1. ABIOGENIC ALTERNATIVE TO THE CHLOROPHYLL-BASED CONVERTER OF SOLAR ENERGY Mikhail S. Kritsky Bach Institute of Biochemistry, Russian Academy of Sciences Moscow, Russia OPARIN-2014 International Conference THE PROBLEM OF THE ORIGIN OF LIFE and Youth Scientific School MOLECULAR AND CELLULAR BASIS OF 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,

2. PHOTOAUTOTROPHY 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 MEMBRANEMEMBRANE Bacteriorhodopsin ΔμH + ATP And yet another hypothetical option ē-transfer Flavin ATP MEMBRANEMEMBRANE Chlorophyll ē-transfer NAD(P)H ΔμH + ATP The currently known versions…

3. PTERIDINES AND BENZOPTERIDINES (FLAVINS) IN THE DARK METABOLISM THESE COMPOUNDS ARE COENZYMES IN ENZYME CATALYSIS Isoalloxazines (Flavins) are benzo-[g ]-pteridines Pteridine 5,10-Methenyl- tetrahydrofolate Lumazine Cyanopterin 7,8-didemethyl-8-hydroxy- 5-deazariboflavin (8-HDF) FAD FMN Fl red -H 2 They also act as chropmophores of photoreceptor proteins

4. EXCITED AND FREE RADICAL FORMS OF FLAVIN ARE HIGHLY ACTIVE IN ELECTRON TRANSFER Flavin (oxidized form) Flavin (oxidized form) Flavin free radical (1ē reduced form) Flavin free radical (1ē reduced form) Dihydroflavin (2ē reduced form) Flavin photoadduct +ē, +H + h  -ē, -H + +ē, +H +  h -ē, -H + +ē, +R donor -ē, -R donor 202 кJ/mol -0,3 +0,4 +1,8 E o ′, V Fl/ HFl  * Fl/HFl  The hjgh E o ′ electron donor(R) ē Fl /HFl  hνhν When excited, flavin can drive the up-hill electron transfer reactions When excited, flavin can drive the up-hill electron transfer reactions

5. PTERINS, TOO, ARE PHOTOCHEMICALLY ACTIVE MOLECULES AND 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 further transformation Irreversible formation of C6- unsubstituted pterin and its further transformation R C6 qН 2 Bpt Н 2 Bpt Н 4 Bpt Bpt + 2ē, Ar + 2ē, Ar + hv -2ē, O 2 -2ē, O 2 + hv - R С6 Spontaneous process -2ē, O 2 No effect of light + 2ē, Ar + 2ē, Ar + hv -2ē, O 2 -2ē, O 2 Low effect of light Photosensitized oxidation of H 4 -form IMPORTANT: Pterins has three 2ē reduction states: oxidized, the dihydro- and tetrahydropterins +O 2

6. 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 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) plus amino acid polymers MICROSPHERES 5 µm THERMOLYTIC PRODUCT 95°C, + Н 2 O, + Н 4 SiO 4 150-200 o C, О 2 –free, H 2 0-free, 4-6 hrs Glu + Lys + Gly (or Ala) (8:3:1) AMINO ACIDS + + + +

7. MODIFICATION OF THE PROTOCOL BRINGS TO FORMATION OF NANOPARTICLES INSTEAD OF MICROSPHERES 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 SiO 2 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. 2  5 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. 2  5 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.

8. WHEN ASSOCIATED WITH A TEMPLATE, ABIOGENIC FLAVINS AND PTERIDINES SENSITIZE PHOTOPHOSPHORYLATON MICROSPHERES 5 µm ADP + P i + hν  ATP + H 2 O mV ADP Dark control ATP After irradiation ADP Retention time, min AMP ATP DETECTION OF ATP 1.Luceferin/Luciferase luminescence method. 2.HPLC-separation of reaction substrates and products. The molar yield of ATP WITH ABIOGENIC FLAVIN Anoxygenic medium 5% With O 2 ( 3х10 -4 М) + EDTA 20 % With H 2 O 2 35 ÷ 40 % WITH ABIOGENIC PTERIDINE With O 2 ( 3х10 -4 М) + EDTA 12 % With H 2 O 2 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 %)

9. 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 HCO 3 - Glycine Formate Ribose-5-phosphate Glutamine Formate Abiosynthesis (Lab Model) Ribose Orthophosphate Aspartate Formate Glycine Bicarbonate Glutamine Abiosynthesis (Lab Model) Ribose Orthophosphate Aspartate Formate Glycine Bicarbonate Glutamine 90  100 о 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  10 10 tones of adenine present in the current biosphere have been formed via this pathway Biosynthesis Riboso-5- phosphate Aspartate Formate Glycine Bicarbonate Glutamine Biosynthesis Riboso-5- phosphate Aspartate Formate Glycine Bicarbonate Glutamine

10. Abiogenic photophosphorylating nanoparticle Flavoproteinoid ATP ADP + P i D red D ox ē hνhν Abiogenic photophosphorylating 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 + P i D red D ox ē hνhν 10 μM10 nM CONDITIONS OF THE EARLY EARTH COULD GIVE RISE TO THE FLAVIN-BASED MICRO- AND NANOSCALE CONVERTERS OF LIGHT ENERGY

11. 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 I ntensity, arb. units Emission, nm Excitation, nm I ntensity, arb. units glycine Glutamic acid, lysine and glycine 150-200 o C, anoxic medium, 4-6 hrs alanine Glutamic acid, lysine and alanine 150-200 o C, anoxic medium, 4-6 hrs THE FLUORESCENCE SPECTRA OF THE PIGMENTS: EMISSION VS. EXCITATION ACTION SPECTRA OF THE FORMATION OF ATP FMN ATP, mmole/einstein, nm A Pterin ATP, mmole/einstein, nm A In chemical evolution: in the absence of genetic control, all depends on the environmental conditions

12. IN MODERN ORGANISMS BENZOPTERIDINES (FLAVINS) AND PTERIDINES ARE ESSENTIAL COENZYMES OF “DARK” METABOLISM 5,10-Methenyl- tetrahydrofolate Lumazine Cyanopterin 7,8-didemethyl-8-hydroxy- 5-deazariboflavin (8-HDF) FAD FMN Fl red -H 2 And also act as chromophores in photoreceptor proteins Main question: Are apoproteins of onophyletic or these photoreceptors monophyletic orpolyphyletic? These families are polyphyletic, FOR THE ROLE OF PHOTON RECEPTORS i.e., BIOLOGICAL EVOLUTION REPEATEDLY SELECTED FLAVINS FOR THE ROLE OF PHOTON RECEPTORS PHOTORECEPTOR FAMILIES DNA-photolyases and cryptochromesDNA-photolyases and cryptochromes LOV-domain photoreceptors LOV-domain photoreceptors BLUF-domain photoreceptors BLUF-domain photoreceptors PHOTORECEPTOR FAMILIES DNA-photolyases and cryptochromesDNA-photolyases and cryptochromes LOV-domain photoreceptors LOV-domain photoreceptors BLUF-domain photoreceptors BLUF-domain photoreceptors

13. 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? 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

14. 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? 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 (E 400 /E 750 ) for non-filtered solar radiation is 1,5. The presence of O 3 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 (E 400 /E 750 ) for non-filtered solar radiation is 1,5. The presence of O 3 in the atmosphere decreases this value to  0,9. Outside atmosphere At sea level Wavelength, nm Solar irradiance, W m -2 nm -1 Chlorophyll b Chlorophyll a Carotenoids Wavelength, nm Relat. efficiency Relat. efficiency, % Absorption, %

15. Thank you for your attention!