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How did life survive Earth’s great oxygenation?

1 1

Woodward W Fischer , James Hemp and

1,2

Joan Selverstone Valentine

Life on Earth originated and evolved in anoxic environments. capable of oxygenic . These organisms like-

Around 2.4 billion-years-ago, ancestors of ly supported themselves largely by means of anoxygenic

invented oxygenic photosynthesis, producing substantial photosynthesis at first, perhaps with only intermittent

amounts of O2 as a byproduct of phototrophic oxidation. production of O2 in relatively small amounts. At this stage

The sudden appearance of O2 would have led to significant in Earth history, life did not have the defenses necessary to

due to incompatibilities with core cellular deal with an oxidant as powerful as O2 [9 ]. How did early

biochemical processes. Here we examine this problem through Oxyphotobacteria survive the intracellular production of O2

the lens of Cyanobacteria — the first taxa to observe significant and make the transition from a strictly anaerobic to aerobic

fluxes of intracellular dioxygen. These early oxygenic organisms ? Here we integrate data from bioinorganic

likely adapted to the oxidative stress by co-opting preexisting chemistry and comparative biology to infer the evolution

systems (exaptation) with fortuitous properties. Over of tolerance in Oxyphotobacteria. Notably, ancestral

time more advanced antioxidant systems evolved, allowing Oxyphotobacteria could have coped with small amounts of

Cyanobacteria to adapt to an aerobic lifestyle and become the O2 during the transition to oxygenic phototrophy by co-

most important environmental engineers in Earth history. opting preexisting systems with fortuitous antioxidant

Addresses properties. This would have allowed time for more modern

1

Division of Geological & Planetary Sciences, California Institute of antioxidant systems to evolve. We discuss non-enzymatic,

Technology, Pasadena, CA 91125, United States

small- solutions to mitigate O stress that were

2 2

Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA

present in early cells, followed by some specific adaptations

90095, United States

that the Oxyphotobacteria developed to enable the transition

Corresponding authors: Fischer, Woodward W (wfi[email protected]) to an oxygenated world.

and Valentine, Joan Selverstone ([email protected])

Geological record of O2

Current Opinion in Chemical Biology 2016, 31:166–178 Today O2 comprises nearly 21% of the atmosphere,

This review comes from a themed issue on Bioinorganic chemistry however a wide array of observations made over the past

sixty years from the geological record illustrates that it was

Edited by R David Britt and Emma Raven

extremely scarce prior to the evolution of oxygenic pho-

tosynthesis [10 ]. How scarce? An exact paleobarometer

for O2 remains out of reach, but there are several types of

http://dx.doi.org/10.1016/j.cbpa.2016.03.013 geological and geochemical data that can be converted

1367-5931/# 2016 Elsevier Ltd. All rights reserved. into O2 concentrations that are thought to be accurate

within an order of magnitude or two (Figure 1). Sedimen-

tary 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

Introduction minerals, such as pyrite (FeS2), uraninite (UO2), and

Data from the geological record indicate that life was siderite (FeCO3) [11]. These minerals are quickly oxi-

present on Earth very early in its history. Intriguing obser- dized and destroyed in the presence of even trace O2, so

5

vations of graphitic carbon in some of the oldest rocks and their presence constrains O2 levels to less than 10 atm

minerals have been proposed to be traces of Earth’s early before 2.4 Ga [11]. Additional independent geochemical

biosphere [1,2]. By 3.4–3.2 billion years ago (giga annum proxies support this view. For example, multiple sulfur

or Ga), a range of observations indicate the presence of isotopes in marine sedimentary rocks older than 2.4 Ga

microbial cells [3,4] with diverse anaerobic display a widespread and unusual type of mass indepen-

[5–8]. Even using the most conservative estimate of 3.2 Ga dent fractionation (MIF) caused by the photochemistry of

5

for the origin of life, it was clearly present long before O2 SO2 in an atmosphere largely devoid of O2 (10 atm

10

appeared in significant amounts in Earth’s atmosphere. and likely closer to 10 atm). This corresponds to

astonishingly low environmental levels of O2 — suffi-

The first organisms to encounter significant and sustained ciently low that the anaerobic microorganisms that

oxidative stress due to O2 were ancestral Oxyphotobac- existed at the time may not have ever encountered

teria — members of the bacterial phylum Cyanobacteria, biologically meaningful amounts of oxygen related stress.

Current Opinion in Chemical Biology 2016, 31:166–178 www.sciencedirect.com

How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 167

Figure 1

animals GOE Charcoal Sulfate deposits redox-sensitive detrital grains Fe in Paleosols & Red beds Oldest sedimentary rocks

Origin of the Earth ~ 4.55 Ga deposits

MIF of S isotopes

Hadean Archean Proterozoic Phan.

104

103

102

101 Log[Mn] (ppm)

100

10–1 4500 40003500 3000 2500 20001500 1000 500 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

5

Paleoproterozoic sandstones and conglomerates illustrates that O2 levels were lower than 10 atm in the atmosphere and Earth surface 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

5 10

levels were exceedingly low, much less than 10 atm, and perhaps closer to 10 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].

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 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

2+

dolomite-bearing (CaMg(CO3)2) sedimentary rocks provides a coarse measure of the amount of Mn 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 irrevers- changes in the biogeochemical cycles of major redox-

ible rise of O2 occurred between 2.4 and 2.35 Ga — a active elements, such as Fe, Mn, and S. At this time MIF

transition known as the Great Oxygenation Event (GOE) [10 ] and redox sensitive detrital grains disappeared [11].

(Figure 1) [10 ,12]. Many of these observations record Ferrous iron in igneous minerals became oxidized and

www.sciencedirect.com Current Opinion in Chemical Biology 2016, 31:166–178

168 Bioinorganic chemistry

was retained in ancient soils [13]. Iron oxide cements crust — evidenced by the loss of detrital pyrite from the

become widespread in fluvial (river) and near-shore ma- record (Figure 1) — sourced and solubilized chalcophilic

rine sandstones, called red beds. Thick deposits of Mn- elements like Cu, which was uniquely suited for the

oxide minerals like those in the Kalahari Manganese evolution of high potential metabolisms and aerobic

Field in South Africa [14] postdate the GOE, with an biology [16].

important exception (more on this below). In addition

sulfate salts begin to accumulate in sedimentary basins, Biological record of O2

recording the oxidative weathering of sulfide-bearing The relative timing of the appearance of O2 from com-

minerals [15]. Because these redox proxies are sensitive parative biology and biochemistry is consistent with

to small amounts of O2 (e.g., ppm levels), we do not have observations from the geological record. These data sug-

good estimates for how high O2 rose during the GOE gest that O2 was not used by early life in either biosyn-

(some hypotheses suggest perhaps only 1% of modern). thetic reactions or respiration. Network analyses of

However it is clear from the geological record that, after metabolic pathways from diverse microbes identified a

the GOE, O2 became widespread across vast swaths of the strictly anaerobic core, with O2 requiring reactions

Earth’s surface and was thereafter an important compo- appearing only in the terminal steps of a small fraction

nent of the atmosphere and surface waters. of [23]. This implies that O2 was not available

to early organisms as a substrate for biochemical reactions

Changes in metal ion availability: Metal use in biology is and was only incorporated into metabolism after the

primarily determined by evolutionary history, which is evolution of oxygenic phototrophy.

the result of dynamic interactions between environmen-

tal availability, biochemical demands, and ecological A similar view is provided by phylogenomic analyses of

flexibility. The metal concentrations in seawater have the distribution of aerobic respiration. The majority of

changed dramatically over Earth history as a function of and Archaea phyla have anaerobic basal mem-

the differential solubility, complexation, and redox be- bers with aerobic members found only in derived (i.e. non

havior of distinct metals — modulated by changes in the ancestral) positions (Figure 2a). For example, in the

redox state of Earth’s surface environments and geo- phylum members of the basal classes Rubro-

chemical budgets set by different rock-forming minerals bacteria and Coriobacteria are anaerobic. Classes with

within the crust [16,17]. Iron and manganese are the first aerobic members (Acidimicrobiia, Nitriliruptoria, and Acti-

and third most abundant transition elements in the crust nobacteria) are derived. Comparisons of the different O2

and substitute for one another in a wide range of rock- reductases used by Actinobacteria for aerobic respiration

forming silicate minerals, dominantly as Fe(II) and ex- shows two major independent acquisitions (once in the

clusively as Mn(II). Consequently, prior to the GOE Thermoleophilia and once in the Acidimicrobiia + Nitrilir-

chemical weathering of silicate minerals would have uptoria + Actinobacteria clade), with a subsequent loss in

2+ 2+

provided substantial fluxes of Fe and Mn into the the Bifidobacteria genus after they adapted to anoxic gut

oceans, resulting in high concentrations in seawater [18]. ecosystems (Figure 2a). Many other phyla exhibit similar

In particular, Archean-age carbonate platforms are loaded phylogenetic patterns, suggesting that the major radiation

with remarkably high levels of Mn(II) — much higher of Bacteria occurred at a time when the Earth was anoxic

than observed in younger carbonate platforms (Figure 1) and that aerobic respiration was acquired after they had

[14,18]. It is challenging to convert these data accurately diverged from one another, likely after the GOE.

into seawater concentrations because the partitioning

depends on the kinetics and mechanisms of carbonate The biological record over the past two billion years

mineral precipitation, but experimental relationships and shows that O2 has driven major revolutions in biology,

2+

solubility constraints suggest that seawater Mn concen- forming a biochemical veil that makes it challenging to

trations prior to the GOE ranged from 5 mM to infer some aspects of early life prior to O2 with a high

120 mM [19]. After the GOE, iron availability dramati- degree of confidence. Deductions from comparative bi-

cally decreased in surface seawater due to removal of ology and biochemistry have tremendous value for study-

insoluble iron oxides, marked by red beds and ferric iron ing this problem, but it is important to remember that

in paleosols (Figure 1). However iron was so thoroughly they present a view biased by extinction. The ‘winners’

integrated into cellular biology by that point that creative wrote the biological record. Mechanisms to cope with O2

strategies were required so that organisms would be able and reactive oxygen species (ROS) toxicity have been

to obtain it in oxygenated environments. Mn concentra- thoroughly integrated into the function of modern

tions appear not to have fallen as precipitously, likely cells — even for what are typically viewed as strictly

2+

because the kinetics of Mn oxidation are substantially anaerobic organisms [24]. All we easily study are the

2+

slower than those of Fe [20]. In contrast to iron, for biological solutions selected after several billion years

2+

example, the bioavailability of Zn does not appear to of evolution. The organisms and biochemistries that

have changed dramatically over time [21,22]. O2-driven did not survive can only be inferred from geological

oxidative weathering of sulfide-bearing minerals in the observations and imagined via evolutionary hypotheses

Current Opinion in Chemical Biology 2016, 31:166–178 www.sciencedirect.com

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 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 , 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 Segre` [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 Cyanobacteria phylum. New members have been found in

systems for dealing with the toxicity of O2 must have many aphotic environments and analysis of genomes

been in place for cells to survive the GOE, it is, however, assembled from environmental metagenomic datasets

just as reasonable that such systems co-evolved in time (and one cultured isolate) demonstrate that these organ-

with dioxygen because they would have had little selec- isms are missing genes for phototrophy [10 ,25 ,26 ,27].

tive advantage prior to the GOE. Instead cells may have Current evolutionary relationships between members of

initially coped with O2 stresses using molecules that the Cyanobacteria phylum are shown in Figure 3; it is

performed other functions. And those that were the most important to note that oxygenic photosynthesis exhibits a

helpful early on were those that had fortuitous antioxidant derived position [10 ]. A new classification scheme has

chemistry. been proposed [26 ] in which the Cyanobacteria are now

comprised of three classes: the Oxyphotobacteria (oxygenic

The transition to oxygenic phototrophy Cyanobacteria), the , and ML635J-21.

New insights into the evolution of Cyanobacteria: We can gain

considerable insights about how life adapted to O2 by After their divergence from the Melainabacteria, it was

examining Oxyphotobacteria, the first organisms to witness stem group members of the Oxyphotobacteria (between

substantial fluxes of intracellular O2. Genomic studies markings 1 and 2 in Figure 3) — perhaps long ex-

over the last couple years have dramatically changed the tinct — that developed oxygenic photosynthesis. Be-

way we think about the diversity and evolution of the tween molecular clock estimates [28] and geological

www.sciencedirect.com 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 therefore have originated by the accumulation of oxidized

2.4–2.35 billion years ago. Mn in the high potential reaction center, with catalytic

Mn oxidation eventually being replaced by water oxida-

Manganese: a gateway to the high potential world: The transi- tion. Importantly, evidence for this transition is found in

tion from anoxygenic phototrophy to oxygenic photosyn- the geological record where O2-independent oxidation of

thesis in Oxyphotobacteria required the evolution of two Mn was observed to have occurred prior to the GOE

important features: a high potential reaction center, and a (Figure 1) [29 ].

CaMn4O5 bioinorganic cluster called the water-oxidizing

complex (WOC) capable of oxidizing water [30]. Modern Initial threats from O2

anoxygenic reaction centers are unable to generate high Early O2 production: fluxes, concentrations, and timescales:

redox potentials (<+500 mV). The highest potential elec- Once oxygenic photosynthesis evolved, the timescale for

tron donor for anoxygenic phototrophy is nitrite O2 to titrate the existing pools of geochemically derived

(+430 mV) [31]. One hypothesis emerging in recent years reductants in Earth’s surface environments, which there-

from both geological and biological data is that Mn- after became oxygenated, is relatively short in the context

oxidizing phototrophy was likely the direct precursor to of geological time — on the order of tens of thousands to a

2+

oxygenic photosynthesis [10 ]. Mn was in abundant in hundred thousand years [32]. A number of anoxic envir-

the early oceans (Figure 1) and has redox potentials near onments would endure through the GOE (e.g., pore fluids

to that of water. Biochemical and structural analyses of in marine sediments), and provide refuge for anaerobic

reaction centers strongly suggest that the ability to oxi- . But for oxygenic phototrophs, O2 was an

dize Mn compounds drove the evolution of high potential undeniable problem molecule. What are the concentra-

reaction centers in early Oxyphotobacteria [30]. In fact, tions and fluxes of O2 that typify photosynthetic cells?

modern Oxyphotobacteria still oxidize Mn using PSII dur- Recent calculations from theory suggest that the O2

ing the photoassembly of their WOCs. The WOC may 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 peroxicin found in crown group Oxyphotobacteria including

than the external environment for solitary planktonic the basal genus [44 ]. Whether or not the

cells, though much higher for cells living in biofilms precursors of these were present in the earliest

and mats, which can achieve highly supersaturated O2 Oxyphotobacteria, it is likely that these cells contained

2+

levels [33 ]. This work highlights an important asym- relatively high levels of Mn , which is itself competent

metry between photosynthetic O2 production and con- to catalyze both the SOD and the reactions [45].

sumption by aerobic respiration: the rates of O2

generation are slow compared to diffusive fluxes. This The early oceans contained relatively high concentrations

2+ 2+

lessens the degree of oxidative stress for the earliest of both Fe and Mn , and it is possible that significant

Oxyphotobacteria [33 ]. Even functioning at or near mod- concentrations of both of these cations were also present

18 1 1

ern photosynthetic rates (10 mol s ), intra- intracellularly in early Oxyphotobacteria. The reactivity of

2+ 2+

cellular concentrations were maximally 250 nM [33 ]; Fe and Mn with and

presumably early Oxyphotobacteria did not produce O2 at are entirely different, with the result that iron functions as

2+

anywhere near these high rates. It is important to note a prooxidant and manganese as an antioxidant [46]. Fe

that these fluxes are still large compared to the concen- reacts with hydrogen peroxide via the Fenton reaction,

3+

trations required for O2 to disrupt the intracellular pools forming highly toxic hydroxyl radicals and Fe as pro-

3+ 2+

of Fe [20], sulfide [34], thiols, cysteine [35 ], flavins [36], ducts. Superoxide can act to reduce Fe back to Fe ,

and metal centers [37]. and both reactions combined constitute the so-called iron

catalyzed Haber-Weiss reaction (Reaction 1). By contrast,

1 2+

O2 as the first oxidative threat: In addition to ROS formed by Mn does not do Fenton-type chemistry but instead

O2 reduction at various points along transport catalyzes (dismutation) of either su-

chains (e.g. Complex I, Complex III, PSI), photoexcited peroxide (Reaction 2) or hydrogen peroxide (Reaction 3).

2+ 2+

singlet O2 presented a unique problem for Oxyphotobacteria In addition, Fe , but not Mn , reacts readily with O2 to

3+

as a substantial and inchoate oxidative threat. produce Fe .

are studded with as the major light harvesting 2þ 3þ

Fe =Fe

molecules. Energy transfer from excited long-lived triplet

O2 þ H2O2 ! OH þ OH (1)

states of to ground state triplet O2 can create

1 2þ

O2 with extremely high quantum efficiency [38]. Further- þMn

1 2O2 þ 2H ! O2 þ H2O2 (2)

more O2 is highly reactive with a wide range of molecules

including and lipids, has a short half-life within

Mn2þ

cells [39], and is one of the most critical sources of oxidative

2H2O2 ! O2 þ 2H2O (3)

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 It has been shown repeatedly that the O2-sensitivity of

mechanisms of non-photochemical quenching (NPQ), and cells entirely missing enzymes can

can be used to dissipate energy from photo- be completely rescued when manganese levels are high,

sensitized chlorophyll; however a good solution is simply to levels well within that observed in early seawater

keep intracellular O2 levels low, particularly near the (Figure 1) [45,47 ,48].

photosystems. This logic appears to have been important

for early Oxyphotobacteria, which appear to have employed Manganese also offered another mode of protection.

flavodiiron proteins to remove O2 and alleviate the produc- Iron(II)-containing non-redox enzymes are frequently

tion of this problematic molecule [41 ]. inactivated by superoxide or hydrogen peroxide which

oxidize the metal center to iron(III) which then dissoci-

3+

Early fortuitous antioxidant systems ates as Fe , leaving behind the inactive apoprotein. If the

Manganese(II) and reactive oxygen species: In modern aerobic iron(II) is replaced by manganese(II), such enzymes

organisms, antioxidant enzymes such as superoxide dis- frequently retain a large fraction of their enzymatic activ-

mutases (SOD), superoxide reductases (SOR), ities and they are relatively resistant to oxidative damage

and peroxidases, play a crucial role in defending cells [46,49,50].

from superoxide, hydrogen peroxide, and other perox-

ides. Based on their widespread distribution and phylo- Iron and sulfur: Modern organisms, both aerobic and

genetic relationships, the iron-containing SOD, FeSOD, anaerobic, rely upon numerous iron–sulfur cluster-con-

appears to be the earliest known form of the SODs, and taining proteins [37], and it has recently become apparent

FeSOR likewise appears to be quite ancient, but there is their numbers may have been seriously underestimated

no evidence that it was in place when the earliest Oxy- in the past [51 ]. Iron–sulfur clusters are generally quite

photobacteria first experienced O2 [42]. The earliest cata- sensitive to oxidants, which oxidize the clusters and cause

lase appears to be the manganese catalase [43], and the them to fall out of the proteins. The fact that this ancient

earliest heme-containing peroxidase known is the short type of metalloproteins survived the rise of O2 in the

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172 Bioinorganic chemistry

atmosphere is a testament to their importance to all forms as Fe(II), thiols, and sulfide remained abundant and did

of life. not become limiting; rapid scavenging of small amounts

of O2 by the Fe(II)–thiolate plus the Fe(II)–sulfide

There are several different complex pathways known for system could have removed O2 and any resulting H2O2

the in vivo assembly of iron–sulfur clusters and their temporarily from the intracellular compartment. With

incorporation into proteins, but the earliest of these higher O2 fluxes, however, excessive consumption of

appears to be the Suf system [52 ]. When it occurs in sulfide and thiolate ligands and the accumulation of

modern aerobic organisms, the Suf system is highly Fe(III) would have certainly been toxic to these anaero-

complex, consisting of multiple components, and it relies bic cells.

2

upon cysteine rather than inorganic sulfide, S , as its

source of sulfur. But it is possible to deduce that in some Greater amounts of O2 could have been tolerated if these

anaerobic organisms, the minimum functional unit is new Fe(II)–sulfide and Fe(II)–thiolate antioxidant sys-

SufB plus SufC — the first is an iron–sulfur scaffold tems were catalytic. Early members of the Oxyphotobac-

protein and the second is an ATPase. This minimal teria may have been capable of recycling sulfite and

system is expected to be sufficient for iron–sulfur cluster sulfate using reductase enzymes [53], and the Fe(III)

assembly on the scaffold and insertion into the apoen- formed could have been reduced by the excess sulfide,

zyme, but only in the case where abundant Fe(II) and making the Fe(II)–sulfide antioxidant system catalytic. In

inorganic sulfide were readily available, as would have addition, the may have acted in a

been the case in early organisms [52 ]. similar fashion in the Fe(II)–thiolate antioxidant system

by catalyzing reduction of disulfide-containing species.

2

Inorganic sulfide (S , HS , or H2S) was relatively abun- Thioredoxin is a class of small proteins present in most or

dant in early anaerobic cells, but the concentrations are all cells that facilitates thiol-disulfide exchange reactions

very low in modern aerobic cells, and instead cysteine acts on other proteins [54,55]. In combination with thiore-

as a source of inorganic sulfur when it is needed. But other doxin reductase, a flavoprotein that uses NADPH to keep

sulfur-containing compounds, in particular relatively con- thioredoxin in its reduced functional state [93], this

centrated pool of thiols, maintain their importance, acting system is used to return cysteine side chains of many

as redox buffers in modern intracellular biochemistry, and proteins to their reduced states. The recent discovery of a

it is likely that the anaerobic ancestors of the Oxyphoto- functional thioredoxin system in the strictly anaerobic

bacteria and other microbial groups used thiols in a similar methanogens, presumably to facilitate thiol-disulfide ex-

fashion prior to the GOE [35 ]. change reactions, suggests that may have

been present in cells long before the rise of O2. This is

In addition to inorganic sulfide, another big difference to be important because it implies that thioredoxins may have

expected between pre-GOE cells and their modern des- already been present in early Oxyphotobacteria and other

cendants lies in the concentration of freely available Fe(II), coeval microbial groups, where they could have contrib-

since early cells would have had little need to sequester uted to the rapid development of a thiol-disulfide antiox-

available iron sources (see discussion of below). idant system by catalyzing the reduction of the disulfides

Photosynthetic cells require a large amount of iron, and we thus returning them to their reduced states [56 ].

can assume that this was true also of the first phototrophic

Oxyphotobacteria; iron would have been abundant and pres- The modern antioxidant most related to this primitive

ent as Fe(II), just as both sulfide and thiols are likewise Fe(II)-sulfide-thiol antioxidant systems is , an

expected to have been abundant. Fe(II) would have readi- antioxidant molecule that appears to have originated in

ly formed Fe(II)–thiolate complexes in addition to Fe(II)– Oxyphotobacteria [57]. Prior to the advent of glutathione,

sulfide complexes, using as ligands free cysteine, cysteine cysteine and then g-Glu-Cys probably were used as

side chains on proteins, and whatever other thiolates were [35 ]. One enormous advantage of g-Glu-

present. When O2 first appeared, the Fe(II)–sulfide and Cys and glutathione over cysteine is that their metal

Fe(II)–thiolate complexes would have been rapidly oxi- complexes are much more slowly oxidized by O2 than

dized to Fe(III). Sulfide ligands are likely to have been most other metal-thiol complexes [35 ]. These other

oxidized to elemental sulfur as well as polysulfides, sulfite, thiol complexes would have been rapidly depleted by

thiosulfate, and sulfate [34], and thiolates would have been higher O2 levels via redox metal-catalyzed reactions. The

oxidized to disulfides, cysteine to cystine, for example, or to evolution of glutathione was enormously important be-

sulfinic acids (RSO2H) or sulfonic acids (RSO3H) [35 ]. cause it allowed for the sequestration of Fe(II), and later

Any reactive oxygen species formed, such as superoxide or Cu(I), as glutathione–metal complexes. These complexes

hydrogen peroxide would likewise be rapidly scavenged by resisted rapid oxidation by O2 and thus protected the

this Fe(II)–sulfide system. other thiols present from redox metal-catalyzed destruc-

tion by O2. It is interesting in this regard to note that the

Thus in early anaerobic Oxyphotobacteria, very small ‘labile iron pool’ present in modern eukaryotic cells

amounts of O2 were not likely to be catastrophic so long consists primarily of Fe(II)–glutathione [58]; cysteine

Current Opinion in Chemical Biology 2016, 31:166–178 www.sciencedirect.com

How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 173

could never have functioned in this manner in aerobic also highly reactive photoexcited singlet O2 [73]. Fortu-

cells because Fe(II)–cysteine complexes would be rapid- nately pigments, which are excellent singlet

ly oxidized by O2 [35 ]. O2 quenchers, were already part of the photosynthetic

apparatus in anoxygenic phototrophs [74]. In modern

Sequestration of iron(III) — and polyphosphate: Dur- Oxyphotobacteria, this role is played by the orange carot-

ing early encounters with O2, microbes may have relied enoid protein (OCP) [75].

upon sulfide and thiolates as antioxidants, but only at

great cost to the budget of sulfides, thiols, and other Evolution of advanced antioxidant systems in

reducing agents present in cells. Moreover, the cellular Oxyphotobacteria

redox potentials would have become more oxidizing as Flavodiiron proteins: Flavodiiron proteins (FDPs) are oxi-

these cells evolved an aerobic lifestyle, until ultimately doreductases that are predominantly found in Oxyphoto-

the soluble Fe(II) would have been converted to insolu- bacteria and anaerobes, where they reduce NO and O2 to

ble Fe(III)oxyhydroxides. The modern solution to this N2O and H2O respectively, with reducing equivalents

problem is the ferritin family of proteins: ferritin, bacter- from NAD(P)H, or, in the case of methanogens, F420H2

ioferritin, and the Dps proteins [59–61]. These proteins [78,80]. All FDPs contain two conserved structural

are soluble hollow polypeptide spheres that are reversibly domains: an N-terminus metallo-b-lactamase-like do-

filled with nanospheres of hydrated ferric oxides (ferrihy- main that contains a non-heme Fe–Fe center, and a C-

drite). Ferritin proteins function as iron storage, but they terminus flavodoxin-like domain. Crystal structures re-

also act as antioxidants. The antioxidant action of ferritins veal that FDPs form a homodimeric head-to-tail arrange-

is probably best illustrated by the Dps proteins. These act ment that places the FMN moiety of one subunit near the

as enclosed nanoreactors in which Fe(II) is oxidized in diiron site of the metallo-b-lactamase-like domain of the

two steps, first by O2 producing hydrogen peroxide, but other for efficient electron transfer during substrate re-

then even faster by hydrogen peroxide without releasing duction [76–79]. While all FDPs have this conserved

any reactive oxygen species in the process [62–64]. flavodiiron compound domain structure, some have gene

fusions of additional redox domains at their C-termini

An additional strategy that early aerobic cells may have (Figure 4) [80].

used to sequester Fe(III) might have been to bind it to

polyphosphate, the synthesis of which is up-regulated in In non-phototrophic organisms, FDPs appear to function

bacteria in response to oxidative stress [65 ]. Coordination in NO and O2 detoxification, commonly showing a pref-

of Fe(III) to polyphosphate stabilizes it in that oxidation erence for one molecule or the other [80,81]. FDPs

state, and thus inhibits its ability to catalyze Fenton provide a biochemically efficient means of removing

chemistry [66]. O2 and alleviating oxidative stress. For example, the

anaerobic protist Giardia lamblia lacks respiratory O2

Quinols: also posed a new kind of reductases and does not contain the typical suite of

threat to the membranes of the ancestral microbes, par- enzymes for dealing with ROS, but it does contain an

ticularly if those membranes contained unsaturated FDP with a high affinity for O2 [82]. FDPs found in

lipids. The membranes of modern aerobic cells would methanogens also display a strong preference for O2 [78].

be susceptible to oxidative damage in the presence of O2

were in not for the presence of quinols such as Interestingly, FDPs are present in many paralogous

and ubiquinol, which act as chain-breakers of the free copies (typically between two and six) in the genomes

process that peroxidizes lipids. Qui- of all extant Oxyphotobacteria, except for losses in non-

nols such as menaquinol were already present in early phototrophic symbionts [83]. These form both a mono-

anoxygenic photosynthetic cells, where they played im- phyletic clade of FDPs and an independent class marked

portant functions as redox-active molecules in electron by the fusion of a flavodoxin-like (RutF) domain at the C-

transport chains [67–70]. Such quinols would have been terminus (Figure 4). This phylogenetic distribution

poised to act as inhibitors of free radical autoxidation of implies a rich evolutionary history of this family of

the membrane components. proteins in the Oxyphotobacteria and suggests that FDPs

were important during the evolution of oxygenic photo-

The modern widespread quinol antioxidant system found in synthesis. The exact function and physiological utility of

aerobic cells is alpha- (), which is only the different flavors of FDPs found in Oxyphotobacteria

synthesized by oxygenic photosynthetic organisms and are still not well known [41 ]. In recombinant studies,

must be consumed in the diets of humans and other animals. FDPs from Oxyphotobacteria are capable of direct reduc-

It is interesting to note that, just like glutathione, this system tion of O2 to water without the production of ROS [80].

appears to have first arisen in Oxyphotobacteria [71,72]. Two genes encoding FDPs, Flv1 and Flv3, promote a

Mehler-like reaction in Oxyphotobacteria, modulating the

Carotenoids and singlet O2: Early Oxyphotobacteria encoun- photoreduction of O2 with downstream of PSI

tered for the first time not only ground state triplet O2 but [84]. Unlike the Mehler reaction in plants and algae,

www.sciencedirect.com Current Opinion in Chemical Biology 2016, 31:166–178

174 Bioinorganic chemistry

Figure 4

(a) (b)

Class A Class B Class C Class D A Oxyphotobacteria B

C O , NO H O , N O O H O 2 2 2 2 2 O2 H2O O2 H2O D

Fe Fe Fe Fe Fe Fe Fe Fe root? FMN FMN FMN FMN Rd Flv Rd 0.1 Rd Flv Flv NAD(P)H NADP+ Flv NAD(P)H NADP+ NADH NAD+ NADH NAD+

(c) crown group Oxyphotobacteria (< 2.0 Ga) environmental O2 (~2.4 Ga) crown group FDPs

fusion of flavodoxin fusion gene duplication & paralogous evolution

non-redox Fe(II) diiron Fe(III) diiron (or Zn) hydrolase oxidoreductase diiron C-terminal flavodiiron flavodiiron flavodiiron flavoprotein diversification O reductase flavodoxin-O2 NAD(P)H-O oxidoreductase O reductase 2 (other taxa) 2 2 homodimer oxidoreductase homodimer complex complex

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 . 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 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 the diversity and omnipresence of FDPs in Oxyphotobac-

does not produce ROS. From observations of the differen- teria supports the view that the FDPs were integral to the

tial mass law fractionations of multiple oxygen isotopes development of oxygenic photosynthesis [41 ].

16 17 18

( O, O, O), Helman et al. [85] estimated that as much as

40% of the O2 leaving PSII was re-reduced by FDPs in Several aspects of the structural biology and biochemistry

photosynthetically active cells, compared with only 6% of FDPs permit one to develop a hypothesis for the

going to respiration at Complex IV. The amount of O2 that evolution of this protein family as an important comple-

flows to FDPs appears to depend on the availability of CO2 ment to oxygenic photosynthesis (Figure 4). Because all

and light and is particularly important during fluctuating FDPs have in common a conserved two-domain struc-

light conditions, providing a harmless mechanism to ture, one might infer that they result from the fusion of

maintain cellular redox balance by dissipating excess elec- proteins that once had little to do with one another.

trons downstream of PSI [86]. Other FDPs in Oxyphoto- Metallo-b-lactamase-like domains appear in proteins

bacteria appear to play roles in of PSII with diverse functions, and on the basis of their distribu-

1

against O2 and may function as heterodimers [84,87]; yet tion in metabolism and the tree of life appear to be

others appear to provide heterocysts with anaerobic envir- exceptionally old [77]. We envision this domain originally

onments conducive to nitrogen fixation [41 ]. Altogether, contained a non-redox metal center comprised of either

Current Opinion in Chemical Biology 2016, 31:166–178 www.sciencedirect.com

How did life survive Earth’s great oxygenation? Fischer, Hemp and Valentine 175

Fe(II) or Zn(II), and probably functioned as a hydrolytic fluxes of intracellular dioxygen. Importantly, manganese

enzyme. With early photosynthetic O2 fluxes, the metal appears to have played major roles in both the production

center became a ferric Fe-Fe site with a high affinity for and early detoxification of O2. Current data also suggest

O2, and formed an early O2 reductase complex in part- that the Oxyphotobacteria were important incubators for a

nership with the flavoprotein. Due to the distances be- wide variety of solutions to deal with oxidative stress (like

˚

tween the FMN moiety and the diiron site (40 A), this glutathione, alpha-tocopherol, and FDPs).

complex could only function as an O2 reductase in the

configuration of a multi-subunit homodimer. The FDPs Going forward to better understand how life responded to

family, thus, was born from the fusion of these two the GOE, we advocate greater focus on non-enzymatic

interacting domains that were probably organized togeth- solutions that were likely present in early cells, because it

er as genes within an operon. The radiation of the FDPs is possible that many of the enzymatic systems important

occurred, providing a useful mechanism for many anaer- today were not available for biology at this time. We think

obic microorganisms to detoxify O2. This co-occured with that efforts using comparative biology to infer the relative

the functional diversification of the C-terminus in a timing of different antioxidant systems will continue to

number of different groups, with lateral gene transfer provide useful insight into this problem. This is a good

playing an important role in the extant distribution of time to be asking these questions because the landscape

FDPs [80]. In stem group Oxyphotobacteria, the fusion of for understanding microbial and metabolic evolution is

another flavodoxin domain occurred before the adaptive rapidly changing, enabled by new single-cell and meta-

radiation of the many different FDPs found within them, genomic sequencing technologies, which probe microbial

because this fusion is observed in all members of crown diversity deeply and make it possible to populate evolu-

group Oxyphotobacteria. Thus based on the phylogenetic tionary analyses with substantial amounts of sequence

distribution of FDPs in Oxyphotobacteria, their general data. We think this approach is promising and will enable

absence or evolutionary incongruence in the Melainabac- integrative hypothesis generation and testing as new

teria, and their diverse roles in protecting cells against O2 groups of microbes are discovered and characterized.

stresses, we hypothesize that FDPs played an key role in

allowing stem group Oxyphotobacteria to complete early

Conflict of interest

experiments in phototrophic O2 production.

The authors declare no conflict of interest.

Ascorbate was not an ancient antioxidant: The ascorbate–

Acknowledgements

ascorbate peroxidase antioxidant system, which is a major

We thank Usha Lingappa, Hope Johnson, Jena Johnson, Dianne Newman,

antioxidant system in plants and most animals, is absent

and two anonymous reviewers for helpful feedback on ideas synthesized in

in Oxyphotobacteria. In fact, it seems likely that prokar- this paper. We acknowledge support from a David and Lucile Packard

yotes in general do not synthesize ascorbate [88]. This is Foundation Fellowship in Science and Engineering (WWF), and the

Agouron Institute (JH and WWF).

in sharp contrast to two other very important modern

antioxidant systems, glutathione and alpha-tocopherol,

both of which appear to have originated in the Cyano- References and recommended reading

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bacteria phylum and were then widely spread to other

have been highlighted as:

species.

of special interest

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