Download Evolution of the citric acid cycle and respiratory

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.
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