ABIOGENIC ALTERNATIVE TO THE CHLOROPHYLL-BASED CONVERTER OF SOLAR ENERGY Mikhail S. Kritsky

1. 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 ABIOGENIC ALTERNATIVE TO THE CHLOROPHYLL-BASED CONVERTER OF SOLAR ENERGYMikhail S. Kritsky Co-Authors: Taisiya A. Telegina, Andrey A. Buglak, Michael P. Kolesnikov, Tamara A. Lyudnikova, Yulia L. Vechtomova, Bach Institute of Biochemistry, Russian Academy of Sciences Moscow, Russia

2. M E MB RANE Bacteriorhodopsin ΔμH+ ATP M E MB RANE Chlorophyll ē-transfer ΔμH+ NAD(P)H ATP PHOTOAUTOTROPHY IN THE OPARINIAN PARADIGM • TWO POSTULATES • 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. CHEMICAL EVOLUTION HETEROTROPHS PHOTOAUTOTROPHS The currently known versions… And yet another hypothetical option Flavin ē-transfer ATP

3. FMN Lumazine FAD Flred-H2 5,10-Methenyl-tetrahydrofolate 7,8-didemethyl-8-hydroxy- 5-deazariboflavin (8-HDF) Cyanopterin PTERIDINES AND BENZOPTERIDINES (FLAVINS) IN THE DARK METABOLISM THESE COMPOUNDS ARE COENZYMES IN ENZYME CATALYSIS Isoalloxazines (Flavins) are benzo-[g]-pteridines Pteridine They also act as chropmophores of photoreceptor proteins

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

5. IMPORTANT: Pterins has three 2ē reduction states: oxidized, the dihydro- and tetrahydropterins RC6 +2ē, Ar +2ē, Ar + hv + hv -2ē, O2 -2ē, O2 Н2Bpt Bpt Н4Bpt + hv + hv Low effect of light -2ē, O2 Spontaneous process - RС6 No effect of light Irreversible formation of C6-unsubstituted pterin and its further transformation qН2Bpt PTERINS, TOO, ARE PHOTOCHEMICALLY ACTIVEMOLECULESAND SENSITIZE THE UP-HILL ELECTRON TRANSFER Photosensitized oxidation of H4-form +O2 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 200945(1) 104-109 (3) Buglak AA; Telegina TA; Lyudnikova TA; Vechtomova YL; Kritsky MSPhotooxidation of tetrahydrobiopterin under UV irradiation: Possible pathways and mechanisms. Photochem Photobiol 2014 90(5) 1017–1026

6. In the History of Life All known types of metabolism use isoalloxazines and pterins as essentialcofactors for “dark” biocatalysis. Flavinsparticipate 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. AMINO ACIDS THERMOLYTIC PRODUCT MICROSPHERES 95°C, +Н2O, +Н4SiO4 plus amino acid polymers + + plus amino acid polymers + 5 µm + Chromoproteinoids, i.e. the complexes of pigments with amino acid polymers (5 12 kDa) Glu + Lys + Gly (or Ala) (8:3:1) 150-200oC, О2–free, H20-free, 4-6 hrs FLAVINS AND PTERIDINESARE EVOLUTIONARY OLD MOLECULES 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.

7. D = 3,4 nm, M = 7,0 kDa D = 4,0 nm, M = 8,4 kDa D = 5,2 nm, M = 26,6 kDa D = 5,8 nm, M = 26,8 kDa MODIFICATION OF THE PROTOCOL BRINGS TO FORMATION OF NANOPARTICLES INSTEAD OF MICROSPHERES NanoparticlesSiO2 Hybrid nanoparticles 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 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. DETECTION OF ATP • Luceferin/Luciferase luminescence method. • HPLC-separation of reaction substrates and products. mV Dark control ADP ATP The hybrid (flavoproteinoid-silicate) nanoparticles, too, sensitize phosphorylation of ADP to form ATP, but with a smaller yield (up to 10-15 %) After irradiation ADP ATP AMP Retention time, min WHEN ASSOCIATED WITH A TEMPLATE, ABIOGENIC FLAVINS AND PTERIDINES SENSITIZE PHOTOPHOSPHORYLATON MICROSPHERES ADP + Pi + hν ATP + H2O 5 µm 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

9. Abiosynthesis (Lab Model) Ribose Orthophosphate Aspartate Formate Glycine Bicarbonate Glutamine Aspartate HCO3- 90100о С Glycine Formate Glutamine Yield AMP per ribose 3 ÷ 4% Formate Ribose-5-phosphate WHAT COULD BE THE ABIOGENIC SOURCE OF AMP? • Cyanide pathway (Oró, Ferris,et al.) • Formamide pathway (Di Mauro) • Pyrimidines: via 2-aminooxazol (Powner and Sutherland) – (but purines…?). 4. According to the abiosynthesis mimicking the scenario of AMP biosynthesis in the cell. Biosynthesis Riboso-5- phosphate Aspartate Formate Glycine Bicarbonate Glutamine All1010tonesof adenine present in the current biosphere have been formed via this pathway KritskyMS; Kolesnikov MP;Telegina TA Modeling of abiogenic synthesis of ATP. DOKLADY BIOCHEMISTRY AND BIOPHYSICS (2007) 417 (1) 313-315

10. 10 μM 10 nM hν ATP ē ADP + Pi Dox Dred CONDITIONS OF THE EARLY EARTH COULD GIVE RISE TO THE FLAVIN-BASED MICRO- AND NANOSCALE CONVERTERS OF LIGHT ENERGY Abiogenic photophosphorylating microsphere Abiogenic photophosphorylating nanoparticle hν ATP ADP + Pi ē Dox Dred Flavo-silico-proteinoid microsphere Flavoproteinoid Thus, prebiotic medium spontaneosly generates microscale and nanoscale converters of light energy, based on the activity of isolloxazines (flavins) and pteridines

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

12. IN MODERN ORGANISMSBENZOPTERIDINES (FLAVINS) AND PTERIDINES ARE ESSENTIAL COENZYMES OF “DARK” METABOLISM And also act as chromophores in photoreceptor proteins Lumazine FMN FAD Flred-H2 5,10-Methenyl-tetrahydrofolate 7,8-didemethyl-8-hydroxy- 5-deazariboflavin (8-HDF) Cyanopterin • PHOTORECEPTOR FAMILIES • DNA-photolyases and cryptochromes • LOV-domain photoreceptors • BLUF-domain photoreceptors

13. Flavin ē-transfer ATP …and yet another hypothetical option А HYPOTHESIS IN EARLY EVOLUTION, A FLAVIN-BASED LIGHT ENERGY CONVERTER, COULD HAVE ARISEN The main facts that form the basis of this hypothesis are: (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. 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. HOW COMPETETIVE IS THIS CONVERTER?

14. Outside atmosphere 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. At sea level Solar irradiance, W m-2 nm-1 Wavelength, nm Chlorophyll b Chlorophyll a Absorption, % Carotenoids Relat. efficiency, % Relat. efficiency Wavelength, nm Wavelength, nm IN THE EARLY HISTORY OF LIFE THE FLAVIN CONVERTER MIGHTHAVE BEEN COMPETETIVE Selective characters of the flavin-based converter? “Pros” • Flavinsare 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.

15. Thank you for your attention!