PHOTOSYNTHETIC MECHANISMS IN BACTERIA AND PLANTS:DEVELOPMENT OF A UNITARY CONCEPT'

R. Y. STANIER

Department of Bacteriology, University of California, Berkeley, California

I. Introduction............................................................................. 1II. Major Variations in the Patterns of Photosynthetic Metabolism........................... 2

III. Recognition of Bacterial Photosynthesis.................................................. 3IV. Concept of the Photolysis of Water ...................................................... 4V. Role of Organic Substrates in Bacterial Photosynthesis.................................... 6VI. Photophosphorylation .................................................................... 9VII. Arnon's Model for Photophosphorylation ................................................. 10VIII. Photosynthetic Hydrogen Evolution and Nitrogen Fixation................................ 10IX. Reinterpretation of Gross Reactions of Bacterial Photosynthesis.......................... 12X. Nature of the Primary Light Reaction ................................................... 13

XI. Conclusion............................................................................... 14XII. References............................................................................... 15

I. INTRODUCTIONAnd here, especially, lies the importance of

these "abnormal" photosynthetic processes,

because a comparison of the factors and condi-tions which are required for their accomplish-ment will enable us to find those characteristicswhich are common to all. It will then be possibleto derive the fundamental laws underlying allphotosynthetic processes and to correlate theseinto a general view.

C. B. van Niel, 1930 (70)

The purple and green bacteria occupy an

enigmatic position in the living world. In termsof their gross morphology, these two small groups

can be regarded as typical unicellular repre-sentatives of the eubacteria; but whereas mosteubacteria depend on chemical sources of energy,

purple and green bacteria are primarily, and inmany cases exclusively, dependent on light as an

energy source. This abrupt intrusion of a photo-synthetic mode of metabolism in a taxonomicgroup distinguished by the unparalleled varietyof its special adaptations to the use of chemicalenergy sources is certainly remarkable, and hasled many bacterial systematists to set off thephotosynthetic bacteria in a special major sub-

' This essay is based on the Mueller MemorialLecture, delivered at Harvard Medical School on

October 29, 1959. Insofar as the views expressedare original, they were developed by the authorduring his tenure (1957-59) of a research pro-

fessorship in the Miller Institute for Basic Re-search in Science of the University of California.

group, a treatment which cannot be justified onthe basis of purely morphological considerations.Considered as members of the photosyntheticcommunity, the purple and green bacteria arealso anomalous. Both their pigment systems andtheir gross photosynthetic mechanisms areunique, and stand out with particular sharpnessin contrast to the basically uniform pigmentsystem and photosynthetic mechanism in algaeand higher plants. It is these striking deviationsfrom the general pattern of photosynthesis thatconfer on the photosynthetic bacteria theircardinal interest as biological entities.The question, "What is photosynthesis?" is a

deceptively simple one. Many different answershave in fact been proposed, and the generallyaccepted one has changed from time to time asunderstanding of the photosynthetic process hasdeepened. It is not improbable that a definitiveanswer can now at last be offered. If so, this greatproblem of cellular physiology will have beensolved in no small measure through studies onthe photosynthetic bacteria. Since the char-acterization of bacterial photosynthesis some 30years ago by van Niel (70, 71), the photosyntheticbacteria have often provided the material forexperimental analyses which could not have beeneasily undertaken, and sometimes could not havebeen undertaken at all, with plant material.Furthermore, the very existence of photo-synthetic processes which differ from those ofgreen plants has greatly facilitated the task, notalways easy, of discriminating between the

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

fundamental and incidental features of thiscomplex metabolic reaction.

This essay will review the development ofideas about photosynthesis during the 30 yearswhich have elapsed since the recognition of thebacterial photosyntheses, and show how workwith bacterial systems has contributed to theformulation of our present concepts. No attempthas been made to provide a comprehensivesurvey of the literature on the metabolism ofphotosynthetic bacteria. References are accord-ingly restricted to those publications which are,in my opinion, directly relevant to the maintheme.As an introduction to the subject proper, the

physiological and structural features whichcharacterize photosynthetic bacteria and distin-guish them from green plants will be summarized.

II. MAJOR VARIATIONS IN PATTERNSOF PHOTOSYNTHETIC METABOLISM

Photosyntheses are metabolic processes whichresult in the conversion of radiant energy intochemical bond energy, specifically, into thephosphate bond energy of adenosine triphosphate(ATP). This is their one common denominator.Under conditions favoring growth, the ATP sogenerated is used for the synthesis of more cellmaterial. Most phototrophs perform a totalsynthesis of cell material from inorganic nutri-ents; accordingly, carbon dioxide is commonlythe sole carbon source. The gross conversion ofcarbon dioxide to cell material requires, inaddition to ATP, a source of reducing power.In all green plants this reducing power is ulti-mately provided by water, through a specialreaction, linked with the photochemical process,which results in the oxidation of water to molec-ular oxygen.The green bacteria and many of the purple

bacteria share with plants the ability to useCO2 as the sole source of carbon for cellularsynthesis. They cannot, however, use water as anultimate reductant, and consequently depend onother inorganic reductants (notably reducedsulfur compounds and molecular hydrogen).Since the oxygen evolution characteristic ofgreen plant photosynthesis is a consequence ofthe oxidation of water, it follows that oxygen isnot evolved in bacterial photosynthesis, and thatthis type of photosynthesis is an anaerobicprocess. Indeed, the green bacteria and many

purple bacteria are strict anaerobes. Since photo-synthetic bacteria cannot use fermentativemechanisms to obtain the energy needed forgrowth, strict anaerobiosis entails as a corollaryobligate phototrophy.The purple bacteria can also use simple organic

compounds as the major carbon source for thephotosynthesis of cell material. In such cases, theexogenous carbon source is similar in oxidationstate to cell material, and the requirement foran exogenous inorganic reductant disappears.However, the bacterial photosyntheses of organicsubstrates are anaerobic processes; accordingly,a strict over-all oxidation-reduction balancemust be maintained, just as in the case of afermentation. When the organic substrate ismore oxidized than cell material, strict oxidation-reduction balance is achieved by the anaerobicoxidation of part of the substrate to C02, whichprovides reducing power for the synthesis of cellmaterial from other substrate molecules. Whenthe organic substrate is more reduced than cellmaterial, strict oxidation-reduction balance isachieved by partial oxidation of the substrate,coupled with reduction and assimilation of C02;in this case, there are two exogenous carbonsources, and assimilation of the organic substrateis mandatorily linked with CO2 assimilation.The major physiological distinctions between

plants, green bacteria, and purple bacteria aresummarized in table 1.

In terms of the chemistry of their pigmentsystems, the purple and green bacteria aresharply distinguished from one another, andalso from all organisms that perform green plantphotosynthesis. No molecular species of chloro-phyll or of carotenoid is common to the photo-synthetic apparatus of all three groups. In allpurple bacteria, there is one molecular species ofchlorophyll, bacteriochlorophyll (74). The char-acteristic carotenoids of purple bacteria arealiphatic (37). In the green bacteria, there occurtwo molecular species of chlorophyll (chlorobiumchlorophylls), only one of which is present inany given strain (68). The characteristic caro-tenoid of green bacteria is y-carotene (38, 80), amonocyclic compound. The pigment system oforganisms that perform green plant photosyn-thesis cannot be so succinctly characterized;although the higher plants are strikingly uniformin this respect, marked systematic differences inthe composition of the pigment system char-

2 [VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

TABLE 1Major physiological distinctions between green plants, green bacteria, and purple bacteria

Green Plants Green Bacteria Purple Bacteria

Source of reducing power H20 H2S, other reduced in- H2S, other reduced inorganic com-organic compounds pounds, organic compounds

Photosynthetic oxygen evolu- Yes No Notion

Principal source of carbon CO2 C02 C02 or organic compoundsRelations to oxygen Aerobic Strictlv anaerobic Strictly anaerobic or facultatively

aerobic

acterize the various groups of algae (69). Never-theless, certain pigments are common to allorganisms that perform green plant photosyn-thesis. These pigments, which accordingly serveas chemical hallmarks of this kind of photo-synthesis, are one molecular species of chloro-phyll, chlorophyll a, and one or both of theclosely related bicyclic carotenoids, a- andf-carotene. The basic features of the photo-synthetic pigment system in plants, purplebacteria, and green bacteria are summarized infigure 1.

Brief mention should be made also of thesingular macromolecular structure of the photo-synthetic apparatus in purple and green bacteria.Unlike most organisms that perform green plantphotosynthesis, photosynthetic bacteria aredevoid of chloroplasts. The pigment system islocalized in much smaller structures known aschromatophores (63) which can be isolated fromcellular extracts. The isolated chromatophoresare 300 to 500 i in diameter, and contain pro-teins, phospholipids, and respiratory pigments,in addition to the entire cellular complement ofchlorophyll and carotenoids (9). The only otherphototrophs which lack chloroplasts are theblue-green algae. A chlorophyll-containing cellfraction similar to the chromatophore fraction ofphotosynthetic bacteria can be isolated fromextracts of these organisms (63, 64). The organi-zation of the photosynthetic apparatus in theintact cell of photosynthetic bacteria and blue-green algae is not yet fully understood. Thiscomplex and somewhat controversial problem(14, 39, 56, 64, 75) will not be discussed furtherhere.

It is customary to recognize two major sub-groups within the purple bacteria: purple sulfurbacteria (family Thiorhodaceae); and nonsulfur

purple bacteria (family Athiorhodaceae). Thedistinguishing properties are relatively minor,and difficult to apply consistently in practice.Purple sulfur bacteria are primarily photo-lithotrophs, which can develop with CO2 as solecarbon source and H2S, other reduced inorganicsulfur compounds, or H2 as reductant. Nonsulfurpurple bacteria are primarily photoorganotrophs,employing organic compounds as the principalcarbon source and source of reducing power. Thevalue of this conventional distinction is dimi-nished by the fact that all purple sulfur bacteriaso far studied in pure culture can also growphotosynthetically with organic substrates.Furthermore, although nonsulfur purple bacteriacannot use sulfide as a reductant for photo-lithotrophic growth, one species can use thiosul-fate for this purpose, and probably all can use H2.A distinction between the two groups can also

be made on the basis of the autotrophy of thepurple sulfur bacteria, as contrasted with theheterotrophy of the nonsulfur purple bacteria,which need vitamins for growth. However,Rhodomicrobium vannielii, although in otherphysiological respects assignable to the nonsulfurpurple bacteria, is an autotroph. Lastly, itshould be mentioned that some nonsulfur purplebacteria can use respiratory metabolism as ameans for aerobic growth in the dark, whereas allpurple sulfur bacteria are strict anaerobes, andconsequently obligate phototrophs.

III. RECOGNITION OF BACTERIALPHOTOSYNTHESIS

Although purple bacteria had been observedsince the earliest days of microbiology and werethe subject of classical physiological investiga-tions by Winogradsky (79) and Engelmann(19, 20) in the late 19th century, the photosyn-

31961.]

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STAN IER

Figure 1. Structure of the characteristic photosynthetic pigments in the three major groups of photo-graphs.

thetic nature of their metabolism was con-

clusively established only in 1930, by van Niel(70). The obstacles to the recognition of bacterialphotosynthesis were intellectual, rather thantechnical: notions, too rigidly held, about thenature of the photosynthetic process. The gross

nature of green plant photosynthesis, a light-catalyzed conversion of carbon dioxide andwater to cell material, with accompanyingevolution of oxygen, had been recognized at thevery beginning of the 19th century, although itwas a good many years before the familiarstoichiometry:

CO2 + H20 lh (CH2O) + 02

was really firmly established. By the end of the19th century, several generations of plant physiol-ogists had been able to convince themselves andthe rest of the scientific world that all plants,from algae to flowering plants, carry out photo-synthesis according to this equation. Theycommitted the classical inductive error, illus-trated in courses on logic by the statement thatall swans are white, and concluded that allphotosynthetic organisms must perform as

prescribed by the equation for green plant

photosynthesis. It then required great intellectualdaring to realize that green plant photosynthesismight be merely a special case of a class ofmetabolic reactions; yet this realization was

essential for the recognition of bacterial photo-synthesis.A second obstacle existed in the case of the

purple bacteria. The invariable association ofchlorophyll with the occurrence of the photo-synthetic process had become clearly recognizedin the 19th century. Although bacteriochlorophyllis in fact closely related chemically to the plantchlorophylls, its spectrum both in cells and inorganic solvents is so radically different as tosuggest a complete lack of chemical relationship.Between 1880 and 1890, Engelmann (19, 20) haddefined the intracellular form of this pigmentspectrally, even to its infrared band (a brilliantphysical achievement for the time), and hadshown that the biological response of purplebacteria to light is mediated through it. Yet thesignificance of these experiments, which rankamong the most elegant researches in cell physi-ology of the 19th century, was completely lost onhis contemporaries and immediate successors.

The reason is obvious: the spectral properties ofpurple bacteria were so different from those of

CHARACTERISTIC PIGMENTS OF THE MAJOR PHOTOSYNTHETIC GROUPS

BIOLOGICAL GROUPALGAE AND | PURPLE GREEN

HIGHER PLANTS BACTERIA BACTERIAMOLECULAR GRWJND PLAN SPECIAL FEATURES

C R2 R3 charphlI g bocteriochorophyll chlorobium chloro-H common to all. common to oll. phylls.L molecular.O RI I R4 R2=VinyI R2=acetyl. two SmpleculaR N N seisol

O )<." Ring IV reduced. Rings 11 and IV one of which0> N N reduced. occurs in anyPNN gi~~~~~~~~~~~~~~venstroin

H structures unknownL~~~~~~~~~~~~~~~~~~~u igsystem

L isofbittr agtype.NOLECULAR OND PLAN SPECIAL FEATURES _

A a-or-carotene various pigments. y-coroteneR present in all. present in all.O p both ends of all open chain one end of chainT t.i&r. JJ chain cyclized. compounds usually cyclized.E with methoxylN ~~~~~~~~~~~~substituents

DS

4 [VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

plants that the functional homology of the twopigment systems appeared most improbable.The purple sulfur bacteria develop char-

acteristically in anaerobic environments exposedto light where hydrogen sulfide is being generated;they are abundant in sulfur springs and ingrossly polluted brackish ponds where sulfide isproduced biologically, as a result of the activitiesof sulfate-reducing bacteria. The fact that suchpurple bacteria are capable of oxidizing sulfide tosulfate had been established by Winogradsky in1888 (79), but before van Niel's time they hadnot been grown in pure culture. Van Niel showed(71) that these purple bacteria, and also thegreen bacteria, will develop in a completelyinorganic medium containing the necessaryminerals, bicarbonate, and sulfide. They arestrict anaerobes, and light is indispensable fortheir growth. Quantitative analyses of thechemical changes that occurred in pure culturesrevealed a close stoichiometric relationshipbetween the oxidation of sulfide and the uptake ofcarbon dioxide.The oxidation of sulfide characteristically

proceeds in a stepwise fashion; sulfide is firstconverted almost quantitatively to elementalsulfur, and the accumulated sulfur is then furtheroxidized to sulfate:

CO2 + 2H2S (CH20) + H20 + 2S(1st step)

3CO2 + 2S + 5H20 -+ 3(CH20) + 2H2SO4(2nd step)

2C02 + H2S +2H20 2(CH20) + H2SO4(Over-all reaction)

As is particularly clearly shown by the equationfor the first step, this reaction is coupled oxida-tion-reduction, in which carbon dioxide serves asthe oxidant and H2S as the reductant. At firstsight, it bears little resemblance to the classicequation for green plant photosynthesis, whichimplies that oxygen is derived in part from waterand in part from carbon dioxide. Van Niel per-ceived, however, that a strict formal homologycould be achieved if the equation for green plantphotosynthesis were rewritten with the additionof a molecule of water to both sides:

C02 + 2H20 - (CH2O)+1H20+02This apparently gratuitous emendation stated

what was then a novel hypothesis about themechanism of green plant photosynthesis;namely, that it is a coupled oxidation-reductionin which CO2 is reduced to cell material whilewater is oxidized to molecular oxygen. As vanNiel saw, both this equation and the variousequations which can be written for bacterialphotosynthesis at the expense of reduced sulfurcompounds can be regarded as special cases of ageneral photosynthetic reaction:

CO2 + 2H2A (CH20) + H20 + 2A

The validity of this interpretation of greenplant photosynthesis was subsequently con-firmed. Tracer studies with 018 demonstratedthat the oxygen evolved originates from water,and not from carbon dioxide (12, 61, 77). Fur-thermore, it was discovered (40) that isolatedchloroplasts can evolve 02 upon illumination inthe absence of C02, if provided with an artificialelectron acceptor. Such a modification of greenplant photosynthesis is now known as a Hillreaction.

IV. CONCEPT OF THE PHOTOLYSISOF WATER

Van Niel's original studies on bacterial photo-synthesis led to a new and more correct inter-pretation of the essential nature of green plantphotosynthesis. They also forced a re-evaluationof the central problem of photosynthesis, namely,the nature of the light-catalyzed reaction. Ifone examines the equation for bacterial photo-synthesis with sulfide, the reason for this isimmediately evident. Of the two half-reactions,the oxidation of hydrogen sulfide to elementalsulfur (and thence to sulfate) was well known tobe a dark reaction, which can be carried out by avariety of nonphotosynthetic bacteria. Further-more, the reduction of C02 to organic compoundscan also clearly proceed in the dark. Even in1930, this was evident from the existence of thechemolithotrophic bacteria, and in the years thatfollowed, many instances of CO2 reduction bychemo-organotrophic organisms were discovered.Light is therefore not intrinsically necessary foreither of the two half-reactions of bacterialphotosynthesis. However, when van Niel (73)made a similar analysis of the two half-reactionsof green plant photosynthesis, a different con-clusion emerged. At least in biological systems,the oxidation of water with the formation of

19611 5

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

molecular oxygen is not known to occur as adark reaction. It was therefore tempting topostulate that light in some way intervenes inthis cleaving of water. Despite repeated attemptsby many investigators, the evolution of oxygenby illuminated photosynthetic bacteria has notbeen demonstrated; in these organisms, photo-synthesis is never accompanied by oxygenrelease. Hence if one wished to assume a commonprimary light reaction in all types of photosyn-thesis, it could not be a cleaving of water withoxygen formation. These considerations led vanNiel, some years after his original work on thepurple and green bacteria, to propose a furthergeneralization about the nature of photosyn-thetic processes (73). He suggested that thecommon primary light reaction in all photo-syntheses is a cleaving of water, with the forma-tion of an oxidized and a reduced moiety, whichare conventionally designated as [H] and [OH].It is not thermodynamically possible for theseentities to be free radicals, and the squarebrackets were therefore symbolic representationsof ignorance concerning their real physicalnature. The beauty of this suggestion was that itpermitted a very simple mechanistic inter-pretation of the photosynthetic process whichapplied alike to green plant and bacterial photo-synthesis, as shown in figure 2.

In all types of photosynthesis, the reducedmoiety from the photochemical cleavage ofwater is ultimately applied to the reductive stepin the conversion of CO2 to cell material. Thebasic difference between bacterial and green plantphotosynthesis must then reside in the fate of[OH], the oxidized moiety. Van Niel assumedthat green plants possess a special enzymesystem capable of generating 02 from this entity;the bacteria, lacking such an enzyme, dispose of[OH] by using it as a terminal oxidant for theexternal electron donor, H2S (or, more generally,HA).

V. ROLE OF ORGANIC SUBSTRATESIN BACTERIAL PHOTOSYNTHESIS

The photosynthetic metabolism of organicsubstrates was first systematically studied byGaffron (28, 29), with nonsulfur purple bacteria,shortly after van Niel's work on the green andpurple sulfur bacteria. Gaffron showed thatmany fatty acids can be rapidly metabolized

anaerobically in the light by nonsulfur purplebacteria, with a relatively small accompanyingmetabolic uptake or release of C02, the sign andmagnitude of which are correlated with theoxidation level of the organic substrate. Carefulmeasurements of the stoichiometry of photo-synthesis with fatty acids as substrates ledGaffron to the conclusion that the metabolicproduct is a cellular reserve material somewhatmore reduced than carbohydrate, with theempirical formula CJH602. He was able to isolatea small amount of polymeric substance withthis empirical formula from the bacterial cells(29), but did not determine its structure. Gaffroninterpreted the photometabolism of organicacids by purple bacteria as a light-catalyzedassimilation of the organic substrate, fixation ofCO2 occurring only when surplus reducingpower is made available, as a result of theassimilatory product being more oxidized thanthe substrate. In effect, according to Gaffron'sinterpretation, CO2 reduction serves as a meansfor preserving the oxidation-reduction balance inthis anaerobic type of metabolism.

His interpretation was vigorously challengedby van Niel (72, 73) who showed that the actualdata could be explained differently by assumingthat the organic substrate acts as a hydrogendonor in accord with the generalized photo-synthetic equation:

CO2 + 2H2A -g > (CH20) + H20 + 2A

Van Niel did not exclude the possibility thatpart of the carbon of the organic substrate mightbe directly assimilated by the cell, but he insistedthat it must always in part serve as an ultimatehydrogen donor for the reduction of CO2. Insofaras an organic substrate serves the function of ahydrogen donor, it will normally be oxidized toCO2. Carbon dioxide will therefore be both areactant and a product of the photosyntheticreaction, and gross analysis of the over-all processcannot be expected to reveal its mechanism.

Gaffron's interpretation of his own datafitted the observations then available just as well,and perhaps slightly better, than the interpreta-tion of van Niel. Nevertheless, Gaffron's hypo-thesis failed to gain general acceptance, and waseventually abandoned even by its author (23).The principal reason for this was undoubtedlythe impossibility of incorporating it into anygeneral interpretation of photosynthetic pro-

6 (VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

hy

ichlorophyll

H20

cell material plants)

(all tphototrophs) [H] [OH]

CO2 (bacteria)to oxidationof external

hydrogen donors

Figure 2. A diagrammatic illustration of the unitary hypothesis of van Niel concerning the mechanismof photosynthetic processes.

cesses, as these were understood at the time. Theconcept of a direct photoassimilation of organiccompounds left no necessary place either for afixation of carbon dioxide or for a photochemicalcleavage of water. Its acceptance would thushave caused the abandonment of van Niel'ssuperb generalizations, the intellectual beautyand logical force of which were evident to all.

There was, furthermore, one experimentalobservation which lent support to van Niel'sinterpretation of the role of organic substrates inphotosynthesis. In 1940 Foster (22), then workingin van Niel's laboratory, isolated a Rhodopseu-domonas strain which performed an incompleteoxidation of isopropanol, with the stoichiometricformation of acetone. Foster was able to showthat in this special case, the photometabolism ofan organic compound conformed closely to thegeneralized equation of van Niel:

CO2 + 2 isopropanol(CH20 + H20 + 2 acetone

Further progress in the understanding ofphotosynthesis with organic substrates was slow.As time went on, however, a number of factsemerged which suggested that the isopropanolreaction of Foster was not a satisfactory generalmodel. When C0402 was used to measure theex-tent of CO2 fixation during the photometab-olism of organic substrates, some surprisingresults were obtained. For example, Glover andKamen (36) and Ormerod (57) observed thatduring the photometabolism of acetate byRhodospirillum rubrum, the rate of CO2 fixationwas actually lower than in control cells not fur-nished with an exogenous organic substrate.

It will be recalled that many nonsulfur purplebacteria can develop aerobically in the dark atthe expense of organic substrates; under thesecircumstances they carry out a purely respiratorymetabolism (73) with the use of the tricarboxylicacid (TCA) cycle as a mechanism of terminalsubstrate oxidation (16). Van Niel's interpreta-tion of the photosynthetic process implies thatwhen the same substrates are metabolizedanaerobically in the light, they undergo a broadlysimilar type of degradation, except that theoxidized product of water cleavage, [OH], re-places molecular oxygen as terminal oxidant.One might therefore expect that the TCA cycleshould operate in a like manner in the respiratoryand photosynthetic metabolism of organicsubstrates. This aspect of the problem wasanalyzed experimentally a few years ago byElsden and Ormerod (18), who studied theeffects of fluoroacetate on the light and darkmetabolism of many organic substrates byRhodospirillum rubrum.

Fluoroacetate, the mode of action of which wasfirst elucidated by the classic studies of Peterset al. with mammalian systems (58), inhibitsterminal respiration as a consequence of itsability to replace acetate in the first step of theTCA cycle, the condensation of acetyl-CoAand oxalacetate to citrate. The product of thefluoroacetyl-CoA condensation, fluorocitrate, in-hibits the enzyme aconitase, which catalyzes theensuing step of the cycle, and thus rapidlybrings terminal respiration to a halt. Elsden andOrmerod showed that the classic effects offluoroacetate poisoning, inhibition of respirationand accumulation of citrate, were readily demon-

719Gl ]

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

strable in purple bacteria during the dark oxida-tion of all organic substrates tested. However, anentirely different picture emerged when theeffect of fluoroacetate on photosynthetic metab-olism of the same organic compounds wasexamined. Only with acetate and pyruvate werethe typical symptoms of fluoroacetate poisoningobserved. The metabolism of propionate, suc-cinate, and malate was scarcely affected. Withbutyrate, an inhibition of photosynthesis wasfound, but this was unaccompanied by theaccumulation of citrate. From these data onemay conclude that the TCA cycle, universallyoperative in the respiratory metabolism ofpurple bacteria, ceases to be a major pathway inthe light except with acetate and closely relatedsubstrates.

Thus, there emerged a series of facts whichcould not be easily reconciled with van Niel'sinterpretation of the photometabolism of organicsubstrates. Dislike of this interpretation wasvoiced (32), but mere dislike is not an adequateweapon for the destruction of a well entrenchedhypothesis. As often happens in science, it waschance discovery rather than deep thought whicheventually provided the solution of this problem.In the course of other studies, our colleague Dr.Germaine Cohen-Bazire observed that nonsulfurpurple bacteria growing photosynthetically withacetate accumulate massive intracellular storesof sudanophilic granules. Isolation and analysisof these granules (13) showed that they consistedlargely of poly-f3-hydroxybutyric acid, (C4H602)n.Gaffron's postulated assimilatory product wasthus rediscovered and identified chemically. Infact, this polyester had been known as a majorbacterial cell constituent for over 30 years. Itwas originally found in a Bacillus species byLemoigne (45), and has subsequently beendetected as a reserve material in many otherchemotrophic bacteria (13, 21, 46, 53).

Studies of the photometabolism of C14-labeledacetate and butyrate by resting cells ofRhodospirillum rubrum (13) showed that thesefatty acids are incorporated into the intracellulardepot of poly-f3-hydroxybutyrate with highefficiency, and virtually without dilution of theirspecific activity. On the physiological level,polymer synthesis can be regarded as a mecha-nism for the intracellular storage of largequantities of fatty acid carbon; as a result ofpolymerization, the acidic substrate is neutralized

and made osmotically inert. Upon removal of theexternal organic carbon source, the cell can drawon this internal carbon store for general bio-synthesis. The process is formally analogous tothe formation of starch as a primary photosyn-thetic assimilate in plants.

Further work (67) showed that Rhodospirillumrubrum accumulates large stores of poly-[3-hydroxybutyrate only when furnished witheven-carbon fatty acids (acetate, /3-hydroxy-butyrate, butyrate). Photometabolism of suchsubstrates as succinate, malate, and propionateby either growing or resting cells leads to theaccumulation principally of a second photo-synthetic assimilate, a glycogen-like polysac-charide. This polysaccharide is also the majorprimary product of photosynthetic C02 assimila-tion in the presence of H2. The paths of carbonassimilation by purple bacteria are consequentlycomplex, the nature of the primary assimilatoryproduct being determined by the chemicalnature of the substrate.The photosynthetic formation of poly-,B-

hydroxybutyrate from acetate and butyrate isbiochemically a relatively simple process whichillustrates in the clearest possible fashion theessential nature of the photometabolism oforganic substrates. The conversion of acetate topolymer is a reductive synthesis:

2n 02H402+ 2nH -+ (CM602)n + 2n H20

It therefore cannot proceed without an inputof electrons. These are normally provided by theoxidation of some acetate via the TCA cycle, thebalanced equation for the gross reaction being:

9n C2H402 > 4(C4H602)n + 2n CO2 + 6n H20This obligatory coupling of synthesis with

oxidation explains the observation of Elsden andOrmerod (18) that the photometabolism ofacetate, unlike that of most other organic sub-strates, is inhibited by fluoroacetate with anaccompanying accumulation of citrate. Far frombeing coupled with a reduction of C02, thephotosynthesis of poly-,B-hydroxybutyrate fromacetate actually competes with C02 reduction forthe limited reducing power available. Thisexplains the observations (36, 57) that the rateof C02 fixation during the photometabolism ofacetate may be less than the endogenous rate.The anaerobic oxidation of some acetate

becomes unnecessary if an external reductant for

8 [VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

polymer synthesis can be provided. Indeed, asGaffron first showed (29), the photometabolismof acetate can be coupled with an uptake ofmolecular hydrogen. We have restudied thekinetics of this interesting photosynthesis andhave found that assimilation of acetate is muchmore rapid in hydrogen than in helium, no doubtbecause the rate of its photosynthetic assimila-tion is normally limited by the rate at whichacetate can be oxidized through the cycle (67).The acetate-hydrogen reaction can be repre-sented by the equation:

2n acetate + n ih2 (C4H6O2). + 2n H20/3-Hydroxybutyrate is rapidly photometabo-

lized by R. rubrum without any uptake or for-mation of CO2 (unpublished observations). Itsphotosynthetic conversion to polymer can berepresented by the equation:

n C4H803 (C4H602)n + n H20This is the purest example of the direct photo-

assimilation of an organic substrate. There isno photoreduction, for the simple reason thatnothing is oxidized or reduced. Hence the reac-tion leaves no obvious place for the photolysisof water with the generation of an oxidation-reduction system, which is the central commonevent of photosynthesis according to van Niel'shypothesis. Yet it is undoubtedly a photosyn-thetic process, since light is indispensable for itsoccurrence. Ten years ago it would not havebeen possible to propose a unitary hypothesiscapable of furnishing a common link betweenthis reaction and the more familiar photosyn-thetic reactions. Fortunately, the situation todayis not so bleak. Since 1950, advances in otherdirections have revealed a new common denomi-nator of photosynthesis, which was almost un-suspected and completely unsupported by ex-perimental evidence when van Niel proposedthe photolysis of water.

VI. PHOTOPHOSPHORYLATION

Between 1945 and 1954, the path of photo-synthetic carbon dioxide assimilation in greenalgae was mapped by tracer methods, largelyby Calvin and his group (8). During the sameperiod, the previously unknown steps of thissequence were independently elucidated at theenzymatic level (60, 78). The details of this

pathway need not concern us here, since thecentral importance of this body of work for theunderstanding of photosynthesis lies not in thebiochemical step reactions so revealed, but ratherin the definitive establishment of the net chemicalrequirements for the reduction of a mole of carbondioxide to the carbohydrate level. These require-ments are 2 moles of reduced pyridine nucleo-tide and 3 moles of ATP. The formation ofreduced pyridine nucleotide did not pose an in-trinsically new problem; in theory, the photo-lysis of water postulated by van Niel couldfurnish an unlimited quantity of reductant atthis potential level. The formation of the re-quired ATP was another matter, however. Forthe first time, the bioenergetics of photosynthesiswas removed from the dense and rather smoggyatmosphere of quantum efficiency measurementsand thermodynamic argumentation in which itwas then shrouded and faced squarely as a bio-chemical problem. We need not retrace here indetail the rather tortuous path which was fol-lowed in the search for the mode of origin ofATP in green plant photosynthesis (4). The cor-rect answer was eventually provided by Arnonet al. (7), with the discovery that plant chloro-plasts perform a hitherto unknown light-inducedsynthesis of ATP from ADP and inorganic phos-phate. During the past few years, this group hassucceeded in demonstrating two distinct ATP-generating reactions catalyzed by chloroplastmaterial (5). The first of these is cyclic photo-phosphorylation, a reaction which can be formu-lated as:

ADP + P -i ATP

and which can occur under strictly anaerobicconditions. in the absence of photosyntheticoxygen evolution. The second type of ATP-generating reaction in chloroplasts can be ex-pressed by the over-all equation:

TPN + 2H20 + ADP + PiTPNH2 + M 02+ ATP + H20

As suggested by the above equation, the esteri-fication of inorganic phosphate is in this caselinked with photosynthetic oxygen evolutionand with the generation of reduced pyridinenucleotide. In effect, it represents a coupledHill reaction.Almost simultaneously with the discovery of

919611

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

photophosphorylation in chloroplast prepara-tions, Frenkel (24) discovered the same phenom-enon in preparations of bacterial chromatophores.Despite considerable subsequent work on photo-phosphorylation with cell-free bacterial prepara-tions (2, 25, 26, 31, 55), the only light-inducedreaction for the synthesis of ATP which has beenfound is cyclic photophosphorylation. Since thesecond, noncyclic reaction characteristic of chlor-oplast preparations is intimately linked withtheir capacity for oxygen evolution, its absencein bacteria is, of course, readily understandable.Cyclic photophosphorylation thus emerges asthe one common biochemical denominator ofall types of photosynthesis.The work that has been done in the past few

years on the mechanism of cyclic photophos-phorylation with both plant and bacterial sys-tems (reviewed in references 6 and 27) suggeststhat formation of ATP is coupled with the trans-port of electrons through a chain of carriersassociated with the photosynthetic apparatus.Cyclic photophosphorylation thus has a certainanalogy with oxidative phosphorylation, theessential difference being that in cyclic photo-phosphorylation both oxidant and reductantare endogenously generated by the primary lightreaction. The detailed structure of the carriersystem is not yet clearly established. However,cytochromes comprise an important component,just as in oxidative electron transport. The firstevidence for the association of cytochromes withthe photosynthetic apparatus was provided in1951 by Hill and Scarisbrick (41), who describeda new plant cytochrome of the c type, cyto-chrome f, which is specifically localized in chloro-plasts. Shortly afterwards, Vernon (76) reportedlarge amounts of a c type cytochrome in extractsof Rhodospirillum rubrum. Similar pigments havesubsequently been found in other photosyntheticbacteria (17, 35, 43, 54), including the obliga-tory anaerobic purple sulfur and green bacteria,in which they obviously cannot play a role inrespiratory electron transport.

Direct evidence for the participation of thesepigments in the events of photosynthesis hasbeen obtained by examination of the spectralchanges that occur when suspensions of algaeor photosynthetic bacteria are illuminated (10,11, 15, 66; summary in 65). The spectral changescaused by illumination are rather complex, sincelight also induces marked shifts in the absorp-

tion bands of the other respiratory carriers andof the carotenoid pigments of the photosyntheticapparatus. However, it is clear that in severalkinds of photosynthetic cells, illumination evokesan immediate oxidation of cytochrome, whichis rapidly reversed when the light is turned off.

VII. ARNON'S MODEL FOR PHOTO-PHOSPHORYLATION

On purely formal grounds, it is possible toreconcile recent observations concerning photo-synthetic ATP generation and cytochrome func-tion with van Niel's hypothesis that the primaryphotochemical event in photosynthesis is thephotolysis of water. The recombination of thereduced and oxidized moieties through an inter-nal transport system could provide the physicalbasis for cyclic photophosphorylation, and thephotosynthetic generation of [OH] could accountfor light-induced cytochrome oxidation. How-ever, Arnon (6) has suggested a more attractivehypothesis. He proposes that the absorption of alight quantum by chlorophyll results in theexpulsion of an electron at a high energy poten-tial, created at the expense of the energy con-tained in the absorbed light quantum. The chloro-phyll molecule thereby becomes electropositiveand immediately accepts an electron from cyto-chrome, which is accordingly oxidized. In cyclicphotophosphorylation, the electron which hasbeen expelled from chlorophyll passes throughthe photosynthetic electron transport chain andis finally captured by the oxidized cytochrome,part of its energy being drained off in passageas ATP (figure 3). This, then, would be the es-sential sequence of events common to both bac-terial and green plant photosynthesis. In greenplants a second possible fate awaits the expelledelectron. It may be accepted by TPN, whichsimultaneously picks up a hydrogen ion fromwater to form reduced TPN. At the same time,the oxidized cytochrome is reduced by the trans-fer of an electron from a hydroxyl ion, a reactionwhich is envisaged as resulting in ATP formationand concomitant oxygen evolution (figure 4).

VIII. PHOTOSYNTHETIC HYDROGEN EVOLUTIONAND NITROGEN FIXATION

As discovered by Gest and Kamen (33, 34),purple bacteria can under certain conditionsevolve molecular hydrogen upon illumination.Such hydrogen production requires the presence

10 [VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

(P.

.?p,I.ATP

LIGHTFigure S. A simplified version of the model of

Arnon (6) to illustrate the mechanism of cyclicphotophosphorylation.

TPN

LIGHTFigure 5. The mechanism postulated by Losada,

Nozaki, and Arnon (49) to account for photo-hydrogen evolution from thiosulfate by purplebacteria.

LIGHTFigure 4. A simplified version of the model of

Arnon (6) to illustrate the mechanism of non-

cyclic photophosphorylation in green plants.

of an oxidizable substrate, and is rigorouslydependent on light. Most of the work on photo-hydrogen production by purple bacteria hasbeen done with organic substrates. Recently,Losada, Nozaki, and Arnon (49) have shownthat in Chromatium a light-dependent evolutionof hydrogen can be obtained at the expense of a

simple inorganic oxidizable substrate, thiosul-fate. They propose that the mechanism involvesa reduction of hydrogen ions by electrons ex-

pelled by light from chlorophyll, oxidation-reduction balance being preserved by electrontransfer to chlorophyll from thiosulfate via thetransport system of the photosynthetic apparatus(figure 5).

Early in the work on photohydrogen produc-tion, it was observed (42) that molecular nitrogenacts as a repressor. This observation led to thediscovery that both purple and green bacteriaare capable of nitrogen fixation when growinganaerobically in the light (47). The capacity fornitrogen fixation by facultatively aerobic purplebacteria under aerobic conditions in the dark is,in contrast, of negligible magnitude (48, 59). Thisphenomenon can also be readily interpreted interms of Arnon's model. The fixation of molecularnitrogen represents, in effect, another possibleway of utilizing the electrons expelled fromchlorophyll by light (Arnon, et al., 7a).Photohydrogen production and photosyn-

thetic nitrogen fixation appear to be of universaloccurrence in photosynthetic bacteria, but arenot strictly confined to these phototrophs. Aswas shown many years ago by Gaffron and Rubin(30), "hydrogen-adapted" green algae (i.e., algaein which formation of the enzyme hydrogenasehas been induced by exposure to molecularhydrogen) are capable of hydrogen evolutionin the light, presumably at the expense of en-dogenous electron donors. It may be supposedthat any photosynthetic organism is potentiallyable to perform this reaction, if it contains hydro-genase as a constitutive enzyme or can form itby induction. Photosynthetic nitrogen fixationis characteristic of many blue-green algae (1).A similar argument can be applied in this case.

II1119611

e-

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

Any phototroph, if capable of synthesizing theenzymatic machinery for nitrogen fixation, willbe able to use the reducing power available fromthe light activation of chlorophyll to reducemolecular nitrogen. The ubiquity of photohydro-gen evolution and photosynthetic nitrogen fixa-tion in photosynthetic bacteria and the rarity ofthese processes in green plants therefore probablyreflect a general difference between these twogroups with respect to the "dark" enzymaticconstitution of the cell, and not a difference withrespect to the photosynthetic machinery itself.

IX. REINTERPRETATION OF GROSS REACTIONSOF BACTERIAL PHOTOSYNTHESIS

The advances in our knowledge of the centralevents of photosynthesis which have been out-lined in the preceding sections permit a newinterpretation of the various gross reactions ofbacterial photosynthesis. Let us reconsider thephotosynthetic conversion of 13-hydroxybutyrateto poly-f-hydroxybutyrate. Merrick and Doudo-roff (52) have recently shown with cell-free prep-arations of Rhodospirillum rubrum that theimmediate substrate for polymer synthesis is f-hy-droxy butyryl-CoA. Polymer formationwith /3-hy-droxybutyric acid as an exogenous substrate thusrequires a minimal input of 1 mole of ATP permole of acid assimilated. The reaction is rigor-ously light-dependent, for the simple reason thatunder anaerobic conditions cyclic photophos-phorylation is the only mechanism available tothe cell for making ATP. This photosynthesiscan therefore be more correctly represented bythe coupled reactions:

lightn ADP + nPi- It ATPand

n ATP+nC4H8,C3A(C4H602)n + n ADP + n Pi + n H20

An additional problem arises in connectionwith those bacterial photosyntheses in whichthe gross synthetic reaction is reductive, whetherthis be the photometabolism of acetate or ofCO2 in the presence of an oxidizable inorganicsubstrate such as H2S or H2. In green plants,reducing power in the form of reduced pyridinenucleotide is provided ultimately by water,through intervention of the photochemicalsystem. In bacteria, however, a chemical reduc-tant other than water must be furnished. Provided

ATP . CH3COOHlight a+Pi ,(+HSCoA

ADP CH3CO-SCoA

CH3COCHCO- SCoA PNred

CH3CHOHCH2CO-SCoA PNox H2-HSCoA

poly- - hydroxybutyric acid

Figure 6. The mechanism postulated by Stanieret al. (67) to account for the acetate-hydrogenreaction in purple bacteria discovered by Gaffron(29).

C02

,S

H2S

(CH20)

Figure 7. A modern interpretation of the grossmechanism of photosynthetic CO2 assimilationwith H2S as reductant by purple and green bac-teria. Compare with the classical interpretationshown in figure 2.

that electrons can be removed from this reductantat the pyridine nucleotide level by purely enzymaticmeans, there is no logical need to invoke the inter-position of the photochemical system for the provi-sion of reducing power. This is illustrated (figure6) by our concept of the acetate-hydrogen reac-tion (67). Here, just as in the photoassimilationof f-hydroxybutyric acid, the only role whichmust be attributed to light is ATP synthesis.The photoassimilation of CO2 with energy-richinorganic electron donors such as H2S and H2can be similarly interpreted, as shown diagram-matically in figure 7.The situation is not so simple in the case of a

donor such as succinate, which transfers electronsat a potential level below that of the pyridinenucleotides. Electrons from succinate cannotserve for the direct reduction of DPN or TPN.Frenkel (26) has observed that bacterial chro-matophores are capable of performing a light-dependent reduction of DPN, coupled with theoxidation of either succinate or reduced flavin

12 [VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS 13

LIGHT LIGHTFigure 8. Mechanisms for the photochemical generation of reducing power in purple bacteria (left)

and in green plants (right).

mononucleotide. This reaction proceeds indepen-dently of cyclic photophosphorylation. Evidently,in this situation radiant energy can interveneto boost the potential of the electrons from donorsthat are incapable of direct coupling with pyridinenucleotide. The Frenkel reaction, interpreted interms of the Arnon model (figure 8), has a certainformal analogy with the water-linked generationof reduced TPN in green plant photosynthesis,and might indeed be regarded as an evolutionaryprecursor of it.

X. NATURE OF THE PRIMARY LIGHT REACTION

Arnon's model of the central events of photo-synthesis represents a major departure from themodel of van Niel, which has dominated mostthinking in the field for the past 20 years. In thenew model, water cleavage is no longer a primaryevent. In fact, the participation of water inphotosynthesis becomes again, as it was assumedto be before 1940, a special feature of green plantphotosynthesis, with the further novel provisothat water is not directly involved in the photo-chemical event. In place of the photolysis ofwater, what is now proposed is a much simplerprimary electronic shift in chlorophyll, closelycoupled with a transfer of electrons from theassociated cytochrome.The electronic nature of the primary event in

photosynthesis was first suggested in 1949 byKatz (44), but this concept had not been in-tegrated into a general formulation of the photo-synthetic process until the model of Arnon was

proposed. We must now consider what experi-mental evidence there is for a primary event ofthis sort. The expulsion of an electron from chlor-

ophyll should cause marked changes in the chloro-phyll absorption bands of the photosyntheticapparatus. However, as we have already seen,

the rather complex spectral changes that followillumination of intact photosynthetic cells appearto reflect changes in the carotenoids and therespiratory pigments.The first clear evidence that illumination

provokes spectral changes attributable to chloro-phyll itself has been obtained recently by Arnoldand Clayton (3), with the use of dried films ofundenatured chromatophores prepared fromRhodopseudomonas spheroides. The effects are

particularly clear with chromatophores preparedfrom a carotenoidless mutant, and involve shiftsin the positions of all the major peaks of bac-teriochlorophyll. With chromatophores preparedfrom cells of the wild type, there are accompany-

ing changes in the carotenoid spectrum; suchchanges can, however, be abolished withoutaffecting the chlorophyll response by brief ex-

posure of the dried chromatophores to ethanolvapor. This light-induced spectral change ofchlorophyll is freely reversible and can be ob-tained repeatedly with a single preparationexposed to successive periods of illumination.The response is independent of temperature;

its magnitude remains unchanged between 1and 300'K. Since no ordinary chemical reactioncan take place at 1VK, the light-induced spectralchanges must reflect a purely electronic event.The participation of water in this reaction isexcluded by the anhydrous state of the biologicalmaterial.The question then arises, "Why cannot a

similar phenomenon be observed with intact

1961]

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

cells?" Arnold and Clayton have also furnisheda plausible answer to this question by showingthat metabolic inhibitors (e.g., azide and hydrox-ylamine) abolish the light-induced changes ofthe cytochrome spectrum in whole cells of Rhodo-pseudomonas spheroides and simultaneously reveallight-induced changes of the chlorophyll spectrumsuch as those observed with dried films of chro-matophores. Probably electron transport issufficiently rapid in the normally functioningphotosynthetic apparatus to prevent the accu-mulation of a pool of excited electrons; the spec-tral changes associated with chlorophyll excita-tion are therefore undetectable.

XI. CONCLUSION

We may now summarize briefly the new pictureof photosynthesis that is beginning to emerge.The only common physical event in all types ofphotosynthesis is the excitation of chlorophyllby light with the expulsion of electrons. This hasone universal chemical consequence: transfer ofthe electrons through a closed carrier systemcoupled to chlorophyll, with a concomitantgeneration of ATP. Several alternative chemicalfates for the electrons are possible (e.g., reductionof TPN in green plants, reduction of DPN inpurple bacteria, hydrogen evolution or nitrogenfixation in photosynthetic bacteria and certainalgae), but none of these is of universal occur-rence.The use that a photosynthetic cell makes of the

ATP generated through photophosphorylationis determined in part by its inherited enzymaticconstitution and in part by the environmentalconditions. In green plants, green bacteria, andpurple sulfur bacteria, the lion's share of theATP will normally be used to drive the synthesisof organic cell constituents from CO2. In non-sulfur purple bacteria (and also in purple sulfurbacteria growing in the presence of organicsubstrates) it will be used to drive the synthesisof organic cell constituents from the externallyprovided organic substrate. It is probable thatmany green plants could partly or entirely re-place CO2 with organic substrates as a source ofcarbon for the photosynthesis of cell material,just as do the purple bacteria, but critical experi-ments to test this point do not seem to have beenundertaken. However, it has been shown thatthe green tissues of higher plants (leaf disks)can perform a light-dependent synthesis of starch

from exogenous glucose (50). In this case, the-plant cell uses photosynthetically generated ATPfor a specific and limited assimilatory metabo-lism of an organic compound.We have recently examined the possible utili-

zation of organic carbon sources for generalcellular synthesis by Chlorobium limicola, agreen bacterium hitherto presumed to be anobligate lithotroph. It was found (62) that thisorganism can use acetate as a major source ofcellular carbon in the light; acetate uptake isstrictly dependent on the simultaneous provisionof CO2 and of an exogenous reductant (H2S).The different requirements for acetate utilizationby C. limicola and by purple bacteria can prob-ably be explained by the absence in C. limicolaof an enzymatic mechanism for the anaerobicoxidation of acetate. If acetate cannot be oxi-dized, it can provide neither the reducing powernor the CO2 required for the over-all processesof biosynthesis, and these requirements must befurnished from other sources.

Recent findings accordingly suggest that theuse of CO2 as the sole source of carbon for thesynthesis of cell material is not really a funda-mental feature of any photosynthetic process.The fact that most phototrophs normally douse CO2 as their sole carbon source is more satis-factorily interpreted as an evolutionary adapta-tion to the scarcity of organic material whichhas long existed on our planet. Photosynthesisfalls into a special metabolic category only interms of the mode of ATP formation; it cannotbe placed in a special metabolic category interms of the use that the cell makes of the ATPso generated.ATP is the common energetic currency of the

cell; it can be used to perform chemical, osmotic,or mechanical work. In the first study ever madeof the reactions of purple bacteria to light (19),Engelmann described a biological response whichcan now be interpreted as a utilization of photo-synthetically generated ATP to perform mechan-ical rather than chemical work. He observedthat purple bacteria suspended in water in asealed cover slip preparation will soon cease allmovement if kept in the dark. When this prepara-tion is illuminated, the cells become motile, andremain in active movement as long as illumina-tion is continued. Since no exogenous substrateis available, the cells cannot perform a grossphotosynthetic reaction, but light triggers the

14 [vo iJ. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

closed cycle of events which results in photo-synthetic phosphorylation. Under these severelyrestrictive conditions, one of the few effectiveuses which the cell can make of the resultingATP is the production of flagellar contractions.

In 1845, Mayer (51) defined photosynthesisas follows: "Die Pflanzen nehmen eine Kraft,das Licht, auf, und bringen eine Kraft hervor;die chemische Differenz."

In retrospect, we can see that work on photo-.synthesis during the intervening century hasbeen largely devoted to clearing away the metab-olic irrelevancies which prevented the restate-ment of this profound insight in more precisephysicochemical terms.

XII. REFERENCES1. ALLEN, M. B. 1956 Photosynthetic nitrogen

fixation by blue-green algae. Sci. Monthly,83, 100-106.

2. ANDERSON, I. C. AND FULLER, R. C. 1958Photophosphorylation by isolated chromato-phores of the purple sulfur bacteria. Arch.Biochem. Biophys. 76, 168-179.

3. ARNOLD, W. AND CLAYTON, R. K. 1960 Thefirst step in photosynthesis: evidence forits electronic nature. Proc. Natl. Acad.Sci. U. S., 46, 769-776.

4. ARNON, D. I. 1956 Phosphorus metabolismand photosynthesis. Ann. Rev. PlantPhysiol., 7, 325-354.

5. ARNON, D. I. 1958 Chloroplasts and photo-synthesis. In The photochemical apparatus:its structure and function. BrookhavenSymposia in Biol., 11, 181-235.

6. ARNON, D. I. 1959 Conversion by lightenergy into chemical energy in photosyn-thesis. Nature, 184, 10-21.

7. ARNON, D. I., ALLEN, M. B., AND WHATLEY,F. R. 1954 Photosynthesis by isolatedchloroplasts. Nature, 174, 394.

7a. ARNON, D. I., LOSADA, M., NOZAKI, M. ANDTAGANA, K. 1960 Photofixation of nitro-gen and photoproduction of hydrogen bythiosulfate during bacterial photosynthesis.Biochem. J., 77, Proc. Biochem. Soc., 23 P.

8. BASSHAM, J. A., BENSON, A. A., KAY, L. D.,HARRIS, A. Z., WILSON, A. T., AND CALVIN,M. 1954 The path of carbon in photosyn-thesis. XXI. The cyclic regeneration ofcarbon dioxide acceptor. J. Am. Chem.Soc., 76, 1760-1770.

9. BERGERON, J. A., 1958 The bacterialchromatophore. In The photochemical appa-ratus: its structure and function. Brook-haven Symposia in Biol., 11, 118-131.

10. CHANCE, B. AND SAGER, R. 1957 Oxygenand light induced oxidations of cytochrome,flavoprotein and pyridine nucleotide in aChlamydomonas mutant. Plant Physiol.,32, 548-561.

11. CHANCE, B. AND SMITH, L. 1955 Respiratorypigments of Rhodospirillum rubrum.Nature, 175, 803-806.

12. DOLE, M. AND JENKS, G. 1944 Isotopiccomposition of photosynthetic oxygen.Science, 100, 409.

13. DOUDOROFF, M. AND STANIER, R. Y. 1959Role of poly-,B-hydroxybutyric acid in theassimilation of organic carbon by bacteria.Nature, 183, 1440-1442.

14. DREWS, G. 1960 Untersuchungen zur Sub-struktur der "Chromatophoren" von Rhodo-spirillum rubrum und Rhodospirillummolischianum. Arch. Mikrobiol., 36, 99-108.

15. DUYSENS, L. N. M. 1954 Reversible photo-oxidation of a cytochrome pigment in photo-synthesizing Rhodospirillum rubrum.Nature, 173, 692-693.

16. EISENBERG, M. A. 1953 The tricarboxylicacid cycle in Rhodospirillum rubrum. J.Biol. Chem., 203, 815-836.

17. ELSDEN, S. R., KAMEN, M. D., AND VERNON,L. P. 1953 A new soluble cytochrome.J. Am. Chem. Soc., 75, 6347.

18. ELSDEN, S. R. AND ORMEROD, J. G. 1956The effect of monofluoroacetate on themetabolism of Rhodospirillum rubrum.Biochem J., 63, 691-701.

19. ENGELMANN, T. W. 1883 Bacterium photo-metricum. Ein Beitrag zur vergleichendenPhysiologie des Licht- und Farbensinnes.Pflugers Arch. ges. Physiol., 30, 95-124.

20. ENGELMANN, T. W. 1888 Die Purpurbak-terien und ihre Beziehungen zum Lichte.Botan. Ztg. 46, 661-669, 677-689, 693-701,709-720.

21. FORSYTH, W. G. C., HAYWARD, A. C., ANDROBERTS, J. B. 1958 Occurrence of poly-,3-hydroxybutyric acid in aerobic gramnegative bacteria. Nature, 182, 800-801.

22. FOSTER, J. W. 1940 The role of organicsubstrates in photosynthesis of purplebacteria. J. Gen. Physiol., 24, 123-134.

23. FRANCK, J., AND GAFFRON, H. 1941 Photo-synthesis, facts and interpretations. Ad-vances in Enzymol., 1, 199-262.

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

25. FRENKEL, A. W. 1956 Photophosphoryla-tion of adenine nucleotides by cell-free

1961] 15

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

R. Y. STANIER

preparations of purple bacteria. J. Biol.Chem., 222, 823-834.

26. FRENKEL, A. W. 1958 Light-induced re-actions of chromatophores of Rhodospiril-lum rubrum. In The photochemical appara-tus: its structure and function. Brook-haven Symposia in Biol., 11, 276-288.

27. FRENKEL, A. W. 1959 Light-induced re-actions of bacterial chromatophores andtheir relation to photosynthesis. Ann.Rev. Plant Physiol., 10, 53-70.

28. GAFFRON, H. 1933 Ueber den Stoffwechselder schwefelfreien Purpurbakterien.Biochem. Z., 260, 1-18.

29. GAFFRON, H. 1935 Ueber den Stoffwechselder Purpurbakterien. II. Biochem. Z.,275, 301-319.

30. GAFFRON, H. AND RUBIN, J. 1942 Fermen-tative and photochemical production ofhydrogen in algae. J. Gen. Physiol., 26,219-240.

31. GELLER, D. M. 1957 Photophosphorylationby Rhodospirillum rubrum preparations.Doctoral Thesis, Harvard University, Cam-bridge, Mass.

22. GEST, H. 1951 Metabolic patterns in photo-synthetic bacteria. Bacteriol. Revs., 15,183-210.

:33. GEST, H. AND KAMEN, M. D. 1949 Photo-production of molecular hydrogen byRhodospirillum rubrum. Science, 109, 558-559.

34. GEST, H. AND KAMEN, M. D. 1949 Studieson the metabolism of photosyntheticbacteria. IV. Photochemical productionof molecular hydrogen by growing culturesof photosynthetic bacteria. J. Bacteriol.,58, 239-245.

35. GIBSON, J. AND LARSEN, H. 1955 Cyto-chromes from Chlorobiurn thiosulfatophiluni.Biochem. J., 60, Proc. Biochem. Soc.,xxvii.

26. GLOVER, J. AND KAMEN, M. D. 1951 Ob-servations on the simultaneous metabolismof acetate and carbon dioxide by restingcell suspensions of Rhodospirillum rubrum.Federation Proc., 10, 190.

.37. GOODWIN, T. W. 1956 The carotenoids ofphotosynthetic bacteria. II. The car-otenoids of a number of non-sulfur purplephotosynthetic bacteria. Arch. Mikrobiol.,24, 313-322.

28. GOODWIN. T. W. AND LAND, D. G. 1956Studies in carotenogenesis. 20. Car-otenoids of some species of (Chlorobiurn.Biochem. J., 62, 553-556.

39. HICKMAN, D. D. AND FRENKEL, A. W. 1959

The structure of Rhodospirillum rubrumn.J. Biophys. Biochem. Cytol., 6, 277-289.

40. HILL, R. 1939 Oxygen produced by isolatedchloroplasts. Proc. Roy. Soc. (London),B127, 192-210.

41. HILL, R. AND SCARISBRICK, R. 1951 Thehaematin compounds of leaves. NewPhytologist, 50, 98-111.

42. KAMEN, M. D. AND GEST, H. 1949 Evi-dence for a nitrogenase system in thephotosynthetic bacterium Rhodospirililmnrubrum. Science, 109, 560.

43. KAMEN, M. D. AND VERNON, L. P. 1955Comparative studies on bacterial cyto-chromes. Arch. Biochem. Biophys., 17,10-22.

44. KATZ, E. 1949 Chlorophyll fluorescence asan energy flow meter for photosynthesis.In Photosynthesis in plants. pp. 287-292.Iowa State College Press, Ames, Iowa.

45. LEMOIGNE, M. 1927 Etudes sur l'autolysemicrobienne. Origine de l'acide f-oxybuty-rique form6 par autolyse. Ann. inst.Pasteur, 41, 148-166.

46. LEMOIGNE M. AND GIRARD, H. 1943 Re-serves lipidiques ,B-hydroxybutyriques chezAzotobacter chroococcum. Compt. rend.acad. sci. Paris, 217, 557-558.

47. LINDSTROM, E. S., BURRIS, R. H., AND WIL-SON, P. W. 1949 Nitrogen fixation byphotosynthetic bacteria. J. Bacteriol., 58,313-316.

48. LINDSTROM, E. S., NEWTON, J. W., AND WIL-SON, P. W. 1952 The relationship be-tween photosynthesis and nitrogen fixa-tion. Proc. Natl. Acad. Sci. U. S., 38,392-396.

49. LOSADA, M., NOZAKI, M., AND ARNON, D. I.1960 Photoproduction of molecular hydro-gen from thiosulfate by Chromatium cells.Symposium on Light and Life, in press.

50. MACLACHLAN, G. A. AND PORTER, H. K. 1959Replacement of oxidation by light as theenergy source for glucose metabolism intobacco leaf. Proc. Roy. Soc. (London),B150, 460-473.

51. MAYER, J. R. 1845 Die organische Bewe-gung. In Mechanik der waerme. J. G.Cotta, Stuttgart, Germany.

52. MERRICK, J. AND DOUDOROFF, M. 1960Enzymatic synthesis of poly-3-hydroxy-butyric acid in bacteria. Nature, in press.

53. MORRIS, M. B. AND ROBERTS, J. B. 1959 Agroup of pseudomonads able to synthesizepoly-,B-hydroxybutyric acid. Nature, 183,1538-1539.

54. NEWTON, J. W. AND KAMEN, M. D. 1956

16 [VOL. 25

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

PHOTOSYNTHETIC MECHANISMS

Chromatium cytochrome. Biochim. etBiophys. Acta, 21, 71-80.

55. NEWTON, J. W. AND KAMEN, M. D. 1957Photophosphorylation by subcellular par-ticles from Chromatium. Biochim. etBiophys. Acta, 25, 462-474.

56. NIKLOWITZ, W. AND DREWS, G. 1957Beitraege zur Cytologie der Blaualgen.Vergleichende Elektromikroskopische Un-tersuchungen zur Substruktur einiger Hor-mogonales. Arch. Mikrobiol., 27, 150-165.

57. ORMEROD, J. G. 1956 The use of radio-active carbon dioxide in the measurementof carbon dioxide fixation in Rhodospirillumrubrum. Biochem. J., 64, 373-380.

58. PETERS, R. A. 1952 Lethal synthesis.Proc. Roy. Soc. (London), B139, 143-170.

59. PRATT, D. C. AND FRENKEL, A. W. 1959Studies on nitrogen fixation and photo-synthesis of Rhodospirillum rubrum. PlantPhysiol., 34, 333-337.

60. RACKER, E. 1954 Alternate pathways ofglucose and fructose metabolism. Advancesin Enzymol., 15, 141-182.

61. RUBEN, S., RANDALL, M., KAMEN, M., ANDHYDE, J. L. 1941 Heavy oxygen (018)as a tracer in the study of photosynthesis.J. Am. Chem. Soc., 63, 877-879.

62. SADLER, W. R. AND STANIER, R. Y. 1960The function of acetate in photosynthesisby green bacteria. Proc. Natl. Acad. Sci.U. S., 46, 1328-1333.

63. SHACHMAN, H. K., PARDEE, A. B., ANDSTANIER, R. Y. 1952 Studies in macro-molecular organization of microbial cells.Arch. Biochem. Biophys., 38, 245-260.

64. SHATKIN, A. J. 1960 A chlorophyll-con-taining cell fraction from the blue-greenalga Anabaena variabilis. J. Biophys.Biochem. Cytol., 7, 583-584.

65. SMITH, L. AND CHANCE, B. 1958 Cyto-chromes in plants. Ann. Rev. Plant Phys-iol., 9, 449-482.

66. SMITH, L. AND RAMIREZ, J. 1959 Absorptionspectrum changes in photosynthetic bac-teria following illumination or oxygenation.Arch. Biochem. Biophys., 79, 233-244.

67. STANIER, R. Y., DOUDOROFF, M., KUNISAWA,R., AND CONTOPOULOU, R. 1959 The role

of organic substrates in bacterial photo-synthesis. Proc. Natl. Acad. Sci. U. S.,45, 1246-1260.

68. STANIER, R. Y. AND SMITH, H. J. C. 1960The chlorophylls of green bacteria.Biochim. et Biophys. Acta, 41, 478-484.

69. STRAIN, H. H. 1949 Functions and prop-erties of the chloroplast pigments. InPhotosynthesis in plants. pp. 133-178. IowaState College Press, Ames, Iowa.

70. VAN NIEL, C. B. 1930 Photosynthesis ofbacteria. In Contributions to marine biology.p. 158. Stanford University Press, PaloAlto, Calif.

71. VAN NIEL, C. B. 1931 On the morphologyand physiology of the purple and greensulphur bacteria. Arch. Mikrobiol., 3,1-112.

72. VAN NIEL, C. B. 1935 Photosynthesis ofbacteria. Cold Spring Harbor SymposiaQuant. Biol., 3, 138-150.

73. VAN NIEL, C. B. 1941 The bacterial photo-syntheses and their importance for thegeneral problem of photosynthesis. Ad-vances in Enzymol., 1, 263-328.

74. VAN NIEL, C. B. 1944 The culture, generalphysiology, morphology and classificationof the non-sulfur purple and brown bacteria.Bacteriol. Revs., 8, 1-118.

75. VATTER, A. E. AND WOLFE, R. S. 1958 Thestructure of photosynthetic bacteria. J.Bacteriol., 75, 480-488.

76. VERNON, L. P. 1953 Cytochrome c contentof Rhodospirillum rubrum. Arch. Biochem.Biophys., 43, 492-493.

77. VINOGRADOV, A. P. AND TEIS, R. V. 1941Isotopic composition of oxygen of differentorigins. Compt. rend. acad. sci., U. S. S. R.33, 490-493.

78. VISHNIAC, W., HORECKER, B. L., AND OCHOA,S. 1957 Enzymic aspects of photosyn-thesis. Advances in Enzymol., 19, 1-71.

79. WINOGRADSKY, S. 1888 Beitraege zur Mor-phologie und Physiologie der Bakterien.Heft I. Zur Morphologie und Physiologieder Schwefelbakterien. Arthur Felix,Leipzig, Germany.

80. WITTMANN, H. 1957 Ueber die Carotinoidevon Chlorochromatium chlorochromatii n.sp. Arch. Mikrobiol., 28, 136-137.

1719611

on April 19, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from