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How did life survive Earth’s great oxygenation?
Woodward W Fischer1, James Hemp1 and
Joan Selverstone Valentine1,2
Life on Earth originated and evolved in anoxic environments.
Around 2.4 billion-years-ago, ancestors of Cyanobacteria
invented oxygenic photosynthesis, producing substantial
amounts of O2 as a byproduct of phototrophic water oxidation.
The sudden appearance of O2 would have led to significant
oxidative stress due to incompatibilities with core cellular
biochemical processes. Here we examine this problem through
the lens of Cyanobacteria — the first taxa to observe significant
fluxes of intracellular dioxygen. These early oxygenic organisms
likely adapted to the oxidative stress by co-opting preexisting
systems (exaptation) with fortuitous antioxidant properties. Over
time more advanced antioxidant systems evolved, allowing
Cyanobacteria to adapt to an aerobic lifestyle and become the
most important environmental engineers in Earth history.
Addresses
1
Division of Geological & Planetary Sciences, California Institute of
Technology, Pasadena, CA 91125, United States
2
Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA
90095, United States
Corresponding authors: Fischer, Woodward W ([email protected])
and Valentine, Joan Selverstone ([email protected])
capable of oxygenic photosynthesis. These organisms likely supported themselves largely by means of anoxygenic
photosynthesis at first, perhaps with only intermittent
production of O2 in relatively small amounts. At this stage
in Earth history, life did not have the defenses necessary to
deal with an oxidant as powerful as O2 [9]. How did early
Oxyphotobacteria survive the intracellular production of O2
and make the transition from a strictly anaerobic to aerobic
metabolism? Here we integrate data from bioinorganic
chemistry and comparative biology to infer the evolution
of oxygen tolerance in Oxyphotobacteria. Notably, ancestral
Oxyphotobacteria could have coped with small amounts of
O2 during the transition to oxygenic phototrophy by coopting preexisting systems with fortuitous antioxidant
properties. This would have allowed time for more modern
antioxidant systems to evolve. We discuss non-enzymatic,
small-molecule solutions to mitigate O2 stress that were
present in early cells, followed by some specific adaptations
that the Oxyphotobacteria developed to enable the transition
to an oxygenated world.
Geological record of O2
Current Opinion in Chemical Biology 2016, 31:166–178
This review comes from a themed issue on Bioinorganic chemistry
Edited by R David Britt and Emma Raven
http://dx.doi.org/10.1016/j.cbpa.2016.03.013
1367-5931/# 2016 Elsevier Ltd. All rights reserved.
Introduction
Data from the geological record indicate that life was
present on Earth very early in its history. Intriguing observations of graphitic carbon in some of the oldest rocks and
minerals have been proposed to be traces of Earth’s early
biosphere [1,2]. By 3.4–3.2 billion years ago (giga annum
or Ga), a range of observations indicate the presence of
microbial cells [3,4] with diverse anaerobic metabolisms
[5–8]. Even using the most conservative estimate of 3.2 Ga
for the origin of life, it was clearly present long before O2
appeared in significant amounts in Earth’s atmosphere.
The first organisms to encounter significant and sustained
oxidative stress due to O2 were ancestral Oxyphotobacteria — members of the bacterial phylum Cyanobacteria,
Current Opinion in Chemical Biology 2016, 31:166–178
Today O2 comprises nearly 21% of the atmosphere,
however a wide array of observations made over the past
sixty years from the geological record illustrates that it was
extremely scarce prior to the evolution of oxygenic photosynthesis [10]. How scarce? An exact paleobarometer
for O2 remains out of reach, but there are several types of
geological and geochemical data that can be converted
into O2 concentrations that are thought to be accurate
within an order of magnitude or two (Figure 1). Sedimentary rocks older than 2.4 Ga, composed of pebbles and
sand eroded from the crust and deposited by rivers, are
distinct from those seen in younger strata because they
contain abundant physically rounded redox-sensitive
minerals, such as pyrite (FeS2), uraninite (UO2), and
siderite (FeCO3) [11]. These minerals are quickly oxidized and destroyed in the presence of even trace O2, so
their presence constrains O2 levels to less than 105 atm
before 2.4 Ga [11]. Additional independent geochemical
proxies support this view. For example, multiple sulfur
isotopes in marine sedimentary rocks older than 2.4 Ga
display a widespread and unusual type of mass independent fractionation (MIF) caused by the photochemistry of
SO2 in an atmosphere largely devoid of O2 (105 atm
and likely closer to 1010 atm). This corresponds to
astonishingly low environmental levels of O2 — sufficiently low that the anaerobic microorganisms that
existed at the time may not have ever encountered
biologically meaningful amounts of oxygen related stress.
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How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 167
Oldest sedimentary rocks
Origin of the Earth ~ 4.55 Ga
Figure 1
GOE
plants
animals
Charcoal
Sulfate deposits
redox-sensitive
detrital grains
Fe in Paleosols & Red beds
Manganese deposits
MIF of S isotopes
Hadean
Archean
Proterozoic
Phan.
103
102
101
Log[Mn] (ppm)
104
100
4500
4000
3500
3000
2500
2000
1500
1000
500
10–1
Today
Age (Ma)
Current Opinion in Chemical Biology
Geological and geochemical data reveal a large first-order, irreversible change in the redox state of Earth surface environments marked by the rise of O2
2.4–2.35 billion years ago. Upper panel: The widespread presence of redox-sensitive detrital grains, like pyrite and uraninite, in Archean and early
Paleoproterozoic sandstones and conglomerates illustrates that O2 levels were lower than 105 atm in the atmosphere and Earth surface waters to
explain their survival through the rock formation processes of weathering, transport, deposition and lithification. A similar pattern is provided by the
mass-independent fractionation (MIF) of multiple S isotopes in Archean and early Proterozoic strata [89,90]. The MIF signal results from photochemistry
involving SO2 in the early atmosphere and models constructed to evaluate the O2 concentrations consistent with these observations suggest that O2
levels were exceedingly low, much less than 105 atm, and perhaps closer to 1010 atm [10]. These observations are juxtaposed by a number of
observations that show a rise of O2 between 2.4 and 2.35 Ga. MIF and redox-sensitive detrital grains disappear from sedimentary rocks [11,12]. Iron
becomes oxidized and retained in preserved soils (paleosols) during weathering of bedrock [13], and it forms abundant hematite (Fe2O3) grains and
cements in sedimentary rocks (red beds). And sulfate salts become conspicuous sulfur cycle-sinks of sulfate derived from weathering of sulfide-bearing
minerals [15]. Though young by comparison, the charcoal record [91] provides an important constraint on atmospheric oxygen because it illustrates that
as long as there was plant biomass around on the land surface that could burn, it did. Mn deposits (Mn-rich sedimentary rocks >1 wt.% Mn) do not
occur until just prior to the rise of O2, an observation that motivates the hypothesis that Mn(II) was a substrate for phototrophy prior to the evolution of
the water-oxidizing complex of PSII, and photosynthetic O2 fluxes [29]. The rise of O2 marks the oldest certain age for the evolution of biological water
splitting by Oxyphotobacteria [10]. The origins of animals and plants are shown for context. Lower panel: Mn(II) content of calcite-bearing (CaCO3) and
dolomite-bearing (CaMg(CO3)2) sedimentary rocks provides a coarse measure of the amount of Mn2+ present in seawater (data from Shields and Veizer
[18]). Before the GOE, carbonates precipitated from seawater are highly enriched in Mn(II), implying high concentrations in seawater. After the rise of
oxygen, Mn-oxide minerals form an important sink of Mn, and the Mn content of seawater subsequently decreased [14]. These measurements exhibit
substantial variation due to post-depositional recrystallization and interaction with later fluids, which tend to enrich carbonates in Mn(II).
The geological record indicates that a rapid and irreversible rise of O2 occurred between 2.4 and 2.35 Ga — a
transition known as the Great Oxygenation Event (GOE)
(Figure 1) [10,12]. Many of these observations record
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changes in the biogeochemical cycles of major redoxactive elements, such as Fe, Mn, and S. At this time MIF
[10] and redox sensitive detrital grains disappeared [11].
Ferrous iron in igneous minerals became oxidized and
Current Opinion in Chemical Biology 2016, 31:166–178
168 Bioinorganic chemistry
was retained in ancient soils [13]. Iron oxide cements
become widespread in fluvial (river) and near-shore marine sandstones, called red beds. Thick deposits of Mnoxide minerals like those in the Kalahari Manganese
Field in South Africa [14] postdate the GOE, with an
important exception (more on this below). In addition
sulfate salts begin to accumulate in sedimentary basins,
recording the oxidative weathering of sulfide-bearing
minerals [15]. Because these redox proxies are sensitive
to small amounts of O2 (e.g., ppm levels), we do not have
good estimates for how high O2 rose during the GOE
(some hypotheses suggest perhaps only 1% of modern).
However it is clear from the geological record that, after
the GOE, O2 became widespread across vast swaths of the
Earth’s surface and was thereafter an important component of the atmosphere and surface waters.
Changes in metal ion availability: Metal use in biology is
primarily determined by evolutionary history, which is
the result of dynamic interactions between environmental availability, biochemical demands, and ecological
flexibility. The metal concentrations in seawater have
changed dramatically over Earth history as a function of
the differential solubility, complexation, and redox behavior of distinct metals — modulated by changes in the
redox state of Earth’s surface environments and geochemical budgets set by different rock-forming minerals
within the crust [16,17]. Iron and manganese are the first
and third most abundant transition elements in the crust
and substitute for one another in a wide range of rockforming silicate minerals, dominantly as Fe(II) and exclusively as Mn(II). Consequently, prior to the GOE
chemical weathering of silicate minerals would have
provided substantial fluxes of Fe2+ and Mn2+ into the
oceans, resulting in high concentrations in seawater [18].
In particular, Archean-age carbonate platforms are loaded
with remarkably high levels of Mn(II) — much higher
than observed in younger carbonate platforms (Figure 1)
[14,18]. It is challenging to convert these data accurately
into seawater concentrations because the partitioning
depends on the kinetics and mechanisms of carbonate
mineral precipitation, but experimental relationships and
solubility constraints suggest that seawater Mn2+ concentrations prior to the GOE ranged from 5 mM to
120 mM [19]. After the GOE, iron availability dramatically decreased in surface seawater due to removal of
insoluble iron oxides, marked by red beds and ferric iron
in paleosols (Figure 1). However iron was so thoroughly
integrated into cellular biology by that point that creative
strategies were required so that organisms would be able
to obtain it in oxygenated environments. Mn concentrations appear not to have fallen as precipitously, likely
because the kinetics of Mn2+ oxidation are substantially
slower than those of Fe2+ [20]. In contrast to iron, for
example, the bioavailability of Zn2+ does not appear to
have changed dramatically over time [21,22]. O2-driven
oxidative weathering of sulfide-bearing minerals in the
Current Opinion in Chemical Biology 2016, 31:166–178
crust — evidenced by the loss of detrital pyrite from the
record (Figure 1) — sourced and solubilized chalcophilic
elements like Cu, which was uniquely suited for the
evolution of high potential metabolisms and aerobic
biology [16].
Biological record of O2
The relative timing of the appearance of O2 from comparative biology and biochemistry is consistent with
observations from the geological record. These data suggest that O2 was not used by early life in either biosynthetic reactions or respiration. Network analyses of
metabolic pathways from diverse microbes identified a
strictly anaerobic core, with O2 requiring reactions
appearing only in the terminal steps of a small fraction
of molecules [23]. This implies that O2 was not available
to early organisms as a substrate for biochemical reactions
and was only incorporated into metabolism after the
evolution of oxygenic phototrophy.
A similar view is provided by phylogenomic analyses of
the distribution of aerobic respiration. The majority of
Bacteria and Archaea phyla have anaerobic basal members with aerobic members found only in derived (i.e. non
ancestral) positions (Figure 2a). For example, in the
Actinobacteria phylum members of the basal classes Rubrobacteria and Coriobacteria are anaerobic. Classes with
aerobic members (Acidimicrobiia, Nitriliruptoria, and Actinobacteria) are derived. Comparisons of the different O2
reductases used by Actinobacteria for aerobic respiration
shows two major independent acquisitions (once in the
Thermoleophilia and once in the Acidimicrobiia + Nitriliruptoria + Actinobacteria clade), with a subsequent loss in
the Bifidobacteria genus after they adapted to anoxic gut
ecosystems (Figure 2a). Many other phyla exhibit similar
phylogenetic patterns, suggesting that the major radiation
of Bacteria occurred at a time when the Earth was anoxic
and that aerobic respiration was acquired after they had
diverged from one another, likely after the GOE.
The biological record over the past two billion years
shows that O2 has driven major revolutions in biology,
forming a biochemical veil that makes it challenging to
infer some aspects of early life prior to O2 with a high
degree of confidence. Deductions from comparative biology and biochemistry have tremendous value for studying this problem, but it is important to remember that
they present a view biased by extinction. The ‘winners’
wrote the biological record. Mechanisms to cope with O2
and reactive oxygen species (ROS) toxicity have been
thoroughly integrated into the function of modern
cells — even for what are typically viewed as strictly
anaerobic organisms [24]. All we easily study are the
biological solutions selected after several billion years
of evolution. The organisms and biochemistries that
did not survive can only be inferred from geological
observations and imagined via evolutionary hypotheses
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How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 169
Figure 2
Rubrobacteria
Thermoleophilia
Coriobacteriia
Acidimicrobiia
Nitriliruptoria
Actinobacteria
Aerobic
Anaerobic
3
2
1
0.1
Aerobic
Anaerobic
(a)
(b)
Current Opinion in Chemical Biology
Observations from comparative biology suggest that O2 and aerobic metabolisms evolved late in Earth history — a pattern that matches
expectations based on the extremely low O2 levels on the early Earth-derived geological observations shown in Figure 1. (a) Phylogenetic tree of
the Actinobacteria phylum based on the conserved marker gene RpoB — a useful phylogenetic marker protein from the b subunit of the bacterial
RNA polymerase. Aerobic respiration was acquired two times independently within this phylum: once in the last common ancestor of the clade
comprised of Acidimicrobiia, Nitriliruptoria, and Actinobacteria (marked 1) classes, and once in the ancestor of the Thermoleophilia (marked 2).
Members of the Bifidobacteria genus lost the capacity for aerobic respiration as they became specialized for anaerobic gut environments (marked
3). The phylogenetic distribution seen here is characteristic of many known bacterial phyla, suggesting that O2 was not present prior to the main
divergences of bacterial phyla from one another. This supports the late evolution of aerobic respiration, with lateral gene transfer playing a
significant role in its modern distribution. (b) A network view of the metabolic networks in the KEGG database containing diverse metabolisms
from all three domains of life from the data reduction by Raymond and Segrè [23]. Nodes mark metabolites and edges reflect reactions. Red
denotes anaerobic metabolism, blue marks areas of the network that are aerobic, and green highlights networks where aerobic reactions are
known to have replaced ones that did not involve O2 (e.g. Raymond and Blankenship [92]). Note that aerobic parts of the network decorate the
outer edges of the network map and are not central to these metabolic processes. These data imply that O2 was not a founding molecule for
cellular metabolism.
from the vestiges we see. While it is often envisioned that
systems for dealing with the toxicity of O2 must have
been in place for cells to survive the GOE, it is, however,
just as reasonable that such systems co-evolved in time
with dioxygen because they would have had little selective advantage prior to the GOE. Instead cells may have
initially coped with O2 stresses using molecules that
performed other functions. And those that were the most
helpful early on were those that had fortuitous antioxidant
chemistry.
The transition to oxygenic phototrophy
Cyanobacteria phylum. New members have been found in
many aphotic environments and analysis of genomes
assembled from environmental metagenomic datasets
(and one cultured isolate) demonstrate that these organisms are missing genes for phototrophy [10,25,26,27].
Current evolutionary relationships between members of
the Cyanobacteria phylum are shown in Figure 3; it is
important to note that oxygenic photosynthesis exhibits a
derived position [10]. A new classification scheme has
been proposed [26] in which the Cyanobacteria are now
comprised of three classes: the Oxyphotobacteria (oxygenic
Cyanobacteria), the Melainabacteria, and ML635J-21.
New insights into the evolution of Cyanobacteria: We can gain
considerable insights about how life adapted to O2 by
examining Oxyphotobacteria, the first organisms to witness
substantial fluxes of intracellular O2. Genomic studies
over the last couple years have dramatically changed the
way we think about the diversity and evolution of the
After their divergence from the Melainabacteria, it was
stem group members of the Oxyphotobacteria (between
markings 1 and 2 in Figure 3) — perhaps long extinct — that developed oxygenic photosynthesis. Between molecular clock estimates [28] and geological
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Current Opinion in Chemical Biology 2016, 31:166–178
170 Bioinorganic chemistry
Figure 3
Melainabacteria
other phyla
Oxyphotobacteria
ML635J-21
0.1
Current Opinion in Chemical Biology
Phylogeny of the Cyanobacteria phylum (shown here from 16S ribosomal DNA) has grown substantially in recent years with advances in genomic
and metagenomic sequencing (e.g., [26]). Current relationships show that all known Cyanobacteria that produce O2 via oxygenic
photosynthesis — a class now termed Oxyphotobacteria — sit in a derived position within the phylum. The Oxyphotobacteria have a close sister
clade: the Melainabacteria [25]. No known Melainabacteria are capable of photosynthesis; most are anaerobic. Basal members of the phylum
form a paraphyletic group only known from environmental samples, currently termed ML635J-21. This topology suggests that oxygenic
photosynthesis in the Oxyphotobacteria is a relatively recent innovation in the context of Earth history [10]. Molecular clock estimates suggest
that the divergence between the Oxyphotobacteria and Melainabacteria (marked 1) occurred between 2.5 and 2.6 Ga, whereas the radiation of
crown group Oxyphotobacteria (marked 2) occurred after 2.0 Ga [28].
data [10,12,29], this appears to have occurred around
2.4–2.35 billion years ago.
Manganese: a gateway to the high potential world: The transition from anoxygenic phototrophy to oxygenic photosynthesis in Oxyphotobacteria required the evolution of two
important features: a high potential reaction center, and a
CaMn4O5 bioinorganic cluster called the water-oxidizing
complex (WOC) capable of oxidizing water [30]. Modern
anoxygenic reaction centers are unable to generate high
redox potentials (<+500 mV). The highest potential electron donor for anoxygenic phototrophy is nitrite
(+430 mV) [31]. One hypothesis emerging in recent years
from both geological and biological data is that Mnoxidizing phototrophy was likely the direct precursor to
oxygenic photosynthesis [10]. Mn2+ was in abundant in
the early oceans (Figure 1) and has redox potentials near
to that of water. Biochemical and structural analyses of
reaction centers strongly suggest that the ability to oxidize Mn compounds drove the evolution of high potential
reaction centers in early Oxyphotobacteria [30]. In fact,
modern Oxyphotobacteria still oxidize Mn using PSII during the photoassembly of their WOCs. The WOC may
Current Opinion in Chemical Biology 2016, 31:166–178
therefore have originated by the accumulation of oxidized
Mn in the high potential reaction center, with catalytic
Mn oxidation eventually being replaced by water oxidation. Importantly, evidence for this transition is found in
the geological record where O2-independent oxidation of
Mn was observed to have occurred prior to the GOE
(Figure 1) [29].
Initial threats from O2
Early O2 production: fluxes, concentrations, and timescales:
Once oxygenic photosynthesis evolved, the timescale for
O2 to titrate the existing pools of geochemically derived
reductants in Earth’s surface environments, which thereafter became oxygenated, is relatively short in the context
of geological time — on the order of tens of thousands to a
hundred thousand years [32]. A number of anoxic environments would endure through the GOE (e.g., pore fluids
in marine sediments), and provide refuge for anaerobic
physiologies. But for oxygenic phototrophs, O2 was an
undeniable problem molecule. What are the concentrations and fluxes of O2 that typify photosynthetic cells?
Recent calculations from theory suggest that the O2
concentrations inside active photosynthetic cells are
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How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 171
much lower than expected — tens of nanomolar greater
than the external environment for solitary planktonic
cells, though much higher for cells living in biofilms
and mats, which can achieve highly supersaturated O2
levels [33]. This work highlights an important asymmetry between photosynthetic O2 production and consumption by aerobic respiration: the rates of O2
generation are slow compared to diffusive fluxes. This
lessens the degree of oxidative stress for the earliest
Oxyphotobacteria [33]. Even functioning at or near modern photosynthetic rates (1018 mol cell1 s1), intracellular concentrations were maximally 250 nM [33];
presumably early Oxyphotobacteria did not produce O2 at
anywhere near these high rates. It is important to note
that these fluxes are still large compared to the concentrations required for O2 to disrupt the intracellular pools
of Fe [20], sulfide [34], thiols, cysteine [35], flavins [36],
and metal centers [37].
1
O2 as the first oxidative threat: In addition to ROS formed by
O2 reduction at various points along electron transport
chains (e.g. Complex I, Complex III, PSI), photoexcited
singlet O2 presented a unique problem for Oxyphotobacteria
as a substantial and inchoate oxidative threat. Photosystems
are studded with chlorophylls as the major light harvesting
molecules. Energy transfer from excited long-lived triplet
states of chlorophyll to ground state triplet O2 can create
1
O2 with extremely high quantum efficiency [38]. Furthermore 1O2 is highly reactive with a wide range of molecules
including proteins and lipids, has a short half-life within
cells [39], and is one of the most critical sources of oxidative
damage in photosynthetic cells [40]. For phototrophic
organisms that generate O2, there was no easy way around
this problem. Light-harvesting can be carefully tuned by
mechanisms of non-photochemical quenching (NPQ), and
carotenoids can be used to dissipate energy from photosensitized chlorophyll; however a good solution is simply to
keep intracellular O2 levels low, particularly near the
photosystems. This logic appears to have been important
for early Oxyphotobacteria, which appear to have employed
flavodiiron proteins to remove O2 and alleviate the production of this problematic molecule [41].
Early fortuitous antioxidant systems
Manganese(II) and reactive oxygen species: In modern aerobic
organisms, antioxidant enzymes such as superoxide dismutases (SOD), superoxide reductases (SOR), catalases
and peroxidases, play a crucial role in defending cells
from superoxide, hydrogen peroxide, and other peroxides. Based on their widespread distribution and phylogenetic relationships, the iron-containing SOD, FeSOD,
appears to be the earliest known form of the SODs, and
FeSOR likewise appears to be quite ancient, but there is
no evidence that it was in place when the earliest Oxyphotobacteria first experienced O2 [42]. The earliest catalase appears to be the manganese catalase [43], and the
earliest heme-containing peroxidase known is the short
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peroxicin found in crown group Oxyphotobacteria including
the basal genus Gloeobacter [44]. Whether or not the
precursors of these enzymes were present in the earliest
Oxyphotobacteria, it is likely that these cells contained
relatively high levels of Mn2+, which is itself competent
to catalyze both the SOD and the catalase reactions [45].
The early oceans contained relatively high concentrations
of both Fe2+ and Mn2+, and it is possible that significant
concentrations of both of these cations were also present
intracellularly in early Oxyphotobacteria. The reactivity of
Fe2+ and Mn2+ with superoxide and hydrogen peroxide
are entirely different, with the result that iron functions as
a prooxidant and manganese as an antioxidant [46]. Fe2+
reacts with hydrogen peroxide via the Fenton reaction,
forming highly toxic hydroxyl radicals and Fe3+ as products. Superoxide can act to reduce Fe3+ back to Fe2+,
and both reactions combined constitute the so-called iron
catalyzed Haber-Weiss reaction (Reaction 1). By contrast,
Mn2+ does not do Fenton-type chemistry but instead
catalyzes disproportionation (dismutation) of either superoxide (Reaction 2) or hydrogen peroxide (Reaction 3).
In addition, Fe2+, but not Mn2+, reacts readily with O2 to
produce Fe3+.
Fe2þ =Fe3þ
O
2 þ H2 O2 ! OH þ OH
Mn2þ
þ
2O
2 þ 2H ! O2 þ H2 O2
Mn2þ
2H2 O2 ! O2 þ 2H2 O
(1)
(2)
(3)
It has been shown repeatedly that the O2-sensitivity of
cells entirely missing superoxide dismutase enzymes can
be completely rescued when manganese levels are high,
levels well within that observed in early seawater
(Figure 1) [45,47,48].
Manganese also offered another mode of protection.
Iron(II)-containing non-redox enzymes are frequently
inactivated by superoxide or hydrogen peroxide which
oxidize the metal center to iron(III) which then dissociates as Fe3+, leaving behind the inactive apoprotein. If the
iron(II) is replaced by manganese(II), such enzymes
frequently retain a large fraction of their enzymatic activities and they are relatively resistant to oxidative damage
[46,49,50].
Iron and sulfur: Modern organisms, both aerobic and
anaerobic, rely upon numerous iron–sulfur cluster-containing proteins [37], and it has recently become apparent
their numbers may have been seriously underestimated
in the past [51]. Iron–sulfur clusters are generally quite
sensitive to oxidants, which oxidize the clusters and cause
them to fall out of the proteins. The fact that this ancient
type of metalloproteins survived the rise of O2 in the
Current Opinion in Chemical Biology 2016, 31:166–178
172 Bioinorganic chemistry
atmosphere is a testament to their importance to all forms
of life.
There are several different complex pathways known for
the in vivo assembly of iron–sulfur clusters and their
incorporation into proteins, but the earliest of these
appears to be the Suf system [52]. When it occurs in
modern aerobic organisms, the Suf system is highly
complex, consisting of multiple components, and it relies
upon cysteine rather than inorganic sulfide, S2, as its
source of sulfur. But it is possible to deduce that in some
anaerobic organisms, the minimum functional unit is
SufB plus SufC — the first is an iron–sulfur scaffold
protein and the second is an ATPase. This minimal
system is expected to be sufficient for iron–sulfur cluster
assembly on the scaffold and insertion into the apoenzyme, but only in the case where abundant Fe(II) and
inorganic sulfide were readily available, as would have
been the case in early organisms [52].
Inorganic sulfide (S2, HS, or H2S) was relatively abundant in early anaerobic cells, but the concentrations are
very low in modern aerobic cells, and instead cysteine acts
as a source of inorganic sulfur when it is needed. But other
sulfur-containing compounds, in particular relatively concentrated pool of thiols, maintain their importance, acting
as redox buffers in modern intracellular biochemistry, and
it is likely that the anaerobic ancestors of the Oxyphotobacteria and other microbial groups used thiols in a similar
fashion prior to the GOE [35].
In addition to inorganic sulfide, another big difference to be
expected between pre-GOE cells and their modern descendants lies in the concentration of freely available Fe(II),
since early cells would have had little need to sequester
available iron sources (see discussion of ferritins below).
Photosynthetic cells require a large amount of iron, and we
can assume that this was true also of the first phototrophic
Oxyphotobacteria; iron would have been abundant and present as Fe(II), just as both sulfide and thiols are likewise
expected to have been abundant. Fe(II) would have readily formed Fe(II)–thiolate complexes in addition to Fe(II)–
sulfide complexes, using as ligands free cysteine, cysteine
side chains on proteins, and whatever other thiolates were
present. When O2 first appeared, the Fe(II)–sulfide and
Fe(II)–thiolate complexes would have been rapidly oxidized to Fe(III). Sulfide ligands are likely to have been
oxidized to elemental sulfur as well as polysulfides, sulfite,
thiosulfate, and sulfate [34], and thiolates would have been
oxidized to disulfides, cysteine to cystine, for example, or to
sulfinic acids (RSO2H) or sulfonic acids (RSO3H) [35].
Any reactive oxygen species formed, such as superoxide or
hydrogen peroxide would likewise be rapidly scavenged by
this Fe(II)–sulfide system.
Thus in early anaerobic Oxyphotobacteria, very small
amounts of O2 were not likely to be catastrophic so long
Current Opinion in Chemical Biology 2016, 31:166–178
as Fe(II), thiols, and sulfide remained abundant and did
not become limiting; rapid scavenging of small amounts
of O2 by the Fe(II)–thiolate plus the Fe(II)–sulfide
system could have removed O2 and any resulting H2O2
temporarily from the intracellular compartment. With
higher O2 fluxes, however, excessive consumption of
sulfide and thiolate ligands and the accumulation of
Fe(III) would have certainly been toxic to these anaerobic cells.
Greater amounts of O2 could have been tolerated if these
new Fe(II)–sulfide and Fe(II)–thiolate antioxidant systems were catalytic. Early members of the Oxyphotobacteria may have been capable of recycling sulfite and
sulfate using reductase enzymes [53], and the Fe(III)
formed could have been reduced by the excess sulfide,
making the Fe(II)–sulfide antioxidant system catalytic. In
addition, the enzyme thioredoxin may have acted in a
similar fashion in the Fe(II)–thiolate antioxidant system
by catalyzing reduction of disulfide-containing species.
Thioredoxin is a class of small proteins present in most or
all cells that facilitates thiol-disulfide exchange reactions
on other proteins [54,55]. In combination with thioredoxin reductase, a flavoprotein that uses NADPH to keep
thioredoxin in its reduced functional state [93], this
system is used to return cysteine side chains of many
proteins to their reduced states. The recent discovery of a
functional thioredoxin system in the strictly anaerobic
methanogens, presumably to facilitate thiol-disulfide exchange reactions, suggests that thioredoxins may have
been present in cells long before the rise of O2. This is
important because it implies that thioredoxins may have
already been present in early Oxyphotobacteria and other
coeval microbial groups, where they could have contributed to the rapid development of a thiol-disulfide antioxidant system by catalyzing the reduction of the disulfides
thus returning them to their reduced states [56].
The modern antioxidant most related to this primitive
Fe(II)-sulfide-thiol antioxidant systems is glutathione, an
antioxidant molecule that appears to have originated in
Oxyphotobacteria [57]. Prior to the advent of glutathione,
cysteine and then g-Glu-Cys probably were used as
antioxidants [35]. One enormous advantage of g-GluCys and glutathione over cysteine is that their metal
complexes are much more slowly oxidized by O2 than
most other metal-thiol complexes [35]. These other
thiol complexes would have been rapidly depleted by
higher O2 levels via redox metal-catalyzed reactions. The
evolution of glutathione was enormously important because it allowed for the sequestration of Fe(II), and later
Cu(I), as glutathione–metal complexes. These complexes
resisted rapid oxidation by O2 and thus protected the
other thiols present from redox metal-catalyzed destruction by O2. It is interesting in this regard to note that the
‘labile iron pool’ present in modern eukaryotic cells
consists primarily of Fe(II)–glutathione [58]; cysteine
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How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 173
could never have functioned in this manner in aerobic
cells because Fe(II)–cysteine complexes would be rapidly oxidized by O2 [35].
Sequestration of iron(III) — ferritin and polyphosphate: During early encounters with O2, microbes may have relied
upon sulfide and thiolates as antioxidants, but only at
great cost to the budget of sulfides, thiols, and other
reducing agents present in cells. Moreover, the cellular
redox potentials would have become more oxidizing as
these cells evolved an aerobic lifestyle, until ultimately
the soluble Fe(II) would have been converted to insoluble Fe(III)oxyhydroxides. The modern solution to this
problem is the ferritin family of proteins: ferritin, bacterioferritin, and the Dps proteins [59–61]. These proteins
are soluble hollow polypeptide spheres that are reversibly
filled with nanospheres of hydrated ferric oxides (ferrihydrite). Ferritin proteins function as iron storage, but they
also act as antioxidants. The antioxidant action of ferritins
is probably best illustrated by the Dps proteins. These act
as enclosed nanoreactors in which Fe(II) is oxidized in
two steps, first by O2 producing hydrogen peroxide, but
then even faster by hydrogen peroxide without releasing
any reactive oxygen species in the process [62–64].
An additional strategy that early aerobic cells may have
used to sequester Fe(III) might have been to bind it to
polyphosphate, the synthesis of which is up-regulated in
bacteria in response to oxidative stress [65]. Coordination
of Fe(III) to polyphosphate stabilizes it in that oxidation
state, and thus inhibits its ability to catalyze Fenton
chemistry [66].
Quinols: Lipid peroxidation also posed a new kind of
threat to the membranes of the ancestral microbes, particularly if those membranes contained unsaturated
lipids. The membranes of modern aerobic cells would
be susceptible to oxidative damage in the presence of O2
were in not for the presence of quinols such as tocopherols
and ubiquinol, which act as chain-breakers of the free
radical autoxidation process that peroxidizes lipids. Quinols such as menaquinol were already present in early
anoxygenic photosynthetic cells, where they played important functions as redox-active molecules in electron
transport chains [67–70]. Such quinols would have been
poised to act as inhibitors of free radical autoxidation of
the membrane components.
The modern widespread quinol antioxidant system found in
aerobic cells is alpha-tocopherol (vitamin E), which is only
synthesized by oxygenic photosynthetic organisms and
must be consumed in the diets of humans and other animals.
It is interesting to note that, just like glutathione, this system
appears to have first arisen in Oxyphotobacteria [71,72].
Carotenoids and singlet O2: Early Oxyphotobacteria encountered for the first time not only ground state triplet O2 but
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also highly reactive photoexcited singlet O2 [73]. Fortunately carotenoid pigments, which are excellent singlet
O2 quenchers, were already part of the photosynthetic
apparatus in anoxygenic phototrophs [74]. In modern
Oxyphotobacteria, this role is played by the orange carotenoid protein (OCP) [75].
Evolution of advanced antioxidant systems in
Oxyphotobacteria
Flavodiiron proteins: Flavodiiron proteins (FDPs) are oxidoreductases that are predominantly found in Oxyphotobacteria and anaerobes, where they reduce NO and O2 to
N2O and H2O respectively, with reducing equivalents
from NAD(P)H, or, in the case of methanogens, F420H2
[78,80]. All FDPs contain two conserved structural
domains: an N-terminus metallo-b-lactamase-like domain that contains a non-heme Fe–Fe center, and a Cterminus flavodoxin-like domain. Crystal structures reveal that FDPs form a homodimeric head-to-tail arrangement that places the FMN moiety of one subunit near the
diiron site of the metallo-b-lactamase-like domain of the
other for efficient electron transfer during substrate reduction [76–79]. While all FDPs have this conserved
flavodiiron compound domain structure, some have gene
fusions of additional redox domains at their C-termini
(Figure 4) [80].
In non-phototrophic organisms, FDPs appear to function
in NO and O2 detoxification, commonly showing a preference for one molecule or the other [80,81]. FDPs
provide a biochemically efficient means of removing
O2 and alleviating oxidative stress. For example, the
anaerobic protist Giardia lamblia lacks respiratory O2
reductases and does not contain the typical suite of
enzymes for dealing with ROS, but it does contain an
FDP with a high affinity for O2 [82]. FDPs found in
methanogens also display a strong preference for O2 [78].
Interestingly, FDPs are present in many paralogous
copies (typically between two and six) in the genomes
of all extant Oxyphotobacteria, except for losses in nonphototrophic symbionts [83]. These form both a monophyletic clade of FDPs and an independent class marked
by the fusion of a flavodoxin-like (RutF) domain at the Cterminus (Figure 4). This phylogenetic distribution
implies a rich evolutionary history of this family of
proteins in the Oxyphotobacteria and suggests that FDPs
were important during the evolution of oxygenic photosynthesis. The exact function and physiological utility of
the different flavors of FDPs found in Oxyphotobacteria
are still not well known [41]. In recombinant studies,
FDPs from Oxyphotobacteria are capable of direct reduction of O2 to water without the production of ROS [80].
Two genes encoding FDPs, Flv1 and Flv3, promote a
Mehler-like reaction in Oxyphotobacteria, modulating the
photoreduction of O2 with electrons downstream of PSI
[84]. Unlike the Mehler reaction in plants and algae,
Current Opinion in Chemical Biology 2016, 31:166–178
174 Bioinorganic chemistry
Figure 4
(a)
(b)
Class A
Class B
Class C
Class D
Oxyphotobacteria
O2 , NO H2O , N2O
H2O
O2
H2O
O2
H2O
O2
Fe Fe
Fe Fe
Fe Fe
Fe Fe
FMN
FMN
FMN
FMN
Rd
Flv
(c)
NAD(P)H NADP
NAD(P)H NADP+
NADH NAD+
crown group Oxyphotobacteria (< 2.0 Ga)
crown group FDPs
environmental O2 (~2.4 Ga)
fusion of
flavodoxin
fusion
non-redox Fe(II) diiron
Fe(III) diiron
(or Zn) hydrolase
oxidoreductase
0.1
Flv
+
Flv
NADH NAD+
root?
Rd
Rd
Flv
A
B
C
D
diiron
flavoprotein
O2 reductase
complex
C-terminal
flavodiiron diversification flavodiiron
O2 reductase (other taxa) flavodoxin-O2
oxidoreductase
homodimer
complex
gene duplication &
paralogous evolution
flavodiiron
NAD(P)H-O2 oxidoreductase
homodimer
Current Opinion in Chemical Biology
(a) Ferric diiron proteins (O2 and NO reductases) comprise a large, and somewhat modular, protein family commonly classified by their domain
structure and diversity of fusions of different redox domains that occur at their C terminus. Cartoons highlight domain structure of the difference
classes. (b) Phylogenetic relationships of ferric diiron proteins (FDPs) constructed using sequence alignments of their common flavodiiron core.
The classes with additional C terminal fusions are well captured by clades in the phylogentic relationships, with positions derived within the
structurally simple Class A FDPs. Note that the fusion of rubredoxin domains appear to have occurred twice independently each in the Class B
and Class D FDPs. These proteins are rather sparsely and variably distributed among Bacteria, Archaea, and a number of eukaryotic protists —
mainly in anaerobes. Importantly all Oxyphotobacteria contain FDPs, often many paralogous copies, where these proteins play an important role in
cell redox balance, removing O2, and protecting photosystems against singlet oxygen. These proteins in Oxyphotobacteria form a diverse clade
highlighting their importance in evolutionary history within the group. They have a unique domain structure with a fusion of a flavodoxin domain,
and they directly oxidize NAD(P)H. These proteins are responsible for a tremendous O2 reduction flux — up to 40% of photosynthetically produced
O2 — in modern Oxyphotobacteria [85]. (c) Evolutionary hypothesis for the origin and early evolution of FDPs in Oxyphotobacteria. Currently there
is no solved crystal structure of a Class C FDP from the Oxyphotobacteria, and the orientation and placement of the C-terminal flavodoxin domain
remains uncertain; it could feed electrons into either active site in the dimer. We view FDPs as important proteins in stem group Oxyphotobacteria
because they would have allowed for the maintenance of low cellular O2 levels — in a sense buying time to explore and test a range of possible
antioxidant systems to cope with aerobic stress.
however, this FDP-dependent reaction in Oxyphotobacteria
does not produce ROS. From observations of the differential mass law fractionations of multiple oxygen isotopes
(16O, 17O, 18O), Helman et al. [85] estimated that as much as
40% of the O2 leaving PSII was re-reduced by FDPs in
photosynthetically active cells, compared with only 6%
going to respiration at Complex IV. The amount of O2 that
flows to FDPs appears to depend on the availability of CO2
and light and is particularly important during fluctuating
light conditions, providing a harmless mechanism to
maintain cellular redox balance by dissipating excess electrons downstream of PSI [86]. Other FDPs in Oxyphotobacteria appear to play roles in photoprotection of PSII
against 1O2 and may function as heterodimers [84,87]; yet
others appear to provide heterocysts with anaerobic environments conducive to nitrogen fixation [41]. Altogether,
Current Opinion in Chemical Biology 2016, 31:166–178
the diversity and omnipresence of FDPs in Oxyphotobacteria supports the view that the FDPs were integral to the
development of oxygenic photosynthesis [41].
Several aspects of the structural biology and biochemistry
of FDPs permit one to develop a hypothesis for the
evolution of this protein family as an important complement to oxygenic photosynthesis (Figure 4). Because all
FDPs have in common a conserved two-domain structure, one might infer that they result from the fusion of
proteins that once had little to do with one another.
Metallo-b-lactamase-like domains appear in proteins
with diverse functions, and on the basis of their distribution in metabolism and the tree of life appear to be
exceptionally old [77]. We envision this domain originally
contained a non-redox metal center comprised of either
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How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 175
Fe(II) or Zn(II), and probably functioned as a hydrolytic
enzyme. With early photosynthetic O2 fluxes, the metal
center became a ferric Fe-Fe site with a high affinity for
O2, and formed an early O2 reductase complex in partnership with the flavoprotein. Due to the distances between the FMN moiety and the diiron site (40 Å), this
complex could only function as an O2 reductase in the
configuration of a multi-subunit homodimer. The FDPs
family, thus, was born from the fusion of these two
interacting domains that were probably organized together as genes within an operon. The radiation of the FDPs
occurred, providing a useful mechanism for many anaerobic microorganisms to detoxify O2. This co-occured with
the functional diversification of the C-terminus in a
number of different groups, with lateral gene transfer
playing an important role in the extant distribution of
FDPs [80]. In stem group Oxyphotobacteria, the fusion of
another flavodoxin domain occurred before the adaptive
radiation of the many different FDPs found within them,
because this fusion is observed in all members of crown
group Oxyphotobacteria. Thus based on the phylogenetic
distribution of FDPs in Oxyphotobacteria, their general
absence or evolutionary incongruence in the Melainabacteria, and their diverse roles in protecting cells against O2
stresses, we hypothesize that FDPs played an key role in
allowing stem group Oxyphotobacteria to complete early
experiments in phototrophic O2 production.
Ascorbate was not an ancient antioxidant: The ascorbate–
ascorbate peroxidase antioxidant system, which is a major
antioxidant system in plants and most animals, is absent
in Oxyphotobacteria. In fact, it seems likely that prokaryotes in general do not synthesize ascorbate [88]. This is
in sharp contrast to two other very important modern
antioxidant systems, glutathione and alpha-tocopherol,
both of which appear to have originated in the Cyanobacteria phylum and were then widely spread to other
species.
fluxes of intracellular dioxygen. Importantly, manganese
appears to have played major roles in both the production
and early detoxification of O2. Current data also suggest
that the Oxyphotobacteria were important incubators for a
wide variety of solutions to deal with oxidative stress (like
glutathione, alpha-tocopherol, and FDPs).
Going forward to better understand how life responded to
the GOE, we advocate greater focus on non-enzymatic
solutions that were likely present in early cells, because it
is possible that many of the enzymatic systems important
today were not available for biology at this time. We think
that efforts using comparative biology to infer the relative
timing of different antioxidant systems will continue to
provide useful insight into this problem. This is a good
time to be asking these questions because the landscape
for understanding microbial and metabolic evolution is
rapidly changing, enabled by new single-cell and metagenomic sequencing technologies, which probe microbial
diversity deeply and make it possible to populate evolutionary analyses with substantial amounts of sequence
data. We think this approach is promising and will enable
integrative hypothesis generation and testing as new
groups of microbes are discovered and characterized.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Usha Lingappa, Hope Johnson, Jena Johnson, Dianne Newman,
and two anonymous reviewers for helpful feedback on ideas synthesized in
this paper. We acknowledge support from a David and Lucile Packard
Foundation Fellowship in Science and Engineering (WWF), and the
Agouron Institute (JH and WWF).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
of special interest
of outstanding interest
Conclusions
The evolution of oxygenic photosynthesis in Oxyphotobacteria led to the GOE and, along with it, to the greatest
interval of environmental change in Earth history. In turn,
O2 fed back on biology with both substantial risk and
substantial reward. While the availability of O2 provided
life with new bioenergetic opportunities, it also produced
significant oxidative stress. Virtually all modern cells —
including strict anaerobes — rely upon diverse mechanisms for coping with oxidative stress, but most of these
systems would not have been in place when O2 first
appeared. Nevertheless, there were likely a number of
preexisting biochemical systems and inorganic reactions
that had fortuitous antioxidant chemistry, which with
time enabled the evolution of more complex enzymatic
antioxidant systems. Oxyphotobacteria provide an interesting test case for understanding how life survived the
GOE, because these cells were the first to deal with large
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