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SOMEASPECTSOFTHE EARLY EVOLUTIONOFPHOTOSYNTHESIS

Z. Masinovsky,” G.I. Lozovaya** and A.A. Sivash**

*Laboratory ofEvolutionaryBiology, CzechoslovakiaAcademyof Sciences,NaFolimance11, 12000Prague, Czechoslovakia**N.G.KcholodnyInstituteofBotany,Ukr. SSRAcademyofSciences,

Repinstr.2,Kiev, U.S.S.R

AB5TRACT

The early evolution of a photocatalytic system of the porphyrin type, able toefficiently collect and utilize solar energy for primary electron transfer isdiscussed. Experimental results concerning some spectral and photochemicalproperties of the porphyrins, biosynthetic precursors of chlorophyll and theircomplexes with polymeric templates are reviewed. Protoporphyrin IX associatedwith pigmented proteinoid is demonstrated to be a favourable candidate fora role of a photosensitizer of the first photosynthetic reaction centers. Theorigin and early evolution of the photosynthetic electron transfer chain andof the phosphorylating mechanism are discussed with emphasis on the energeticmechanisms of archaebacteria.

INTRODUCTION

5olar energy has probably played a key role in the synthesis of complex orga-nic compounds on the primitive Earth as well as in space. The question underdiscussion concerns the source of free energy for pre—biotic systems and thefirst living cells. There are two alternative conceptions of primitive energe—tics which correspond to two different approaches to the problem of the originof life in general.— The traditional Oparin—Urey conception /1/ postulated that the pool of thereduced organic compounds synthesized during chemical evolution was the mainsource of energy for pre—biotic systems constructed by the macromolecules fromthe same pool of organics (protoheterotrophy).— An alternative conception was proposed by Granick /2/. He claimed that thefirst organized units which originated from some common minerals could performthe elementary processes of photosynthesis based on metal ions as chromopho—res. 5uch mineral particles would serve both as co—ordinating templates andcatalysts for the synthesis of organic molecules which gradually became orga-nized into the particles (photoautotrophy). This theory was developed byCairns—5mith /3/, Hartman /4,5/ and some others.Hartman /5/ suggested the following three—stages scenario for the evolution ofphotosynthesis: Ci) The synthesis of sugars which is based on the carbon re-duction cycle coupled with the Fe~’ Fe~ reversible transformations (ironcycle) driven by light on the surface of clay particles. Formaldehyde yieldedfrom this process is then polymerized to give sugars. (2) 5ulfur is introducedinto the evolving clay—Fe—sugar system giving rise — probably through acetylthin esters — to fatty acids (i.e. to lipids and membranes), quinones and Ca—rotenoids. Fe.5. and Fe~5, clusters capable of an electron transfer role simi-lar to that of ferredoxins are formed in an environment of a lipid bilayer.(3) Introduction of molybdenum into a system containing an Fe,5~ cluster al-lows the formation of the active center of nitrogenase. Nitrogen is reduced toammonia, which allows the synthesis of amino acids, purines and pyrimidines,flavins and nicotinamide. Condensation of glycine with a succinyl thio esteryields i—amino levulinic acid, the starting substrate for the synthesis ofporphyrins and linear tetrapyrrols. A similar ~autotrophic~ theory of earlyenergetics and the origin of life was proposed by Woese /~i, who suggestedthat inorganic photosynthesis was the source of energy for the synthesis oforganics within aqueous microdroplets dispersed in the primitive atmosphere.

it is not a purpose of this paper to compare the two theories. Nevertheless,

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a few points should be noted. Even if the first pre—biotic systems were hete—rotrophic, solar energy might have been used in their metabolism. One possi-bility was through the photoactivation of catalysts, as has been demonstratedwith many contemporary enzymes (for review see /7/) as well as with proteino—ids /8,9/. Another pathway might have been through the donation to primary he—terotrophs of free electrons produced by external inorganic sensitizers /10/.

On the other hand, let us suppose that the primary photosynthesis~ was sensi-tized by inorganic compounds, which is consistent with the demonstratedUV—induced reduction of carbon dioxide to formaldehyde by ferrous ion /11/ aswell as with the UV—induced splitting of water coupled with the reduction ofelectron acceptors by titanium, zinc or tungsten oxides /12/. However, eventhe most primitive contemporary organisms do not utilize inorganic sensitizersin their metabolism. Therefore, the transfer from inorganic to organic photo—sensitizers probably occurred very early in evolution. Another transfer (fromphotosensitive co—enzymes or other non—specific photosensitizers to the highlyeffective contemporary pigments) probably occured during the development ofphotosynthesis from initially heterotrophic metabolism. 80th evolutionary pro-cesses probably passed through several stages. Finally, chlorophyll became theuniversal transformer of solar energy in bioenergetics. 5tructural and funct-ional advantages of the chlorophyll molecule that may have determined its se-lection in evolution have been analyzed in detail by Mauzerall /13/.

ORIGIN OF A PHOTOCATALYTIC SY5TEM BA5ED ON PORPHYRIN5

Our own exprimental work modelling the early stages of the evolution of theporphyrin—containing photocatalytic systems is actually based on the idea ofGaffron /14/, Granick /15/ and Mauzerall /13/ that the first photosensitizersmight have been porphyrins analogous to the contemporary biosynthetic precur-sors of chlorophyll. Mercer—5mith et al. ii&/ have shown that the first cyclictetrapyrrol of this biosynthetic pathway, colourless uroporphyrinogen, may bephotooxidized by shortwave ultraviolet ( < 220 nm), and that this oxidation isaccompanied by the release of molecular hydrogen. Uroporphyrin, which is theproduct of this photochemical reaction, is already coloured and is able (aswell as other porphyrins corresponding to the subsequent intermidiates ofchlorophyll biosynthesis) to sensitize the photocatalytic oxidation of reducedorganic substrates coupled with the release of the molecular hydrogen upon il-lumination in the visible region /17/. According to Mercer—Smith and Mauzerall/18/ such processes could have played a role analogous to that of contemporaryphotosynthesis.

We have compared some spectral and photochemical properties of porphyrins suchas the biosynthetic precursors of chlorophyll, as well as of their natural andsynthetic analogs from the point of view of their possible participation inearly photosynthesis /19/. The pigments of contemporary photocatalytic systemsare associated with the proteins forming the photosynthetic reaction centersand the light—harvesting antenna, and this association strongly influencestheir functions /20,21/. Therefore, we have also studied the properties of mo-del complexes of various porphyrins with polymeric templates such as the natu-ral non—specific proteins, bovine serum albumin (85A) and human serum albumin(H5A), as well as with synthetic abiogenic polymers synthesized by thermal po—lycondensation of amino acids (basic proteinoids) /22/.

The photochemical activity increased from the less advanced biosynthetic pre-cursors of chlorophyll to the more advanced ones (uroporphyrin or coproporphy—rin -. protoporphyrin IX - chlorophyllide a) /19/. However, this increase couldnot be detected with the phytol—containing porphyrins, chlorophyll a and pheo-phytin a, due to aggregation in water which results in a strong decrease ofthe lifetime of their triplet excited states and thus of their photochemicalactivity. In the presence of proteinoid or H5A the activity of phytol—freeporphyrins was not much higher when compared with the aqueous environment (nomore than by 50’/.), whereas in the case of chlorophyll a and pheophytin a itincreased dramatically (20—30 times). These effects might result from the f or-mation of complexes of porphyrins with the polymeric template which preventstheir aggregation in water. This suggestion is consistent with the correspond-ing changes of the position of the specific absorption maxima of porphyrins indifferent microenvironments /19/.

Some properties of such complexes have been studied with the use of 85A asa polymeric template /23/. Both in the aqueous environment and in the complex-es a change of PH from 1 to B resulted in a change of the absolute intensityof the red~ peaks of porphyrins, while their position remained practicallyunchanged. The PH—dependent shift of the absorption maxima in the Soret band

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(about 400 rim) was generally detected at lower pt-s values with theporphyrin—BSA complexes than in the case of free porphyrins, which might bea result of microbuffering caused by the protein moiety within theporphyrin—protein complex. The value of the ionic strength had practically noinfluence on the position of the absorption maxima of the porphyrins when as-sociated with BSA; so one may claim that other than electrostatic interact-ions, probably hydrophobic ones, determine the character of the complexes andtheir spectral properties.

our data are consistent with the possibility that in the early stages of evo-lution compounds related to the contemporary biosynthetic precursors of chlo-rophyll might have played a role as photosensitizers with a gradual transitionfrom less advanced porphyrins to the more advanced ones during evolution. Cri-teria for the selection should be: the light—absorbing ability, photochemicalactivity and the ability to form supramolecular structures. Protoporphyrin IX

PPIX), the key compound of the biosynthesis of all biogenic porphyrins, hasall these abilities. It is the first biosynthetic precursor of chlorophyllthat has a strong absorption in the visible region and a sufficient photoche-mical ability. It has been suggested as a photosensitizer of the first photo-synthetic reaction centers /24/.

our comparative study of the absorption spectra as well as of the fluorescenceemission and excitation spectra of protoporphyrin IX in various microenviron—ments /25/ supports this suggestion. it also demonstrates the role of a macro—molecular template in the improvement of the processes of transformation ofthe excitation energy by protoporphyrin IX molecules. The formation ofprotoporphyrin—template complexes is accompanied by an increase of the fluo-rescence quantum yield (and of the total intensity of emission) in parallelwith the increase of the photochemical activity. Since protoporphyrin IX isable to form complexes with basic proteinoid and the properties of such com-plexes are essentially the same when compared to those of the contemporaryprotein (l-55A), proteinoids that would not only have increased absorbing andphotochemical abilities of PPIX, but also have enabled its incorporation intothe cytoplasmic membrane can be suggested as possible components of thepre—biotic photosynthetic reaction centers, One more possible function of pro—teinoids in pre—biotic photosynthesis should be noted. Proteinoids are charac-terized by the presence of melanoidins. These heterogenous pigments havea strong absorbance in the uv— and visible region and fluoresce just in theinterval of the absorption peaks of protoporphyrin IX /25/, which might enablethe energy transfer from the proteinoid melanoidins to PPIX by thedipol—dipol mechanism. Actually, we have obtained some experimental evidencefor such transfer /25/. Additionally, a strong E5R—signal has been detected inthe proteinoid melanoidins /26/, reflecting the presence of free radicals. Ifproteinoid melanoidins served as primitive light—harvesting antenna, they mayalso have protected the sensitive reaction centers from the high level of Uv

irradiation on the early Earth. During the gradual evolution of the photosyn-thetic apparatus, the polyfunctional pigmented abiogenic templates would the-refore have been replaced by highly specialized colourless proteins, and bothlight—harvesting and the light—protective functions have been taken over byspecial photosynthetic pigments like carotenoids.

Further evolutionary improvement of the protoporphyrin IX molecule was basedon its ability to form chelates, i.e. to be inserted by an ionically boundeddivalent metal at the vacant hole in the porphyrin ring. This stage of the de-velopment of the porphyrin structure led to the functional specialization ofporphyrins as dark catalysts of redox—transformations on one hand(Fe—porphyrins) and as photosensitizers on the other hand (Mg—porphyrins). Al-though hemin is photochemically inactive either alone or in complex with pro—teinoid, hemoproteinoid, in which the heme(s) is firmly bound with theprotein—like moiety, is already capable of photocatalysis of electron trans-fer, and its activity is comparable with that of chlorophyll a in aqueous en-vironment /19/. Such sensitizers could probably have taken part in pre—biotic

photosynthesis~’. However, their photochemical activity would not have beensufficiently high as to compete with chlorophyll—proteinoid orchlorophyll—protein complexes. Therefore, the extremely high light—absorbingand photochemical potential of the chlorophyll molecule was fully realized inevolution in the form of chlorophyll—protein complexes (light—harvesting an-tenna and the reaction centers) , while heme—containing proteins have evolvedas dark catalysts of redox processes.

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EARLY EVOLUTION OF THE PHOTOSYNTHETIC MACHINERY

Besides the development of the photocatalytic system of a porphyrin type ca-pable of efficient collection of solar energy and its utilization in primaryredox—transformatione (1) , at least three other evolutionary processes shouldbe discussed in the context of the early evolution of photosynthesis: the ori-gin and evolution of the electron transfer chain (2), the development of thephosphorylating mechanism (3) and the improvement of the spatial arrangementof the photosynthetic apparatus (4).

The autotrophic theory suggests the joint evolution of components 1—3 of thephotosynthetic apparatus. According to Hartman /5/, organic sensitizers wouldevolve from inorganic ones (ferric—ferrous system) through quinones which ab-sorb in UV and carotenoids and phycobilins which absorb in the blue and yellowregion to chlorophyll which absorbs in the red. The electron transfer chainwould evolve from the ferric—ferrous system through the ferredoxin cores tothe hemes. The high energy esters should evolve from silicates through sulfa-tes to phosphates, ATP being the principle one.

Heterotrophic theories suggest a relatively independent evolution of compo-nents 1—3. The gradual transition from glycolysis to primary photosynthesismight have started by the light activation of individual glycolytic enzymes.This is indicated e.g. by the data of Shugar /27/, in which up to a five—foldincrease of the activity of glyceraldehyde—3--phosphate NADI-$ dehydrogenase byultraviolet irradiation was demonstrated in vitro. NAD with an absorption ma-ximum at about 340 nm might serve as sensitizer of this photosynthesis~. Thephotochemical activation of NADH by uv radiation in vitro was demonstrated byKrasnovsky and 51-in /26/. irradiation resulted in an accelerated oxidation ofNADH by various electron acceptors, e.g. viologenes or ferredoxin. As theseacceptors have a more negative redox potential than NADH, the reaction may beaccompanied by the accumulation of energy of UV photons. Thus, the radiationenergy might have been utilized for the reversion of glycolytic transformat-ions of NAD as a coenzyme of glyceraldehyde—3—phosphate dehydrogenase, whichcould have been the first step in the transition from protoheterotrophy tophotoautotrophy.

In terms of spatial arrangement the primordial photosynthesis probably did notsubstantially differ from glycolysis, i.e. it proceeded in the cytoplasm andATP was produced by phosphorylation at the substrate level /29/. However, itsefficiency would have been very low due to spontaneous reverse reactions bet-ween the primary products of the photoinduced electron transfer. ThiS problemwas solved by the arrangement of pigments at the membrane in such a way thatthe reduced products of the primary photochemical reaction formed on one sideof the membrane and the oxidized ones on the other. This might have been thefirst step in the development of the photosynthetic electron transfer chain.Individual porphyrins might have been attached to the primitive redox systemon the basis of their ability to enhance electron transfer reactions.

The evolution of the biological electron transfer chain can be explained bytwo alternative hypotheses:— The conversion hypothesis /29/ assumes that the electron transfer chainfirst developed in anaerobic photosynthetic bacteria. After completion by ad-ditional carriers with a higher redox potential this chain could have beenused in oxygenic photosynthesis. The respiratory chain is of secondary (andpolyphyletic) origin and was formed from the photosynthetic chain.— The splitting hypothesis /30/ assumes that the so—called short chain of an-aerobic respiration (with nitrate or sulphate as electron acceptors) developedfirst. Chlorophyll photosystems became associated with this chain later. Dur-ing the third phase the original short chain was joined by the oxidoreductivecomplex of cytochrome oxidase and, thus, the aerobic respiratory chain of nor-mal length was formed.

The photosynthetic electron transfer chain produces a proton gradient acrossthe membrane, which is a driving force for the synthesis of macroergic phos-phates catalyzed by ATPase. Nevertheless, some authors assume that membranescapable of vector reactions might have existed already in protoheterotrophicpredecessors of archaic photosynthetic bacteria, where they facilitated theactive transport of nutrients into the cell. According to Mitchell /31/, theoxidoreductive system of proton (or electron) transfer and proton translocat—ing ATPase might have developed independently of each other. Only their simul-taneous occurence in a single cell made it possible to utilize freeoxido—reductive energy for ATP synthesis by the originally hydrolytic ATPase.

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Raven and Smith /32/ assume that H~ extrusion by ATPase preceeded proton tran—slocation driven by redox reactions induced by light. It is generally assumedthat the ATPase proton channel (F,,) originated earlier in evolution than thecoupling factor catalyzing phosphate transfer to ADP (F

1). According to Balt—scheffsky /33/, a protonophore predecessor was bound to the membrane alreadyduring pre—biotic evolution, and functioned as a primordial proton transloca—tor in the cell. The ancient role of F., is indicated by the high proportion ofglycine and alanine residues, which were probably among the first coded aminoacids /34/. saltscheffsky /35/ also assumed that a pyrophosphatase (PPase) wasassociated with the original primitive channel. Later it could ~co—operateboth with PPase and ATPase and, finally, PPase was gradually eliminated fromthe system of membraneous phosphorylation in most organisms.

Thus, it can be argued that the mechanism of membraneous phosphorylation andthe biological electron transfer chain developed in parallel and relativelyindependently of each other, at least during the early stages of evolution. Inour opinion, this suggestion is also consistent with the analysis of the twosystems in archaebacteria. In halophilic bacteria a special mechanism of pro-duction of proton gradient via photoinduced transformations (conformationalchanges) of bacteriorhodopsin developed independently of the respiratory elec-tron chain. Since halophiles have been suggested to have originated from me—thanogenic bacteria /36/, it can be speculated that the bioenergetic mecha-nisms of these two groups of archaebacteria are mutually related. In methano—genic bacteria ATP is produced both by phosphorylation at substrate level andby membrane phosphorylation driven by a proton gradient. However, this gradi-ent is not always produced by the activity of the respiratory chain. Zehnderand Brock /37/ assume that in Methanosarcina barkeri growing on acetate it canalso be formed by direct proton uptake, resulting from conformational changesof a hypothetical membraneous protein caused by decomposition of acetate at-tached to this protein at the outer side of the membrane. Thus, a primordialorganism resembling methanogenic bacteria could have provided a possibilityfor the evolutionary development of both mechanisms of formation of the photo-synthetic proton gradient known in contemporary organisms: the “chlorophyllmechanism, based on the activity of the electron transfer chain and the “bac—teriorhodopsin one, in which solar radiation replaced an organic energy sour-ce and chemically induced conformational changes of a hypothetical photoinertprotein were replaced by bacteriorhodopsin phototransformations. It may be as-sumed that under conditions in which the transition from hetero— to autotrophytook place, both these mechanisms began to develop simultaneously. However,the “bacteriorhodopsin~ type of photosynthesis did not play a role in furtherevolution due to the sensitivity of the biosynthesis of bacteriorhodopsin tooxygen. There may have been other attempts at such a transition, but only thehalophilic bacteria survived (probably due to the specific conditions of theirenvironment where the competition for resources is minimal) and transformedtheir energetics to heterotrophy based on the respiratory chain.

The development of the photosynthetic apparatus continued on the basis of thespatial and functional association of primary photochemical transformations ofporphyrins (up to chlorophyll a) with the electron transfer chain whose acti-vity leads to the membrane proton gradient and then to the macroergic phospha-tes.

REFERENCES

1. A.I.Oparin, Vozniknovenie Zhizni na Zemle, Izd. AN 555R, Moscow, 1957.

2. 5.Granick, 5peculations on the origin and evolution of photosynthesis,r~nn.N.Y.Acad.5cj. 69, 292 (1957).

3. A.G.Cairns—Smith, Precambrian solution photochemistry, inverse segregat-ion, and banded iron formations, Nature 276, 607 (1970).

4. H.Hartman, Speculations on the origin and evolution of metabolism,

3.Mol.Evol. 4,359(1975).

5. H.Hartman, origin and evolution of photosynthesis, in: The Origin of Life,ed. Z.Masinovsky, Czechoslovak Acad. Sci., Prague 1969, p. 346.

6. C.R.Woese, An alternative to the Oparin view of the primeval sequence, in:The Origins of Life and Evolution, eds. H.O.Halvctrson and K.E.Van Holde,

(4)204 Z. Masinovskye:aL

A.R.LiSs, 1960, p. 65.

7. D.H.Hug, Photoactivation of enzymes, Photochem.Photobiol.Rev. 6, 87(1961)

8. A.WOod and H.G.Hardebeck, Light enhanced decarboxylation by proteinoids,in: Molecular Evolution. Prebiological and Biological, eds. D.L.Rohlfingand A.I.Oparin, Plenum Press, New York— London, 1972, p. 233.

9. Z.Masinovsky, Model studies of some aspects of prebiotic and early biolo-gical energetics, in: EBEC Reports, Patron editore, Bologna 1980, p. 409.

10. V.V.Nikandrov, M.A.5hlyk, N.A.Zorin, i.N.GOgOtov and A.A.Krasnovsky, Effi-

cient photoinduced electron transfer from inorganic semiconductor TiO,, tobacterial hydrogenase, FEDS Letters 234, 111 (1988).

11. N.GetOff, Reduktion der kohlensaure in vasseriger losung unter einwirkungvon UV licht, Z.Naturforsch. 178, 87 (1962).

12. A.A.Krasnovsky, tcchimicheskaya evolyuciya fotosinteza: modeli i gipotezy,in: Proiskchozhdenie zhizni i evolyuciorsnaya biokchimiya, eds. G.Deborin,T.Pavlovskaya, K.Dose and 5.W.Fox, Nauka, Moscow 1975, p. 133.

13. D.Mauzerall, Why chlorophyll?, Ann.N.Y.Acad.Sci. 206, 483 (1973).

14. H.Gaffron, On dating stages in photochemical evolution, in: Horizons ofBiochemistry, eds. M.)casha and B.Pullman,Academic Press, New York, 1962,p. 59.

15. 5.Granick, Evolution of heme and chlorophyll, in: Evolving Genes and pro-teins, eds. V.Bryson and H.L.Vogel, Academic Press, New York, 1965, p. 67.

16. 3.A.Mercer—Smith, A.Raudino and D.C.Mauzerall, A model for the origin ofphotosynthesis — III. The ultraviolet photochemistry of uroporphyrinogen,Photochem.Photobiol. 42, 239 (1985).

17. 3.A.Mercer—Smith and D.Mauzerall, Photochemistry of porphyrins: a modelfor the origin of photosynthesis, Photochem.Photobiol. 39, 397 (1964).

16. J.A.Mercer—Smith and D.Mauzerall, Molecular hydrogen production by uropor—phyrin and coproporphyrin: a model for the origin of photosynthetic funct-ion, Photochem.Photobiol. 34, 407 (1981).

1?. 2.Masinovsky, G.I.Lozovaya, A.A.Sivash and M.Drasner, Porphyrin—proteinoidcomplexes as models of prebiotic photosensitizers, BioSystems 22, 305(1989)

20. G.Britton, The Biochemistry of Natural Pigments, Cambridge UniversityPress, Cambridge — London, 1963.

21. 3.M.Anderson, Photoregulation of the composition, function and structureof thylacoid membranes, Annu.Rev.Plant Physiol. 37, 93 (1966).

22. 5.W.Fox and K.Dose, Molecular Evolution and the Origin of Life, MarcelDekker, New York, 1977.

23. Z.Masinovsky, M.Drasner and G.I.Lozovaya, 5ome physicochemical propertiesof porphyrin — bovine serum albumin complexes, in:t4-thinternat.Congr.Diochem. , Czechoslovak Acad. Sci., Prague 1988, v.V, p. 69.

24. 3.M.Olson and B.K.pierson, Origin and evolution of photosynthetic reactioncenters, Origins of Life 17, 419 (1987).

25. G.I.Lozovaya, Z,Masinovsky and A.A.Sivash, Protoporphyrin Ix as a possibleancient photosensitizer: spectral and photochemical studies, origins ofLife, in press (1990).

26. T.A.Telegina, z.Masinovsky, A.A..Sivash, T.E.Pavlovskaya, V.Liebl andL.Bejsovcova, Proteinoids as complexes of polyamino acids with melanoi—dm5, Origins of Life, in press (1990).

27. D.5hugar, Ultra—violet irradiation of triose phosphate dehydrogenase,

EarlyEvolutionofPhotosynthesis (4)205

Biochim.Diophys.Acta 6, 548 (1951).

28. A.A,Krasnovsky and G.P.Brin, Fotokchimiya sistem piridinnukleotidy—kchlo—rof ill i razvitie cepi fotosinteticheskogo perenosa elektronov, in: prob—lemy Evolyucionrioi I Tekchnicheskoi biokchimii, eds. V.L.Kretovich andT.E.Pavlovskaya, Nauka, Moscow 1964, p. 221.

29. E.Uroda, The Evolution of the Dioenergetic Processes, Pergamon Press, Ox-ford, 1975.

30. W.Schwemmler, Mechanismen der Zellevolution, W. de Gruyter, Berlin, 1979.

31. P.Mitchell, Chemiosmotic Coupling and Energy Transduction, Bodmin, London,1968.

32. .1.A.Raven and F.A.Smith, H~transport in the evolution of photosynthesis,Biosystems 14, 95 (1981).

33. M.Baltscheffsky, H.Daltscheffsky and J.Boork, Evolutionary and mechanisticaspects on coupling and phosphorylation in photosynthetic bacteria, in:Electron Transport and Phosphorylation, ed. J.Barber, Elsevier BiomedicalPress, 1982, p. 249.

34. M.Eigen and p.schuster, Hypercycle. A principle of natural self—organi-zation. Part C: The realistic hypercycle, Naturwiss. 65, 341 (1978).

35. H.Baltscheffsky, Stepwise molecular evolution of bacterial photosyntheticenergy conversion, Dio5ystems 14, 49 (1981).

36. E.Stackebrandt and C.R.Woese, The evolution of prokaryotes, in: Molecularand Cellular Aspects of Microbial Evolution, eds. M.J.Carlile,J.F.COllins and B.E.B.Moseley, Cambridge University Press, London 1981, p.1.

37. A.J.B.Zehnder and T.D.Brock, Biological energy production in the apparentabsence of electron transport and substrate level phosphorylation, FEDSLetters, 107, 1 (1979).