Models for protocellular photophosphorylation

Models for protocellular photophosphorylation

BioSystems, 14 (1981) 3--14 © Elsevier/North-Holland Scientific Publishers Ltd, 3 MODELS FOR PROTOCELLULAR PHOTOPHOSPHORYLATION PETER R. BAHN and S...

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BioSystems, 14 (1981) 3--14 © Elsevier/North-Holland Scientific Publishers Ltd,

3

MODELS FOR PROTOCELLULAR PHOTOPHOSPHORYLATION

PETER R. BAHN and SIDNEY W. FOX

Laboratory o f the National Foundation for Cancer Research at the Institute for Molecular and Cellular Evolution, University o f Miami, Coral Gables, FL 33134, U.S.A. (Received June 30~h, 1980) (Revision received December 22nd, 1980) Several photoreactions for transducing light energy have been analyzed for their relevance as models for protocellular photophosphorylation. Inorganic ions and compounds could have played a role in protocellular photophosphorylation. Organic catalysts may have been the next significant agents used by protocells for photophosphorylation. Membranous photophosphoryhtion probably became the most recent type of photoenergy transduction to be acquired by protocells; it is still used by modern cells although components of the other types of pho:~phorylation are found in present day cells. Recorded yields of energy-rich phosphates from the model reactions discussed are small. Arguments are advanced that such yields could have been sufficient to have fueled protocellular metabolism, which was probably very slow compared to modern cellular metabolism. Future prospects for research in this area are discussed.

One of the key questions composing the problem of the origin of life is how evolving cells acquired the ability to convert sunlight into chemical energy. Much of the present day biota depends on photosynthesis as an ultimate source of energy. Without sunlight, the biosphere as we know it would quickly collapse. Although sun]~ight has spurred the proliferation of life over the Earth it is not a requirement for the maintenance of all life. Despite the fact that many books and reviews unqualifiedly treat life as requiring solar energy, early non-photochemical autotrophs probably inhablited the primitive Earth in abundance (Broda, 1975). Modern descendants of the l~irst chemoautotrophs most likely include many bacteria which live in completely dark hot springs and consume H2S as their sole energy source (Kluyver and Van Niel, 1956). New types of sulfur bacteria are still being discovered (Waldrop, Chemical and Engineering News, March 24, 1980). While all of the first cells may have been chemoautotrophs, some cells did acquire the

capacity for photosynthesis at least 2 billion years ago (Brasier, 1979). The progeny of these cells are thought to have generated the Earth's oxidizing atmosphere by their photosynthetic production of molecular oxygen. A detailed knowledge of cellular photosynthesis is likely to yield important practical benefits as well as theoretical concepts. Our current shortage of combustible fuels makes it necessary that we develop effective sources of solar power as rapidly as possible. In this technological effort, photosynthetic cells provide an admirable example of efficiency from which we have already learned a great deal (Bolton and Hall, 1979}. At present, a fairly sophisticated picture of how photophosphorylation occurs in modem cells is available. This picture owes much to the concepts pioneered by Peter Mitchell (1961) in his Chemiosmotic Theory. The clues to how photophosphorylation occurs in modem cells have come mostly from analytical dissection of the m o d e m cell itself. Analytical biochemistry may be limited, however, in what it can tell us about the first

4 cellular photophosphorylation because the nature of the first cell is left undiscovered by such a reductionistic method (Fox, 1977). A more effective method of understanding the origin of cellular photophosphorylation is the constructionistic one of allowing simple molecules to self-associate into a system which, although less complex than a m o d e m cell, can participate in photophosphorylation. Two main types of photochemical energy capture occur in living organisms: (a) the photoreduction of carbon dioxide into oxidizable fuel molecules; and (b) photophosphorylation -- the principal topic of this article. The core reaction of photophosphorylation is O-

O-

R--O-P--O- + HO--P--O- + H÷

H

II

0 hv X

0

OOI z R--O--P-'O--P--O- + H20 II

0

II

ground-state. Instead, the electrons are routed into a path that compels the physical movement and connection of the negatively charged phosphorylating molecules. The process of photophosphorylation is a multistage energy transduction which can be symbolized: electromagnetic energy -* electrical energy -* mechanical energy -~ chemical energy Exactly how this energy transduction occurs on the quantum mechanical level remains very much a mystery. Several types of phosphorylation may have been tested by natural experiments in the course of cellular evolution. These categories include non-photochemical, inorganic photochemical, organic photochemical, and membranous phosphorylation. The phosphorylations listed are in the direction of increasing molecular complexity of the catalyst involved.

Non-photochemical phosphorylation

0

in which R can be hydrogen or any chemical group and X is a light-sensitive catalyst such as chlorophyll, which initially absorbs the electromagnetic" energy, lZ is usually AMP; the reaction occurs with a positive standard free energy change of 7.30 kcal/mol. The chemical energy is essentially in the phosphodiester bond connecting one phosphate group to another. Phosphoester bonds may be highly energetic in part because the negatively charged phosphate groups tend to repel each other. During the formation of the phosphodiester bond, the phosphate groups are oxidized while the proton is reduced to water. The reaction therefore involves a flow of electrons in some manner. The light energy excites ground-state electrons of the catalyst into higher energy molecular orbitals. The catalyst in its particular microenvironment, prevents the excited electrons from flowing directly back to their

The first type of phosphorylation to occur on the primitive Earth may well have been a combination of thermal and chemical rather than photochemical phosphorylation (Waehneldt and Fox, 1967; Calvin, 1969; Kenyon and Steinman, 1969). In this type of phosphorylation, water is removed by heat or by chemical dehydration of orthophosphate groups to yield oligophosphates. Principal energy-rich compounds which would have resulted from thermal or chemical phosphorylation are polyphosphates including pyrophosphate. Nucleotide energy such as ATP may not have been advantageous for protolife although it obviously became advantageous in the evolutionary era of modem life. Clues to the non-photochemical generation of condensed phosphates have been provided by several types of model reaction. For example, Ferris (1968) produced pyrophosphate from orthophosphate by using cyanoacetylene as a dehydrating

agent. Pyrophosphate (Ponnamperuma and Chang, 1971) and other polyphosphates (Osterberg and Orgel, 1972) have also been produced by the direct heating of orthophosphates at temperatures below 100°C. Once oligophosphates are formed, they easily phosphorylate nucleosides to form nucleotides. Waehneldt and Fox {1967), as indicated, obtained substantial yields of nucleotides, including ATP, by warming adenosine with polyphosphoric acid at modern ambient temperatures. The oligop]hosphates drive endergonic reactions in aqueous solution by sequentially splitting off terminal phosphate groups to activate metabolites (Hulsof and Ponnamperuma, 1976). Indications of the importance for evolution of polyphosphate and pyrophosphate can be found in some primitive bacteria. Kulaev (1974), for example, reported that a number of phylogeneticaUy ancient bacteria possess polyphosphate hexokinase acl~ivity. Baltscheffsky (1971) found that Rhodospirillum rubrum photochemically converts orthophosphate to pyrophosphate which then serves as the main energy source for this organism. Such phosphate condensing agents as cyanide derivatives which have been suggested by Calvin (1969) and others would have been rather quickly depleted from the surface of the Earth. The "acquisition of solar-powered phosphorylation at this stage would have greatly increased protocellular independence from special chemical and thermal factors in the environment.

Inorganic photophosphorylation Even before the first organic compounds originated on the primitive Earth, various metals, metallic ions, metal oxides, metal sulfides, and semiconducting elements were undoubtedly present on the planet. These compounds could then have been involved in the protocellular acquisition of energy. Many inorganic cofactors such as metals in

porphyrins and iron in ferredoxins remain intimately involved in the energy metabolism of modern cells (Baltscheffsky and Baltscheffsky, 1974). Ever since the discovery of the photoelectric effect (cf. Richards et al., 1960), it has been known that some elements and inorganic compounds produce electrical charge separations when irradiated with light. In fact, the charge separation can be directly harnessed as in solid state photovoltaic cells to do useful work (Perez-Albuerne and Tynan, 1980). Recent investigations have demonstrated that inorganic compounds in aqueous suspension or solution are also capable of producing charge separations and storing the resulting energy in chemical form (Wrighton, 1979). Most of the inorganic reactions studied so far are not phosphorylation reactions but they could be used as a potential first step in a more complicated set of reactions leading to phosphorylation. For example, Fujishima and Honda reported direct photoelectrolysis of water for the first time in 1972 on a TiO2 electrode, although no yields were given. Krasnovsky (1974, 1976) has reported splitting water into hydrogen ions and oxygen with the use of ultraviolet light, Fe 3÷ as an electron acceptor, and one of the following: titanium, zinc, or tungsten oxides. Krasnovsky has also used Fe 2+ ion in solution to photoreduce organic dyes such as viologen blue. This compound can reduce other compounds, thereby returning the dye to its original state. Yields were not reported for these reactions. Inoue et al. (1979) photolyzed CO2 in aqueous suspensions of TiO and other semiconductor particles to get formaldehyde and methanol. This photoreduction was carried out over 7 h to give approximately 0.01% yields or a conversion rate of 0.001% yield/h of illumination. A different type of experiment has been performed by Fan et al. (1976, 1978), who have reported observing direct photophosphorylation of ADP with 32Pi to ATP in aqueous suspensions of semiconductors. They

used ZnO with UV radiation for 30 min to obtain 2000 cpm of 32p esterified or a rate of 4000 cpm/h of illumination. In another experiment, they photolyzed CdS for 60 min with visible light to obtain a yield of 8000 cpm of 32p. esterified or a rate of 8000 cpm/h of illumination. Dark controls gave about half of these cpm of esterified 32p. Unfortunately, they did not report the total cpm of 32p used in the experiments so the actual yields cannot be quantified. The authors do state that their yields of ATP synthesized are extremely low. The results are essentially qualitative and inferential. A chromatogram which does not quite align the supposed photoproducts with known standards from chloroplasts is presented as evidence that photophosphorylation has occurred. Therefore, these potentially dramatic results must be regarded as preliminary until they are reported from other laboratories. This type of direct photophosphorylation, however, could provide a very useful mechanism whereby evolving protocells could have generated ATP in an aqueous environment. The protocells would obtain ATP by complexing a semiconductor catalyst or possibly by using ATP generated by aqueous suspensions of semi-conductors outside of their own boundaries.

Organic photocatalysts Several organic compounds are capable of directly catalyzing photophosphorylation in onon-aqueous environments. In combination with metal cofactors, they begin to approach in design the photocatalytic components found in m o d e m cells. These compounds include quinones, flavins, imidazole, and porphyrins. Fox et al. (1978) photophosphorylated ADP to ATP in 20% yield in a reaction catalyzed by the quinone, chloranil, and ferrous ion in dimethylformamide solution with white fight. This maximum yield was reached in an irradiation time of 18 h to give an approximate rate of 1% yield/h of illumination. The photophosphorylation of nucleo-

tides catalyzed by quinones probably occurs via a phosphorylated quinol ester as an intermediate (Stillwell, 1977). Quinones could have been important direct organic photocatalysts in protobiotic phosphorylations. They are universally present today in mitochondria and chloroplasts as ubiquinone and plastoquinones, respectively (Jones, 1976}. The quinones were probably first used to transport phosphate groups in early direct catalytic phosphorylation but are now used to transport hydrogen atoms across membranes during chemiosmotic phosphorylation. In the generation of quinones under model primitive terrestrial conditions, tyrosine may have been a key starting point. Tyrosine itself has been produced in the laboratory by Hayatsu et al. {1971) by a Fischer-Tropsch synthesis from CO, H2, and NH3 at temperatures of 200--700°C or less; this suggests its plausibility as a prebiotic compound. Chloranil can result from the reaction of tyrosine with KC103 and HC1 (St~ideler, 1860). Potassium chlorate and HCI, in turn, could have resulted from the reaction with potassium ion and water of C12 and CO found in volcanic gases (Rubey, 1964, p. 42). Tyrosine might also have been oxidized under prebiotic conditions to form dihydroxyphenylalanine (DOPA) and then been oxidized to produce DOPAquinone (Mason, 1948). One attraction of DOPA-quinone is that, as an amino acid, it can be directly incorporated into polyamino acid membranes. In a related development, Heinz et al. (1979) found that flavins were produced as side products when mixed amino acids were thermally polymerized to make proteinoids. Flavins act like quinones in oxidationreduction reactions since they contain two reversibly reducible carbonyl oxygens attached to an aromatic carbon ring (Lehninger, 1975}. Wang (1970) and his colleagues (Tu et al., 1970) have reported that imidazoles will function in solution as phosphate group carriers in various oxidation-reduction systems. For example, Brinigar et al. (1967}

photophosphorylated AMP to ATP in 1% yield by using hematoporphyrin and imidazole in dimethylacetamide. The reaction was carried out for 7 days to give a rate of 0.006% yield/h of illumination. Porphyrins are prebiotically plausible as they have been synthesized by Hodgson a n d Baker (1967) from pyrrole and formaldehyde under geochemical condil~ions. The trend of results obtained so far suggests that organic photocatalysts perform direct photophosphorylation only in a solution that is non-aqueous. No successful attempts to utilize organic catalysts for efficient direct photophosphorylation of ADP to ATP in aqueous solution have yet been reported in the literature. It may be possible to obtain efficient direct photophosphorylation by inserting organic catalysts into hydrophobic membranes. Such experiments are still in a preliminary stage of development.

Membranous phosphorylation In modern ,organisms, photophosphorylation of ADP to ATP occurs indirectly in a multistep mechanism. First, light energy is absorbed by a photosensitive pigment to produce a charge separation across a hydrophobic membrane. Electrons are transported toward the exterior surface of a membranesurrounded compartment and protons are transported in the opposite direction. In a part of the membrane separated from the photosensitive :pigment and charge carriers, the protons from this proton gradient flow through a membrane-bound ATPase. The protons drive the ATPase reversibly to make ATP from ADP and Pi. These statements form the main postulates of the Chemiosmotic Theory developed by Mitchell (1979). Although this l~heory was initially rejected by most researchers working on phosphorylation when it w~s proposed in 1961, it is now widely accepted (Boyer et al., 1977). The theory proposes a similar mechanism for both photosynthetic phosphorylation in chloro-

plasts and oxidative phosphorylation in mitochondria. The main differences are that the chloroplast produces a proton gradient by photochemical charge separation and the mitochondrion produces a proton gradient by fuel-consuming redox reactions. In the mitochondrion, the proton flow and the phosphorylating membrane are oriented in the reverse direction to that of the chloroplast. When fuel molecules are oxidized in the mitochondrion, a charge separation occurs; this places electrons inside the ~hosphorylating membrane and protons outside the same membrane. The reason for this reversal in membrane orientation and proton flow between chloroplast and mitochondrion is that photoreduction and oxidation of fuel molecules are opposing processes. The ATPases from chloroplast and mitochondrial membranes have been isolated and studied in great detail by Racker (1976) and his colleagues. The ATPases consist partly of a hydrophobic stalk segment, F0, which is securely embedded in the membrane and can be removed from it only by disruption with detergents (Fig. 1). The F0 part attaches to a hydrophilic head. F,, which extends out from the membrane into the aqueous medium. This protein structure for the ATPase is clearly seen on electron micrographs of isolated chloroplast and mitochondrial membranes. The spherical F~ part of the ATPase has been cleaved from the F0 part by proteolysis. In aqueous solution, the separate F~ protein hydrolyses ATP to ADP and Pi with the production of a proton. Apparently, it will not run backwards in this isolated state to produce ATP from ADP and Pi. When the intact F0--F1 ATPase is inserted into a phospholipid membrane and an artificial pH difference is instituted on the two sides of the membrane, the ATPase consumes protons while phosphorylating ADP with Pi to ATP. A widely cited example of cellular photophosphorylation is the organism Halobacterium halobium. Stoeckenius (1976) and Stoeckenius et al. {1979} have extensively studied the protein bacteriorhodopsin from

hv

s

'÷-I

ADP+Pi

Fo

' ATP Fig. 1. C h e m i o s m o t i c p h o t o p h o s p h o r y l a t i o n in lipid vesicles. TABLE 1 ENERGY TRANSDUCING PHOTOREACTIONS Reference

Reaction

Yield

Fujishima and H o n d a (1972)

h~ H2OT-~2 H 2 + ½0~

Krasnovsky ( 1974, 197 6)

hv, Fe 3÷ ~ 2H ÷ + % 0 2 H 2 0 TiO~, Z n O , or WO3

I n o u e et al. (1979)

CO 2

hv, TiO

~ H C H O , CH3OH

Illumination time

Approx. conversion rate

__

0.01%

7 h

0.001%/h

1 h

8000 cpm/h a

7 days

0.006 %/h

H20 Fan et al. (1976, 1978)

hv, H 2 0 ~ ATP ADP + Pi ZnO or CdS

Brinigar et al. (1967)

hv, Im AMP + 2 P i p o r p h ' D M A * ATP

1%

F o x et al. (1978)

ADP + Pi

hv, Chloranil F e 2÷, D M F ~ A T P

20%

Racker and Stoeckeniu s (1974)

ADP

hv, b R + Pi F0 _ FI ' M e m b

~ ATP

8000 cpm a

1.5%

18 h

1%/h

15 min

6%/h

a A p p r o x i m a t e l y 2)< t h e c p m of 32p f o u n d in a dark control. Ira, i m i d a z o l e ; Porph, p o r p h y r i n ; D M A , d i m e t h y l a c e t a m i d e ; D M F , d i m e t h y l f o r m a m i d e ; b R , b a c t e r i o r h o d o p s i n ; F 0 - F 1, A T P a s e ; M e m b , m e m b r a n e .

9 the purple membrane of this bacterium. Oesterheldt and Stoeckenius (1973) found that the iUumination of the bacteri0rhodopsin causes it to pump protons out of the bacterium. This observation supported the Chemiosmotic Theory. In a further test of the theory, :Racker and Stoeckenius (1974) combined the bacteriorhodopsin and the F0--FI ATPase from mitochondria into the same lipid vesicles to form an inside-out model of the bacterium. The vesicles phosphorylated ADP with Pi to ATP in 1.5% yield during 15 min of photolysis. This gives a rate of 6% yield/h of illumination, the highest seen in this literature search (Table 1). In this model, the bacteriorhodopsin injects protons into the vesicle after being illuminated by light. The protons then run out through the ATPase, causing it to phosphorylate ADP to ATP (Fig. 1). The photosynthetic thylakoid membranes from chloroplasts use the same fundamental proton gradient mechanism for photophosphorylation as is operative in the vesicles of Stoeckenius and Racker (Avron, 1977). For example, Jagendorf and Uribe (1966) found ATP production in acidified spinach chloroplasts that were suddenly subjected to a basic medium. In a related experiment, Reid et al. (1966) produced ATP in rat liver mitochondria by suddenly transporting them from a basic to an acid medium. Numerous electron microscope and topological labeling studies have been conducted on the thylakoid membrane of chloroplasts (Staehelin et al., 1978) and the inner membrane of mitochondria (Harmon et al., 1974). The re~sults show that these membranes possess highly organized, asymmetrical structures which are much more complicated than the Halobacterial membrane. In contrast to the Halobacterial membrane, the eukaryotic organelles use multiple loops of electronic and protonic movement through their membranes to power phosphorylation (Hinkle and McCarty, 1978).

Discussion The preceding modes of photophosphoryla-

tion all serve as mechanisms by which cells originally may have synthesized ATP. Some researchers in the field of molecular evolution might be concerned with the seemingly low yields of ATP obtained in these model studies. Yields have often been a concern of those who have simulated the prebiotic synthesis of amino acids and other small molecules. Photophosphorylation, however, has more of the quality of cellular synthesis and yields do not necessarily have such significance in cells as in batch reactions. Atkinson (1977), for example, has emphasized that continuous reactions between low concentrations of intermediates constitute the most important factor in the rate of cellular energy metabolism. In modern physiological conditions, ATP has a high turnover rate even though it is present in low steady state concentrations. In the case of protocells, the turnover rate of ATP logically would have been much lower than it is in modern cells because primitive enzymes which used ATP whould have had a lower turnover rate than modern enzymes. This view emerges from experiments in which, for example, proteinoids catalyze the hydrolysis o f esters several orders of magnitude more slowly than do modern enzymes (Dose and Zaki, 1971). Most prominent theories of protocellular evolution have emphasized that living organisms arose somewhat gradually (Oparin, 1938) or in a stepwise manner (Fox and Dose, 1977) from assembly of organic polymers rather than instantaneously. A necessary corollary of this idea is the assumption that protocellular metabolism must have been slower than modern cellular metabolism. This assumption seems quite logical in the sense that something half way between "non-living" and "living" might be expected to possess a metabolic rate (related mostly to turnover) half way between zero and the modern cellular level. In addition, the results just presented indicate that model precellular conditions generated small amounts of ATP by photochemical reactions. Putting these two thoughts together, we infer that protocells could operate on very small but continuously generated amounts of ATP. :Larger

10 concentrations of ATP, if they occurred prebiotically (geologically), may simply have returned largely to ADP and AMP by hydrolysis before the ATP could be incorporated into protocellular metabolism. Large concentrations of ATP may also have been inhibitory to some protoenzymic functions. For example, Atkinson {1977, p . 88) has pointed out that large amounts of ATP actually inhibit certain modern enzymes. The yield of ATP from photophosphorylation by protocellular catalysts may have been extremely small but nevertheless adequate for protocells. The yield may even initially have been mostly that from a photocatalyzed dismutation reaction such as

2 ADP

hp~ catalyst

ATP + AMP

If the ATP were continuously bled off and its energy utilized by the metabolizing protocell, then by Le Chatelier's Principle, more ATP would continue to be made by the resulting shift in equilibrium to the right. This mechanism might initially require some metabolic coupling in the protocell with the oxidative phosphorylation of photochemically reduced carbon to obtain a net photoenergy input. It is possible that the low yields of ATP such as that obtained in the experiments of Fan et al. (1976, 1978) may be explained on the basis of the even simpler dismutation mechanism. A number of plausible protobiological processes can be proposed which could have promoted weakly yielding photophosphorylation reactions by the use of storage of the high-energy products. Examples of these processes include polynucleotide synthesis, phosphate-mediated peptide synthesis, and phosphate priming of sugars either for glycolysis or for storage as glycogen and starch. Jungck and Fox (1973) showed that acidicbasic proteinoid microparticles in aqueous

suspension catalyze the formation of oligonucleotides from nucleotides. Fox et al. {1974) then used microparticles composed of basic proteinoid and poly(A) to make peptides from phenylalanine in the presence of ATP. More recently, Nakashima and Fox (1980) have synthesized peptides from amino acids with lysine-rich proteinoids in aqueous solution. The reaction depends on the phosphate bond energy of ATP or of pyrophosphate. Little information is available at present on protobiological kinase activities which would phosphorylate sugars as a priming of the glycolytic pathway, but it can be expected that such activities will be found in further modeling of protobiological systems. The glycolytic pathway is believed to be one of the oldest metabolic pathways (Gest, 1980). Glycolysis occurs anaerobically and most evidence supports the view that the atmosphere of the primitive Earth lacked molecular oxygen. Glycolysis is universally present in modem cellular metabolism. The structure of glycolysis is relatively simple, both in terms of pathway (linear vs. cyclic) and architecturally (soluble enzymes vs. membrane-bound). Even the relative inefficiency of ATP energy production from glycolysis points to its early origin. This same pathway probably provided protoceUs with the first genuine catabolic substrate-level phosphorylation which could have regenerated ATP from ADP (Quayle and Ferenci, 1978) or ADP from AMP. Sugars, themselves, have been synthesized from the base-catalyzed condensation of formaldehyde in aqueous solution (Mizuno and Weiss, 1974). Like the phosphate kinase activities, the phosphate mediated polymerization of sugars to form starch and glycogen in the protobiological context is also unexplored at present but it is likely to be identified as an early evolutionary process. Therefore, future research emphasis on primitive kinase activities that produce phosphorylated sugars and polymerization of the sugar-phosphates

11 will be awaite] with interest. In passing, it can be noted that sugars have been thermally polymerized by acid catalysis (Mora and Wood, 1958) but without the mediation of phosphate bond energy. Future prospects The photophosphorylating vesicles of Racker and Stoeckenius (1974) provide a simple model of photophosphorylation which could conceivably be imitated with prebiotically available materials. For example, Epps et al. (1979) have synthesized glycerol from glyceraldehyde. Hargreaves et al. (1977) have synthesized phospholipids and prepared vesicular membranes under prebiotic conditions from glycerol, fatty acids, and

phosphate. Fox et al. (1965) and Durant and Fox (Fed. Proc. 25 (1966) 352) found that some of the numerous thermal proteinoids containing zinc as a cofactor will hydrolyze ATP to ADP and Pi. Perhaps an appropriate proteinoid having ATPase activity .could be inserted into a phospholipid vesicle membrane (Stoeckenius, 1978) and be run in reverse by a proton gradient to produce ATP much as has been done by Racker and "Stoeckenius (1974). An alternative possibility is that proteinoid microspheres were the first protocells which carried out photophosphorylation with the aid of catalysts bound within the microsphere structure (Fox and Dose, 1977). The lightcatalyzed reactions would be expected to have taken place in the hydrophobic zones of the microsphere membrane. Since micro-

B

Phospholipids

Spontaneous aggregation

Ph osphol ipid vesicle membrane

Spontaneous aggregation

l

Proteinoids

Proteinoid

\ %aT:yo'

Spontaneous insertion of ~

Modern membrane (50% protein - 50% lipid)

Fig. 2. Two convergent evolutionary pathways to modern membranes.

microsphere membrane

12 spheres have been shown to catalyze many simulated protometabolic reactions (Fox, 1980), there would have been tight coupling between the energy-yielding and energy-using reactions. In the course of evolution of proteinoid microspheres (Fox and Nakashima, 1980), lipids would have inserted themselves into the double-layer outer membrane of proteinoid microspheres (Fox et al., 1969}. The lipids could have been geochemically synthesized outside the microspheres or metabolically synthesized within them. These two possible evolutionary routes to modem membranes are shown in Fig. 2. In one path (A), proteinoids spontaneously insert into a lipid vesicle membrane. In the other path (B), lipids spontaneously insert into a proteinoid microsphere membrane. The two paths converge to a modem membrane containing roughly equal parts of lipid and protein. To decide which path was followed by evolution may be an irrelevant issue because both pathways could have occurred simultaneously on the primitive Earth. In either case, proteinoids in the membrane region of the protocell eventually could have evolved into the efficiently organized proteins of modem phosphorylating, membranes.

Acknowledgement Experimental work referred to was supported in part by the National Aeronautics and Space Administration Grant No. NGK 10007-008. Publication No. 339 of the Institute for Molecular and Cellular Evolution.

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