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FEMS Microbiology Letters 12 ( 1981 ) 209-215 Published by Elsevier/North-Holland Biomedical Press 209 Evolution of the citric acid cycle and respiratory energy conversion in prokaryotes Howard Gest Photosynthetic Bacteria Group, Department of Biology, Indiana Uni~;ersio', Bloomington, IN 47405, U.S.A. Received 17 July 1981 Accepted 21 July 1981 The citric acid cycle (CAC) is the basis of energy metabolism of numerous cell types, principally those of aerobic character. By means of the cycle, acetyl units are completely converted to CO 2 and reducing equivalents which serve as the fuel of the energy conversion apparatus per se. The flow of reducing equivalents to Oz via specialized elect r o n a n d H atom carriers embedded in membranes is the driving force for production of ATP and other forms of utilizable energy. This kind of energy conversion is conveniently referred to as "electrophosphorylatiofi", which is defined as any process in which electron flow in membranes drives phosphorylation of ADP [ 1]. Several intermediates of the CAC (Fig. 1)--notably c~-ketoglutarate, succinyl-CoA, and oxaloacetate ( O A A ) - - a r e important precursors of essential cell components and, accordingly, certain segments of the cycle have two potentially different functions, namely, bioenergetic and biosynthetic. The simplest interpretation of this very remarkable metabolic economy is that portions of the CAC arose in early anaerobic prokaryotes as biosynthetic devices, and that the aerobic energy-yielding CAC was the final result of gradual and closely intermeshed evolutionary improvements in both biosynthetic and bioenergetic mechanisms. It is likely that some of these improvements occurred by "recruitment" and modification of previously existing processes. The foregoing overall view and relevant information from comparative biochemistry and bio0378-1097/81/0000-0000/$02.75 geochemistry provide the background for a more specific scenario for evolution of the modern CAC and respiratory energy conversion in prokaryotes. This hypothesis, outlined in the present communication, takes into account the following considerations: (i) Fermentative anaerobes do not have a complete complement of CAC enzymes, but employ segments of the CAC for biosynthesis (e.g., [2]) or for facilitating achievement of oxidation/reduction balance in fermentation [1]. (ii) aspartate ~, malate pyruvate / acetyl CoA citrate fum(rate ~ aconitate •'i l, I succinate isocitrate l ! rSUCCINYLI ....................................... I a-KETO-I I CoA ,I BChl i cytochromes aspar tate - family amino acids ,".N~ NH 3 m/ /m glutg, ine ................ IGLUTARATEII( glutamate Fig. 1. The modern citric acid cycle and related metabolic sequences. Oxidative reactions are represented by hatched arrows, and reductive steps or sequences by dotted arrows. Major biosynthetic products derived from cycle intermediates are indicated. OAA, oxaloacetate; BChl, bacteriochlorophyll. t' 1981 Federation of European Microbiological Societies 210 2H 2H The CAC segment: OAA --> malate--> fumarate --> succinate operates in the reductive ("reverse") direction during anaerobic metabolism of numerous organisms as an "electron sink" device and the reduction of fumarate, which involves participation of cytochrome b, is frequently associated with phosphorylation of A D P [1,3]. (iii) In prokaryotes capable of obtaining energy from the aerobic CAC or some alternative anaerobic process, mutants no longer able to use the CAC for bioenergetics still employ segments of the cycle for biosynthesis or other purposes [4]. (iv) Aerobic chemosynthetic autotrophs that are unable to utilize organic substrates as energy sources use segments of the CAC for biosynthesis and usually lack or show low activity of one or more enzymatic steps of the classical energy-yielding CAC; this is also true for certain methylotrophs [5]. (v) Despite its great efficiency as a cycle, the dehydrogenase activities of the aerobic CAC do not conform to a unitary plan, but rather represent a heterogeneity of reaction types. (vi) A maximally efficient bioenergetic C A C could operate only after the O z content of the Earth's atmosphere increased to appreciable levels; this presumably occurred relatively late in respect to prokaryotic evolution and required "full" development of the cytochrome system. (vii) The respiratory electron transport systems of higher forms are strikingly similar in fine structure. In contrast, the corresponding systems of prokaryotes show much diversity, especially in respect to "branching" of electron transport pathways [6]. Such branching may represent relics of stages in evolution of efficient electrophosphorylation. The "C 4 dicarboxylic acid electron s i n k " It seems likely that the earliest evolutionary root of the CAC was a reductive reaction sequence that emerged as an adjunct to hexose fermentation, namely: OAA NADH 2 ~ malate H20 NADH 2 ~ fumarate ~ succinate The conversion of OAA to succinate in this fashion requires the equivalent of 4 H atoms and is employed by numerous contemporary prokaryotes (and some eukaryotes) as a means of achieving oxidation/reduction balance in sugar fermentation ([1,7]; note that in such fermentations, succinate accumulates in the medium in substantial quantities.) In classical anaerobic glycolysis, the generation of ATP requires reduction of N A D and reoxidation of N A D H 2 as follows: C6Ht206 +2 ADP+2 P~ + 2 N A D ~ 2 CH3COCOOH + (pvruvate) 2 ATP+2 NADH 2 2 CH3COCOOH+2 NADH 2 ~ 2 CH3CHOHCOOH (lactate) + 2 NAD Thus, pyruvate functions as the terminal oxidant. Alternatively, if N A D H 2 is reoxidized using the "C 4 electron sink" sequence, p y r u v a t e - - o r its fermentative precursor, phosphoenolpyruvate--is potentially spared for use in biosynthetic metabolism. In anaerobes that lack an oxidative CAC the usual source of OAA is: p y r u v a t e + C O 2 + A T P ~ O A A + A D P + P, o r p h o s p h o c n o l p y r u v a t e + C O 2 ~ O A A + Pi Exploitation of the "C 4 electron sink" mechanism can have the effect of sparing one of the two C 3 moieties produced per hexose molecule catabolized. Cells containing this variation of hexose fermentation could have had a significant advantage over those obliged to use pyruvate as the sole electron acceptor for achievement of redox balance. Effective utilization of fumarate as a terminal electron acceptor depends on the activity of a "reductase" with appropriate kinetic properties. The existence of an essentially unidirectional "fumarate reductase" (FR) was first clearly recognized in studies with the strict anaerobe Micrococcus lactilyticus (now known as Veillonella alcalescens [8]). Extracts of this bacterium were shown to contain a soluble, flavin-linked fumarate reductase activity that clearly could not be ascribed to conventional succinate dehydrogenase (SD) acting in the "reverse" (reductive) direction. Accordingly, it was suggested that the reductase was similar to a "succinate dehydrogenase", but represented a catalyst "modified to meet the physiological requirements of anaerobic growth". In subsequent research with a variety of anaerobes and 211 "amphiaerobes" it was found that FR and other components of the fumarate reduction system are almost always membrane-bound ([3]; an amphiaerobe is defined as an organism or cell that can use 0 2 as the terminal electron acceptor for energy conversion or some alternative energy transduction process that is independent of O 2 [9]). In early fermentative systems that used fumarate as an accessory oxidant, the electron transfer catalysts presumably were soluble (cytoplasmic) and served only to ensure redox balance (various other accessory oxidants including certain inorganic compounds could have functioned in similar fashion [10]). I have proposed [1] that the soluble fumarate reduction mechanism gradually evolved by addition of intermediary electron carriers and incorporation of most of the system into the cytoplasmic membrane, eventually yielding systems in which reducing equivalents are transferred according to the general pattern: NADH 2 ~ Fe/S proteins ~ menaquinone cytochrome b ~ fumarate A number of prokaryotic systems show the capacity to produce one ATP per N A D H 2 oxidized in this way [3]. In other words, various organisms that obtain the bulk of their ATP by conventional fermentation (substrate-level phosphorylation) also can generate ATP by anaerobic electrophosphorylation based on operation of a CAC sequence operating in the reductive direction. The idea that reduction of OAA to succinate was first employed as an improved means of achieving redox balance in sugar fermentation [1] has recently been elaborated by Wilson and Lin [11] into a more detailed scheme for the role of FR in the evolution of a redox-proton pump. It should be noted that in anaerobes and amphiaerobes the "C 4 electron sink" pathway also can fulfill a biosynthetic purpose in furnishing succinate for conversion to succinyl-CoA; the latter is a precursor of metalloporphyrins and corrins, and also participates in synthesis of several amino acids. Pyruvate, crossroads of metabolism Pyruvate can justifiably be viewed as a hub of metabolism, and its conversion to acetyl-CoA is of special import. Numerous anaerobes have the unique capacity to metabolize pyruvate according to the equation: C H 3 C O C O O H + C o A S H ~ C H 3 C O S C o A + C O 2 ~- H 2 (acetyI-CoA) This type of pyruvate decomposition involving the generation of hydrogen gas is virtually restricted to prokaryotes ([12,13]; the only exception is its occurrence in several parasitic protozoa [14]), and probably was an early innovation in anaerobic energy metabolism. The overall reaction is the sum of two partial reactions, namely: CI-13COCOOH + CoASH + Ferredoxin C H 3C O S C o A + C O 2 + F e r r e d o x i n ( r e d u c e d ) Ferredoxin (reduced) + 2 H + ~ Ferredoxin + H 2 Formation of H 2 in this way represents disposal of electrons released in energy-yielding oxidations without the necessity for special terminal electron acceptors. In respect to function, the Hz-evolving system of anaerobes has been described [13] as being analogous to the cytochrome oxidase of aerobes. It seems that the " H 2 formation valve" in clostridia and other anaerobes also serves regulatory purposes [13,15]. Thus, the "valve" adjusts under different conditions so as to discard (in the form of H2) any excess electrons in the oxidation/reduction "balance sheet". The terminal catalyst in biological H 2 formation is either a hydrogenase or, alternatively, a nitrogenase complex supplied with a reservoir of suitable reductant and ATP [12,15-17]. The capacity of nitrogenase to produce H 2 (especially in the absence of N 2 ) provides the basis for an anaerobic variant of the CAC in purple photosynthetic bacteria [18]. This process is energy (light)-dependent (rather than energy-yielding) and is characterized by the dissimilation of organic substrates to CO 2 and H 2. "Photoproduction" of H 2 is interpreted as a fine-tuning mechanism for energy-idling when this is required by the temporal balance between energy, conversion activity and overall biosynthetic rate [19,20]. Note should be made of the interesting suggestion 212 [21] that enzyme complexes with nitrogenase activity evolved from H2-producing hydrogenases that originally functioned only as a means of electron discard. Acetyl-CoA resulting from pyruvate cleavage presumably was utilized by early anaerobes in several ways. Increase in fermentative ATP yield was made possible by the reactions: C H ~ C O S C o A + P~ ~ C H 3 C O O P O 3 H 2 + C o A S H CH 3COOPO3 H 2 + ADP ~ CH 3COOH + ATP According to Barker [22], "The most frequently used phosphoryl donor for ATP synthesis in anaerobic bacteria is probably acetyl phosphate". Acetyl-CoA is also widely employed by anaerobes as an oxidant for facilitation of oxidation/reduction balance in fermentations. Clostridia, for example, commonly use the conversions [23]: 2 CH3COSCoA CH3COSCoA 4H ~ CH3CHzCH2COOH+CoASH 4H ~ CH3CH2OH+CoASH Another major use of acetyl-CoA is in the biosynthesis of various cell constituents. In the present context, production of a-ketoglutarate (KG) is of particular significance. It is suggested that the "C 6 branch" of the CAC first evolved as a purely biosynthetic reaction sequence for the production of KG, the direct precursor of glutamic acid. The "C 6 branch" consists of the reactions: O A A + a c e t y l - C o A + H 2O ~ citrate + C o A S H citrate~ cis-aconitate,-.isocitrate the membrane-bound electron transport system that drives phosphorylation of ADP. Fumarate reductase versus succinate dehydrogenase The possibility that SD originated by modification of FR after O 2 began to accumulate in the biosphere is consistent with studies of these catalysts in amphiaerobes such as Escherichia co#. SD and FR in E. coli are membrane-associated flavoproteins that catalyze succinate~ fumarate interconversions, and a strict difference in their physiological functions has been demonstrated in various ways [24]. In accord with the functions implied by the names of these flavoproteins, the SD is induced aerobically and repressed during anaerobic growth; in contrast FR is repressed aerobically and derepressed anaerobically. In connection with the notion that FR was an evolutionary precursor of SD, it is of considerable interest that Guest [24] has recently shown that in genetically manipulated strains of E. coli, SD function can be replaced, in part, by FR. Regearing of the succinate,--, fumarate interconversion in aerobes to operate in the direction succinate--, fumarate can be interpreted as an example of the necessity for evolution of "unidirectionality" in metabolism. Atkinson [25] has pointed out that "nearly every metabolic sequence is paired with a sequence that carries out the same conversion in the opposite direction", and suggests that oppositely directed sequences must have evolved "specifically because of the advantages of kinetic control of metabolic direction as well as of rate". isocitrate + NAD(P) ~ a-ketoglutarate + CO 2 + NAD(P)H 2 A primitive cell (in the anaerobic biosphere) that could catalyze the reductive sequence: OAA-~ succinate as well as the biosynthetic conversion: O A A - , a-ketoglutarate would have had most of the major elements needed for construction of a bioenergetic CAC. What was still missing? The short answer is: (a) a connecting link between the linear pathways noted, (b) availability of a suitable electron acceptor--namely, O 2 - - f o r the reducing equivalents generated by oxidative cycle reactions, (c) regearing of catalysts to operate in the oxidative direction only, and (d) further elaboration of Regulation of a-ketoglutarate dehydrogenase The final link in assembly of the CAC mechanism apparently was the "insertion" of aketoglutarate dehydrogenase (KGD), which catalyzes the reaction: a-ketoglutarate + CoASH + NAD succinyl-CoA + CO 2 + NADH 2 In contemporary cells, K G D occurs as a complex of large molecular size (consisting of three protein components), which appears to be closely similar to the pyruvate dehydrogenase complex of aerobes 213 in all basic respects [26], suggesting evolutionary relatedness. K G D could well have originated in anaerobes with the exclusive function of producing the succinyl-CoA needed for biosynthesis. In cells that acquired this enzyme activity, the continued operation of a fumarate reductase system would have become superfluous, at least as far as biosynthesis is concerned. Assuming that the final catalyst needed for completion of the CAC was indeed KGD, we can ask what design characteristics this enzyme should have to ensure effective operation of a bioenergetic cycle. Information of interest in this connection is provided by recent studies [4,27] with the photosynthetic bacterium Rhodopseudomonas capsulata. This remarkable organism has the capacity to use several alternative bioenergetic systems to support growth under different environmental conditions [28]. Anaerobically, energy can be obtained either from light, or from dark ("accessory oxidantdependent") fermentation of sugars. If suitable organic compounds and O 2 are provided, R. capsulata can also grow readily as an aerobic heterotroph in darkness using the CAC and associated electrophosphorylation as the energy conversion system. In other words, R. capsulata appears to embody an unusually large amount of biochemical evolutionary history. Wild-type R. capsulata cells grown anaerobically on organic carbon sources with light as the energy source contain K G D which, in this growth mode, functions only for biosynthesis of succinylCoA; fumarate reductase activity is not detectable in this organism [27]. Mutants of R. capsulata deficient in K G D retain the capacity to grow photosynthetically (anaerobically) on malate or on C O 2 ÷ H 2, but lose the ability to grow as aerobic heterotrophs owing to failure of the energy conversion function of the CAC [4]. The K G D activity level required to support bioenergetic function of the CAC is evidently much higher than that necessary to satisfy biosynthetic demands; thus the low rate of succinyl-CoA formation needed for photosynthetic growth of K G D mutants can be accounted for by assuming a slow leak through the K G D "block" (it is possible that biosynthesis of 8-aminolevulinic acid, a precursor of porphyrins and corrins, in such mutants may also occur via a pathway alternative to the classical "succinyl-CoA + glycine" route; see [29] for recent experiments in this connection). It is evident that chemosynthetic autotrophs or methylotrophic bacteria that lack K G D [5] (or produce low levels of the enzyme requisite only for biosynthetic purposes) would be unable to obtain energy from organic substrates metabolized via the CAC; such organisms are obliged to use special sources of energy, namely, inorganic compounds (autotrophs) or C I compounds (methylotrophs). In regard to the effects of O 2, it is of considerable interest that regulation of K G D synthesis shows features in common with the controls affecting succinate~ fumarate interconversions. Thus, in amphiaerobes, K G D synthesis appears to be induced by O 2 and is repressed during anaerobic growth [30-32]. Keevil et al. [33] have recently reported that even trace concentrations of 02 (approx. 20 ppm) in the gas phase can lead to detectably increased K G D levels in Citrobacter freundii cells growing in "anaerobic" cultures. Similar results have been obtained with wild-type R. capsulata [4], suggesting that the phenomenon may be general in amphiaerobes. With increased concentrations of O 2, large effects are observed in R. capsulata; K G D activities in extracts of cells grown aerobically (20% O2) are 3-5-fold higher than in extracts from cells grown photosynthetically (02 absent) on the same organic carbon sources. For organisms that can oscillate between aerobic and anaerobic bioenergetic modes, regulatory controls based on 02 partial pressure might well be expected. Final stages in organization of respiratory energy cont~ersion Redox reactions in early heterotrophic cells (during the anaerobic Precambrian period) were probably poised in the E~ range approx. - 0 . 4 to - 0 . 3 V. Electron transfer catalysts included F e / S proteins that participated in utilization and production of H 2, and in N 2 fixation; for some time, only one kind of pyridine nucleotide, NAD,.was used in redox reactions. The pyridine nucleotide, NADP, derived from NAD, appeared later and facilitated regulation of metabolism by making 214 possible a separation of functions; thus, in more highly evolved cells NAD was employed for bioenergetics and N A D P was used primarily in biosynthetic metabolism [34]. Fig. 2 depicts the evolutionary roots of the organic carbon transformations of the aerobic CAC of contemporary organisms. In essence, the diagram shows the general features of two energyyielding systems, side-by-side. At left is the overall PY~TE f biosynthesis c ,+co~+zH = ~NADH 2 C ~= --NNADH E HEXOSE ~'COz C02 Fig. 2. Evolutionary roots of the citric acid cycle. It is suggested that the modern aerobic CAC originated from three major "roots" (I, II, III). I. The anaerobic reductive sequence 2H OAA ~ M ~ F 2H ~ SU [OAA, oxaloacetate; M, malate; F, fumarate; SU, succinate]. This sequence evolved as a mechanism that could achieve redox balance in sugar fermentation (left part of diagram) with the effect of minimizing the amount of pyruvate required to serve as terminal electron acceptor; a small fraction of the succinate formed was used for production of the biosynthetic intermediate succinyl-CoA [SU-CoA]. II. Invention of the conversion: pyruvate+CoASH~acetyl-CoA + C O 2 + H 2 led to several potential advantages (see text) including the construction of a pathway for biosynthesis of glutamate, namely, OAA + acetyl-CoA ~ Cit ~ IC ~ KG glutamate [Cit, citrate; IC, isocitrate; KG, a-ketoglutarate]. After 0 2 appeared in the biosphere, sequence I was modified so as to operate in the oxidative direction [SU-CoA ~ SU ~ F ~ M ~ O A A ; right part of diagram]. III. With addition of the reaction: KG + CoASH + N A D ~ SU-CoA + CO 2 + N A D H 2 and elaboration of Oz-sensitive controls for eliciting high activity of a-ketoglutarate dehydrogenase, the scheme of the cycle was completed. Reducing equivalents from oxidative cycle reactions were fed into a membrane-associated electrophosphorylation system originally used in connection with the electron transport sequence: N A D H 2 ~ cytochrome b ~ fumarate. The terminal catalyst for the earliest aerobic electron transport system was probably an autooxidizable b type cytochrome; cytochrome c and its oxidase were later additions that extended the effective redox span for energy conversion. Many contemporary amphiaerobes have the capacity to metabolize organic compounds by both of the patterns depicted. scheme of a hexose fermentation mechanism in which fumarate, generated from OAA, serves as the terminal H acceptor. As 02 began to accumulate in the Earth's atmosphere (due to oxygenic photosynthesis), the sequence: OAA ~ succinate was regeared to perform in the oxidative direction, and with the eventual addition of the K G D reaction the CAC was completed (Fig. 2, right). Use of 02 as the ultimate electron acceptor substantially extended the redox range of energy-yielding electron flow and much greater efficiency in the utilization of organic compounds as energy sources was in prospect. I suppose that early versions of the aerobic CAC were not as effective as the modern cycle, partly because the membraneassociated electron transport system used for oxidation of N A D H 2 with O 2 was still undergoing evolutionary refinement. Certain bacterial cytochromes of the " b " type are rapidly autooxidizable with 02 [35,36] and proteins of this kind were likely to have been terminal catalysts of respiration for an extended period during the early history of aerobic metabolism. An electron transport chain of the kind employed by many heterotrophic anaerobes in connection with use of fumarate as a terminal oxidant is assumed to have been recruited for primitive aerobic respiration. The switch to 02 reduction is envisaged as the consequence of modification of an ancient cytochrome b that could react only with fumarate (or certain inorganic nitrogen compounds) to a form autooxidizable with 02. Individual species of prokaryotes frequently contain a multiplicity of cytochromes (especially of the b type), many of still unknown function. Perhaps some of these proteins are vestiges of early electrophosphorylation systems that used various alternative terminal inorganic oxidants. The foregoing scenario suggests that cytochrome c and its oxidase were the most recent elements added to the basic blueprint for the elaborate multi-stage electron transport systems now observed in membranes of aerobes. If electron carriers with oxidation/reduction potentials near the 02 end of the redox scale evolved relatively late, it follows that c type cytochromes would have limited value as indicators of cellular evolution during the lengthy anaerobic phase of Earth's 215 history (for a recent s u m m a r y of attempts to construct bacterial phylogenetic trees based on structures of cytochromes c, see [37]). ACKNOWLEDGEMENT Research of the author is supported by a grant from the U.S. National Science Foundation. REFERENCES [I] Gest, H. (1980) FEMS Microbiol. Lett. 7, 73-77. [2] Stern, J.R. and Bambers, G. (1966) Biochemistry 5, 11131118. [3] Thauer, R.K., Jungermann, K. and Decker, K. (1977) Bacteriol. Rev. 41, 100-180. [4] Beatty, J.T. and Gest, H. (1981) J. Bacteriol., in press (Nov.). [5] Smith, A.L and Hoare, D.S. (1977) Bacteriol. Rev. 41, 419-448. [6] Cole, J.A. (1976) in Advances in Microbial Physiology, Vol. 14 (Rose, A.H. and Tempest, D.W., Eds.), pp. 1-92. Academic Press, London. [7} Gest, H. and Schopf, J.W. (1982) in Origin and Evolution of Earth's Earliest Biosphere: an Interdisciplinary Study (Schopf, J.W., Ed.), Princeton University Press, in press. [8] Peck Jr., H.D., Smith, O.H. and Gest, H. (1957) Biochim. Biophys. Acta 25, 142-147. [9] Chapman, D.J. and Gest, H. (1982) in Origin and Evolution of Earth's Earliest Biosphere: an Interdisciplinary Study (Schopf, J.W., Ed.). 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