, but has been identified by Luderitz and Klemme (20) in photosynthetically grown cells. (Data in this laboratory with light-grown mutant C supported their findings, which were obtained by using various R. rubrum strains.) Results of our studies also showed that pyruvate:lipoate oxidoreductase activity was not affected by light. In addition, when mutant C was grown photosynthetically, the cells developed succinatelinked ATP-dependent NAD reduction activity. The role of the light (or ATP)-dependent reaction has been generally accepted as the most important, if not the exclusive (22), source for NADH in photosynthesizing, anoxygenic bacteria (8, 12). This, however, may not be entirely correct, since data in this laboratory indicated that, in mutant C, light-independent NADH formation via pyruvate:lipoate oxidoreductase occurred at about an 18-fold-faster rate than the light-dependent reaction. Consequently, from these observations and with the results of other studies on ATP-dependent H2-nitrogenase in light-grown R, rubrum mutant C (Gorrell and Uffen, in presws), we suggest that ATP-dependent reactions such as these contribute to the regulation of the adenylate nucleotide pool size or, specifically, maintain a proper energy charge value (1) in photosynthesizing cells. Additionally, if this notion is correct, ATP might play a novel role in regulating the process of photosynthetic differentiation. When this differentiation occurs in dark-grown R. rubrum mutant C upon exposure to light (29, 30), ATP-dependent NAD reduction activity, H2-nitrogenase (Gorrell and Uffen, in press; 8, 35), and, presumably, nicotinanide nucleotide transhydrogenase (8, 12, 15) develop in additiQn to the fermentative (ATPindependent) reactions. The possibility that changes in the adenylate nucleotide pool size and/or the energy charge value in the cells serve to regulate these events is being studied. ACKNOWLEDGMENTS We are grateful to R. J. Patterson for the use of his spectrophotometer. T.E.G. thanks M. P. Dashkevicz for helpful discussions. This research was sponsored by National Science Foundation grant BMS 7202066. T.E.G. was supported by Public Health Service training grant GM 01911-09 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Atkinson, D. E. 1968. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7:4030-4034. 2. Bosshard-Heer, E., and R. Bachofen. 1969. Synthese

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