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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 ENERGY
Mikhail 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
PHOTOAUTOTROPHY IN THE OPARINIAN PARADIGM
CHEMICAL EVOLUTION
HETEROTROPHS
PHOTOAUTOTROPHS
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.
The currently known versions…
M
E
M
B
R
A
N
E
Chlorophyll
ē-transfer
ΔμH+
ATP
NAD(P)H
M
E
M
B
R
A
N
E
And yet another
hypothetical option
Bacteriorhodopsin
ΔμH+
ATP
Flavin
ē-transfer
ATP
PTERIDINES AND BENZOPTERIDINES (FLAVINS)
IN THE DARK METABOLISM THESE COMPOUNDS
ARE COENZYMES IN ENZYME CATALYSIS
Isoalloxazines (Flavins)
Pteridine
4a
3
are benzo-[g ]-pteridines
5
4
N
N
O
6
3
N
8a
O
N
2
9
O
HN
N
HN
N
FAD
O
O
N
N
OH
N
H
N
+
HN
N
O
OH
O
P
O
O
O
O
HO
H
N
N
H
N
H2N
HO
O
OH OH
HO
O
O
OH
O
OMe
N
N
R
Flred-H2
N
OH
O
N
N
H
N
N
H
H
N
HO
N
Cyanopterin
O
HO
O
N
HN
HO
O
HN
N
N
O
OH
O
N
H
5,10-Methenyltetrahydrofolate
H2N
8
O
O
H2N
7
FMN
Lumazine
O
5a
N109a
H
10a
1
HN
6
N
4a
N
8
1
5
HN
7
2
4
N
O
O P O
O
O PO
O
7,8-didemethyl-8-hydroxy5-deazariboflavin (8-HDF)
O
C
HN
HO
OH
CO2H
N
O
N
HO
HO
They also act as chropmophores
of photoreceptor proteins
OH
HO
OH
EXCITED AND FREE RADICAL FORMS OF FLAVIN
ARE HIGHLY ACTIVE IN ELECTRON TRANSFER
hν
Eo′, V
Fl/HFl
-0,3
The hjgh Eo′
electron
donor(R)
202
кJ/mol
+0,4
When excited, flavin can
drive the up-hill electron
transfer reactions
Fl/HFl
ē
*Fl/HFl
+1,8
O
h

O
N
HN
O
+ē, +H+
+ē,
O
+H+
+
N
HN
N
N
R
Flavin
(oxidized form)
-ē,
-H+
H
O
-ē, -H+

h
N
N
R
Flavin free radical
(1ē reduced form)
HN
N
O
N
R
Dihydroflavin (2ē
reduced form)
+ē, +Rdonor
-ē, -Rdonor
H
N
O
N
HN
O
R Donor
N
N
R
Flavin
photoadduct
PTERINS, TOO, ARE PHOTOCHEMICALLY ACTIVE MOLECULES
AND SENSITIZE THE UP-HILL ELECTRON TRANSFER
IMPORTANT:
Pterins has three 2ē reduction states: oxidized, the dihydro- and tetrahydropterins
Photosensitized
oxidation of H4-form
RC6
+O2
O
4
3
HN
2'
N
6
7
H2N
2
N
1
+ hv
OH
5
N
8
3'
1'
+2ē, Ar
OH
Bpt
O
OH
5
4
3
2'
N
HN
6
3'
1'
-2ē, O2
H2N
N
1
8
N
H
Н2Bpt
HN
H2N
Low effect
of light
O
4
3
N
6
5
2'
N
6
N
1
N8
H
2
N
1
N8
H
3'
1'
OH
qН2Bpt
3'
OH
Н4Bpt
+ hv
OH
7
2
2'
1'
7
-2ē, O2
+ hv
H2N
4
3
OH
H5
N
OH
7
2
O
+ hv
+2ē, Ar
- RС6
Irreversible
formation of C6unsubstituted
pterin and its
further
transformation
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
FLAVINS AND PTERIDINES
ARE EVOLUTIONARY OLD MOLECULES
• In the History of Life
• The Prehistory (Chemical Evolution).
• 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
Flavins and pteridines
AMINO ACIDS
can emerge
abiogenically
• 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.
THERMOLYTIC PRODUCT
O
HO
O
H2N
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 photoreceptors. BioSystems 14(1):33–
40.
O
+
O
N
H
plus amino acid
polymers
95°C,
+ Н2O,
+ Н4SiO4
HO
HO
NH2
HO
O
O
HO
N
O
H2N
O
N
HN
+
H2N
HO
H2N
NH2
MICROSPHERES
N
N
+
+
CH3
H2N
H2N
O
N
N
plus amino acid
polymers
O
HO
HO
Glu + Lys + Gly (or Ala)
(8:3:1)
150-200oC, О2–free,
H20-free, 4-6 hrs
Chromoproteinoids, i.e. the complexes
of pigments with amino acid polymers
(5 12 kDa)
5
µm
MODIFICATION OF THE PROTOCOL BRINGS TO FORMATION
OF NANOPARTICLES INSTEAD OF MICROSPHERES
Hybrid nanoparticles
D = 4,0 nm,
M = 8,4 kDa
-
2.0+0.1 S
0,4
Nanoparticles SiO2
0,12
D = 3,4 nm,
M = 7,0 kDa
-
2.1 + 0.3 S
0,10
0,08
D = 5,8 nm,
M = 26,8 kDa
0,2
0,1
c(s)
c(s)
0,3
0,06
0,04
-
4.2+0.1 S
0,02
0,0
0,00
0
2
4
6
8
10
12
14
16
sedimentation coefficient (S)
The AFM analysis (Aist-NT, Zelenograd)
has revealed particles with a diameter of
approx. 3 nm
(in collaboration with Dr. I.V.Safenkova) .
-
5.5 + 0.3 S
D = 5,2 nm,
M = 26,6 kDa
0
2
4
6
8
10
12
14
16
sedimentation coefficient (S)
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.
WHEN ASSOCIATED WITH A TEMPLATE, ABIOGENIC FLAVINS
AND PTERIDINES SENSITIZE PHOTOPHOSPHORYLATON
1000
MICROSPHERES
DETECTION OF ATP
1. Luceferin/Luciferase luminescence method.
0
2. HPLC-separation
of5 reaction
and
0
10
15 substrates
20
25
30
products. mV
ADP + Pi + hν  ATP + H2O
5
µm
The molar yield of ATP
ADP
ATP
0
0
5
1000
10
15
ADP
ATP
WITH ABIOGENIC FLAVIN
Anoxygenic medium
5%
With O2 ( 3х10-4М) + EDTA
20 %
With H2O2
35 ÷ 40 %
Dark control
1000
20
25
30
After
irradiation
AMP
0
0
5
10
15
20
Retention time, min
25
30
1000
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
0
0
5
10
The hybrid
(flavoproteinoid-silicate)
nanoparticles, too,
sensitize phosphorylation
of ADP to form ATP, but
with a smaller yield
(up to 10-15 %)
15
20
25
30
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
Biosynthesis
Riboso-5phosphate
HCO3Formate
Glutamine
Aspartate
Formate
Glycine
Bicarbonate
Glutamine
All 1010 tones of adenine present
in the current biosphere have been
formed via this pathway
Glycine
Formate
Abiosynthesis
(Lab Model)
Ribose-5-phosphate Ribose
Orthophosphate
Aspartate 90100о С
Formate
Glycine
Yield AMP
Bicarbonate per ribose
Glutamine
3 ÷ 4%
Kritsky MS; Kolesnikov MP; Telegina TA
Modeling of abiogenic synthesis of ATP.
DOKLADY BIOCHEMISTRY AND
BIOPHYSICS (2007) 417 (1) 313-315
CONDITIONS OF THE EARLY EARTH COULD
GIVE RISE TO THE FLAVIN-BASED MICRO- AND
NANOSCALE CONVERTERS OF LIGHT ENERGY
Abiogenic
photophosphorylating
Abiogenic
photophosphorylating
microsphere
nanoparticle
10 μM
hν
10 nM
hν
ATP
ATP
ADP + Pi
ē
Dox
Dred
Flavo-silico-proteinoid
microsphere
ADP + Pi
ē
Dox
Dred
Flavoproteinoid
Thus, prebiotic medium spontaneosly generates microscale
and nanoscale converters of light energy, based
on the activity of isolloxazines (flavins) and pteridines
THE ACTION SPECTRA OF ATP FORMATION CORRESPOND
TO THE ABSORPTION SPECTRA OF ABIOGENIC PIGMENTS
Glutamic acid, lysine and alanine
Glutamic acid, lysine and glycine
150-200o C, anoxic medium, 4-6 hrs
150-200o C, anoxic medium, 4-6 hrs
THE FLUORESCENCE SPECTRA OF THE PIGMENTS: EMISSION VS. EXCITATION
10
10
5
5
600
500
400
300
500
450
600
500
400
250
300
350
400
300
500
450
250
300
350
400
ACTION SPECTRA OF THE FORMATION OF ATP
1,5
ATP,
mmole/einstein
FMN
A
200
Pterin
ATP,
mmole/einstein
800
150
1,0
100
400
0,5
After:
Telegina TA.,
Kolesnikov MP.,
Vechtomova YL.
Buglak AA.,
Kritsky MS
Abiotic photophosphorylation
model based on
A abiogenic flavin
and pteridine
0,2 pigments. J Mol
Evol (2013) 76(5),
332-342.
0,1
50
0
300
350
400
l, nm
450
500
0,0
550
0
300
350
400
450
500
0,0
550
l, nm
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
And also act as chromophores in photoreceptor proteins
Lumazine
FMN
O
HN
O
HN
H2N
Cyanopterin
HN
N
H
N
N
H
O
HO
OH OH
HO
O
O
N
OH
N
H
N
+
N
O
O
O
N
OH
HO
O
O P O
O
N
N
H2N
O
O
O P O
O
O PO
O
OMe
N
CO2H
N
Flred-H2
OH
7,8-didemethyl-8-hydroxy5-deazariboflavin
(8-HDF)
O
C
HN
HO
OH
PHOTORECEPTOR FAMILIES
•DNA-photolyases and cryptochromes
• LOV-domain photoreceptors
• BLUF-domain photoreceptors
Main question:
Are apoproteins of
these photoreceptors
monophyletic or
polyphyletic?
N
R
N
H
N
HO
HO
N
H
OH
O
HO
O
H
N
HN
N
HN
N
N
O
O
O
OH
O
N
H
5,10-Methenyltetrahydrofolate
H2N
O
O
N
HN
N
FAD
O
N
N
O
N
OH
HO
HO
OH
HO
These families are polyphyletic,
i.e., BIOLOGICAL EVOLUTION
REPEATEDLY SELECTED FLAVINS FOR
THE ROLE OF PHOTON RECEPTORS
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:
…and yet another
hypothetical option
Flavin
ē-transfer
ATP
(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?
Solar irradiance, W m-2 nm-1
IN THE EARLY HISTORY OF LIFE THE FLAVIN
CONVERTER MIGHT HAVE BEEN COMPETETIVE
Outside atmosphere
At sea level
Relat. efficiency
Relat. efficiency, % Absorption, %
Wavelength, nm
Wavelength, nm
Chlorophyll b
Chlorophyll a
Carotenoids
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.
Wavelength, nm
Selective characters of the flavin-based converter?
“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 shortwavelength” area.
• Low absorptivity of main pigments.
Thank you for your attention!