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Curtis Cecchi
Biology 650
Dr. Ward Watt
Essay #2

An evaluation of the possible origins of the Krebs Cycle

based on contemporary literature

The tricarboxylic acid cycle (TCA) or Krebs cycle is the major biochemical
process used by almost all organisms on earth to oxides carbonaceous compounds to
form high energy metabolites, among other compounds for use in a variety of metabolic
pathways that are key to life as we know it. This process forms carbon dioxide, water,
amino acid precursors, reducing agents such as Nicotinamide Adenine Dinucleotide
Hydrogen (NADH), and high energy bonds most commonly in the form of adenosine
triphosphate (ATP). The reducing agents like NADH are then fed into the oxidative
phosphorylation pathway to generate more ATP to meet the energy requirements of the
organism and at the same time regenerate NAD+ for use in the Krebs cycle. The Krebs
cycle is dependent on oxidative phosphorylation for its continued function due to its
dependance on proton receptors like NAD+. The Krebs cycle and oxidative
phosphorylation work in concert and are essential for present day organisms to carry
out their day to day metabolic functions. However, oxidative phosphorylation requires
oxygen to be carried out. Thus, the Krebs cycle can not run without oxygen because of
its dependence on oxidative metabolites such as NAD+ regenerated by oxidative
phosphorylation. This leads to a major question; if there was little to no atmospheric
oxygen until almost 2 billion years ago (1.5-2 billions years after life had appeared), how
did the Krebs cycle and its link to oxidative phosphorylation become essential to almost
all life on the planet?
Due to its central function in almost all organisms on earth, its beginnings must
have originated early on in the evolution of life. This conclusion can be drawn from the
theory that these traits must have been passed down via genetic inheritance from a
common ancestor that all modern life must share.This suggests that the modern Krebs
cycle, oxidative phosphorylation system must not have evolved to its current state until
after the establishment of contemporary levels of atmospheric oxygen, but the major
components and precursors of the modern processes must have been sufficiently
established by that time in order to incorporate oxygen as a more efficient means of
metabolism. Current literature suggests that indeed the Krebs cycle or at least many of
its major components and pathways did exist during the pre atmospheric oxygen era.
Research into alternative pathways of the TCA cycle, chemolithoautotrophic organisms,
and organisms that live under anaerobic conditions; has shed light onto the possible
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origins of the Krebs cycle, although a consensus on its true origin has not yet been
reached by the scientific community.
The first major insight into the possible origins of the Krebs cycle was brought
about by the work of P. W. Hochachka and T. Mustafa. Their research was some of the
first work to glean insight into the fact that the TCA cycle could be carried out in
alternative pathways under anoxic conditions at the substrate level. They also found
evidence to suggest that the genes that enabled these modifications were potentially
widespread across the kingdoms of life. The work of Hochachka and Mustafa became
the basis for speculation and research into the possible origins of the Krebs cycle as it
would have existed under anoxic conditions before the evolution of photosynthesis and
the oxygenation of the earths atmosphere. In the absence of oxygen the Krebs cycle
can be modified to undergo reductive reactions in order to generate usable high energy
molecules and other essential metabolites. This is known as the reductive tricarboxylic
cycle (rTCA). This type of tricarboxylic acid cycle still generates ATP and NADH, but
also has pathways incorporated into it at the substrate level to use and regenerate
NAD+ among other metabolites of similar function to keep the cycle turning in the
absence of an oxidative sink such as oxidative phosphorylation. Hügler and colleges as
well as E. Smith and H. J. Morowitz both present reviews on literature and research to
suggest how the rTCA could have been the precursor metabolic pathway to the modern
Krebs/oxidative phosphorylation cycle.
In their study of normally aerobic invertebrates under anaerobic conditions,
Hochachka and Mustafa observed that helminths and mollusks were able to sustain
themselves for long periods of time in the absence of oxygen. (Hochachka et. al, 1972)
Helminths were long known to be able to survive anaerobic conditions via a form of the
rTCA, but not mollusks. Some species of mollusks, such as the species Hochachka and
Mustafa studied (Crassostrea gigas), are frequently exposed to anoxic conditions.
These intertidal bivalves will dry out if they are exposed to air, which occurs on a regular
basis as the tide recedes and exposes the rocks on which they live. In order to cope
with this, the muscles seal themselves in their shells and they can only access the
oxygen in the little bit of water that they have sealed within their shells. This presents an
immediate problem because the tide can be out for several hours and there is not nearly
enough oxygen in the small amount of water trapped within the shell to sustain the
organism oxygen requirements. Then the question becomes how do they live through
this period of oxygen deprivation. Hochachka and Mustafa discovered that under
aerobic conditions, intertidal bivalves store high amounts of glycogen in their tissues.
Under aerobic conditions the catabolism of glycogen leads to the formation of pyruvate
which is then fully oxidized via the Krebs cycle reactions. (Hochachka et. al, 1972)
However, under anoxic conditions the glycogen undergoes a different set of pathways.
The glycogen is broken-down to the level of phosphoenolpyruvate (PEP), which is
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similar to what occurs in vertebrate muscle, but then differs strongly in that the final
product of catabolism in intertidal bivalves is alanine and succinate. (Hochachka et. al,
1972) Note that succinate is one of the major metabolic steps in the middle of the
oxidative Krebs cycle. Also it was found that the amount of succinate that was produced
under anoxic conditions was proportional to the work that was done by the organism
over the time. This suggests that succinate production is the major energy yielding
reaction for the intertidal bivalves during anoxic conditions. (Hochachka et. al, 1972)
This ability to cause the TCA cycle to run in a different direction to yield high energy
molecules and restore pools of NAD+ for coenzyme activity to sustain the organism
under anoxic conditions has some major implications for the possible origin of the
modern oxidative Krebs cycle. Not only is the organism sustained by this alternative
TCA pathway, but one of its major byproducts at the end of the reaction is an
intermediate in the oxidative TCA pathway that is coupled to oxidative phosphorylation.
This observation did not go over the heads of scientists and investigations were soon
launched into the possibility of alternative pathways of the TCA cycle.
In papers by Micheal Hügler and colleges as well as Smith and Morowitz both
parties present insight into the possible origins of the modern Krebs cycle based on an
earlier reductive form. Hügler’s research group looked at the presence of the reductive
TCA cycle in contemporary organisms as grounds to suggest the rTCA cycle evolved in
proteobacteria at some point in the anoxic era in order to fix carbon dioxide and power
metabolic functions. While Smith and Morowitz argued that the earliest forms of the
rTCA cycle formed on the early earth under reductive and energy stress conditions that
would driven the formation of such reaction pathways in the presence of hydrogen and
carbon dioxide. Smith and Morowitz suggest that some of the major components of life
could have arisen from the formation of this cycle at the beginning of life’s history. We
shall take a look at both suggested forms of the rTCA cycle suggested by each group,
discuss the modifications needed to alter their rTCA cycle to the modern Krebs cycle.
Hügler and his team studied the rTCA cycle in chemolithoautotrophic sulfur
oxidizing proteobacteria. They concentrated their work on proving that the rTCA was the
primary pathway that this class of organisms used to fix CO2 for metabolic use. They
were able to prove that rTCA cycle was the primary way in which this organisms fix
carbon dioxide and that they could sustain themselves indefinitely with these reactions
based on an analysis of the presences of TCA cycle related enzyme activity and the
absence of enzymatic activity of other know alternative carbon fixation pathways. They
presented a figure representing the alternative form of the TCA cycle as they believed to
be functioning in these proteobacteria. Their model follows as such, “ Fig 1: Outline of
the reductive citric acid cycle for autotrophic CO2 fixation. The reactions catalyzed by
key enzymes are indicated by bold arrows. Enzyme activities: 1, malate
dehydrogenase; 2, fumarate hydratase (fumarase); 3, fumarate reductase; 4, succinyl-
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CoA synthetase; 5, 2-oxoglutarate:ferredoxin

oxidoreductase; 6, isocitrate dehydrogenase; 7,
aconitate hydratase (aconitase); 8, ATP citrate
lyase; and 9, pyruvate:ferredoxin oxidoreductase.
Fdred, reduced ferredoxin.” (Hügler et. al, 2005)
They suggest that the rTCA cycle in this form
could be the reductive pre-aerobic predecessor
of the modern oxidative Krebs cycle. In order for
this rTCA cycle to move from its suggested
reductive form to its modern oxidative form,
several additional intermediates and their
coenzymes need to be formed in order for this to
occur. Obviously to go from reductive to oxidative
all the arrows representing enzyme activity need
Figure 1 (Hügler et. Al, 2005)
to be reversed. Acetyl-CoA would then be the input for
the system formed from pyruvate. to form citrate. Aconitase would need to be
synthesized in order to go from citrate to cis-Aconitate and from there to D-Isocitrate.
Isocitrate dehydrogenase would then need to be formed in order to convert D-Isocitrate
to a-ketoglutarate instead of 2-oxoglutarate. With those additions the Hügler team’s
rTCA cycle can be converted to the modern form of the Krebs cycle. The rTCA cycle
they suggest is about 75% to the modern Krebs cycle, but still would require minimal
additional enzymes and enzyme modifications to achieve it.
Smith and Morowitz taken a metabolism first approach to the origin of the Krebs
cycle. They suggest that a form of the rTCA cycle appeared potentially before the
appearance of cellular life on earth. Their version of the early rTCA cycle was catalyzed
not by enzymes, but by the theory that topology, rate kinetics, and the free energetic
stability of molecules together would push the reactions. (Smith et. Al, 2004) Thus, they
anticipated that an energetically stressed environment should be populated by a
distribution of reaction currents, of which the rTCA cycle is a plausible core.(Smith et.
Al, 2004) Essentially Smith and Morowitz suggest that rTCA has the potential to be self
establishing and can catalyze itself from any point in the cycle in the presence of
reductive conditions. Their implications suggest that the rTCA cycle could be a major
part of the story of the origin of life. Their model suggests fixation of CO2 as well as
hydrogen gas as the reductive agent. This type of rTCA model is uses the reductive,
energy stressed nature of the early earth to catalyze itself. This further suggests that
this early form of the rTCA cycle could have appeared very early on if not before the first
cellular life appeared. The implications that their paper could have on the story of the
origin of life is fascinating, but unfortunately this is not the focus of this paper. In figure 3
pulled from the Smith and Morowitz paper shows their model for how the rTCA cycle
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would have worked under pre-enzymatic conditions. Note that all the major players of
the contemporary Krebs cycle are present, but their order is slightly different and
hydrogen gas is the major reducing agent. Their are only a few steps one would need to
take in order to convert this model into the
contemporary Krebs cycle. Obviously because
the nature of their model is reductive the
arrows indicating enzymatic conversion from
one metabolite to next would be reversed.
NAD and NADH would be the major proton
carriers in the process instead of elemental
hydrogen. Starting from citrate the cycle can
be reversed as is with the addition of the
appropriate enzymes up until the suggested
formation of oxaiosuccinate, which would not
be necessary in the contemporary oxidative
Krebs cycle. The cycle can then be reversed
as is with the appropriate enzymes from the
contemporary Krebs cycle with the exception of the Figure 3 (Smith et. Al, 2004)
direct interaction with acetate. Smith and Morowitz’s
model of the early is well supported as a form of primordial metabolism. Due to the fact
that all life undergoes some variation of the TCA cycle, their suggestion that it arose in
the primordial era would make a lot of sense.
Based on the evidence presented in both of these papers Smith and Morowitz’s
suggested origin of the TCA cycle is more likely than the model that Hügler and
colleagues suggest. Smith and Morowitz put the appearance of the early rTCA cycle at
the base of the phylogenic tree, while Hügler and his associate’s work suggests that the
TCA cycle came about towards the bottom of the phylogenic tree as well, but higher up
the tree than Smith and Morowitz. Both agree that the TCA cycle existed in a reductive
form prior to the oxygenation of the atmosphere. However, the very early appearance of
the TCA cycle seems to be imperative because of its essence in all forms of life. Smith
and Morowitz’s suggested way in which the TCA cycle originated is thus the more
appealing of the two suggestions. The rTCA cycle that Hügler and colleagues suggest is
more than likely a derivative of the earlier rTCA cycle that Smith and Morowitz suggest.
Hügler’s rTCA cycle is closer to the contemporary Krebs cycle than Smith and
Morowitz’s model in the sense that it has already reached the enzymatic conditions. The
rate of reaction with which Smith and Morowitz’s rTCA cycle would have gone would
have been much slower, but the appearance of more enzymes Hügler’s rTCA cycle
would arise. Hügler’s rTCA cycle could potentially be an intermediate step towards the
contemporary Krebs cycle with Smith and Morowitz’s model being the original system
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that appeared on earth in conjunction with the appearance of the first forms of life. Thus
neither paper presents a stronger case for the origin of the Krebs cycle, but are both
potential parts of the bigger picture of the evolution of biochemistry.
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Bibliography

Hochachka, P.W. and T.Mustafa. 1972. Invertebrate facultative

anaerobiosis. Science 178: 1056- 1060.
Hugler, M, et al. 2005. Evidence for autotrophic CO2 fixation via the
reductive tricarboxylic acid cycle by members of the ε subdivision of
protebacteria. J. Bact. 187: 3020-3027.
Smith, E., and H.J. Morowitz. 2004. Universality in intermediary
metabolism. Proceedings of the National Academy of Sciences of the USA
101: 13168-13173.