ABIOGENIC ALTERNATIVE TO THE CHLOROPHYLL-BASED CONVERTER OF SOLAR ENERGY

Mikhail S. Kritsky

Bach Institute of Biochemistry, Russian Academy of Sciences Moscow, Russia

OPARIN-2014International Conference

THE PROBLEM OF THE ORIGIN OF LIFEand

Youth Scientific SchoolMOLECULAR AND CELLULAR BASISOF THE EARLY EVOLUTION OF LIFE

Moscow – September 22-26, 2014

Co-Authors:Taisiya A. Telegina, Andrey A. Buglak,

Michael P. Kolesnikov,Tamara A. Lyudnikova,Yulia L. Vechtomova,

PHOTOAUTOTROPHY IN THE OPARINIAN PARADIGMPHOTOAUTOTROPHY IN THE OPARINIAN PARADIGM

TWO POSTULATES

(1) The primitive photosynthesis was built from the products of «dark» metabolism and/or abiogenic molecules.

(2) The evolution Is a selection of the fittest from a variety of options.

TWO POSTULATES

(1) The primitive photosynthesis was built from the products of «dark» metabolism and/or abiogenic molecules.

(2) The evolution Is a selection of the fittest from a variety of options.PHOTOAUTOTROPHS

HETEROTROPHS

CHEMICAL EVOLUTION

M E MB RANE

Bacteriorhodopsin

ΔμH+

ATP

And yet another hypothetical option

ē-transfer

Flavin

ATPATP

M E MB RANE

Chlorophyll

ē-transfer

NAD(P)HNAD(P)H

ΔμH+

ATPATP

The currently known versions…

PTERIDINES AND BENZOPTERIDINES (FLAVINS) IN THE DARK METABOLISM THESE COMPOUNDS

ARE COENZYMES IN ENZYME CATALYSIS

NH

N NH

N

O

O1

2

3 45

6

7

8

910

4a 5a

9a10a

Isoalloxazines (Flavins)are benzo-[g ]-pteridines

N

N N

N

1

2

3

5

6

7

88a

44a

Pteridine

NH

N NH

N

O

O

NH

N

N

N

NH

NH2

O

NH

O

OH

O

OHO

+

5,10-Methenyl-tetrahydrofolate

Lumazine

NH

NH

N

NH

NH2

O

OO

OHOH OH

OOH

OHOMe

CO2HO

Cyanopterin

NH

N N

N

O

O

O

PO

PO

OH

OHOH

OO

OO

OHOH

O

N N

NNNH2

NH

N N

N

O

O

O

PO

OH

OHOH

OO

7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF)

NH

N N

C

O

O

OH

OH

OHOH

OH

FAD

FMNNH

NH

N

NH

O

O

R Flred-H2

They also act as chropmophores of photoreceptor proteins

EXCITED AND FREE RADICAL FORMS OF FLAVIN ARE HIGHLY ACTIVE IN ELECTRON TRANSFER

EXCITED AND FREE RADICAL FORMS OF FLAVIN ARE HIGHLY ACTIVE IN ELECTRON TRANSFER

NH

N N

N

O

O

R

NH

N N

N

O

O

R

Flavin(oxidized form)

Flavin(oxidized form)

NH

N N

N+

O

O

R

H

NH

N N

N+

O

O

R

H

Flavin free radical

(1ē reduced form)

Flavin free radical

(1ē reduced form)

NH

N N

NH

O

O

R

NH

N N

NH

O

O

R

Dihydroflavin (2ē reduced form)

Dihydroflavin (2ē reduced form)

NH

N N

N

O

O

R

RDonor

NH

N N

N

O

O

R

RDonor

Flavin photoadduct

Flavin photoadduct

+ē, +H+

h

-ē, -H+

+ē, +H+

h

-ē, -H+

+ē, +Rdonor

-ē, -Rdonor

202 кJ/mol

-0,3

+0,4

+1,8

Eo′, V Fl/HFl

*Fl/HFl

The hjgh Eo′electron donor(R)

ē

Fl/HFl

hνWhen excited, flavin can drive the up-hill electron

transfer reactions

When excited, flavin can drive the up-hill electron

transfer reactions

PTERINS, TOO, ARE PHOTOCHEMICALLY ACTIVE MOLECULESAND SENSITIZE THE UP-HILL ELECTRON TRANSFER

PTERINS, TOO, ARE PHOTOCHEMICALLY ACTIVE MOLECULESAND SENSITIZE THE UP-HILL ELECTRON TRANSFER

The slide illustrates the light-induced transformations within biopterin family. After: (1) Kritsky MS; Lyudnikova TA; Mironov EA; Moskaleva IV. The UV radiation-driven reduction of pterins in aqueous

solution. J Photochem Photobiol B-Biol 1997 39(1) 43-48 (2) Lyudnikova TA; Dashina OA; Telegina TA; Kritsky MS. Investigation of the photochemical properties of biopterin and its reduced forms. Appl Biochem Microbiol 2009 45(1) 104-109 (3) Buglak AA; Telegina TA; Lyudnikova TA; Vechtomova YL; Kritsky MS Photooxidation of tetrahydrobiopterin under UV irradiation: Possible pathways and mechanisms. Photochem Photobiol 2014 90(5) 1017–1026

The slide illustrates the light-induced transformations within biopterin family. After: (1) Kritsky MS; Lyudnikova TA; Mironov EA; Moskaleva IV. The UV radiation-driven reduction of pterins in aqueous

solution. J Photochem Photobiol B-Biol 1997 39(1) 43-48 (2) Lyudnikova TA; Dashina OA; Telegina TA; Kritsky MS. Investigation of the photochemical properties of biopterin and its reduced forms. Appl Biochem Microbiol 2009 45(1) 104-109 (3) Buglak AA; Telegina TA; Lyudnikova TA; Vechtomova YL; Kritsky MS Photooxidation of tetrahydrobiopterin under UV irradiation: Possible pathways and mechanisms. Photochem Photobiol 2014 90(5) 1017–1026

Irreversible formation of C6-

unsubstituted pterin and its

furthertransformation

Irreversible formation of C6-

unsubstituted pterin and its

furthertransformation

RC6

NH

N

N

N

NH2

O OH

OH

1

2

3 45

6

8

7

1'2'

3'

qН2Bpt

NH

N

N

NH

NH2

O OH

OH

1

2

3 45

6

8

7

1'2'

3'

Н2Bpt NH

NH

N

NH

NH2

O OH

OH

1

2

3 4 5

6

8

7

1'2'

3'

Н4BptN

N

N

NH

NH2

O OH

OH

1

2

3 45

6

8

7

1'2'

3'

Bpt

+2ē, Ar

+2ē, Ar

+ hv+ hv

-2ē, O2

-2ē, O2

+ hv+ hv+ hv+ hv+ hv+ hv - RС6

Spontaneous

process-2ē, O

2

No effect

of light

+2ē, Ar

+2ē, Ar+ hv+ hv

-2ē, O2

-2ē, O2

Low effect of light

Photosensitized oxidation of H4-form

IMPORTANT: Pterins has three 2ē reduction states: oxidized, the dihydro- and tetrahydropterins

+O2

Flavins and pteridines can emerge abiogenically

Heinz, B, Ried, W, Dose, K (1979) Thermische Erzeugung von Pteridinen und Flavinen aus Aminosäueregemischen. Angew Chem 91(6):510–511 Heinz, B, Ried, W (1981) The formation of chromophores through amino acid thermolysis and their possible role as prebiotic photo-receptors. BioSystems 14(1):33–40.

FLAVINS AND PTERIDINES ARE EVOLUTIONARY OLD MOLECULES

FLAVINS AND PTERIDINES ARE EVOLUTIONARY OLD MOLECULES

• In the History of Life • All known types of metabolism use isoalloxazines

and pterins as essential cofactors for “dark” biocatalysis.

• Flavins participate in ē and H+ transfer and are an Interface between 2ē and 1ē transfer reactions.

• Pterins H4-biopterin – in 1ē transfer (e.g., hydroxylation in metabolism of aromatic amino acids).

H4-Folates – in C1-units transfer

• The Prehistory (Chemical Evolution).• Isoalloxazines and pteridines are formed in

simulated prebiotic environment • Thermolysis of amino acid mixtures gives rise to

flavins and pteridines conjugated with amino acid polymers. Then, in aqueous medium and in the presence of silicate ions, these conjugates aggregate to form micro- and nanoparticles.

Chromoproteinoids, i.e. the complexes of pigments with amino acid polymers

(5 12 kDa)

N

N N

N

O

O

H

H

plus amino acid polymers

N

N N

N plus amino acid polymers

MICROSPHERES

5 µm

THERMOLYTIC PRODUCT95°C, + Н2O,

+ Н4SiO4

150-200oC, О2–free, H20-free, 4-6 hrs

Glu + Lys + Gly (or Ala)(8:3:1)

AMINO ACIDS

NH2 O

O

OH

OH

NH2 O

NH2

OH

NH2 O

OH

O

OH

NH2

CH3

NH2 O

O

OH

OH

NH2 O

NH2

OH

++

++

MODIFICATION OF THE PROTOCOL BRINGS TO FORMATION OF NANOPARTICLES INSTEAD OF MICROSPHERES

0 2 4 6 8 10 12 14 16

0,00

0,02

0,04

0,06

0,08

0,10

0,12

-5.5 + 0.3 S

c(s)

sedimentation coefficient (S)

2.1 + 0.3 S-D = 4,0 nm, M = 8,4 kDa

D = 5,8 nm, M = 26,8 kDa

Hybrid nanoparticles

D = 5,2 nm, M = 26,6 kDa

D = 3,4 nm, M = 7,0 kDa

Nanoparticles SiO2

The AFM analysis (Aist-NT, Zelenograd) has revealed particles with a diameter of

approx. 3 nm (in collaboration with Dr. I.V.Safenkova) .

The AFM analysis (Aist-NT, Zelenograd) has revealed particles with a diameter of

approx. 3 nm (in collaboration with Dr. I.V.Safenkova) .

The analytical ultracentrifugation data (60000 rpm, 1 hr) processed with program SEDFIT.

(In collaboration with Prof. N.A.Chebotareva)

The analytical ultracentrifugation data (60000 rpm, 1 hr) processed with program SEDFIT.

(In collaboration with Prof. N.A.Chebotareva)

The smallest particles have a diameter ca. 25 nm and M 8 kDa. They show a tendency to aggregate to form clusters ca. 60 140 nm.

The smallest particles have a diameter ca. 25 nm and M 8 kDa. They show a tendency to aggregate to form clusters ca. 60 140 nm.

The 8 kDa particle contains ca. 80-90 silicate tetrahedrons. Assume that the particle consists of two layers, then each layer should contain 40-45 tetrahedrons. Considering the

geometry of hexagonal structures, the diameter of 45 tetrahedrons is about 3 nm, what well fits the size of the

structures determined by sedimentation and AFM.

WHEN ASSOCIATED WITH A TEMPLATE, ABIOGENIC FLAVINS AND PTERIDINES SENSITIZE PHOTOPHOSPHORYLATON

WHEN ASSOCIATED WITH A TEMPLATE, ABIOGENIC FLAVINS AND PTERIDINES SENSITIZE PHOTOPHOSPHORYLATON

MICROSPHERES

5 µm

ADPADP + + PPi i + h+ hνν ATPATP + + HH22OO ADPADP + + PPi i + h+ hνν ATPATP + + HH22OO mV

ADP

Dark control

ATP

After irradiationADP

Retention time, min

AMP

ATP

DETECTION OF ATP1. Luceferin/Luciferase luminescence method.2. HPLC-separation of reaction substrates and

products.

The molar yield of ATP WITH ABIOGENIC FLAVIN

Anoxygenic medium 5% With O2 ( 3х10-4М) + EDTA 20 %With H2O2 35 ÷ 40 %

WITH ABIOGENIC PTERIDINE

With O2 ( 3х10-4М) + EDTA 12 %With H2O2 21 ÷ 24 %

After: Kolesnikov MP, Telegina TA, Lyudnikova TA, Kritsky MS (2008) Abiogenic photophosphorylation of ADP to ATP sensitized by flavoproteinoid microspheres. Orig Life Evol Biosph 38(3):243–255

The hybrid (flavoproteinoid-silicate) nanoparticles, too, sensitize phosphorylation of ADP to form ATP, but with a smaller yield(up to 10-15 %)

The hybrid (flavoproteinoid-silicate) nanoparticles, too, sensitize phosphorylation of ADP to form ATP, but with a smaller yield(up to 10-15 %)

WHAT COULD BE THE ABIOGENIC SOURCE OF AMP? WHAT COULD BE THE ABIOGENIC SOURCE OF AMP?

1. Cyanide pathway (Oró, Ferris, et al.)2. Formamide pathway (Di Mauro)3. Pyrimidines: via 2-aminooxazol (Powner and Sutherland) – (but

purines…?). 4. According to the abiosynthesis mimicking the scenario of AMP biosynthesis in the cell.

Aspartate

HCO3-

Glycine

Formate

Ribose-5-phosphate

Glutamine

Formate

Abiosynthesis (Lab Model)RiboseOrthophosphate

AspartateFormateGlycineBicarbonateGlutamine

Abiosynthesis (Lab Model)RiboseOrthophosphate

AspartateFormateGlycineBicarbonateGlutamine

90100о С90100о С

Yield AMP per ribose

3 ÷ 4%

Yield AMP per ribose

3 ÷ 4%

Kritsky MS; Kolesnikov MP; Telegina TA Modeling of abiogenic synthesis of ATP. DOKLADY BIOCHEMISTRY AND BIOPHYSICS (2007) 417 (1) 313-315

All 1010 tones of adenine present in the current biosphere have been

formed via this pathway

All 1010 tones of adenine present in the current biosphere have been

formed via this pathway

Biosynthesis

Riboso-5-phosphate

AspartateFormateGlycineBicarbonateGlutamine

Biosynthesis

Riboso-5-phosphate

AspartateFormateGlycineBicarbonateGlutamine

Abiogenicphotophosphorylating

nanoparticle

Flavoproteinoid

ATP

ADP + Pi

DredDox

ēē

hν

Abiogenicphotophosphorylating

microsphere

Thus, prebiotic medium spontaneosly generates microscale and nanoscale converters of light energy, based

on the activity of isolloxazines (flavins) and pteridines

Flavo-silico-proteinoid microsphere

ATP

ADP + Pi

DredDox

ēē

hν10 μM 10 nM

CONDITIONS OF THE EARLY EARTH COULD GIVE RISE TO THE FLAVIN-BASED MICRO- AND NANOSCALE CONVERTERS OF LIGHT ENERGY

CONDITIONS OF THE EARLY EARTH COULD GIVE RISE TO THE FLAVIN-BASED MICRO- AND NANOSCALE CONVERTERS OF LIGHT ENERGY

THE ACTION SPECTRA OF ATP FORMATION CORRESPOND

TO THE ABSORPTION SPECTRA OF ABIOGENIC PIGMENTS

THE ACTION SPECTRA OF ATP FORMATION CORRESPOND

TO THE ABSORPTION SPECTRA OF ABIOGENIC PIGMENTS

After: Telegina TA., Kolesnikov MP., Vechtomova YL. Buglak AA., Kritsky MS Abiotic photo-phosphorylation model based on abiogenic flavin and pteridine pigments. J Mol Evol (2013) 76(5), 332-342.

Excitation , nm

Emission, nm

Inte

nsi

ty, a

rb. u

nit

s

Emission, nmExcitation , n

m

Inte

nsi

ty, a

rb. u

nit

s

Glutamic acid, lysine and glycineglycine150-200o C, anoxic medium, 4-6 hrs

Glutamic acid, lysine and alaninealanine150-200o C, anoxic medium, 4-6 hrs

THE FLUORESCENCE SPECTRA OF THE PIGMENTS: EMISSION VS. EXCITATION

ACTION SPECTRA OF THE FORMATION OF ATP

FMN

AT

P,

mm

ole/

eins

tein

, nm

A

0,0

0,1

0,2

300 350 400 450 500 5500

50

100

150

200

PterinA

TP

, m

mol

e/ei

nste

in

, nm

A

In chemical evolution: in the absence of genetic control, all depends on the environmental conditions

IN MODERN ORGANISMS

BENZOPTERIDINES (FLAVINS) AND PTERIDINES ARE ESSENTIAL COENZYMES OF “DARK” METABOLISM

NH

N NH

N

O

O

NH

N

N

N

NH

NH2

O

NH

O

OH

O

OHO

+

5,10-Methenyl-tetrahydrofolate

Lumazine

NH

NH

N

NH

NH2

O

OO

OHOH OH

OOH

OHOMe

CO2HO

Cyanopterin

NH

N N

N

O

O

O

PO

OH

OHOH

OO 7,8-didemethyl-8-hydroxy-

5-deazariboflavin (8-HDF)NH

N N

C

O

O

OH

OH

OHOH

OH

NH

N N

N

O

O

O

PO

PO

OH

OHOH

OO

OO

OHOH

O

N N

NNNH2

FAD FMN

NH

NH

N

NH

O

O

R

Flred-H2

And also act as chromophores in photoreceptor proteins

Main question: Are apoproteins of these photoreceptors monophyletic or onophyletic or polyphyletic? polyphyletic?

These families are polyphyletic, i.e., BIOLOGICAL EVOLUTION REPEATEDLY SELECTED FLAVINS FOR FOR THE ROLE OF PHOTON RECEPTORSTHE ROLE OF PHOTON RECEPTORS

PHOTORECEPTOR FAMILIESPHOTORECEPTOR FAMILIES •DNA-photolyases and cryptochromesDNA-photolyases and cryptochromes

• LOV-domain photoreceptorsLOV-domain photoreceptors• BLUF-domain photoreceptorsBLUF-domain photoreceptors

PHOTORECEPTOR FAMILIESPHOTORECEPTOR FAMILIES •DNA-photolyases and cryptochromesDNA-photolyases and cryptochromes

• LOV-domain photoreceptorsLOV-domain photoreceptors• BLUF-domain photoreceptorsBLUF-domain photoreceptors

А HYPOTHESIS А HYPOTHESIS

(1) Flavins are evolutionarily ancient molecules;

(2) The flavin photocycle can lead to the accumulation of free energy in the products, and this cycle (in the chemical model) can provide the formation of high-energy phosphate: ATP;

(3) Evolution repeatedly selected flavins to function in photoreceptors, and, as a result, organisms today utilize several families of flavoprotein photoenzymes and sensory photoreceptors.

IN EARLY EVOLUTION, A FLAVIN-BASED LIGHT ENERGY CONVERTER, COULD HAVE ARISEN

HOW COMPETETIVE IS THIS CONVERTER?HOW COMPETETIVE IS THIS CONVERTER?

The main facts that form the basis of this hypothesis are:

After: Kritsky, MS, Telegina, TA., Vechtomova, YL., Buglak, AA. (2013) Why flavins are not competitors of chlorophyll in the evolution of biological converters of solar energy Int J Mol Sci 14(1), 575-593.

…and yet another hypothetical option

ē-transfer

Flavin

ATP

IN THE EARLY HISTORY OF LIFE THE FLAVIN CONVERTER MIGHT HAVE BEEN COMPETETIVE

IN THE EARLY HISTORY OF LIFE THE FLAVIN CONVERTER MIGHT HAVE BEEN COMPETETIVE

“Pros”• Flavins are easily available. • They are active redox photocatalysts• Flavins are well adapted to photic environment of early Earth. • The system can develop an antenna.• (?) Simple structural organization (?)• (?) The system does not need lipid membrane (?)

“Pros”• Flavins are easily available. • They are active redox photocatalysts• Flavins are well adapted to photic environment of early Earth. • The system can develop an antenna.• (?) Simple structural organization (?)• (?) The system does not need lipid membrane (?)

“Contras”• The absorption spectrum is narrow and is situated in a “ too short- wavelength” area. • Low absorptivity of main pigments.

“Contras”• The absorption spectrum is narrow and is situated in a “ too short- wavelength” area. • Low absorptivity of main pigments.

Selective characters of the flavin-based converter?Selective characters of the flavin-based converter?

The absorption spectrum of flavins well fits the spectrum of native Solar radiation. Such a spectrum could provide for flavins a selective advantage untill ozone shield has emerged. The (E400/E750) for non-filtered solar radiation is 1,5. The presence of O3 in the atmosphere decreases this value to 0,9.

The absorption spectrum of flavins well fits the spectrum of native Solar radiation. Such a spectrum could provide for flavins a selective advantage untill ozone shield has emerged. The (E400/E750) for non-filtered solar radiation is 1,5. The presence of O3 in the atmosphere decreases this value to 0,9.

Outside atmosphere

At sea level

Wavelength, nm

Sol

ar ir

radi

ance

, W m

-2 n

m-1

Chlorophyll b

Chlorophyll aCarotenoids

Wavelength, nm Wavelength, nm

Rel

at. e

ffici

ency

Re

lat.

eff

icie

ncy

, %A

bso

rptio

n,

%

Thank you for your attention!