INORGANIC ENZYME: FROM

HYDROLYSIS OF PHOSPHATE ESTER

PROMOTED BY AGED INORGANIC

IRON SOLUTIONS

-------- A NEW APPROACH FOR ORIGIN

OF LIFE

Xiao-Lan Huang, Ph D

GLUCOSE, GLUCOSE PHOSPHATE AND GLYCOLYSIS

– glycolysis does not require O2.

– glycolysis is common to all organisms, suggesting it evolved early in life.

D-Ribose 5-phosphate

Glycerol -phosphate

Guanosine triphosphate (GTP)

D-Fructose 1-phosphate

PHOSPHATE ESTER

ATP: ENERGY SOURCES OF ALL

LIFE

KINETICS OF CATALYSIS: HYDROLYSIS OF PHOSPHATE ESTER

Time (h)

0 2 4 6

Ph

osp

horu

s c

on

ce

ntr

ation

(M

)

0

2

4

6

8

10

12

14

16

18

20

IP

G6P

( a )

Hydrolysis of G6P in a 16.5 nM Fe(NO3)3 solution aged for 14 months

at room temperature (22 2 C)

TIME COURSES OF FORMATION OF PHOSPHORANTIMONYLMOLYBDENUM BLUE

COMPLEX FROM PHOSPHATE RELEASED FROM HYDROLYSIS OF 20 µM G6P IN AN AGED

(4-MONTH) 1.0 µM INORGANIC IRON SOLUTION AT ROOM TEMPERATURE (22 ± 2°C) AT

G6P HYDROLYSIS TIMES OF 0, 1, 3, AND 6 HOURS, RESPECTIVELY.

Time after addition of the mixed reagent (min)

0 10 20 30 40 50 60

Abso

rbance

(890 n

m)

0.00

0.02

0.04

0.06

0.08

0 h

1 h

3 h

6 h

KINETICS OF CATALYSIS: FIRST ORDER

Time (h)

0 2 4 6

Lo

g [

G6

P] t (

M)

-5.05

-5.00

-4.95

-4.90

-4.85

-4.80

-4.75

-4.70

Log [G6P]t =-4.7188 -0.0000131t

r2=0.999

( b )

KINETICS OF CATALYSIS: LINEWEAVER-BURK PLOT

1/[G6P]o ( M-1)

-0.1 0.0 0.1 0.2

1/v

0 (

S n

M-1

)

0

1

2

3

4

PGoov 6

137085.998

1

max

1

vmK

1

( c )

where [G6P]o is in M and vo is in M s-1

KINETICS OF CATALYSIS: MICHAELIS-MENTEN EQUATIONS

[G6P]o ( M)

0 50 100 150 200 250

vo (

nM

S-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

vmax = 1.0 nM S

-1

Km = 13.7 M

( d )

AGED PROCESS AND RATE CONSTANT

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

1.20E-05

1 5 50 100 200 500 1000

Hydro

lysis

rate

consta

nt

(S-1

)

Fe (nM)

1-mo

2-mo

3-mo

4-mo

Hydrolysis rate constant (k) of 20 µM G6P as a function of

aging time in solutions of different iron concentrations.

INHIBIT BEHAVIOR (I)

Time (h)

0 1 2 3 4 5

[G6

P] t c

once

ntr

atio

n (

M)

0

5

10

15

20

Black: No inhibitorRed: 1 µM MoO4Blue: 10 µM PO4Green: 1 µM WO4

(a)

Glucose-6-phosphate hydrolysis in an aged 10-month, 1000

nM Fe(NOR3R)R3R solution at room temperature (22 2 C).

INHIBIT BEHAVIOR (II)

Tetrahedral oxyanions concentration (µM)

0 10 20 30 40

Perc

enta

ge o

f th

e

hydro

lysis

ra

te c

onsta

nt

k (

%)

0

20

40

60

80

100

WO4 : 1.373 0.993

MoO4: 0.493 0.974

PO4: 0.0649 0.981

(b)

axy

1

100r2

Percentage reduction of the hydrolysis rate constant (k) of initial 20µM G6P as a

function of the concentration of different tetrahedral oxyanions.

[G6P]0 ( M)

0 20 40 60 80 100 120 140

vo (

nM

S-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

No inhibitor

1 M MoO4

1 M WO4

10 M PO4

5 M PO4

(a)

Effect of initial concentration of G6P on the initial hydrolysis velocity

of G6P.

(5)

1/[G6P]o ( M-1)

0.00 0.05 0.10 0.15 0.20

1/v

o (

S-1

nM

)

0

1

2

3

4

5

6

7

No inhibitor

1 M MoO4

1 M WO4

5 M PO4

10 m PO4

(b)

Lineweaver-Burk plot of aged iron solutions in the absence and presence of

tetrahedral oxyanions.

Michaelis-Menten equations Without additions

with 1µM MoO4:

with 1 µM WO4:

with 5µM PO4:

with 10µM PO4:

The activity of aged iron solution did not changed with the addition of F

in solution.

The activity of aged iron solution was significantly decreased in the Tris

buffer system.

However, the natural PAPs are sensitive to F and it was reported that F

can significantly reduce their activity.

BACKGROUNDS:

Purple acid phosphatases

(PAPs) are metalloenzymes

that hydrolyze phosphate

esters and anhydrides under

acidic condition.

RCH2OPO32- + H2O RCH2OH + HPO4

2-

Purple acid phosphatase

(PAP )

Stereoview of the di-nuclear iron center in rat purple acid phosphatase.

μ-(hydr)oxo bridge and a

bound solvent molecule

at the di-iron center. The di-iron center and

the active-site of rat purple acid

phosphatase

Y. Lindqvist, E. Johansson, H. Kaija, P. Vihko, G. Schneider, Journal of Molecular Biology 291, 135 (1999)

Active site of red kidney bean PAP.

Fe(III) binds to the high-affinity metal site M1,and Zn(II) is bound to

the low-affinity site M2.

R. Than, A. A. Feldmann, B. Krebs, Coord. Chem. Rev. 182, 211 (1999)

G. Schenk et al., Proceedings of the National Academy of Sciences of the United States of America 102, 273 (2005)

Stereodiagram of the active-site residues in sweet potato PAP

Stereodiagram of the active-site residues in sweet potato PAP. Side chains that coordinate directly to

the metal ions are shown as stick models. Side chains that form the active site are shown as ball-and-

stick models. Carbon atoms colored gray represent side chains that are conserved between red kidney

bean and sweet potato PAP. Carbon atoms colored yellow are nonconserved, and the name of the

equivalent residue in red kidney bean PAP is in brackets. Primed amino acid residues are from the

neighboring subunit.

Sweet potato PAP is

homodimeric; each subunit

contains two domains, an N-

terminal domain (in blue) of

unknown function and a

catalytic C-terminal domain (in

magenta). The structure was

solved complexed with

phosphate (not shown). The

metal ions are colored orange

for Fe(III) and blue for Mn(II).

Mammalian PAPs are

monomeric and contain only one

domain. In panel B, the two Fe

ions are shown as orange

spheres and the bound

phosphate is shown as a stick

model.

Sweet

potato

pig

Proposed mechanism of

hydrolysis by PAPs.

(A) Substrate binding;

(B) nucleophilic attack and

release of leaving group;

(C) regeneration of the

active site; phosphate is

replaced by two or three

water molecules

BIOMIMETICS OF PAPS

C. Belle, J.-L. Pierre, Eur. J. of Inorg. Chem. 23, 4137 (2003)

Molecular structure of the cation in

Fe2(tbpo)(O2PO(OH))2]ClO4 · CH3OH · 8.5H2O

R. Than, A. A. Feldmann, B. Krebs, Coord. Chem. Rev. 182, 211 (1999)

Molecular structure of the cation in

[Fe2(tbpo)(O2P(OPh)2)(Cl)2(CH3

OH)](ClO4)2 · 3CH3OH

From Roberto Than, Arnold A. Feldmann and Bernt Krebs ,1999

a trans-[(OH2)Fe(μ-OR)2Fe(OH2)]

[Fe2III(BPClNOL)2OAc]+

[Fe2III(BPClNOL)2(H2O2)]2+

A. Horn Jr et al., Inorganica Chimica Acta 358, 339 (2005)

Schematic formation of the ferrous polycation

[Fe4(OH)4(OH2)12]4+ from complexes in solution.

Oxo bridges formation

(oxolation mechanism):

Hydroxo bridges formation

(olation mechanism)

J.-P. Jolivet, E. Tronc, C. Chaneac, C. R. Geosci. 338, 488 (2006)

Iron oxides:

from molecular clusters to

solid.

Possible pathway of the formation of goethite α-FeOOH and electron

micrograph of particles.

J.-P. Jolivet, E. Tronc, C. Chaneac, C. R. Geosci. 338, 488 (2006)

CHANGES IN PARTICLE SIZE, PHYSICOCHEMICAL

PROPERTIES, AND PHASE ABUNDANCES AS A FUNCTION OF

AGING.

Michel F M et al. PNAS 2010;107:2787-2792

Changes in particle size, physicochemical properties,

and phase abundances as a function of aging.

(A) Average particle size from TEM analysis. Bars

indicate standard deviations.

(B) Density (Open Diamonds) from pycnometry and

specific surface area (Light Shade) from N2-BET

as a function of aging.

(C) Total Fe (Solid Squares) of solid phase and

hydration weight losses by TGA (Shaded). Medium

and dark shaded regions indicate weight losses

after heating to 1,000 °C and 125 °C, respectively.

Note that data for t = 14 h are representative of

single-phase hematite.

(D) Percent phase abundances of precursor ferrihydrite

(fh), ferrimagnetic ferrihydrite (ferrifh), and hematite

(hm) from chemometric analysis

Aged Effects on the change of iron oxide: A case study

EVIDENCE FOR FE VACANCIES IN FERRIHYDRITE.

Michel F M et al. PNAS 2010;107:2787-2792

Evidence for Fe vacancies in ferrihydrite. (A) Intensity increases for specific correlations (Red Arrows) during initial 8 h aging time are attributed to filling of vacant Fe sites. Shifts in other correlations (Blue *) are due to the decreasing a-dimension of the unit cell during the fh → ferrifh transition.

(B) Element-specific and (C) site-specific partials calculated from the refined structure of ordered ferrifh (Fig. 3B). (Inset) Refined occupancies (%) from PDF analysis normalized for total Fe for three Fe sites in ferrihydrite during aging.

POSSIBLE ELECTRON SPIN ORIENTATIONS AND MAGNETIC MOMENTS IN ORDERED

FERRIHYDRITE.

Possible electron spin orientations and magnetic moments in ordered ferrihydrite.

Based on the Michel et al. 2007 structure of ferrihydrite (6), 6 Fe3+ with spins in one direction and 4 Fe3+ opposing results in a magnetic moment of 30 B . Alternatively, aligning the spins of the Fe3 (tetrahedral) and Fe2 (octahedral) sites antiparallel to one another results in a magnetic moment of 30 B .

Michel F M et al. PNAS 2010;107:2787-2792

FE SOLUTION AGED PROCESS

HYDROLYZED FE SOLUTION CHEMISTRY

N. Panina, A. Belyaev, A. Eremin, P. Davidovich, Russ. J. Gen. Chem. 80, 889 (2010)

HYDROLYZED FE SOLUTION CHEMISTRY

N. Panina, A. Belyaev, A. Eremin, P. Davidovich, Russ. J. Gen. Chem. 80, 889 (2010)

HYDROLYZED FE SOLUTION CHEMISTRY

N. Panina, A. Belyaev, A. Eremin, P. Davidovich, Russ. J. Gen. Chem. 80, 889 (2010)

HYDROLYZED FE SOLUTION CHEMISTRY

N. Panina, A. Belyaev, A. Eremin, P. Davidovich, Russ. J. Gen. Chem. 80, 889 (2010)

HYDROLYZED FE SOLUTION CHEMISTRY

The product of hydrolysis of aqua-complex cations

[Fe(H2O)6]2+ in the gas phase and in solution are binuclear

dihydroxobridging compound [FeII(H2O)4( -OH)2FeII(H2O)4]2+.

For aqua-complex cations [Fe(H2O)6]3+, the most stable

species in solutions are the dihydroxobridging binuclear cations

[FeIII(H2O)4(μ-OH)2·FeIII(H2O)4]4+.

N. Panina, A. Belyaev, A. Eremin, P. Davidovich, Russ. J. Gen. Chem. 80, 889 (2010)

FE3O4 MAGNETIC NANOPARTICLES AS

PEROXIDASE MIMETICS

L. Gao et al., Nat Nano 2, 577 (2007).

L. Gao et al., Nat Nano 2, 577 (2007).

Left: Typical photographs of 24 L of 60 mM ABTS reaction solutions

catalytically oxidized by the as-prepared Fe3O4 MNPs in the presence of

H2O2 incubated at 45 C in 185 L of 0.2 M pH 4.0 acetate buffer (from left

to right: 0 mM H2O2 with Fe3O4, 10 mM H2O2 without Fe3O4, 10 mM

H2O2 with Fe3O4.).

(A) A dose response curve for H2O2 detection using the as-prepared Fe3O4 MNPs as artificial enzymes and (B) the linear

calibration plot for H2O2. The error bars represent the standard deviation of three measurements.

Fe3O4 Magnetic Nanoparticles as Peroxidase Mimetics

H. Wei, E. Wang, Anal. Chem. 80, 2250 (2008).

“The specificity of the physiological activity of

substances is determined by the size and shape of

molecules, rather than primarily by their chemical

properties, and that the size and shape find

expression by determining the extent to [which]

these regions of the two molecules are

complementary in structure... The enzyme is closely

complementary in structure to the "activated

complex" for the reaction catalyzed by the enzyme”

by L. Pauling (Nature, 1948, 161, 707-709)

All organisms depend upon metallo-enzymes. The

dependence arises from the inability of individual organic

side-chains of proteins to activate molecules such as H2,

N2, CH4 and CO and their weakness in hydrolysing many

simple compounds such as many peptides, phosphates,

even urea. The metal ion sites have been found

to be 'designed' for selective uptake and

catalytic activity. By Williams, R. J. P. (Metallo-

enzyme catalysis." Chem. Commun.2003 (10): 1109-1113.)

Ribozymes Catalytic RNA

G-12 (Red part): base in the self-cleavage reaction

G-8 (dark blue) : general acid catalysis

phosphate

Science 31 January 1986:

Vol. 231 no. 4737 pp. 470-475

DOI: 10.1126/science.3941911

CONCLUSION

Aged, acid-forced hydrolysed nanomolar inorganic iron solutions were found to have phosphoesterase activity, which significantly promoted the hydrolysis of phosphate ester following Michaelis-Menten kinetics.

The catalysis was inhibited by tetrahedral oxyanions with inhibition strength in an order of WO4 >MoO4 > PO4.

The activity was related to the aging process and total iron concentrations, but the detailed mechanism is still unknown.

Further work is needed to understand the nature of the (hydr)oxo-bridged Fe-Fe structure in water and its potential role in organic phosphorus transformation in geochemistry.

IMPLICATIONS

This observation and reported intrinsic peroxidase-like activity of ferromagnetic nanoparticles demonstrates that “The chain of life is of necessity a continuous one, from the mineral at one end to the most complicated organism at the other”, as proposed by Leduc.

The hydrolysed iron solutions might be just one of ubiquitous sets of undiscovered inorganic biocatalysts (enzymes) in nature.

Inorganic enzymes might act as a bridge between the inorganic and organic worlds and would have played important roles in the origin of life.

Discovery of further inorganic enzymes might provide clues on the emergence of life and a potential solution to the puzzle of “chicken and egg” in life’s evolution.

When did life arise?

Although the oldest microfossils in Apex chert (3.465 b. y. old, above) are

currently being questioned, additional isotopic evidence suggests that life

was present by 3.5 b. y. ago and may have originated even earlier.

Russell, M. J., and Kanik, I. (2010). Why Does Life Start, What Does It Do, Where

Will It Be, And How Might We Find It? Journal of Cosmology, 5, 1008-1039.

Russell, M. J., and Kanik, I. (2010). Why Does Life Start, What Does It Do, Where Will It Be, And How

Might We Find It? Journal of Cosmology, 5, 1008-1039.

M. J. Russell, A. J. Hall, J. Geol. Soc. 154, 377 (1997).

M. J. Russell, A. J. Hall, J. Geol. Soc. 154, 377 (1997).

M. J. Russell, A. J. Hall, J. Geol. Soc. 154, 377 (1997).

MOLECULAR CLUES

TO THE

ORIGIN OF LIFE

DNA

Nucleotide

DNA Sugars and Phosphate

Bases

Three Dimensional Structure of tRNA

RNA

PROTEINS AND ENZYMES

PASTEUR’S

EXPERIMENT ON

SPONTANEOUS

GENERATION (1862)

THE RNA WORLD HYPOTHESIS

ROLE OF MINERAL MINERAL IN BIOCHEMICAL

EVOLUTION

Sorption and Exchange

Catalyze chemical reactions

Promote & propagate chiral molecules

Provide scaffolding for organic synthesis

Protect fragile molecules

Create semi-permeable microenvironments of cell-

like dimensions (mimicking a lipid membrane)

Inorganic Enzyme

(e.g., Al)

ADSORPTION MECHANISMS OF AMINO ACIDS ON MINERAL SURFACES

• Adsorption

of glycine on

silica through

covalent bond

formation

• Adsorption

of glycine on

titania

through

coordinative

bond

• Adsorption of glycine on Al

oxyhydroxides through

formation of a ternary

coordination complex

• Adsorption

of aspartate

on kaolin

through the

formation of a

hydrogen-

bonded adduct

Lambert (2008) Adsorption and Polymerization of Amino Acids on Mineral Surfaces: A

Review. Origins of Life and Evolution of Biospheres, 38(3), 211-242.

MINERAL SURFACES CAN SERVE AS TEMPLATES FOR CHIRAL MOLECULES

Hazen (2001) Sci. Am., April 2001: 77-85

MINERAL SURFACES CAN PROTECT FRAGILE MOLECULES

Hazen (2001) Sci. Am., April 2001: 77-85

IRON SULFIDE MICROCOMPARTMENTS: Mimicking a lipid membrane

Russell & Martin, Phil Trans RS (B), 2003, 358, 59

WACHTERSHAUSER’S IRON–SULFUR WORLD THEORY

• Iron-sufide minerals catalyze production of pyruvate* & other

biomolecules under conditions common in hydrothermal vent systems.

*Pyruvate = branch point for many biosynthetic pathways

G. Wachtershauser, Prog. Biophys. Mol. Bio. 58, 85 (1992).

G. Wächtershäuser, Chemistry & Biodiversity 4, 584 (2007).

MINERAL-SURFACES CAN CATALYZE

ORGANIC SYNTHESES UNDER

HYDROTHERMAL CONDITIONS

GRAHAM CAIRNS-SMITH’S HYPOTHESIS:“GENETIC TAKEOVER”

Cairns–Smith (1982) proposed that clay minerals were life’s ultimate ancestor.

characteristics of clay minerals:

products of processes of weathering and diagenesis

main component of soils and sediments

extensive structural variation

high affinity for adsorption of H2O and organics

high ion exchange capacity

clay minerals could provide the basis for an evolution through natural selection

A. G. Cairns-Smith, Genetic Takeover and the Mineral Origins of

Life. (Cambridge University Press, Cambridge, UK, 1982), pp. 488

• the yellow regions represent phenotypes - G1 is the primary

genetic substrate, and G2 is the secondary one. Arrows within

organisms indicate paths of genetic expression

• a simple organism with genetic substrate G1 produces

substance G2 as a component of its metabolic processes

• G2 is inherited - and comes to carry heritable information.

Gradually, G2 displaces G1 as the primary genetic material for

the organism

• secondary genetic material arises not as a modification of the primary one, but rather from molecules synthesized under its control

Genetic Takeover

A. G. Cairns-Smith, Genetic Takeover and the Mineral Origins of

Life. (Cambridge University Press, Cambridge, UK, 1982), pp. 488

WHICH CAME FIRST, THE CHICKEN OR THE

EGG ?

Inorganic

Enzyme

No DNA, No RNA, No Protein,

Only Mineral.

Not surface, but the structure!

斯特凡 勒杜克

THE MECHANISM OF LIFE

1911

In the waters of the ancient world, an at the present time, very small

masses of mucilaginous matter were collected. Under the influences of

light, certain elements, caloric and electric, entered these little bodies.

These corpuscles became capable of taking in and exhaling gases;

vital movements began, and thus an elemental plant or animal sprang

into existence. Possibly higher forms of life, such as infest the

intestines, originate in this way. Nature is thus always creating."

LAMARCK,1802

“the chain of life is of necessity a continuous one, from the

mineral at one end to the most complicated organism at the

other”…..by Dr. Stéphane LEDUC

“It is often said that all the conditions for the first production of a living organism are

now present, which could ever have been present. But if (and oh! what a big if!) we

could conceive in some warm little pond, with all sorts of ammonia and phosphoric

salts, light, heat, electricity, &c., present, that a proteine (sic) compound was

chemically formed ready to undergo still more complex changes, at the present day

such matter would be instantly absorbed, which would not have been the case before

living creatures were found.” Darwin, 1871

Pasteur did not disprove the origin of life by natural means, and the saying "all cells from cells" was

not intended to cover the initial period of life on earth. Darwin did not propose a theory of the origin of

life in the beginning.

John. S. Wilkins, 2004,Spontaneous Generation and the Origin of Life

http://www.talkorigins.org/faqs/abioprob/spontaneous-generation.html

让-巴蒂斯特·拉马克

查尔斯·罗伯特·达尔文

Nature has formed and shaped every day things as simple by

spontaneous generation .

OTHER NON-HEME DIIRON ENZYME

The diiron family consists of soluble and membrane bound

desaturases/hydroxylases.

All non-heme diiron enzymes are capable of carrying out a desaturase and/or

hydroxylase reaction.

Members of this family of proteins include the oxygen carrier Hemerythrin (oxygen

carrier in marine invertebrates), methane monooxygenase (converts methane to

methanol in methanogenic organisms), ribonucleotide reductase and purple

phosphatase.

CARBOXYLATE-BRIDGED DINUCLEAR IRON CLUSTERS-ENZYME

These proteins play

essential roles in such

crucial biochemical

processes as nitrogen

fixation, photosynthesis,

oxidative phosphorylation

and ribonucleotide

reduction.

CH4 + O2 + NAD(P)H + H+ -> CH3OH + NAD(P)+ + H2O (MMO)

RIBONUCLEOTIDE REDUCTASE (RNR)

Ribonucleotide reductase

(RNR, also known as

ribonucleoside diphosphate

reductase) is an enzyme that

catalyzes the formation of

deoxyribonucleotides from

ribonucleotides.

Deoxyribonucleotides in turn

are used in the synthesis of

DNA. The reaction catalyzed

by RNR is strictly conserved in

all living organisms.

Furthermore RNR plays a

critical role in regulating the

total rate of DNA synthesis so

that DNA to cell mass is

maintained at a constant ratio

during cell division and DNA

repair.