Photosynthesis Research 24: 47-53, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.
A reverse KREBS cycle in photosynthesis: consensus at last Bob B. Buchanan & Daniel I. Arnon Division of Molecular Plant Biology, University of California, Berkeley, CA
Received 3 July 1989; accepted I 1 September 1989
Key words: CO2 assimilation, photosynthetic bacteria, ferredoxin
The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible. It liberates CO2 and generates N A D H whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation. In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle. Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al. 1966) (now known as Chlorobium limicola forma thiosulfatophilum). Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source. In the ensuing years, the new cycle was viewed with skepticism. Not only was it in conflict with the prevailing doctrine that the 'one important prop e r t y . . , shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged. It is only now that, in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic
C O 2 assimilation pathway distinct from the pentose cycle. (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al. 1986). Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.
Origin of the concept of the new cycle
The concept of the carboxylic acid cycle had its origin in the discovery of ferredoxin-linked reductive carboxylation reactions. In the pentose cycle the reductant is NADPH (Era, 7 = - 3 2 0 m V ) . When our laboratory determined that ferredoxin and not NADPH is the first stable carrier of photosynthetically generated reducing power and that its midpoint potential is about equal to that of molecular hydrogen (Era, 7 = - 4 2 0 m V ) (Tagawa and Arnon 1962; Arnon 1988) the possibility arose that reduced ferredoxin may serve directly as a reductant in reductive carboxylations instead of participating indirectly by way of pyridine nucleotides with an attendant 100mV loss in reducing potential. The first substantiation of this possibility was the
48 discovery (Bachofen et al. 1964) that reduced ferredoxin could drive a net reductive synthesis of pyruvate from acetyl-CoA and CO2 (Eq. 1). Acetyl-CoA +
+ 2Fdred + 2H +
Pyruvate + CoA + 2Fdox
The reaction was in essence a reversal of the oxidative decarboxylation of pyruvate through which acetyl-CoA is supplied to the Krebs cycle. The enzyme catalyzing the new reaction was detected in cell-free extracts of the fermentative anaerobe, Clostridium pasteurianum. Just prior to our work, Mortenson et al. (1962) showed that the breakdown of pyruvate by C. pasteurianum depends on ferredoxin. Our findings (Bachofen et al. 1964) demonstrated that the strong reducing potential of reduced ferredoxin could drive this reaction in the reverse (carboxylation) direction (Eq. 1 ) - - a feat only marginally accomplished previously with the nonphysiological reductant, sodium hydrosulrite (dithionite) (Mortlock and Wolfe 1959). The enzyme catalyzing the new reaction, named pyruvate synthase (Bachofen et al. 1964) (now also known as pyruvate ferredoxin oxidoreductase), was later purified to homogeneity from a related organism, Clostridium acidi-urici (Uyeda et Rabinowitz 1971). After initial experiments with ferredoxin-dependent carboxylations in fermentative bacteria that live in the soil independently of light, we found such carboxylations also in photosynthetic bacteria that live autotrophically in the light by means of an anaerobic (anoxygenic) type of photosynthesis. Here reducing equivalents are supplied not by water but by reductants such as hydrogen sulfide or thiosulfate. We found pyruvate synthase in cell-free preparations from each of the three then known groups of photosynthetic bacteria: the already discussed Chlorobium (Evans et al. 1966) representing the green sulfur group; Chromatium strain D (now called Chromatium vinosum) representing the purple sulfur group (Buchanan et al. 1964); and Rhodospirillum rubrum representing the purple non-sulfur group (Buchanan et al. 1967). Furthermore, the activity of pyruvate synthase explained previously puzzling 14C-labelling results in bacterial photosynthesis obtained by other investigators with whole cells (reviewed by Buchanan et al. 1967).
The next important development that led directly to the formulation of the carboxylic acid cycle was the discovery of e-ketoglutarate synthase (Eq. 2) in Chlorobium (Buchanan and Evans 1965) and later (with lower activity) in R. rubrum (Buchanan et al. 1967). Succinyl-CoA + CO2 + 2Fdred + 2H + ~
c~-Ketoglutarate + CoA + 2Fdox). ~-ketoglutarate synthase aroused special interest because it exemplified the use of the reducing power of ferredoxin to reverse the step in the Krebs cycle hitherto considered to be irreversible, i.e. the decarboxylation of e-ketoglutarate to succinyl-CoA and CO2. Its action was similar to that of pyruvate synthase (Eq. 1) in that both enzymes catalyzed reductive ferredoxin-dependent carboxylations of an acyl-CoA derivative to form the corresponding ~-keto acid (Eq. 3). R-CO-S-CoA + CO2 + 2Fdred + 2H + ~ (3) R - C O - C O O H + CoA-SH + 2Fdox This similarly raised the question whether pyruvate synthase and e-ketoglutarate synthase were one or two distinct enzymes. Later studies with purified preparations showed that e-ketoglutarate synthase is indeed distinct from pyruvate synthase with respect to molecular mass and chromatographic properties (Gehring and Arnon 1972).
The reductive carboxylic acid cycle The new ferredoxin-dependent enzymes, pyruvate synthase and e-ketoglutarate synthase, formed the cornerstones of the reductive carboxylic acid cycle. Krebs (1981) defined metabolic cycles 'as processes in which an overall chemical change is brought about by continuing cyclic reaction sequences.' In the carboxylic acid cycle the overall chemical change is the net synthesis from CO2 (i) of acetylCoA starting with oxaloacetate, or (ii) of oxaloacetate starting with acetyl-CoA. The continuing cyclic reaction sequences are the formations of mono-, di- and tricarboxylic acids. Beginning with oxaloaeetate, two sequential additions of CO2 (C4 ~ C5 --* C6) give citrate. Cleavage by citrate lyase yields acetyl-CoA as the net product and regenerates oxaloacetate which gives
Fig. 1. A. Schematic overview of the net synthesis of a C 2 product via the reductive carboxylic acid cycle. Heavy arrows indicate carboxylation or cleavage steps. B. Schematic overview of the net synthesis of a C 4 product via the reductive carboxylic acid cycle. Heavy arrows indicate carboxylation or cleavage steps.
rise to a four-carbon CO2 acceptor (Figs. 1A and 2). Beginning with acetyl-CoA, the cycle operates by four sequential additions of CO2 ( C 2 --~ C 3 ---+ C 4 --)- C 5 -~ C6) producing citrate. The citrate is cleaved by citrate lyase, yielding oxaloacetate as product and regenerating acetyl-CoA as CO2 acceptor (Fig. 1B and 2). The carboxylic acid cycle is well suited to provide directly or indirectly building blocks for all other cellular constituents (Buchanan and Arnon 1970). For example, acetyl-CoA may be used directly for lipid synthesis or be converted to pyruvate which in turn may give rise, with or without additional carboxylations by the cycle, to amino acids (e.g. alATP CoA
anine, aspartate, glutamate) or to sugar phosphates (glucose, fructose) via gluconeogenesis. Participating in the latter pathway in photosynthetic bacteria is pyruvate-Pi-dikinase (Buchanan 1974), an enzyme that also functions in certain higher plants. Evidence for the operation of the carboxylic acid cycle as a full-fledged photosynthetic path for CO2 assimilation operating independently of the penrose cycle was assembled most completely for Chlorobium. The original evidence for the cycle included (i) demonstration in cell-free preparations, by measurements of enzymatic activity and 14C tracer distribution, of all the enzymes needed to catalyze the reactions of the cycle proper and of the
/ UWTE Y [ Fdred~
PHOSPHOENOL ~, PYRUVATE ~ ~ N
SUC~NATF FU~ARATE Z~_.~
Fig. 2. The reductive carboxylic acid cycle.
50 associated reactions needed for biosynthesis (ii) identification of the expected metabolic products in short-exposure ~4CO2 experiments with growing cells, and, as discussed below, (iii) evidence that Chlorobium lacks the key enzymes of the pentose cycle for photosynthetic CO2 assimilation. Moreover, it had just been demonstrated (Evans and Buchanan 1965) that reduced ferredoxin which drives the cycle is photochemically generated by Chlorobium. Evidence for the cycle was soon materially strengthened by independent inhibitor and 14Clabeling studies elsewhere with washed suspensions of Chlorobium cells (Sirev~tg and Ormerod 1970a; 1970b). This support, however, did not avert the ensuing controversy.
The controversy The pentose is uniquely characterized by the operation of two key enzymes: ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBP [formerly RuDP] carboxylase, or rubisco) and phosphoribulokinase (PRK). In fact, advocates of the pentose cycle as the exclusive pathway for autotrophic CO2 assimilation proposed that 'the aquisition.., of RuDP carboxylase and phosphoribulokinase may have triggered (or corresponded with) the emergence of autotrophic life' (McFadden 1973). CO2 assimilation in Chlorobium, a strict autotroph, directly contradicted this generalization. One way to resolve this contradiction was to test for the presence in Chlorobium of the two marker enzymes of the pentose cycle, rubisco and PRK. Their presence would reaffirm the universality of the pentose cycle for autotrophic CO2 assimilation and relegate the new carboxylic acid cycle to an 'ancillary role' (Fuller 1978). Conversely, the absence of rubisco and PRK in Chlorobium would indicate that an alternate pathway for CO2 assimilation such as the carboxylic acid cycle must indeed operate to account for the strictly autotrophic growth of this organism. At first it appeared that despite our doubts to the contrary (Evans et al. 1966), rubisco may be present in Chlorobium. Other investigators isolated the enzyme from Chlorobium and described certain of its properties (Smillie et al. 1962; Tabita et al. 1974). These results, still lacking confirmation, were found in other studies to be due to errors
introduced by contaminating microorganisms (Buchanan and Sirev~tg 1976). Furthermore, extensive attempts in several laboratories (SirevSg, 1974; Buchanan and Sirev~tg 1976; Buchanan et al. 1972; Chernajiev et al. 1974; Takabe and Akazawa 1977) yielded no evidence for the activity of either rubisco or PRK in Chlorobium. Similar results were obtained by other investigators who used both enzymatic (Quandt et al. 1977) and molecular genetic approaches (Shively et al. 1986). Finally, despite continuing reservations (McFadden 1978), mass ratio ~3C analyses (Sirevfig et al. 1977; Quandt et al. 1977; Bondar et al. 1976) and ~4C labelling studies with growing Chlorobium cells ruled out a significant role for the pentose cycle (Fuchs et al. 1980b) in this organism and produced strong evidence for the operation of the carboxylic acid cycle (Fuchs et al. 1980a), as the main pathway of CO2 assimilation in Chlorobium (Ormerod and Sirevgtg 1983; Fuchs and Stupperich 1985; Gottschalk 1985). Questions were also raised about the presence in Chlorobium of some of the key enzymes required for the carboxylic acid cycle, specifically, the ATPlinked citrate lyase. Some early work contradicted our initial findings (Evans et al. 1966) and suggested its absence (Beuscher and Gottschalk 1972) but later investigations showed that activity of this enzyme in Chlorobium preparations was greatly stimulated by dithiothreitol (Ivanowsky et al. 1980). When proper precautions were exercised, ATP-linked citrate lyase was consistently found in Chlorobium preparations (Ormerod and Sirevgtg 1983), also in laboratories that had earlier reported negative findings (Antranikian et al. 1982). In sum, the original documentation of the cycle has been validated and greatly enlarged by extensive studies in other laboratories. The operation of the cycle is now generally accepted and has ceased to be a subject of controversy (Tabita 1988; Ormerod and Sirevfig 1983; Fuchs and Stupperich 1985; Gottschalk 1985; Gest 1987).
Other carboxylations, other organisms The ability of reduced ferredoxin to drive reductive carboxylations of acyl-CoA derivatives to form corresponding c~-keto acids (Eq. 3) is not limited to pyruvate and ~-ketoglutarate synthase. It was also found to include the carboxylation of (i) propionylCoA yielding ~-ketobutyrate (Buchanan 1969;
51 Bush and Sauer 1976), (ii) phenylacetyl-CoA yielding phenylpyruvate (Allison and Robinson 1967; Gehring and Arnon 1971) and (iii) isobutyl-CoA yielding ~-ketoisovalerate (Allison and Peel 1971). These reductive carboxylations are involved in assimilatory processes of diverse microorganisms ranging from methane producing rumen bacteria to other photosynthetic bacteria, including even oxygenic cyanobacteria (Bothe et al. 1974), that inhabit a wide range of habitats. There is also growing evidence that in some nonphotosynthetic obligate anaerobes, reduced ferredoxin functions in a variant of the Krebs cycle, which jointly with its anaplerotic reactions requires both pyruvate synthase and ~-ketoglutarate synthase for the degradation of acetyl-CoA (Jungerman et al. 1970; Thauer 1988). Finally, according to recent evidence, certain nonphotosynthetic sulfate reducing bacteria resemble Chlorobium in lacking the reductive pentose phosphate cycle and in utilizing the reductive carboxylic acid cycle for autotrophic CO2 assimilation (Schauder et al. 1987; Pruess et al. 1989).
Evolutionary implications So long as the reductive pentose cycle was thought to be the only mechanism for autotrophic CO2 assimilation, the emergence of autographic life would have to coincide with the emergence of that cycle. The recognition of the reductive carboxylic acid cycle in a photoautrophic organism like Chlorobium suggests another scenario that seems more probable (cf. Ormerod and Sirev~tg 1983). On an evolutionary scale, ferredoxin is an ancient, low potential (equivalent to molecular hydrogen) metalloprotein electron carrier, that probably functioned in the earliest living organisms. These, it is generally agreed, were anaerobes. Then as now, ferredoxins were key participants in oxidoreductions that involve strong reducing potentials. As discussed above, clostridia and other anaerobic bacteria, both photosynthetic and nonphotosynthetic, possess ferredoxin-dependent reductive carboxylation enzymes, pyruvate synthase and ~ketoglutarate synthase, that are the cornerstones of the reductive carboxylic acid cycle uncovered in Chlorobium. Earlier work in this laboratory (Arnon et al.
1961) buttressed the concept that as molecular hydrogen vanished from the primitive atmosphere, ancient phototrophs became dependent on the photosynthetic apparatus for the generation of strong reductants like reduced ferredoxin from such electron donors as thiosulfate. Support for this view came from similarities found among Chlorobium, Chromatium and clostridial ferredoxins (Buchanan et al. 1969). From this point of view, the reductive carboxylic acid cycle would have evolved in an organism like Chlorobium that already had ferredoxin and a complement of enzymes capable of catalyzing ferredoxin-dependent carboxylations. Hence, the reductive carboxylic acid cycle rather than the reductive pentose phosphate cycle, would have been the more likely pathway for CO2 assimilation in primitive photosynthesis typified by Chlorobium (Ormerod and Sirev~tg 1983; Fuchs and Stupperich 1985). The reductive pentose phosphate cycle would be a later development (it seems absent in Archaebacteria, cf. Fuchs and Stupperich 1985), that has probably evolved by a reversal of the oxidative pentose cycle and the acquisition of the rubisco and RuPK enzymes (Broda 1975; Quayle and Ferenci 1978). Is there an evolutionary connection between ancient pathways of CO2 assimilation characteristic of obligate anaerobes and the Krebs cycle that is the hallmark of aerobic metabolism? Krebs himself seemed to think so. In the last year of his life, he wrote (Krebs 1981) 'in the evolution of the citric acid cycle use was made of mechanisms already existing in connection with other functions' and 'pathways leading to citrate evolved long before oxygen appeared in the atmosphere' (our emphasis). Oxygen appeared in the atmosphere as a result of oxygenic photosynthesis, which, on the scale of evolution, had evolved later than the anoxygenic photosynthesis typified by Chlorobium. From this perspective, the carboxylic acid cycle may indeed have an ancestral relation to the Krebs cycle. In the course of evolution, the biosynthesis of acetate (acetyl-CoA) from CO2 by the carboxylic acid cycle may have been transformed into the citric acid cycle that degrades acetate into CO2 (Weitzman 1985). Such transformation would be in harmony with 'a general principle of evolution that multiple use is made of given resources' (Krebs 1981) i.e., of mechanisms that had evolved in earlier epochs in connection with other functions.
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