Published by AAAS

A. D. Anbar et al., Science 297, 1137 -1142 (2002)

Fig. 1. During the Archean, oceans

Basic Basic Premises/HypothesisPremises/Hypothesis

1.1. In the first ca. 2.5 Ga of Earth’s In the first ca. 2.5 Ga of Earth’s history, nature invested heavily in history, nature invested heavily in R&D from which a “core” set of R&D from which a “core” set of metabolic machines that evolved. metabolic machines that evolved.

2.2. There are approximately 1500 There are approximately 1500 core metabolic genes that make core metabolic genes that make the world go aroundthe world go around

3.3. This period of “metabolic This period of “metabolic innovation” is characterized by innovation” is characterized by machinery that has been retained machinery that has been retained virtually without change to the virtually without change to the present time (“frozen metabolic present time (“frozen metabolic accidents”). accidents”).

4.4. All of the key metabolic processes All of the key metabolic processes were developed in prokaryoteswere developed in prokaryotes

5. These metabolic sequences are 5. These metabolic sequences are coupled on local and planetary coupled on local and planetary scales to facilitate an electron scales to facilitate an electron market between C, N, O, and S. market between C, N, O, and S.

6. 6. Most of the metabolic sequences Most of the metabolic sequences were rapidly appropriated by a were rapidly appropriated by a large number of groups of large number of groups of microbes – and some (not all) microbes – and some (not all) subsequently were subsumed into subsequently were subsumed into eukaryotic lineages via primary eukaryotic lineages via primary and secondary symbioses.and secondary symbioses.

7. The eukaryotes derived secondary 7. The eukaryotes derived secondary metabolic adapations during the metabolic adapations during the 22ndnd half of Earth’s history – the era half of Earth’s history – the era of “metabolic adaptation”, but of “metabolic adaptation”, but did not invent any new did not invent any new fundamental process.fundamental process.

8. However, the dispersal of the 8. However, the dispersal of the core metabolic processes to core metabolic processes to large numbers of widely large numbers of widely differing taxa helped to ensure differing taxa helped to ensure their continuity (resiliance).their continuity (resiliance).

9. All these metabolic 9. All these metabolic sequences are observable in sequences are observable in the modern world – but many the modern world – but many are extremely “inefficient”.are extremely “inefficient”.

10. Despite these 10. Despite these inefficiencies, alternatives have inefficiencies, alternatives have not been selected. Why not?not been selected. Why not?

H2 or C-mediated

Photosynthesis

O2

Mn4+

NO3-

N2

Fe3+

SO42-

CO2

H2O

Mn2+ NH4+ Fe2+ HS- CH4 H2 [CH2O]

+CO2

Respiration

O2-mediated

Mn -mediated

Fe-mediated

S-mediated

N-mediated

N2-fixation

Falkowski, Fenchel and Delong, Science, 2008

Three examples of Three examples of frozen metabolic frozen metabolic

accidentsaccidentsCarbon fixation (C)- RubiscoCarbon fixation (C)- RubiscoNitrogen fixation (N) - Nitrogen fixation (N) -

NitrogenaseNitrogenaseOxygen evolution (O)- The Oxygen evolution (O)- The

reaction center of reaction center of Photosystem IIPhotosystem II

Example 1: Example 1: Carbon Fixation Carbon Fixation

and the and the evolution of evolution of

RuBisCORuBisCO

UniversalAncestor

Bacteria

Archaea

Eukarya

Pyrodictium

Thermoproteus Thermococcus

Methanococcus

MethanobacteriumHalobacterium

Thermoplasma

Methanopyrus

Aquifex

Thermotoga

Flavobacteria

Cyanobacteria

Proteobacteria

Gram -Positive Bacteria EntamoebaeSlime Molds

Animals

Fungi

Plants

Ciliates

MicrosporidiaDiplomonads

Archaeoglobus

Green Non -Sulfur Bacteria

Pathways of autotrophic CO2

fixation

Reductive citr ic acid cycle

Reductive acetyl CoA pathway

Reductive hydroxypropionate pathway

Calvin -Benson cycle

SulfolobusEuglena

THREE DOMAINS OF LIFE

RUBisCORUBisCO

Rubisco arose from a methionine Rubisco arose from a methionine “salavge” pathway long before it was “salavge” pathway long before it was appropriated for use in the Calvin-appropriated for use in the Calvin-Benson cycle.Benson cycle.

The enzyme is catalytically challenged, The enzyme is catalytically challenged, and can barely figure out what its and can barely figure out what its substrate looks like (blind and slow). substrate looks like (blind and slow).

In an oxygen rich world Rubisco is In an oxygen rich world Rubisco is notoriously inefficient (dumb).notoriously inefficient (dumb).

However, there is very little selection However, there is very little selection pressure on Rubisco active sites. Why pressure on Rubisco active sites. Why not?not?

Remove the selection Remove the selection pressurepressure

Cells can make a lot of Rubisco – Cells can make a lot of Rubisco – but don’t reinvent the technology but don’t reinvent the technology (hire lots of dumb, blind, slow (hire lots of dumb, blind, slow workers), orworkers), or

They developed a secondary set of They developed a secondary set of adaptations that removed or reduce adaptations that removed or reduce the selection pressure – e.g., the the selection pressure – e.g., the Carbon Concentrating MechanismCarbon Concentrating Mechanism

Carbon concentrating Carbon concentrating mechanismmechanism

Example 2: NitrogenaseExample 2: Nitrogenase

A detour into the rise of oxygen the A detour into the rise of oxygen the coupling between C,N and O cycles on coupling between C,N and O cycles on EarthEarth

2N2 + 4H+ + 3CH2O 4NH4+ + 3CO2

A 6 electron transfer reaction

NitrogenaseNitrogenase

The Biological Nitrogen Cycle

1G20 -- Nitrogenase MoFe protein only

Fe7MoS9

Fe8S7Fe7MoS9

Fe8S7

DigressionDigression

Evolution of core structural motifsEvolution of core structural motifs The paradox of structure/sequence The paradox of structure/sequence

divergencedivergence

Science. 1966 Apr 15;152(3720):363‐366.Science. 1966 Apr 15;152(3720):363‐366. Evolution of the Structure of Ferredoxin Based on Evolution of the Structure of Ferredoxin Based on

Living Relics of Primitive Amino Acid Sequences.Living Relics of Primitive Amino Acid Sequences. Eck RV, Dayhoff MO.Eck RV, Dayhoff MO. The structure of present‐day ferredoxin, with its simple, inorganic active The structure of present‐day ferredoxin, with its simple, inorganic active

site and its functions basic to photon‐energy utilization, suggests the site and its functions basic to photon‐energy utilization, suggests the incorporation of its prototype into metabolism very early during incorporation of its prototype into metabolism very early during biochemical evolution, even before complex proteins and the complete biochemical evolution, even before complex proteins and the complete modern genetic code existed. Ferredoxin has evolved by doubling a modern genetic code existed. Ferredoxin has evolved by doubling a shorter protein, which may have contained only eight of the simplest shorter protein, which may have contained only eight of the simplest amino acids. This shorter ancestor in turn developed from a repeating amino acids. This shorter ancestor in turn developed from a repeating sequence of the amino acids alanine, aspartic acid or proline, serine, sequence of the amino acids alanine, aspartic acid or proline, serine, and glycine. We explain the persistence of living relics of this primordial and glycine. We explain the persistence of living relics of this primordial structure by invoking a conservative principle in evolutionary structure by invoking a conservative principle in evolutionary biochemistry: biochemistry: The processes of natural selection severely inhibit The processes of natural selection severely inhibit any change a well‐adapted system on which several other any change a well‐adapted system on which several other essential components depend.essential components depend.

1FXR – Ferredoxin I from Desulfovibrio 1FXR – Ferredoxin I from Desulfovibrio AfricanusAfricanus

Ferredoxin protein with Fe4S4 clusterFerredoxin protein with Fe4S4 cluster

Image Courtesy: Dr. Vikas Nanda Image Courtesy: Dr. Vikas Nanda (UMDNJ)(UMDNJ)

1FXR – Ferredoxin I from 1FXR – Ferredoxin I from Desulfovibrio Desulfovibrio africanusafricanus Fe4-S4 cluster in ferredoxin tightly Fe4-S4 cluster in ferredoxin tightly

held by four cystein groups via thiolate held by four cystein groups via thiolate bondsbonds

Beta carbon distribution (385 Beta carbon distribution (385 structures)structures)

385 Structures from Protein Data Bank385 Structures from Protein Data Bank

22 High Potential Iron Proteins (amide)22 High Potential Iron Proteins (amide)

21 Fe Protein of Nitrogenase (amide)21 Fe Protein of Nitrogenase (amide)

HiPIP and Fe NitrogenaseHiPIP and Fe Nitrogenase NN

Number of Cysteine peaks….Signature of different environment for different redox potential?

HPiP

Fe Nitrogenase

High potential protein vs Fe protein of High potential protein vs Fe protein of Nitrogenase (cont’d)Nitrogenase (cont’d)

-number of CYS peaks-Hydrophilic group contrast

HPiP

Fe Nitrogenase

Signature of different environment for different redox potential?

The basic FeS bindingThe basic FeS binding ~ 30% of all FeS clusters are bound to a ~ 30% of all FeS clusters are bound to a

C XX C XX(X) C motif with a final C in a C XX C XX(X) C motif with a final C in a variable position further along the protein.variable position further along the protein.

The most common X residues are neutral The most common X residues are neutral aa’s (especially A, L and I).aa’s (especially A, L and I).

These motifs are virtually all chiral!These motifs are virtually all chiral!

Chronically Crippled Chronically Crippled NitrogenaseNitrogenase

Example 3: The oxygen Example 3: The oxygen evolving complex and evolving complex and

the evolution of the evolution of Photosystem IIPhotosystem II

Example 3: The oxygen Example 3: The oxygen evolving complex and evolving complex and

the evolution of the evolution of Photosystem IIPhotosystem II

PSII type Reaction PSII type Reaction CenterCenter

The Mn cluster The Mn cluster in PS IIin PS II

All oxygenic photosythetic organisms All oxygenic photosythetic organisms share common centers.share common centers.

The protein, D1, in photosystem II was The protein, D1, in photosystem II was inherited from purple sulfur bacteria. inherited from purple sulfur bacteria.

In all oxygenic organisms this protein In all oxygenic organisms this protein is damaged and replaced (not simply is damaged and replaced (not simply repaired) every 30 min during the day.repaired) every 30 min during the day.

Despite this inefficiency, there is Despite this inefficiency, there is almost no change in the primary almost no change in the primary sequence of this protein for the past 3 sequence of this protein for the past 3 Ga (86% identity at the aa level) Ga (86% identity at the aa level)

Lesson learned: If it works, keep Lesson learned: If it works, keep using the old technology. Just pay the using the old technology. Just pay the costs and fix the machinery.costs and fix the machinery.

Hypothesis: Hypothesis: The core metabolic machines are The core metabolic machines are usually multimeric protein complexes usually multimeric protein complexes that bind prostetic groups. The that bind prostetic groups. The tempo of evolution of the core tempo of evolution of the core complexes is constrained by protein-complexes is constrained by protein-protein interactions. protein interactions. The “Rubic cube paradox” – Don’t The “Rubic cube paradox” – Don’t mess with it unless you know how to mess with it unless you know how to get the thing back to the original get the thing back to the original configuration.configuration.

Why doesn’t this core Why doesn’t this core metabolic machinery metabolic machinery

improve with age?improve with age?

Photosynthetic gene clusters in Photosynthetic gene clusters in cyanobacteriacyanobacteria

C K J

ndh

B D

pet

E G I A

ndh

L N

chl

D F

ndh

B A C

cox

A B

psa pet

A C

psb

C D BD

crtpsb

A B

psapsb

CD

psb

N L

chl

BAC

cox

N L

chlpsb

CD

EGIA

ndh

F D

ndh pet

A C CKJ

ndh

B D

pet

B A C

cox

BD

crt

EGIA

ndh

B D

petpsa

A BD F

ndh psbpet

AC B D

crt

B A C

coxpsb

C D BD

pet

N B L

chl pet

AC

psbpsa

B A B D

crt

DF

ndh

E G I A

ndh

C K J

ndh

B D

crt

DF

ndh

E G I A

ndh

CKJ

ndhpsa

B A

psb

B D

pet pet

A C L B N

chl

AC

cox

B

psb

CD

BD

pet psa

B A CKJ

ndh psb

B D

crt psb

CD N B L

chl pet

ACDF

ndh

E G I A

ndh

C

cox

AB

1 kbSynechocystis sp. PCC6803

Anabaena sp. PCC7120

Thermosynechococcus elongatus BP-1

Synechococcus sp. WH8102

Prochlorococcus marinus MED4

Prochlorococcus marinus MIT9313

EFLJ

EFLJ

JLFE

EFLJ

EFLJ

EFLJ

PS II PS Ib6f

D1 D2

CP47 CP43 BA

QB

PQH2

P700

PC

Fd

PC

H2O O2+2H+

PQ

2H+ 2H+

2H+

ISP f

b6IV

V P'

4H+

P680

21

βα

ε

ab

b'

CF1

CF0e−

A0

A1

Fx

FA|FB

YZ

e−

Phe

QA

e−

bHci

e−

e−

e−

f

O

γ

δ

ndh

NAD++H+NADH

Cyt co

orCytc553

Cytc553

or

A

B C

NADPHNADP + H+

C6H12O6

L S

Rubisco

e−

FNR

APPC

PE

A

O2+2H+2

1 H2O

2H+

H+

B

PQH2

e−

bL

Photosystem II Cytoxhrome b6f complex

NADH dehydrogenase

Photosystem ICytoxhrome oxidase

Phycobilisome

ATP synthase

C D

Protein-protein interactions in linked photosystems revealed by the co-evolutionary analysis. Red lines represent predicted interactions with coefficient values better than 0.8. Also shown is a network of protein-protein interactions in the ATPase complex. The pattern of protein-protein interactions strongly suggests co-evolution of photosynthetic genes driven by electron transport and redox state of the primary photochemistry. Black arrows, electron transfer; blue arrows, proton transfer.

So how can such So how can such apparently inefficient apparently inefficient

machinery be both machinery be both robust and resilient?robust and resilient?

Spread the risk (The “Microsoft” Spread the risk (The “Microsoft” approach)approach)

Select secondary adaptive Select secondary adaptive featuresfeatures

Rhodophyta

Glauco-cystophytaCyanophora

ChlorophytaEuglenophyta

Euglena

CryptophytaGuillardia

BacillariophytaOdontella

atpIcemAminDodpB

accAaccBargB bas1carA clpCcpcGcpeAcpeBdnaBdfrdsbDfabHfdxftrBftsH

acpPapcAapcBapcDapcEapcF atpDatpGcpcAcpcB dnaKgroELhisHpetFpetMpreA psaEpsaF

cysAcysTftsWinfAminDndhA-IndhKPorphyra

Cyanidium

clpPftsW psbMrbcLg

chlNcpcAcpcBcpcGdfrglnBgltBhisHinfCnblA

cemAcpeBftrBilvBilvHinfBminDpbsApsaKrnetsf

thiGbas1

accAaccBaccDapcAapcBapcDapcEapcFargBcarA

glnBgltBilvBilvHinfBinfCmoeBnblAntcAodpApbsApetJpgmApsaDpsaKpsaLrbcLr

accDccsAcemAchlBchlLchlNclpP

chlBchlLcpeAdsbDfabHfdxmoeB

ndhJ odpBrpl33rps15rps16

Ancestralphotosynthetic

prokaryote

> 90% of genes lost

ntcAodpAodpBpetJpreArpl28trpAtrpGtrxA

Streptophyta

Secondary endosymbiosis

psbVpsbWpsbXrbcRrpl1rpl3rpl6 rpl11 rpl18rpl28rpl34rpl35rps5rps6rps10rps13rps17rps20secYtrpG

rnerpl23rpl32

Mesostigma

NephroselmisChlorella psbM

petApetDpetLpsaIrnerpl19rpoA

Secondary endosymbiosis

Primary endosymbiosis

bas1 cpeBinfBminDpbsApsbXrps20

hisHminD

accD

rpl22

psaM

rbcSr rpl4rpl9rpl13rpl24rpl27rpl29rpl31rps1secAsyfBsyhthiGtrpAtrxAtsfupp

chlIftsWminDodpBrne rpl5rpl12rpl19rps9tufA

rpl21

ftsW ndhA-IndhK

pgmArpl9rps1syfBsyhupp

cystAcystT infAndhA-Krps15 bioY

crtEgroEShemAmntAmntBnadArbcSg

Marchantia

Pinus

Nicotiana

Zea

Oryza

cysA, cysTrpl21, ndhA-K

accD

rps16

cysA, cysT, rpl21chlB,L,N, psaM

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

mut

atio

n ra

te (s

ubst

itut

ion/

site

/OT

U) proteins in all chloroplasts

proteins in at least one but not all chloroplasts

proteins in nucleus only

PS genes retained in chloroplasts are very highly conserved

How does this How does this inform us about N inform us about N limitation in the limitation in the ocean or other ocean or other

aquatic aquatic ecosystems?ecosystems?Reflection of elemental N/P Reflection of elemental N/P

ratios from organic matter in ratios from organic matter in the soluble inorganic pool of the soluble inorganic pool of fixed N and P. fixed N and P.

Is the Redfield “paradigm” Is the Redfield “paradigm” for the ocean a coincidence for the ocean a coincidence or a true biological feedback?or a true biological feedback?

Variations in N:P

Analyzed deep water DIN, DIP, and O2 measurements from 104 observational data sets for 33 water bodies

Water bodies ranged from ocean basins to freshwater lakes, from 109 km2 to 0.075 km2

Seawater N:P averages 15-16

Freshwater N:P range from 0.005 (Lake Lugano (Barbieri and Simona,

2001)) to 8700 (Lake Superior (Sterner et al., 2007)).

Restricted basin N:P range from ~20 (Med and Red Seas) to 1 (Caspian (Sapozhnikov et al., 2007))

Distribution of N:P Data

Canonical Redfield ratio is anomalous

N:P vs. O2

N:P linearly correlated to O2 when O2 is < 100M

Loss of N linked to denitrification under low O2

Variance in N:P vs. Basin Area

More variability in N:P for small basins

Larger basins less volatile with respect to variations in nutrient input, productivity, etc.

What did this exercise reveal?Deep water N:P ratios are linearly correlated to O2 when O2 is less

than 100M

This correlation breaks down at O2>100M

N:P ratios are generally higher, but no simple link

This may be due to anthropogenic influences (e.g. N loading)

There is less variability in N:P ratios amongst larger basins (not a surprise)

Small basins more affected by changes in input, productivity and seasonal cycles

N:P in the soluble pools is not simply controlled by organic matter remineralization, but also by REDOX state of the water body

What about on a What about on a molecular level?molecular level?

Assume protein N:rRNA P = 16:1Assume protein N:rRNA P = 16:1

In 1 ribosome, there are 5732 P, so then there are 83792 In 1 ribosome, there are 5732 P, so then there are 83792 protein N per ribosomeprotein N per ribosome

Assume 1.4 N/aa, so then 59851 aa/ribosome Assume 1.4 N/aa, so then 59851 aa/ribosome

If that protein turns over every day, then translation rate per If that protein turns over every day, then translation rate per

ribosome = 0.69 aa/sribosome = 0.69 aa/s

This is >10% of the average ribosomal translation capacityThis is >10% of the average ribosomal translation capacity

Tentative conclusion - about 90% of the time, ribosomes in Tentative conclusion - about 90% of the time, ribosomes in the ocean are “idling” - because they are waiting for a charged the ocean are “idling” - because they are waiting for a charged tRNA - N limitation on a cellular/global leveltRNA - N limitation on a cellular/global level

Resiliency on a Global Resiliency on a Global ScaleScale

There is ~ 10 Gg of nitrogenase in the There is ~ 10 Gg of nitrogenase in the oceans.oceans.

There is ~ 10,000 x more RubiscoThere is ~ 10,000 x more Rubisco

What limits N fixation over geological What limits N fixation over geological time? Is Fe really the “ultimate” time? Is Fe really the “ultimate” limiting nutrient? – the untestested limiting nutrient? – the untestested hypothesis.hypothesis.

ConclusionsConclusions On a planetary scale, the key metabolic On a planetary scale, the key metabolic

pathways that sustain life are all based on pathways that sustain life are all based on old, inefficient technologies that have been old, inefficient technologies that have been widely dispersed.widely dispersed.

Evolutionary history suggests that selection Evolutionary history suggests that selection for body plans and secondary adaptive for body plans and secondary adaptive strategies has permitted the continuation of strategies has permitted the continuation of the core machinery without need for de novo the core machinery without need for de novo invention of metabolism. invention of metabolism.

Some pathways (e.g., NSome pathways (e.g., N22fixation) appear to fixation) appear to be less functionally redundant (more be less functionally redundant (more vulnerable but also more effective in vulnerable but also more effective in regulating biogeochemical processes) than regulating biogeochemical processes) than others (e.g., photosynthesis).others (e.g., photosynthesis).

Conclusions ContinuedConclusions Continued Small changes in efficiency in one pathway can Small changes in efficiency in one pathway can

alter planetary chemistry. These changes are alter planetary chemistry. These changes are primarily regulated at the POST-primarily regulated at the POST-TRANSLATIONAL LEVEL and appear to be TRANSLATIONAL LEVEL and appear to be driven by the presence of MOLECULAR OXYGEN.driven by the presence of MOLECULAR OXYGEN.

The feedback on RuBisCO primarily affects The feedback on RuBisCO primarily affects terrestrial ecosystems while the feedback on terrestrial ecosystems while the feedback on nitrogenase primarily affects aquatic nitrogenase primarily affects aquatic ecosystems.ecosystems.

Despite the inefficiencies, the old technologies Despite the inefficiencies, the old technologies work under many different environmental work under many different environmental conditions and appear to have co-evolved into a conditions and appear to have co-evolved into a network of very strong feedbacks on Earths network of very strong feedbacks on Earths metabolic cycles (robust and resilient) .metabolic cycles (robust and resilient) .