symposium on metabolism of inorganic compounds' iv ... · symposium on metabolism of inorganic...

SYMPOSIUM ON METABOLISM OF INORGANIC COMPOUNDS'

IV. HYDROGEN PHOTOSYNTHESIS AND ALTERNATIVE METABOLIC PATHWAYS IN

PHOTOSYNTHETIC BACTERIA2

JOHN G. ORMEROD3 AND HOWARD GEST

The Henry Shaw School of Botany and the Adolphus Busch III Laboratory of Molecular Biology,Washington University, St. Louis, Missouri

I. Introduction...................................................................II. Photoreduction of Carbon Dioxide with Molecular Hydrogen...............................

III. Photometabolism of Organic Compounds.................................................A. Photoproduction of Molecular Hydrogen...............................................B. The Stoichiometry of Hydrogen Production............................................C. An Anaerobic Light-dependent Citric Acid Cycle.......................................D. C02 Fixation during Photometabolism of Citric Acid Cycle Intermediates...............E. Alternative Pathways of Carbon Metabolism .........................................

IV. Functions of the Accessory Electron Donor in Bacterial Photosynthesis; Alternative Path-ways of Electron Transfer................................................................

V. Concluding Remarks.....................................................................VI. Addendum................................................................................VII. Literature Cited............... ...............................

I. INTRODUCTION

"Now although it is true that many funda-mental chemical reactions are common to animals,plants and microbes, it is easy to overestimate theunity of nature when bacteria are being consideredin relation to animals and plants. It is probablytrue to say that whilst most chemical changes oc-

curring in the latter are either duplicated or closelyparalleled in some microorganism or other, yet theconverse is far from being true. The number andvariety of chemical reactions already known to becatalised by bacteria far exceed those attrib-utable to the animal and the plant together.Moreover amongst heterotrophs it is as anaerobesthat bacteria specially excel; this implies that thisgroup has elaborated special enzymes for oxidisingtheir substrate molecules without having recourse

to oxygen; in other words it is in the use of hydro-

1 This symposium was organized by Dr. HowardGest under the auspices of the Division of GeneralBacteriology and presented at the Annual Meetingof the American Society for Microbiology inChicago, Ill., on April 26, 1961. Dr. Jacques C.Senez served as honorary convener.

2 Research of the authors supported by grantsfrom the National Institute of Allergy and Infec-tious Diseases, U.S. Public Health Service (grantno. E-2640), and the National Science Foundation.

3Present address: Norsk Institutt for Vann-forskning, Blindern, Norway.

51

gen acceptors that bacteria are specially developedas compared with animals and plants."

Marjory Stephenson, 1947 (73).

In a sense, the hydrogen-evolving enzyme

present in many anaerobic microorganisms can

be thought of as a special "hydrogen acceptor,"with the unique capacity to combine hydrogenatoms, or electrons and hydrogen ions, to produceH2. The ability to catalyze hydrogen formation,and to utilize H2 as an electron donor is, in fact,surprisingly widespread among microorganismsof very different metabolic types (24), and hasthus far not been observed in higher plants or

animals.There is little doubt that the electron-transfer

pathways involved in production and oxidationof molecular hydrogen will prove to have many

similarities with more familiar electron transportsequences in which an external acceptor is re-

duced or a more complex metabolite oxidized.The possession of enzymes which can evolve or

oxidize H2, however, endows many microor-ganisms with significantly increased potentialitiesfor growth or for obtaining energy under unusualconditions. Thus, these variations on a basicmetabolic theme have important biological im-plications, including a probable usefulness as

"indicators" in tracing the development of bio-chemical evolution.

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ORMEROD AND GEST

Photosynthetic microorganisms, as a physio-logical group, show a remarkable degree ofmetabolic versatility of the kind suggested byMarjory Stephenson. In the present paper, evi-dence for and the significance of alternativemechanisms of light-activated electron transferand carbon metabolism of photosynthetic bac-teria are considered, in particular connectionwith photosyntheses in which molecular hydrogenis utilized as an electron donor or produced as aresult of photometabolism.

II. PHOTOREDUCTION OF CARBON DIOXIDEWITH 1IOLECULAR HYDROGEN

For the purpose of initiating the present dis-cussion, it is convenient to consider bacterialphotosynthesis in an over-all sense as the anaero-bic light-dependent conversion of carbon dioxideor organic compounds to cell materials. Theutilization of CO2 as the sole carbon source forgrowth by such photosyntheses depends on thepresence of a suitable oxidizable compound, whichwe designate as the "accessory electron or hydro-gen donor" (22). In 1935, Roelofsen (61) reportedthat molecular hydrogen could serve as theaccessory donor for photoautotrophic growth ofpurple sulfur bacteria; other studies (10, 18, 40,49, 50, 80) disclosed that resting suspensions ofrepresentative strains of all types of photosyn-thetic bacteria also can catalyze the light-de-pendent utilization of CO2 in the presence of H2.Van Niel (76) has reported that certain non-

sulfur purple bacteria can also grow photoauto-trophically with H2 as the accessory donor; amedium containing yeast extract was employedand negligible growth was observed in the absenceof H2. We considered it desirable that this ob-servation be confirmed, particularly since recentgrowth experiments (47) with Desulfovibrio de-sulfuricans suggested the possibility of someuncertainty in the interpretation of Van Niel'sundocumented report. In a putatively auto-trophic environment containing hydrogen, bi-carbonate, and limiting amounts of yeast extract,the growth of Desulfovibrio was observed byAMechalas and Rittenberg (47) to be dependentupon the presence of H2, but it was also foundthat most of the cell carbon originated from com-ponents of the yeast extract. We have observed(Fig. 1) unambiguous growth of Rhodospirillumrubrum on H2 + CO2 in a completely syntheticmedium, but further work is required for defini-tion of the optimal requirements for reproducible

300 _CIMALATE +CO,,OHe H

2 20000

100

a_0

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0 100 200 300HOURS

FIG. 1. Photoautotrophic growth of Rhodospiriltum rubrum (Si) on C02 and H2 in a completelysynthetic medium. The basal medium and conditionsof incubation were similar to those described byOrmerod et al. (52) except that malate was omittedand NaHCO3 added at a concentration of 0.6mg per ml. The nitrogen source was 0.05%0 (NH4)2SO4. Gas phases: 5% CO2 + 95% H2 or 5% C02 +95%7 He, as indicated. The inoculum (1%) consistedof washed cells derived from a malate + (NH4)2SO4medium. For comparative purposes, a growth curvewith 0.6% DL-malate is also shown. Optical densitywas measured with a Klett-Summerson photometerusing filter no. 66.

growth under these circumstances. This observa-tion provides added evidence that separation ofthe photosynthetic bacteria into distinct meta-bolic categories is arbitrary and artificial (77).The pathway of carbon in photoreduction of

CO2 with hydrogen has not been investigated ingreat detail but several studies (31, 75) suggestthat the major pathways are probably verysimilar to those believed to be operating in othertypes of autotrophic CO2 reduction. Assumingthis to be the case, it might be argued that thespecial features of this "hydrogen photosyn-thesis" will be concerned with unique mechanismsby which reducing power and adenosine triphos-phate (ATP) are generated.Hydrogenase preparations from various bac-

teria will reduce pyridine nucleotides and flavins(36, 53, 55, 81) in the presence of H2, and Arnon(1) has recently reported that extracts of sul-fur purple bacteria, genus Chromatium, willsimilarly catalyze diphosphopyridine nucleotide(DPN) reduction in the dark, but only whensupplemented with the one-electron dye benzyl

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METABOLIC PATHWAYS IN PHOTOSYNTHETIC BACTERIA

viologen. Proper evaluation of this preliminaryreport is difficult; data for controls in whichhydrogen is replaced by an inert gas are not givenand only 10% of the DPN added was reduced inan unspecified time interval. In view of theseconsiderations, it is premature to exclude thepossibility that the ultimate oxidant for H2 inphotoreduction of C02 is an entity generated bythe photochemical apparatus.

It is known that, in certain nonphotosyntheticorganisms, ATP can be generated by the oxida-tion of H2 with inorganic oxidants such as oxygen

(62) and sulfate (54). Thus the possibility existsthat phosphorylation may also result from theanaerobic oxidation of H2 with a photooxidant.The properties of the hydrogenases of photo-

synthetic bacteria have been studied to a limitedextent (23, 34, 56, 57) and are still not well under-stood. A start has been made on questions con-

cerned with localization (3), and the influence ofgrowth conditions on synthesis of the enzyme

(52), but much remains to be investigated, par-

ticularly in connection with the basic problem ofthe fate of electrons derived from oxidation ofH2. It might be added that there is a possibility,suggested by preliminary experiments in our

laboratory, that several hydrogenases with dif-ferent functions may be produced by Rhodo-spirillum species.

III. PHOTOMETABOLISM OF ORGANIC COMPOUNDS

Purple bacteria, such as members of the generaRhodospirillum and Chromatium, grow readily andabundantly when illuminated under anaerobicconditions in synthetic media containing malateand an ammonium salt as the sole carbon andnitrogen sources, respectively. In other words,addition of C02 or inorganic accessory donors isunnecessary when a suitable organic compoundis provided. All of the evidence accumulated todate4 indicates, furthermore, that purple bacteriagrow much more rapidly with organic carbonsources such as C4 dicarboxylic acids than withCO2 (see also Fig. 1).

In an early investigation, Muller (48) madequantitative carbon balance studies on the utiliza-tion of a number of organic compounds by grow-

ing cultures of purple sulfur bacteria. These ex-

4 The extensive literature on metabolism oforganic compounds and C02 by photosyntheticbacteria is discussed in a number of earlier reviews(8, 22, 29, 76); see also references (52) and (72).

TABLE 1. Over-all carbon dioxide metabolism ingrowing cultures of purple sulfur bacteria*

Moles C02 produced orSubstrate utilized, per mole

substrate consumedt

Acetate...................... +0.17Lactate ...................... +0.29Succinate .................... +0.70Malate ...................... +1.22Butyrate ..................... -0.74

* After Muller (48).t Mean values of at least six determinations. A

positive value denotes CO2 production; negativevalue, CO2 utilization.

periments disclosed a net production of CO2 dur-ing growth with acetate, lactate, succinate, andmalate (Table 1).

It can be seen that the C02 yield varied fromapproximately 0.17 mole per mole of acetate usedto 1.2 in the case of malate. With butyrate as theorganic substrate, addition of C02 was requiredfor growth and was in fact consumed to the ex-tent of about 0.7 mole per mole of butyrateutilized. Similar results with "resting" suspen-sions of nonsulfur purple bacteria have been re-ported by other investigators (9, 17, 76). Mullerinterpreted his observations to indicate extensiveassimilation of carbon intermediates, generatedby oxidation of the organic substrate, into cellmaterials. Direct reductive assimilation of ace-tate carbon was also indicated by experimentsof Gaffron (18), who demonstrated light-de-pendent utilization of H2 with acetate by non-sulfur purple bacteria.

Subsequent studies, between 1935 and 1949,on the metabolism of organic compounds by pur-ple bacteria were largely concerned with sub-strates (e.g., fatty acids, isopropanol) with whicha net utilization of C02 was characteristicallyobserved. The results of these investigations wereinterpreted by Van Niel (76) to support theconcept that the photosynthesis of purple bac-teria in the presence of organic substrates isfundamentally a photochemical C02 reductionwith organic hydrogen donors; direct assimilationof carbon from the organic substrate was accord-ingly viewed as a secondary phenomenon. Com-parative biochemical reasoning also led Van Niel(78) to postulate that photosynthetic C02 re-duction could be explained on the basis ofreversal of the conventional citric acid cycle.

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The discovery (28) of light-dependent produc-tion of molecular hydrogen by purple bacteria aswell as a number of other observations indicatedthe necessity for a re-evaluation of Van Niel'shypothesis. A survey of the information availablein 1951 concerning the photometabolism of or-ganic compounds led one of the present authors(22) to the following conclusions:"The accessory (organic) 'hydrogen donor' re-

quired in bacterial photosynthesis ordinarily doesnot undergo a simple one-step oxidation; it gener-ally supplies carbon intermediates, other than CO2,which are directly used by the cell for synthetic pur-poses. This is apparently true for practically allclasses of organic 'hydrogen donors."'

Moreover, it was suggested (30, 33) that underconditions in which organic compounds supplyintermediates for direct assimilation, photosyn-thetic CO2 fixation is not obligatory and mayin fact be bypassed or suppressed. Additionalstrong support for these views was provided bylater investigations, notably by Siegel's excellentstudies (65-68, 70) on the light-dependent con-version of acetone to cell materials by Rhodo-pseudomonas gelatinosa.4Thus from the standpoint of carbon metabo-

lism, the anaerobic utilization of organic com-pounds by purple bacteria is essentially hetero-trophic in character, i.e., organic substrates areconverted to intermediates which are assimilated(through syntheses requiring ATP) in the form ofcarbohydrate, lipid, protein, etc. In addition tothis highly developed carbon heterotrophy, it isalso apparent that photosynthetic bacteria pos-sess (i) specialized systems for generation of ATPthrough light-activated electron transfer (11, 12,21) and (ii) a "photosynthetic carbon cycle"presumably similar to that occurring in greenplants. As has been noted previously (29), theinterplay between the CO2 reduction cycle andthe heterotrophic utilization of organic com-pounds might be expected to result in complexpatterns of electron transport as well as of carbontransformations during photosynthetic metabo-lism.

It can reasonably be assumed that the specificpathways used by photosynthetic bacteria forsynthesis of cell materials will depend on a num-ber of factors, which include the particular en-zymatic capacities of the cell and the status ofthe endogenous reserves. Equally important con-siderations are: whether the cell is growing or

resting, and the nature of the utilizable substratesprovided. It seems obvious that results obtainedwith a given substrate and a particular organismgrown under a special set of circumstances cannotbe readily extrapolated to generalizations defining"the role" of organic compounds in bacterialphotosynthesis. Failure, on the part of some in-vestigators, to recognize the significance of thefactors noted above has without doubt beenresponsible to a large degree for the tortuoushistory of discussions on the photometabolism oforganic compounds by purple bacteria.

A. Photoproduction of Molecular Hydrogen

The occurrence of major alternative pathwaysin the disposition of electrons generated duringphotometabolism in purple bacteria is clearly in-dicated by the phenomenon of light-dependentproduction of molecular hydrogen. Purple bac-teria produce large quantities of H2 during photo-synthetic growth in media containing malate asthe carbon source, and molecular nitrogen oramino acids such as glutamate or aspartate serv-ing as the nitrogen source (26-28). Resting cellsderived from media of this kind rapidly producehydrogen when illuminated in the presence ofvarious organic compounds ((28, 30, 35); see Fig.2A). Depending on the exact composition of thegrowth medium and "age" of the cells at time ofharvest, photoproduction of hydrogen is some-times observed as an endogenous process. Itappears that any oxidizable substrate may evokelight-dependent H2 production under appropriateconditions (29, 52, 69). The range of compoundseffective has recently been extended by thedemonstration that resting Chromatium cellsshow photoproduction of H2, dependent uponaddition of thiosulfate (45, 52). Considering thegreat variation in chemical nature of the sub-strates which have given positive results, we re-gard photoproduction of hydrogen as a funda-mental metabolic activity intimately associatedwith light-activated electron transfer in all bac-teria of this group.'Many earlier studies, however, had indicated

that molecular hydrogen was not produced dur-

5 Green sulfur bacteria have not yet been tested,under appropriate conditions, for ability to photo-produce hydrogen. It seems very likely that suchorganisms will also show the phenomenon whengrown under circumstances in which formation ofthe hydrogen-evolving system is not repressed.

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0 20 40 60 80 100 0 100 200 300

MINUTES MINUTES

FIG. 2A (left). Photoproduction of hydrogen by cells of Rhodospirillum rubrum grown on 0.6% DL-malicacid + 0.05% L-glutamic acid. Details of growth and manometric methods can be found in reference (52).The cells were harvested when the optical density of the culture was 125 Klett-Summerson units. Five micro-moles of L-malate were added, where indicated, to 10 mg of cells (dry weight) at zero time. The gas phasewas 1% C02 in He, and diethanolamine, a CO2 buffer, was present in the center well. The Warburg vesselswere illuminated at 30 C.

FIG. 2B (right). Photoproduction of hydrogen by cells of R. rubrum grown on 0.6% DL-malic acid +0.125% (NH4)2S04 . The cells were harvested while ammonia was still present in the culture medium (atan optical density of 240 photometer units). At zero time, 40 jrmoles of L-malate were added. Other condi-tions as in Fig. 2A.

ing growth of purple bacteria on malate or otherorganic compounds when ammonia was thenitrogen source. Resting cells from such mediareadily photometabolized organic substrates, butthey appeared to lack the capacity to producehydrogen. These observations led to the sugges-tion (26, 74) that a specific electron carrier ortransport system required for hydrogen formationis not synthesized when ammonia constitutes thesole nitrogen source. Our more recent investiga-tions (52), in which completely synthetic mediawere used, have disclosed that the system respon-sible for photoproduction of hydrogen can alsodevelop in Rhodospirillum cells grown on am-monia.Development of the hydrogen-evolving system

in ammonia-grown cells requires light and ametabolizable substrate such as malate, and oc-curs only after ammonia has been exhausted bymetabolic or other means. Cells harvested a num-ber of hours after exhaustion of the nitrogensource from a malate medium (initially contain-ing limiting amounts of ammonia) show imme-diate photoproduction of hydrogen. On the otherhand, cells obtained from the medium before theammonia has disappeared show activity onlyafter an appreciable induction period (Fig. 2B).Using cells of the latter kind, experiments withinhibitors such as chloramphenicol and aminoacid analogues have shown that appearance of

hydrogen photoevolution activity is contingenton protein synthesis. Since addition of an ex-ternal nitrogen source is unnecessary, it is likelythat the amino acids required for synthesis ofenzymatic components of the system can be de-rived from turnover of existing proteins. Additionof a small amount of glutamate, however, stimu-lates the development of the hydrogen-producingsystem, and incubation of the cells under molecu-lar nitrogen during the induction period causesan even more pronounced acceleration (activitymeasured after replacement of the N2 withhelium). Our interpretation of these and relatedexperiments (52) is that ammonia, or a metab-olite derived from ammonia, represses forma-tion of one or more specific enzymes required forhydrogen production. Accordingly, this systemis highly developed during growth only withnitrogen sources that do not give rise to a meas-

6 The repression of synthesis of the hydrogen-evolving system of R. rubrum by ammonia is pos-sibly another example of "feedback repression."Since ammonia can be assumed to be the end prod-uct of N2 fixation, this interpretation would sug-gest that both H2 production and N2 activation inR. rubrum are catalyzed by the same enzyme orenzyme complex. Accordingly, the inhibition ofphotoproduction of H2 by N2 (28) may be due, inpart, to combination of the latter gas with an enzy-matic site which is also concerned with hydrogenformation.

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urable steady state level of ammonia in themedium (i.e., N2 and certain amino acids (4, 26,27)).From our present knowledge it seems that the

operation of metabolic pathways resulting inevolution of molecular hydrogen may be the rule,rather than the exception, in the anaerobicphotometabolism of Rhodospirillum rubrum. Con-sidered from the standpoint of nitrogen nutrition,H2 appears to be always produced by truly restingcells, regardless of the nitrogen growth source,and is lacking among photosynthetic growthproducts only when ammonia is present.

B. The Stoichiometry of Hydrogen Production

The over-all properties of the hydrogen-pro-ducing system which has developed in ammonia-grown cells appear to be generally similar to thoseobserved with cells grown with glutamate as thenitrogen source (52). For example, in both in-stances the evolution of H2 is completely light-dependent and is (reversibly) inhibited by mo-lecular nitrogen or ammonium salts. There is,however, one significant difference between thetwo cell types, viz., the yields of H2 and CO2observed as the result of photometabolism of agiven quantity of substrate are usually muchhigher with glutamate-grown cells. Even withthe latter nitrogen source, the particular growthconditions may have a marked influence on thequantity of hydrogen produced by the harvestedorganisms. This is illustrated in Fig. 3.

It is evident that as the initial concentrationof glutamate in the medium is decreased, theamount of hydrogen produced by the correspond-ing cells increases. On the other hand, the ratesof H2 formation are relatively constant, suggest-ing that the hydrogen-evolving system may bedeveloped to the same extent in each case. Thedata of Fig. 3 illustrate the common experience(29) that the stoichiometry of 12 production canvary widely, depending upon the physiologicalstate of the cells. Detailed interpretation of thisparticular experiment is somewhat complicatedby the fact that the cell concentrations in thevarious cultures at the time of harvesting weresignificantly different; this in turn means that,due to shading, the cells were growing with some-what different light intensities in each case andtherefore could not be said to be strictly com-parable. It is obvious that study of cells grown

0

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FIG. 3. Rates and stoichiometries of photoproduc-tion of hydrogen by cells of Rhodospirillum rubrumgrown in 0.6% DL-malic acid media with differentinitial concentrations of L-glutamic acid. The cul-tures were inoculated simultaneously and harvestedafter 67 hr of growth in media initially containing thepercentages of glutamic acid indicated. Each vesselcontained 10 mg (dry weight) of cells, and lO,molesof L-malate were added at zero time (except for theendogenous curves). Other details as in Fig. 2A.

in continuous culture under steady state condi-tions is required for a more penetrating analysis.The data at hand, however, do suggest thatmalate can be simultaneously utilized by atleast two competitive pathways, only one ofwhich results in hydrogen evolution.

In earlier studies on the quantitative aspectsof malate photometabolism by resting cells ofglutamate-grown R. rubrum, Bregoff and Kamen(4) found that the amount of hydrogen producedby different suspensions varied considerably.Numerous experiments, however, indicated thatthe stoichiometry during the early stages ofmetabolism could be represented by the equation:

C461.05 -- "031H403" + C02 + H2where "C3H403" is the assumed elementarycomposition of the assimilatory products. Inlater phases, malate was utilized at a decreasedrate, whereas the rates of H2 and C02 productionremained constant or increased. The eventualmolar ratios of H2 and C02 produced, per mole

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of malate consumed, were in approximate accordwith the equation:C4H605 + H20 -* "C2H402" + 2CO2 + 2H2

Stoichiometries of this kind are suggestive ofincomplete carbohydrate fermentations typical ofsaccharolytic Clostridium species, but it is alsoapparent that the balances observed by Bregoffand Kamen could equally well be interpreted asthe resultant of two simultaneous processes: (i)conversion of part of the malate to CO2 and a C3fragment, which is assimilated as carbohydrate,and (ii) extensive conversion of the remainder toCO2 and H2. Depending on the ratio of (i) to(ii), any one of a number of values of stoichi-ometry might be observed with a particular cellsuspension. An increased degree of assimilationof substrate carbon into cell materials by route(i) would obviously diminish the quantity of H2and CO2 produced (cf. discussion of this point byGest, Kamen, and Bregoff, (30)).The hydrogen yield can, in fact, exceed 2 moles

per mole of malate added; yields of 3 were ob-served on occasion by Siegel and Kamen (69) andsimilar yields have been noted in the photo-metabolism of lactate (22). It is evidently dif-ficult to reconcile these high yields with "normal"heterotrophic fermentation patterns.

Using the observations recorded in Fig. 3, bysystematic study we have developed a procedurefor obtaining cell suspensions which will rapidlyphotometabolize malate and succinate accordingto the approximate values of stoichiometry:

lightC4H605 + 3H20 - 4CO2 + 6H2

lightC4H604 + 4H20 -t 4CO2 + 7H2

These balances indicate complete conversion toCO2 and H2, and, on a formal basis, suggest thatat least one half of the hydrogen originates fromwater.

C. An Anaerobic Light-dependent Citric Acid CycleThere is convincing evidence that the dark

aerobic oxidation of organic compounds by Rho-dospirillum rubrum occurs to a considerable ex-tent through the conventional citric acid cycle.4Thus, Eisenberg (7) observed that all of theintermediates of the cycle were oxidized by cell-free extracts, and the individual steps of thecycle were demonstrated using either extracts ordried cells. Additional evidence was provided by

experiments with the inhibitor fluoroacetate (9).In mammalian systems which oxidize substratesvia the cycle, fluoroacetate is rapidly convertedto fluorocitrate, which is a potent inhibitor ofaconitase (58). Inhibition by fluoroacetate, ac-companied by accumulation of citrate, is accord-ingly taken as strong evidence for operation ofthe citric acid cycle. Elsden and Ormerod (9)found that fluoroacetate caused a significant ac-cumulation of citrate in intact cell suspensions ofR. rubrum which were aerobically oxidizing C4dicarboxylic acids. Fluoroacetate greatly de-pressed the rate of 02 utilization with acetate,pyruvate, propionate, and butyrate, but citratedid not accumulate in these instances, perhapsbecause an adequate supply of oxaloacetate wasnot available.The effect of fluoroacetate on the anaerobic

photometabolism of various substrates was alsoinvestigated by Elsden and Ormerod (9). Inthese experiments, the cells were grown withan ammonium salt as the nitrogen source,and exposed to substrates and fluoroacetatein bicarbonate buffer under an atmosphere of5% CO2 + 95% N2. Since nitrogen was pres-ent in the gas phase, photoproduction of H2 couldnot occur, even if the hydrogen-evolving systemhad been present (28, 30). Fluoroacetate causedaccumulation of citrate from acetate, pyruvate,and oxaloacetate, but not from the other C4dicarboxylic acids. Consequently, the conclusionwas drawn that the citric acid cycle was not in-volved in the photometabolism of succinate,fumarate, and malate. The difference in be-havior of oxaloacetate and the other C4 acids isprobably related to the fact that the latterare more reduced. Thus, the reducing powerresulting from oxidation of malate and its con-geners to oxaloacetate, in cells which do notproduce hydrogen, probably is responsible fordiversion of carbon from the citric acid cyclesequence into an assimilatory pathway.The quantitative conversion of malate to CO2

and H2 by glutamate-grown cells of R. rubrum isin net effect equivalent to the complete oxidationof malate by the conventional (aerobic) citric acidcycle. This consideration prompted experimentsto test the effect of fluoroacetate on photoproduc-tion of hydrogen. Fluoroacetate in concentrationsas low as 104 M markedly inhibits evolution ofH2 in the presence of malate and it can be seen

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TABLE 2. Effect of fluoroacetate on gas productionand citrate formation from L-malate byilluminated cells of Rhodospirillum rubrum

No fluoroacetate fluoroacetate

Endo- +5 Endo- p+egenous ma1ates genous maoltes

Gas (H2 + C02) pro-duced,pumoles ..... 3.2 40.6* 0.1 8.4*

Citrate, jumoles ...... 0.03 0.07 0.07 1.51

* These values are approximate because they donot include C02 bound in the liquid phase.

Experimental conditions: The cells were grownas for Fig. 2A and harvested when the opticaldensity was 195 photometer units (53 hr). EachWarburg vessel contained a suspension equivalentto 1.2 mg cell nitrogen and the gas phase was 1%C02 in He; temperature, 30 C. Where indicated,fluoroacetate was added 15 min before addition ofL-malate. Sixty-seven minutes after substrate ad-dition, the reaction was terminated by additionof sulfuric acid. The vessel contents were thencentrifuged and aliquots of the supernatant fluidswere analyzed for citric acid by the pentabromo-acetone method.

from Table 2 that this inhibitor also causes amassive accumulation of citrate.These observations. together with results of ex-

periments with other substrates to be detailedelsewhere (30a), have provided strong evidencethat C4 dicarboxylic acids and acetate can be com-pletely converted to C02 and H2 by means of ananaerobic light-dependent citric acid cycle. It isconceivable that the transformations of carbonin this process might occur through a modifiedcycle involving isocitratase, but this seems un-likely since the formation of this enzyme is usuallyunder repression control and it is not present insignificant quantity in cells of R. rubrum grownin the malate or acetate media used by Kornbergand Lascelles (38). To our knowledge, this is thefirst unambiguous evidence for operation of acomplete citric acid cycle to a quantitatively sig-nificant extent under anaerobic conditions.7

Intact cells of Rhodospirillum rubrum are notpermeable to a-ketoglutarate and the tricarboxylicacids (7); it is therefore not possible to study theutilization of these intermediates directly. Indica-tions suggesting operation of the anaerobic citricacid cycle during photometabolism of certain sub-strates can be found in data reported by Elsden

It is obvious that an anaerobic citric acid cyclerequires a special mechanism for disposal of theelectrons or hydrogen atoms released in theoxidative steps. This could be accomplished byformation of molecular hydrogen, either directlyor indirectly.

D. C02 Fixation during Photometabolism of CitricAcid Cycle Intermediates

The anaerobic citric acid cycle might be ex-pected to function without hydrogen productionif a suitable electron acceptor is provided, orgenerated by the metabolism of added substrates.In this sense, C02 itself may act, to some extent,as an electron acceptor, after conversion tophosphoglyceric acid by the ribulose diphosphatecarboxylase reaction. In Ormerod's (51) experi-ments with ammonia-grown cells incapable ofevolving H2 (under a N2 atmosphere), succinatewas metabolized with the net production of 0.7mole of C02 and isotopic data showed that 0.5mole of C02 was simultaneously fixed. This resultimplies (51) that the electron pair derived fromoxidation of succinate to fumarate can be con-verted in some manner to reducing equivalentsin the form of reduced pyridine nucleotide, whichis then used for the reductive conversion of phos-phoglyceric acid to triose phosphates However,the fact that glutamate-grown cells can convertsuccinate almost quantitatively to C02 and H2indicates that the succinic dehydrogenase step isnot obligatorily coupled to C02 fixation.

It is noteworthy that Lascelles (41) has re-centlyobserved high levels of ribulose-1 ,5-diphos-phate carboxylase in purple bacteria grownphotoheterotrophically (e.g., with malate). Atfirst glance, this finding might be considered tobe somewhat unexpected, since in certain faculta-tive autotrophs such as Micrococcus denitrificans

and Ormerod (9), from experiments with ammonia-grown cells of R. rubrum incubated in the presenceof N2 . It may also be remarked that an elegant,but not necessarily correct, case could be readilydeveloped designating the anaerobic light-de-pendent cycle as the evolutionary predecessor ofthe exergonic aerobic cycle of nonphotosyntheticheterotrophs.

8 It should be noted, however, that C02 fixationexperiments of the kind under discussion are in-variably complicated by the possibility of ex-change reactions which would permit introductionof labeled carbon from C1402 into a variety of com-pounds in the absence of net C02 fixation.

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the carboxylase is present in significant quantitywhen the cells are grown with CO2 as the solecarbon source, but not when assimilable organiccompounds provide the growth substrates (37).From the foregoing, however, it would appear

that under certain conditions purple bacteria can

simultaneously fix CO2 via the carboxylase reac-

tion and photoassimilate fragments derived fromadded organic compounds. The quantitative ex-

tent of CO2 fixation occurring through the"photosynthetic carbon cycle" during themetabolism of organic substrates is uncertain,and without doubt is strongly influenced by a

variety of factors.It is known that continuous removal of CO2

can cause a significant decrease in the amount ofH2 evolved by purple bacteria (69), suggestingthat CO2 (or operation of the "photosyntheticcarbon cycle") has the potentiality of influencingthe direction of electron transfer. The possibilitythat this particular effect of CO2 is catalyticcannot be excluded, especially since Fuller et al.(16) have recently described a mechanism for theconversion of malate to oxaloacetate (in Chroma-tium cells) in which CO2 plays such a role.

E. Alternative Pathways of Carbon MIetabolism

Direct analyses indicate that polysaccharide isproduced as a major reserve material in R.rubrum during photosynthetic growth on malate,with an ammonium salt as the nitrogen source

(52). Photometabolism of malate by suspensionsof cells obtained from such cultures results in theformation of approximately 1 mole of CO2 per

mole of substrate added (51), when the develop-ment of the hydrogen-evolving system is pre-

vented or its activity inhibited. These facts sug-

gest (9) that, when H2 is not produced, a majorpathway of malate utilization is conversion toCO2 and a C3 fragment which is assimilated as

polysaccharide, according to the followingsequence:

2H C02

Malate oxaloacetateATP

phosphoenolpyruvate

3-phosphoglyceric acidATPDPNH

triose phosphate -* hexose -- polysaccharide

Since the reduced pyridine nucleotide requiredfor reduction of phosphoglyceric acid to triosephosphate could, in principle, be derived fromthe oxidation of malate to oxaloacetate, the onlyobvious net requirement is for the nucleosidetriphosphate. The latter can evidently be fur-nished by cyclic photophosphorylation (11, 12,21), and under these particular conditions it ispossible that the necessity for light could beexplained primarily on this basis.The photoassimilatory sequence outlined above

would result in the efficient conversion of ap-proximately three of the four carbons of malateto cell materials. As in media containing am-monium salts, sizable polysaccharide reserves arealso produced by R. rubrum during growth onmalate + glutamate, presumably via the samepathway. At the same time, molecular hydrogenappears to be continuously evolved (unpublishedobservations). The control mechanisms governingthe "split" of carbon flow into assimilation versusthe anaerobic cycle are still not understood, butin all probability are related to the temporalenergy requirements of the organism. Duringnonsteady-state growth, it is likely that the ATPrequirement of the cell can vary significantly,depending on metabolic circumstances. For ex-ample, it can be expected that when a nitrogensource such as ammonia is exhausted, the ender-gonic syntheses of various macromolecular cellconstituents will abruptly cease, thereby alteringthe demand for ATP appreciably. If it is furtherassumed that ATP continues to be produced un-der conditions of constant illumination, then itmight be expected that the supply of ATP willsoon exceed the diminished ATP requirement ofsuch cells (i.e., resting cells). Thus, the partialtransition from assimilation of organic inter-mediates to anaerobic oxidation to CO2 and H2which occurs in R. rubrum upon exhaustion ofammonia may well be a reflection of a drasticchange in "energy balance." From a physio-logical viewpoint, the complete conversion of avail-able organic substrates to CO2 and H2, whichcan be demonstrated in resting suspensions.might be considered to be an extreme, observableonly in cells of a particular physiological state.This may be so, but it is also probable that furtherstudy of such metabolically exaggerated systemsmay provide insights into the mechanisms oflight-activated electron transfer and the natureof the metabolic control devices used by photo-synthetic cells.

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IV. FUNCTIONS OF THE ACCESSORY ELECTRONDONOR IN BACTERIAL PHOTOSYNTHESIS; AL-TERNATIVE PATHWAYS OF ELECTRON TRANS-FER

Current theories concerning the action of lightin photosynthesis are largely based on the premisethat reducing equivalents (either electrons orhydrogen atoms) are created by the interactionof light with the photochemical apparatus, andthat oxidizing equivalents (oxidized chlorophyll(Chl+) or OH) are simultaneously produced. Adescription of the photochemical basis of thephotoelectric theory of photosynthesis, accordingto which the oxidant and reductant are Chl+ ande, respectively, can be found in papers by Levitt(42-44). The alternative postulation of H andOH as the reactive moieties generated by light isan old hypothesis (60, 63, 64, 76) and is in neteffect equivalent to the photoelectric theory.9Light-induced electron spin resonance signalshave been detected in photosynthetic systems(e.g., see reference (6)), but interpretation ofthese observations in terms of specific electron orhydrogen atom transfer processes is still notpossible.

Detailed consideration of the available datasuggests that the transport routes of photoreduc-ing equivalents and of electrons derived from theaccessory donor are not invariant, but ratherthat major alternative pathways are utilized, de-pending upon metabolic circumstances and thechemical nature of the accessory electron source.This view predicts that any attempt to explainlight-activated electron transfer and associatedreactions on the basis of any one specific patternwill inevitably require a number of qualificationsand assumptions to cover "special cases."The investigations of Siegel (65-68, 70) on the

utilization of acetone by Rhodopseudomonasgelatinosa provided the first significant evidencefor a partial equivalence of light and ATP in thephotometabolism of organic compounds. Si-multaneous-adaptation experiments with cellsgrown on a variety of substrates suggested thefollowing sequence for photoassimilation ofacetone:

Acetone + ) acetoacetate -2 acetate - cell materials.

See Gaffron (20) for an extensive account andcritique of various alternative theories that havebeen proposed.

Strong supporting evidence for this pathway wasobtained from experiments with C14-labeledacetone and C"402. Cell suspensions incubatedanaerobically in the light with C1402 and a mix-ture of unlabeled acetone and acetoacetate in-corporated C14 into the carboxyl group of thelatter compound in the predicted manner. Fur-thermore, at certain light intensities the ac-cumulation of almost 2 moles of acetate, per moleof acetone used, was demonstrated. Additionalexperiments also indicated that the energy re-quired (AF = +10 kcal) for the carboxylation ofacetone to acetoacetate could be derived eitherfrom light or from energy-yielding dark metabo-lism, viz., oxidation of acetone or fermentation ofadded acetoacetate. Direct experimental evidenceincluded the demonstration that cell-free extractsof R. gelatinosa rapidly produce ATP from aden-ylic acid anaerobically in the dark, upon additionof acetoacetate and coenzyme A. Siegel inter-preted his results to indicate that ATP was thecommon component required for the endergoniccarboxylation of acetone and that this could beprovided by several alternative mechanisms, viz.,photophosphorylation, oxidative phosphoryla-tion, or substrate-level phosphorylation resultingfrom the dark anaerobic cleavage of acetoacetate.A more extreme version of Siegel's proposal is

the recent hypothesis (46, 72) that the function oflight in bacterial photosynthesis is "primarily"limited to the formation of ATP by cyclic photo-phosphorylation (i.e., phosphorylation resultingfrom the recombination of light-generated reduc-ing and oxidizing equivalents). Reducing power(reduced pyridine nucleotide), according to thispostulation, is normally produced, when required,by dark oxidation of the accessory hydrogendonor. This formulation can, in principle, ac-count for several light-dependent metabolic con-versions observed in photosynthetic bacteria,e.g., certain conversions which do not involveoxidation-reduction, such as the carboxylation ofacetone and the synthesis of succinate frompropionate + CO2 (39), and several processeswhich do, viz., the formation of carbohydratefrom malate and the reductive synthesis of f-hy-droxybutyrate polymer from acetate (18, 72).However, its general application is seriouslyhampered by the lack of an unequivocal experi-mental basis to rationalize: (i) light-dependentreduction of DPN by chromatophores incubatedin the presence of succinate or reduced flavinmononucleotide (13-15), and (ii) photoproduc-

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"Photochemical apparatus"

Carotenoid and bacteriochlorophyll

IPhotoreductant

\\RHP Cytochrome C2

DPNH TPNH

IPhotooxidant

FIG. 4. Electron transfer scheme suggested by Horio and Kamen (32). RHP is a specific heme proteinpresent in Rhodospirillum rubrum and other photosynthetic bacteria.

tion of H2 from thiosulfate, C4 dicarboxylic acids,and other organic substrates.

Clear-cut experimental evidence that electronscan be transported to an appreciable extentagainst the thermodynamic gradient in photo-synthetic cells, through the intervention of ATP(in the dark), has not been described. Accordingto Pratt, Frenkel, and Hickman (59), addition ofATP does not appear to promote DPN reductionby chromatophores exposed to succinate in dark-ness. Frenkel (14) apparently prefers to interpretthe succinate-dependent photoreduction of DPNby chromatophores as the manifestation of a"noncyclic" electron transport system, in whichlight-generated reducing equivalents are utilizedfor production of reduced DPN (DPNH), bal-anced by transfer of electrons from succinate tothe photooxidant. He has also pointed out (15)that there is as yet no definitive evidence avail-able to permit an unambiguous choice betweenthis interpretation and the alternative thatDPNH is generated by dark oxidation of theaccessory donor (facilitated by ATP), nor isthere evidence to permit the assumption thatonly one of these mechanisms can be operative.Whether or not the photometabolism of succinatecan be considered as an isolated exceptional caseremains to be seen, particularly since there is solittle knowledge concerning the nature of theprimary acceptors involved in the oxidation ofthe large variety of accessory electron donorseffective in bacterial photosynthesis.The theory of complete equivalence of light

and ATP (which is already undergoing revision(2) to accommodate alternative pathways) as-sumes, furthermore, that electrons from the acces-sory donor cannot enter the transport sequencenormally concerned with cyclic photophosphoryla-

tion. There are, however, several indications thatthis may occur (14, 79). In this connection it maybe noted that recent experiments by Horio andKamen (32) were considered to be consistent withthe following scheme (Fig. 4).

Photoproduction of molecular hydrogen can beconsidered to be analogous to the substrate-de-pendent photoreduction of DPN catalyzed bychromatophores. Thus, mechanisms similar tothose discussed in connection with DPN photo-reduction can be postulated to account for hy-drogen formation. A direct "upgrading" by ATPof electrons derived from reduced pyridinenucleotide (and from oxidation of succinate tofumarate) to the level required for H2 productionis not excluded by available data,10 but we are in-clined to favor the view that photoproduction ofH2 represents a major alternative pathway ofhydrogen transport, in which the photoreductantis the precursor of the electrons or hydrogenatoms of the H2 molecule. This concept is out-lined in Fig. 5; the scheme proposed is in essencethe same working hypothesis which has servedas a guide for experimentation for many years(see, e.g. (19, 25, 26)).According to the representation in Fig. 5B,

molecular hydrogen is produced as the result oftransfer of photochemically generated electronsor hydrogen atoms to a hydrogen-evolving en-

10 Chance (5) interprets his experiments withmammalian mitochondria to indicate that ATP-activated reversal of electron transfer may occurover a large redox potential range. It might beadded that attempts to evoke H2 production byaddition of reduced pyridine nucleotides and ATPto cell-free preparations from Rhodospirillumrubrum have thus far given negative results (un-published experiments).

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hydrogen - evolving - Henzyme

PR PR

Reductive synthesis,

\43 _ {includingCOt

PA RHP PA RHP

fl 1 A~~~~~~~~c ccss

|t > ~~~~~~~~~~~electron)

cyt. C2 cyt. C2

P0 PO

A. CYCUC ELECTRON B. NON-CYCUC ElECTRON TRANSFERTRANSFER

FIG. 5. Schematic representation of (postulated)alternative light-dependent electron transfer path-ways in bacterial photosynthesis. (A) represents theelectron transfer chain responsible for cyclic photo-phosphorylation. Light-dependent phosphorylationmay also occur during noncyclic electron transfer(B) when pyridine nucleotide is the acceptor. Theinhibition of photoproduction of H2 by N2 is inter-preted (26, 30) as the result of competition for photo-reductant, between the nitrogen-reducing system andthe hydrogen-evolving enzyme; it is assumed thatthe competition is greatly in favor of the former.Molecular nitrogen may also inhibit H2 productionby an additional mechanism in which N2 acts cataly-tically (4). As implied by Fig. 4, electrons de-rived from the oxidation of accessory donors may

enter the "cyclic" electron transfer chain, possiblyat different points; dark oxidation of certain acces-

sory electron donors would be expected to producereduced pyridine nucleotides or other reduced co-

enzymes. PA = photochemical apparatus, PO =

photooxidant (Chl+, OH), PR = photoreductant(e, H), RHP = heme protein, cyt. C2 = cytochromeC2

zyme. An equivalent number of electrons fromthe oxidative steps of the citric acid cycle (orfrom oxidation of other accessory electron donors)are transferred through a chain of oxidation-re-duction carriers to the photooxidant. Thisscheme indicates that a completely anaerobiccitric acid cycle (without added oxidants) ismade possible by two important factors. One ofthese is a photochemical mechanism which pro-

vides an internal oxidant for electrons releasedby the oxidative reactions of the cycle. This, inturn, would require continuous removal ofphotoreductant, which can be achieved, withoutthe necessity for exogenous electron acceptors,by transfer to a hydrogen-evolving enzyme.

MNany basic aspects of the cyclic photophos-phorylation process are still unclear. Thus, the

oxidation-reduction carriers involved in trans-port of the photoreductant to the heme proteinRHP have not been identified. The yield ofATP per reducing equivalent generated by lightand the actual sites of phosphorylation are alsostill unknown. Although it has been suggested(71) that phosphorylation may occur during elec-tron transfer from reduced cytochrome to thephotooxidant, definitive data are lacking.Our interpretation of the mechanism and sig-

nificance of photoproduction of hydrogen sug-gests that phosphorylation may not occur duringelectron transfer from RHP to the photooxidant,or as a consequence of conversion of photore-ductant to H2. This view implies that phos-phorylation in cyclic electron transfer is associ-ated with steps between the photoreductant andRHP. The possibility that phosphorylation alsocan occur during noncyclic electron transferwhen pyridine nucleotide is the acceptor cannotbe excluded (79).

V. CONCLUDING REMARKS

The calculated free energy changes associatedwith the conversion of citric acid cycle inter-mediates to CO2 and H2 indicate that this typeof dissimilation probably cannot be a mechanismfor net energy production. This suggests thatphotoevolution of hydrogen may represent analternative pathway of electron transfer whichserves a regulatory function in energy metabolism.Thus it could be envisioned that evolution of hy-drogen is a mechanism for circumventing phos-phorylation (or for utilizing excess ATP) underconditions where the cell has a diminished re-quirement for ATP. In analogy with current con-cepts of the control of electron flow and oxidativephosphorylation in aerobic systems, it might besupposed that the ratio of ATP to adenosine di-phosphate in critical sites could be a key factorin determining whether the photoreductant isused as a "substrate" for cyclic photophos-phorylation, for production of reduced pyridinenucleotides, or shunted to the hydrogen-evolvingenzyme.

It is evident that the considerable degree ofspeculation inherent in current discussions of themechanism of bacterial photosynthesis resultsfrom the fact that many basic questions remainunanswered. Nevertheless, the progress whichhas been made in our understanding of light-activated electron transfer and associated proc-

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esses has provided the basis for a more refineddefinition of the basic issues and has also un-

covered experimental systems of value for theirstudy. The recognition of major alternativepathways of electron transport and carbonmetabolism is clearly a prerequisite for furtherstudy of the control mechanisms governing theiroperation and integration, and of their true sig-nificance in the metabolic economy of the cell.

VI. ADDENDUM

Several investigators (Frenkel (14), Vernonand Ash (79), and more recently, Nozaki,Tagawa, and Arnon (50b)) have studied "non-cyclic" photo-reduction of DPN by chromato-phores of Rhodospirillum rubrum and the re-

lationship of this process to light-dependentphosphorylation. In the studies cited, different ex-

perimental conditions of varying complexitywere used, and the data reported do not permitan unambiguous assessment of the relationshipin question. It appears that clarification of thisimportant point will require further investigation.Note should also be made of recent experimentsby Nishimura (50a) which suggest that "photo-phosphorylation" by R. rubrum chromatophorescan occur in darkness, i.e., during dark periodsbetween intermittent flashes of infrared illumina-tion.

VII. LITERATURE CITED1. ARNON, D. I. 1961. Cell-free photosynthesis

and the energy conversion process, p. 489-565. In W. D. McElroy and B. Glass, [ed.],A symposium on light and life. Johns Hop-kins Press, Baltimore.

2. ARNON, D. I., M. LOSADA, M. NOZAKI, AND K.TAGAWA. 1961. Photoproduction of hydro-gen, photofixation of nitrogen and a unifiedconcept of photosynthesis. Nature 190:601-606.

3. BosE, S. K., H. GEST, AND J. G. ORMEROD.1961. Light-activated hydrogenase activityin a photosynthetic bacterium: a permeabil-ity phenomenon. J. Biol. Chem. 236:PC13.

4. BREGOFF, H. M., AND M. D. KAMEN. 1952.Studies on the metabolism of photosyntheticbacteria. XIV. Quantitative relations be-tween malate dissimilation, photoproduc-tion of hydrogen, and nitrogen metabolismin Rhodospirillum rubrum. Arch. Biochem.Biophys. 36:202-220.

5. CHANCE, B. 1961. The interaction of energy

and electron transfer reactions in mito-chondria. II. General properties of adeno-

sine triphosphate-linked oxidation of cyto-chrome and reduction of pyridine nucleotide.J. Biol. Chem. 236:1544-1554.

6. COMMONER, B., J. J. HEISE, AND J. TOWNSEND.1956. Light-induced paramagnetism inchloroplasts. Proc. Natl. Acad. Sci. U. S.42:710-718.

7. EISENBERG, M. A. 1953. The tricarboxylic acidcycle in Rhodospirillum rubrum. J. Biol.Chem. 203:815-836.

8. ELSDEN, S. R. 1954. The utilization of organiccompounds by photosynthetic bacteria, p.202-223. In B. A. Fry and J. L. Peel, [ed.],Autotrophic micro-organisms. CambridgeUniversity Press, Cambridge.

9. ELSDEN, S. R., AND J. G. ORMEROD. 1956. Theeffect of monofluoroacetate on the metabo-lism of Rhodospirillum rubrum. Biochem. J.63:691-701.

10. FRENCH, C. S. 1937. The quantum yield of hy-drogen and carbon dioxide assimilation inpurple bacteria. J. Gen. Physiol. 20:711-735.

11. FRENKEL, A. W. 1954. Light induced phos-phorylation by cell-free preparations ofphotosynthetic bacteria. J. Am. Chem. Soc.76:5568-5569.

12. FRENKEL, A. W. 1956. Photophosphorylationof adenine nucleotides by cell-free prepara-tions of purple bacteria. J. Biol. Chem.222:823-834.

13. FRENKEL, A. W. 1958. Simultaneous reductionof diphosphopyridine nucleotide and oxida-tion of reduced flavin mononucleotide byilluminated bacterial chromatophores. J.Am. Chem. Soc. 80:3479-3480.

14. FRENKEL, A. W. 1959. Light-induced reactionsof chromatophores of Rhodospirillum rubrum.Brookhaven Symposia in Biol. 11:276-287.

15. FRENKEL, A. W. 1961. Reflections on recenttheories concerning the mechanism of bac-terial photosynthesis, p. 587-592. In W. D.McElroy and B. Glass, [ed.], A symposiumon light and life. Johns Hopkins Press,Baltimore.

16. FULLER, R. C., R. M. SMILLIE, E. C. SISLER,AND H. L. KORNBERG. 1961. Carbon metab-olism in Chromatium. J. Biol. Chem. 236:2140-2149.

17. GAFFRON, H. 1933. Uber den Stoffwechsel derschwefelfreien Purpurbakterien. Biochem.Z. 260:1-17.

18. GAFFRON, H. 1935. t'ber den Stoffwechsel derPurpurbakterien. II. Biochem. Z. 276:301-319.

19. GAFFRON, H. 1944. Photosynthesis, photore-duction and dark reduction of carbon diox-ide in certain algae. Biol. Rev. CambridgePhil. Soc. 19:1-20.

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23. GEST, H. 1952. Properties of cell-free hydro-genases of E8cherichia coli and Rhodospiril-lum rubrum. J. Bacteriol. 63:111-121.

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25. GEST, H. 1958. Some aspects of electron trans-port in microorganisms with special re-ference to oxidation and formation of molec-ular hydrogen, p. 250-255. In K. Ichihara,led.], Proceedings of the international sym-posium on enzyme chemistry (Tokyo andKyoto, 1957). Academic Press, Inc., NewYork.

26. GEST, H., J. JUDIS, AND H. D. PECK, JR. 1956.Reduction of molecular nitrogen and rela-tionships with photosynthesis and hydrogenmetabolism, p. 298-315. In W. D. McElroyand B. Glass, [ed.], Inorganic nitrogenmetabolism. Johns Hopkins Press, Balti-more.

27. GEST, H., AND M. D. KAMEN. 1949. Studies onthe metabolism of photosynthetic bacteria.IV. Photochemical production of molecularhydrogen by growing cultures of photosyn-thetic bacteria. J. Bacteriol. 58:239-245.

28. GEST, H., AND M. D. KAMEN. 1949. Photo-production of molecular hydrogen by Rho-dospirillum rubrum. Science 109:558-559.

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30. GEST, H., M. D. KAMEN, AND H. M. BREGOFF.1950. Studies on the metabolism of photo-synthetic bacteria. V. Photoproduction ofhydrogen and nitrogen fixation by Rhodo-spirillum rubrum. J. Biol. Chem. 182:153-170.

30a. GEST, H., J. G. ORMEROD, AND K. S. ORME-ROD. 1962. Photometabolism of Rhodospiril-lum rubrum: light-dependent dissimilationof organic compounds to carbon dioxide andmolecular hydrogen by an aerobic citric acidcycle. Arch. Biochem. Biophys. (in pres8).

31. GLOVER, J., M. D. KAMEN, AND H. VAN GEN-DEREN. 1952. Studies on the metabolism of

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32. HoRmo, T., AND M. D. KAMEN. 1960. Somehaem protein-linked pyridine nucleotideoxidation systems in Rhodospirillum rubrum.Biochim. et. Biophys. Acta 43:382-392.

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35. KOHLMILLER, E. F., JR., AND H. GEST. 1951.A comparative study of the light and darkfermentations of organic acids by Rhodo-spirillum rubrum. J. Bacteriol. 61:269-282.

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