Metabolism Origin

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Annu. Rev. Earth Planet. Sci. 2004. 32:56999
doi: 10.1146/annurev.earth.32.101802.120225
Copyright c 2004 by Annual Reviews. All rights reserved
First published online as a Review in Advance on January 20, 2004

TRANSITION METAL SULFIDES AND THE
ORIGINS OF METABOLISM

George D. Cody
Geophysical Laboratory, Carnegie Institution of Washington,
Washington, D.C. 20815; email: g.cody@gl.ciw.edu

Key Words sulfides, prebiotic chemistry, hydrothermal metabolism
Abstract The history of the theory and experimental evidence that the natu-
ral catalytic and reactive qualities of transition metal sulfides are linked to primitive
metabolism is reviewed. In the late 1980s, a hypothesis arose that proposed that tran-
sition metal sulfides (in particular pyrrhotite and pyrite) might play a significant role
promoting abiotic organic chemistry. As an outgrowth of this hypothesis, elaborate
theories were presented, including proposals for earliest life being structurally distinct
from extant prokaryotic life. During the 1990s and into the twenty-first century, exper-
imental evidence has emerged that supports certain aspects of these theories; in other
cases, the experiments reveal chemistry that diverges significantly from that which was
proposed theoretically. In either case, however, there is clear evidence that transition
metal sulfide minerals exhibit catalytic qualities for the promotion of reactions that
have obvious metabolic utility and, therefore, could have provided the primitive Earth
with valuable biochemical intermediates.

INTRODUCTION

Throughout the nineteenth and twentieth centuries the majority of theories regard-
ing the origins of life were tied to the concept that life arose from a prebiotic
broth. Essential to this concept was the belief that the primitive Earth provided
an abundance of organic molecules either through endogenous synthesis or ex-
ogenous delivery. Within this paradigm, hypotheses have been proposed that life
emerged by natural chemistry that exploited this bounty (see for example Oparin
1953, Dyson 1985, Gilbert 1986, Orgel 1986, Joyce 1989, de Duve 1991, as well
as reviews by Chyba & McDonald 1995 and Brack 1998). First life was, by defini-
tion in these hypotheses, heterotrophic (i.e., first life would have used the abundant
organic molecules as fuels to support both anabolic and catabolic processes).
The heterotrophic hypotheses for lifes origins arose quickly following advances
in biochemistry, e.g., Oparins hypothesis of a metabolic proto-organism (Oparin
1953) emerged from the explosion of research into metabolic biochemistry in the
early twentieth century. The RNA world hypothesis (e.g., Gilbert 1986, Orgel

0084-6597/04/0519-0569$14.00 569
570 CODY SULFIDE CATALYZED ABIOTIC CHEMISTRY 570

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1986, Joyce 1989) arose from the scientific advances in molecular biology that
solved the function of polynucleotides.
In general, the heterotrophic hypotheses focused minimal attention on the geo-
chemical constraints that must have allowed life to emerge and evolve on the
primitive planet. Minerals were invoked to serve the function of catalyst for the
oligomerization of activated amino acids and/or nucleotides (Ferris & Ertem 1993,
Ferris et al. 1996, Orgel, 1998); Cairns-Smith (1982) invoked the imaginative hy-
pothesis that clays could have served as genetic precursors to DNA. Beyond this,
however, the issue of the source of the essential monomers that such heterotrophic
life required was buried in the unknown (and perhaps unknowable) environmental
conditions of the most primitive Earth. Lurking in this shadow was a disconcert-
ing question. How would life have originated if the Hadean Earth did not have a
concentrated reservoir of useful organic molecules?

TRANSITION METAL SULFIDES AND THE ORIGINS
OF AUTOTROPHIC LIFE

In the last two decades of the twentieth century, two hypotheses were presented
that set out to explain lifes origins in a primitive world without a natural abundance
of organic compounds. These are Wachtershausers iron-sulfur world hypothesis
(Wachtershauser 1988a, 1990, 1992, 1994, 1998) and Russell & Halls iron-sulfur
membrane hypothesis (Russell et al. 1988, 1989, 1997, 1998). Both proposals
predicted that life emerged as a direct consequence of chemical reactions derived
from reduced fluids (crustal and/or mantle derived) interacting with reactive and
catalytic transition metal sulfides. One of the essential differences between these
hypotheses and those that preceded them was the idea that Earths first organism
was an autotroph, i.e., a life form capable of synthesizing all of its biomolecular
constituents from simple inorganic compounds, such as CO
2
, NH
3
, H
2
S, and PO
1
.

Pyrite Formation as a Primordial Energy Source

Wachtershauser (1988b) proposed that pyrite formation could have provided a vi-
able energy source for Earths first life. The specific reaction involved iron mono-
sulfide reacting with hydrogen sulfide to produce pyrite and hydrogen, i.e.,
FeS + H
2
S FeS
2
+ H
2
. (1)
At standard state, the Gibbs free energy (1G
o
) for this reaction, calculated
by Wachtershauser, was exergonic, i.e., 1G
o
= 41.9 kJ/mol. Subsequent cal-
culations with more recent thermodynamic data yielded 1G
o
= 38.4 kJ/mol
(Wachtershauser 1990). Schoonen et al. (1999) reported a slightly less negative
1G
o
of 31.2 for this reaction, using the program SUPCRT92 (Johnson et al. 1992)
and the thermodynamic database of Helgeson et al. (1978). Both Wachtershauser
and Schoonen et al. (1999) noted that at pHs greater than the first dissociation

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constant of H
2
S, the reducing power associated with pyrite formation drops signif-
icantly, thus the reducing power associated with Reaction 1 is realized under acidic
conditions (Wachtershauser 1988b, 1990). Regardless of the thermodynamic data
used, Reaction 1 is significantly exergonic. Highly exergonic reactions have the
potential to drive otherwise endergonic reactions when coupled with them.
For example, the reduction of CO
2
to form formic acid is endergonic (e.g.,
Wachtershauser 1988b):
CO
2
(aq.) + H
2
HCOOH, G
o
= +30.2 kJ/mol. (2)
Combining the formation of pyrite (Reaction 1) with the reduction of CO
2
(Reac-
tion 2) yields, e.g.,

CO
2
(aq.) + FeS + H
2
S HCOOH + FeS
2
+ H
2
O, G
o
= 8.2 kJ/mol. (3)

Note that in Schoonen et al.s (1999) calculation, 1G
o

for Reaction 2 was
13.69 kJ/mol; hence, Reaction 3 would have a 1G
o
of 17.51. In either calcu-
lation, the coupling of pyrite formation and CO
2
reduction is thermodynamically
favorable at standard state conditions.
Aside from the favorable thermodynamics of Reaction 3 at low temperatures,
there remains the question of kinetics. Wachtershauser (1990, 1992) favored slug-
gish kinetics in the case of pyrite formation. Ideally, Reaction 1 would not proceed
unless coupled to CO
2
reduction (e.g., Reaction 3) or other significant organic
reactions (Wachtershauser 1988a, 1990, 1992), thus, not wasting precious reduc-
tion potential by producing excessive H
2
(which would be rapidly lost from the
system). Although Schoonen et al. (1999) agreed that reactions such as those de-
scribed above are thermodynamically viable, they concluded that direct electron
transfer from FeS to CO
2
was not feasible; hence, these reactions would not occur.
The relevance of this argument is difficult to assess, as it requires considerably
more detailed information about the reaction mechanism. Such details are not
provided by either Wachtershauser (1988b) or Schoonen et al. (1999). It is known
that the direct electroreduction of CO
2
requires large negative over potentials (e.g.,
less than 2.0 V versus a mercury electrode). It is interesting to note, however, that
the presence of soluble [Fe
4
S
4
(SR)
4
]
2
clusters (where R corresponds to a phenyl
group) reduces the size of the over potential considerably (Tezuka et al. 1982).

Pyrite-Pulled Metabolism

At the core of both Wachtershausers and Russell & Halls hypotheses is the
concept that primitive carbon fixation reaction mimics the reductive citrate (or
citric acid) cycle (RCC) [the RCC is used by some extant micro organisms, e.g.,
Thermoproteus, an anaerobic archea (Buckel 1999)] (Figure 1) (Wachtershauser
1988a, 1990, 1992). Hartman (1975) had proposed earlier that given the ubiq-
uity of the citric acid cycle, it might have provided a primitive chemical back-
bone from which all extant metabolic strategies ultimately evolved. Morowitz
(1992) theorized that intermediary metabolism recapitulates prebiotic chemistry.

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Figure 1 Schematic diagram of the reverse (or reductive) citrate cycle, RCC. ATP,
ADP, and AMP refer to adenosine tri-, di-, and monophosphate, respectively; CoA
refers to the coenzyme acetyl CoA. The cycle fixes carbon (CO
2
) in a counter-clockwise
sense of rotation along the various biochemical reactions. Upon achieving the synthesis
of citrate, this molecule is cleaved to form one molecule of oxaloacetate and acetyl
CoA. The RCC is network autocatalytic, i.e., the final products are the cycles initiators.

The attraction of the RCC is that it is network autocatalytic (see Figure 1); it doubles
with every turn.
Critical to Wachtershausers hypothesis is the proposal that there would in-
evitably have been a long induction period for the initiation of any given abiotic
carbon fixation pathway, including the RCC. Given that the product of the RCC
upon one turn is a vital initiator for the cycle, once the RCC began, it was proposed
that it would continue unabated. In a relatively short period, the RCC pathway
would completely dominate the chemical landscape of the prebiotic environment.
One of the intriguing postulates of Wachtershausers hypothesis
(Wachtershauser 1990) is that many of the reactions within the extent RCC would
initially have used thioanalogues of the metabolic intermediates, e.g., thiomalic

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acid in place of malic acid (Figure 2). In Wachtershausers view, this afforded two
distinct advantages. First, for certain carboxylations (e.g., the beta carboxylation
of pyruvic acid to form oxaloacetic acid), the energetics and kinetics are highly
unfavorable (Wachtershauser 1988b). Wachtershauser hypothesized that the thio-
lated intermediates, e.g., thiopyruvate, might exhibit more facile kinetics. Second,
Wachtershauser hypothesized that certain reactions involving thiolated interme-
diates would inevitably couple with pyrite formation leading a favorable thermo-
dynamics (Wachtershauser 1988a, 1990). This is the essence of Wachtershausers
pyrite-pulled intermediary metabolism.
For Wachtershausers thio-analog RCC to run, certain classes of reactions were
postulated to occur, some of which that have yet to be shown to be viable in organic
chemistry. The first class of reaction, A, involves the formation of monothioke-
tals, dithioketals, and aminothioketals (when excess NH
3
is present) from keto
carbonyls, e.g.,
R (C O) R
0
+ H
2
S R(HO

C

SH) R
0
. (4)
Reaction 4 is straightforward and expected under conditions of high concentrations
of H
2
S; it is not pyrite pulled. An example of a pyrite-pulled reaction class, B,
involves the reductive carboxylation of a thioacid to yield an alpha keto-acid. For
example, Wachtershauser proposed that starting with thioacetic acid and CO
2
,
pyruvic acid would form utilizing the sulfur in FeS as the electron donor, e.g.,
R COSH + CO
2
+ FeS R (C O) COOH + FeS
2
. (5)
Another pyrite-pulled reaction class, C, involves a postulated reductive desulfur-
ization reaction, e.g.,
R (HS

C

SH) R
0
+ FeS R (H

C

SH) R
0
+ FeS
2
. (6)
The final postulated reaction class, D, involves a beta carboxylation of 2-enethiol
carboxylic acid analogue of pyruvic acid (Wachtershauser 1990) and is not pyrite
pulled, but may be less kinetically inhibited than the equivalent reaction involving
the 2-keto carboxylic acid (Wachtershauser 1988a). Reaction class D has not been
verified experimentally, but can be written as
HOOC (C SH) CH
2
+ CO
2
HOOC (C SH)CH
2
COOH. (7)
Reaction classes AD coupled with thioacid-carboxylic acid equilibria, H
2
S
addition and elimination reactions, and a retro-aldol cleavage reaction serve to
drive the thio-analogue of the RCC. These reactions are shown schematically in
Figure 2. Note that the pyrite-pulled reactions depicted in Figure 2 are replaced in
the extant RCC (Figure 1) by iron-sulfur proteins and adenosine triphosphate.

Surface Metabolism and Two-Dimensional Life

The formation of pyrite is postulated to drive carbon fixation as well as a broad
range of essential proto-biochemical reactions (Wachtershauser 1988a, 1992).

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Figure 2 Wachtershausers thio-analogue of the reverse citrate cycle. Note that each
of the molecular intermediates is rendered in a simplified fashion as stick structures.
Here, only the carbon skeleton of a given molecule is revealed along with the het-
eroatoms oxygen and sulfur. Where doubled bonding occurs, a pair of lines is used
to designate the unsaturated linkage. The reactions designated A, B, C, and D are dis-
cussed in the text. Note that where a given reaction is highlighted with an asterisk, the
reaction is coupled to the oxidative transformation of FeS to FeS
2
.

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In Wachtershausers theory, however, pyrite is not simply a by-product; rather,
it provides the essential template for surface metabolism. An essential aspect of
the surface metabolism theory is that all of the metabolic intermediates are anions
linked to the cationic surface of pyrite. Wachtershauser (1988a) proposed that the
oceans of the early Earth would have been essentially infinitely dilute; thus, any
organic molecule synthesized via pyrite-pulled metabolism (Figure 2) must remain
adsorbed on the product pyrite. If the molecule desorbed it was forever lost from
the system.
Several advantages are proposed for such a scenario (Wachtershauser 1988a,
1990). The first is that the pyrite surface will select for particular products based
on preferential adsorption characteristics. Thus, competitive adsorption on pyrite
surfaces gives the surface metabolic system the capacity of chemical evolution.
The second advantage proposed for pyrite surface metabolism is enhanced rates
of condensation reactions (polymerization) involving anionic monomers:
A + A AA, AA + A AAA, AAA + A AAAA, . . . (8)
The condensation of biological macromolecules, such as peptides, involves
the initial condensation of a pair of amino acids to form a dimer, followed by
successive condensations with monomers (e.g., Reaction 8). In any hypothetical
abiotic scenario, condensation reactions compete with hydrolysis reactions, hence
the equilibria indicated in Reaction 8. The first condensation step is slightly en-
dergonic under standard state conditions (Shock 1992). Furthermore, the kinetics
of condensation under dilute conditions obviously suffer from low probability of
monomer-monomer collisions.
Extant enzymes drive condensation reactions by linking monomers held in close
proximity. In essence, binding of substrate within the active site of an enzyme
raises the effective substrate molarity, in some cases many orders of magnitude
(see, for example, discussions by Abeles et al. 1992), and drives the reaction toward
condensation even when the solution molarity of the substrate is too low to favor
reaction. Ferris and collaborators (Ferris & Ertem 1993, Ferris et al. 1996) have
shown that clays enhance the rate of oligomerization reactions against hydrolysis,
suggesting a similar phenomenon.
Wachtershausers surface metabolism theory hinges on pyrites surface having
a positive charge. Bebie et al. (1998) explored the relationship between solution
pH and surface charge for pyrite and other transition metal sulfides, and observed
that at low pH (<2.0) pyrite does have a positive surface charge. At higher pHs,
however, pyrites surface is negatively charged. Subsequent experiments revealed
that the isoelectric of pyrite in the presence of an excess of Fe
2+
ions in solution
shifts to a considerably higher pH (5.0), and in the case of an excess of H
2
S, to
much lower pHs (Bebie et al. 1998).
Many of Wachtershausers reactions require a high activity of H
2
S and low pH
to exploit pyrite formation as an energy source (e.g., Figure 2). Wachtershausers
surface metabolism hypothesis also requires strong interaction between organic
anions and the pyrite surface. The extent to whether these combined requirements

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can be met given the complex natures of pyrites surface charge is not known.
Evidently, at very low pH, pyrites surface will be positively charged regardless of
the solution chemistry (Bebie et al. 1998). However, organic acids are, in general,
weak acids; for example, the pK
1
of citric acid at room temperature is 3.14 (Yadav
et al. 1989). Most of the intermediary metabolic organic acids have pK
1
s in the
range of 3.0 to 4.0. Consequently, at very low pH, many (if not all) of the organic
acids involved in Wachtershausers theoretical chemical schemes will be proto-
nated. This observation notwithstanding, Bebie & Schoonen (2000) explored the
adsorption of various organic acid anions and discovered that acetate adsorption
did occur on pyrite at a pH of 4.0; their conclusion was that the specific anion-
surface metal interaction can overcome a net columbic repulsion from the average
surface charge.
Perhaps the most imaginative aspect of Wachtershausers iron-sulfur world
hypothesis was the proposal that Earths first organism existed not encapsulated
in a roughly spherical cellular membrane, but rather existed on the surface of
pyrite (Wachtershauser 1988b, 1992) enclosed from the environment by a semi
or half membrane. It was proposed that such an organism, the progenitor of all
organisms, eventually detached from the pyrite surface, taking with it the metabolic
functionality (in the form of protoenzymes with transition metal sulfur clusters) it
learned from the surface metabolism on the pyrite surface. Needless to say this
theory generated both excitement and skepticism (see, for example, a critique by
de Duve & Miller 1991).

Life in a FeS Membrane

Russell & Hall (1997) approach the origins of life question from a decidedly
geochemical perspective, specifically guided by their earlier studies of exhalative-
sedimentary base metal deposits. The essence of their theory involves the postulate
that the early Hadean oceans were warm (T 90
C), mildly acidic (pH 5.5 owing

to high concentrations of CO
2
in the primitive atmosphere), and relatively rich in
dissolved Fe
2+
and Ni
2+
. Russell & Hall (1997) envisioned that hydrothermal
fluids generated within the ancient Hadean oceanic crust would have been highly
alkaline (pH 9.0), hot (T 150
C), rich in bisulfide (HS-), and reduced (H

2
bearing). Where these hydrothermal exhalations penetrated the sea floor and mixed
with the oceanic fluids, reaction of the bisulfide with dissolved base metals would
have resulted in the rapid precipitation of transition metal sulfides (predominantly
mackinawite).
Based on comparison with recent ore bodies, Russell & Hall (1997) surmised
that FeS bubbles would have formed at the sites of mixing on the ancient sea
floor. These bubbles, it is proposed, served as primitive membranes. The purported
advantage afforded to the FeS membrane theory is that the membrane naturally
separates a low pH exterior fluid from a high pH reduced interior fluid. Appeal-
ing to the comparison of cell membrane function in mitochondria and certain
prokaryotes, Russell & Hall noted that the presence of both pH gradients and

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Eh gradients would have allowed these membranes to support processes akin to
electron-transport phosphorylation. Critical, of course, to the viability of the FeS
membrane hypothesis is the requirement of electron diffusion outward through the
membrane as well as diffusion of CO
2
(perhaps as HCO
3
) back into the interior
of the membrane.
One of the most significant differences between the FeS membrane hypothesis
and Wachtershausers pyrite-pulled metabolism is that Russell & Hall (1997) do
not consider the oxidation of FeS as an energy source nor pyrite as a catalytic
agent for organosynthetic reaction. Rather, in the FeS membrane hypothesis, the
membrane is anticipated to undergo geochemical transformation to form mixed
valance catalytic phases that use H
2
as the primary electron donor. Russell & Hall
favored the mineral phases greigite (Fe
3
S
4
) and violarite (FeNi
2
S
4
) as particularly
favorable catalysts based on the fact that both minerals contain Fe
4
S
4
(cubane)
structures, superficially similar in structure to four iron ferredoxins. In the FeS
membrane hypothesis, it is these potentially catalytic phases that promote the
crucial redox chemistry to drive the RCC and other anabolic syntheses.

EXPERIMENTAL EXPLORATION OF TRANSITION METAL
SULFIDES AND THEIR POTENTIAL ROLE IN
PROTOMETABOLISM

Transition Metal Sulfide Catalyzed Biomimetic Chemistry

Interest in the possibility that abiotically synthesized transition metal clusters might
provide insight into biocatalysis began early in the 1960s following the discovery
of iron-sulfur clusters in a variety of enzymes. It was soon discovered that various
iron sulfur clusters (e.g., ferredoxins) could be synthesized by self-assembling
mechanisms using a variety of reaction schemes (see, for example, Schrauzer
et al. 1966, Herskovitz et al. 1972). There currently exists an enormous body
of literature covering the synthesis of model ferredoxins ([Fe
n
S
n
(SR)
4
]
2
, n =
2,4) and rubredoxins ([Fe(SR)
4
]
2
), review of which is far beyond the scope of
this article (see Hagen et al. 1981, 1983 for overviews). Readers interested in
the structure, characterization, and function of iron-sulfur clusters in enzymes are
directed to reviews by Howard & Rees (1992) and Cammack (1992), as well as a
recent review by Beinert et al. 1997.
Nakajima et al. (1975) provided one of the first examples of the potential
catalytic role of transition metal sulfide clusters for protometabolic chemistry.
In the course of an attempt to create biomimetic photosynthetic reaction cen-
ters, it was discovered that amino acids could be synthesized via a carbon fixa-
tion reaction that appeared to mimic the function of a critical enzyme, pyruvate
synthase. The experiment started with gaseous CO
2
bubbled into a solution of
tetrahydrofuran, methanol, and water (4:2:1) that contained synthetic iron-sulfur
complex [Schrauzers complex, Fe
2
S
4
C
4
Phenyl
4
; Schrauzer et al. (1966)], sodium
hydrosulfite, and sodium bicarbonate. To this solution, Nakajima et al. added a

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thioester, n-octyl thiol phenylacetate, a source of reactive and transferable nitro-
gen (pyridoxamine dihydrochloride), potassium hydroxide, and zinc acetate. After
nine hours of reaction at room temperature the amino acid phenylalanine was de-
tected. Subsequent experiments with the thioesters n-octyl thiol acetate and n-octyl
thiol 3-methyl butanoate yielded alanine and leucine, respectively.
This was a remarkable result. To synthesize amino acids, it is required that CO
2
is fixed via a carbonyl insertion to form alpha-keto acid intermediates. Nakajima
et al. (1975) reported the detection of phenyl pyruvate, thus clearly identifying the
critical intermediate. The formation of amino acids required the formation of an
imine intermediate by reaction with pyridoxamine followed by reduction to form
the amino acid. Overall, the yield of phenylalanine and phenyl pyruvate was low,
0.3% and 0.66%, respectively. However, it is worthwhile to consider the individual
reaction steps in order to place this yield into perspective.
First, Schrauzers FeS complex (designated C) must be reduced by sulfite (note
that the steps leading to the synthesis of Schrauzers complex are not included).
If it is assumed that the complex can accept two electrons, this reaction may be
written as

3C
ox
+ S
2
O
2
+ 4H
2
O 2SO
2
+ 8H
+
+ 3C
red
. (9)

4 4

A likely key first step involves the transfer of the phenylacetyl group to the re-
duced complex, C
red
, presumably through a reaction involving the thioester and
the complex, e.g.,
R (C O) SR
0
+ H
+
+ C
red
R (C O) C
red
+ R
0
SH. (10)
Here, R (C 0) is the phenyl acetyl group and R is the octyl group. Now CO
2

must be reduced to CO either by the sulfite or the reduced complex. Methanogenic
organisms that live utilizing CO
2
as their primary carbon source utilize a ferredoxin-
based enzyme for the reduction of CO
2
to CO (e.g., Lindahl et al. 1990); thus, it
is attractive to assume that it was the reduced Fe
2
S
2
complex that reduced CO
2
to CO in Nakajima et al.s reaction. It is also possible that the alpha-keto acid
formed via a reductive carboxylation, i.e., similar to the reaction promoted by
the enzyme pyruvate synthase [see, for example, Furdui & Ragsdale (2000) and
references therein]. Reaction of the phenyl acetylated Fe
2
S
2
complex with CO in
the presence of water presumably yields the alpha-keto acid, e.g.,
R (C O) C
red
+ CO
2
+ H+ R (C O) COOH + C
OX
. (11)
The final steps toward the synthesis of phenylalanine involve performing a
reductive amination of the keto acid to yield the amino acid. Diagrammatically,
this involves first nucleophilic attack by the pyridoxamine (a nucleophile, under
basic conditions) to form an unstable pyridoxamino-ketal intermediate,
e.g.,

R (C O) COOH + Pyr NH
2
R (Pyr NH C OH) COOH. (12)

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It is interesting to note that the role that pyridoxamine plays in this reaction is
similar to its role in extant biochemistry (see, for example, Abeles et al. 1992). The
hypothetical amino-ketal intermediate is very unstable and can either decompose
back to the alpha-keto acid plus pyridoxamine or decompose to an imine and a
hydroxylated pyridoxine, e.g.,
R (Pyr NH C OH) COOH R (C NH) COOH + Pyr OH. (13)
The final step involves reduction of the imine to form the amino acid. Note that
it is during this step that the chiral center of the amino acid is formed. If the imine
reduction is nonstereo-selective, then both L and D amino acid enantiomers will be
formed in equal abundance and the mixture is defined as racemic. However, if this
reaction is performed using a chiral catalyst (e.g., rhodium complex with chiral
phosphine ligands; see discussions in Carey & Sundberg 1990) then a high degree
of enantiomeric selectivity could occur. In the case of Nakajima et al.s reaction,
no attempt was made to analyze potential enantiomeric excesses in the amino
acid products. It is also not known whether the reduction occurs on the reduced
Schrauzers complex or with a reduced species in solution (e.g., formic acid).
From a protometabolic perspective, however, it is clearly intriguing to consider
the possibility that the final reduction reaction occurred in a concerted way via
electron transfer from the reduced complex, e.g.,

R (C NH) COOH + 2H
+
+ C
red
R (C NH
2
) COOH
L,D
+ C
ox
.
(14)

The apparently low yields of synthesized amino acids is not surprising when
placed in context of the relatively large number of reactions that must operate in
the scheme of Nakajima et al. (1975). Clearly, the experimental conditions used
in this experiment deviate far from what would be expected in a terrestrial setting.
Nevertheless, Nakajima et al.s reaction suggested the tantalizing possibility that
similar abiotic carbon fixation reactions could have occurred on the early Earth
provided there existed a source of reducing power and active catalysts. Nakajima
et al. (1975) used sodium hydrosulfite as the source of reducing power. Many years
later, Drobner et al. (1990) demonstrated a potentially more geologically relevant
source of strong reducing power.

Hydrogen Production Following Pyrite Formation
from FeS + H
2
S
In 1990, Drobner et al. provided the first experimental results that supported
Wachtershausers (1988b) conjecture that pyrite formation from FeS could gener-
ate significant quantities of hydrogen. In this experiment, 2 mg of pyrrhotite (99%
pure) or amorphous FeS (produced by the sulfidization of an aqueous solution of
FeSO
4
) was reacted in the presence of 2 mmol H
2
S at 100
C for 14 days under

a CO
2
atmosphere. After reaction, the headspace gas was analyzed and between
15 to 40 mol of H
2
were detected via gas chromatography (GC).

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The solids were analyzed with X-ray diffraction. In the case of the pyrrhotite
reactions, only pyrite was detected along with unreacted pyrrhotite. They noted
that pyrite appeared to coat and replace pyrrhotite. When amorphous FeS was
used, pyrite and mackinawite were detected, suggesting that the amorphous FeS
recrystallized during the incubation period. The pyrite formed in these experiments
appeared as primary, cubic crystals. In either case, no effort was made to determine
a mass balance, i.e., to show that the amount of pyrite formed correlated with
abundance of H
2
detected in the headspace.

Carbon and Nitrogen Reduction Coupled with Pyrite Formation

The compelling results of Drobner et al. (1990) in relation to Wachtershausers
pyrite-pulled hypothesis led to a series of experiments designed to test the utility
of such reducing power on a number of oxidized organic molecules (Blo chl et al.
1992). In each experiment, FeS was either synthesized by sulfidization of FeSO
4
with H
2
S or procured from chemical suppliers. Following the protocol of Drobner
et al. (1990), 2 mmol FeS and 2 mmol of H
2
S were employed, except in the control
experiments. In the first experiment, Blo chl et al. demonstrated that starting with
FeS and H
2
S in the presence of sodium nitrate (480 mol) and at a pH of 4.0 upon
reaction for three days at 100
C, nitrate was reduced to ammonia at nearly 42%

(200 mol) yield. In control experiments, where H
2
S was absent, the yield of
ammonia dropped to 11%, and in the presence of H
2
S only, no ammonia was de-
tected. In the case of the FeS-alone reaction, the source of the reducing power is not
precisely known, but could have been derived from either iron or sulfur oxidation.
It is interesting to note that 200 mol of NH
3
corresponds to 300 mol H
2
produced, a 15% yield based on the starting amount of H
2
S. In the previous study
of Drobner et al. (1990), wherein nearly identical reaction conditions were used,
the yield of H
2
relative to H
2
S initial was only 1% after 14 days. It is possible that
the presence of nitrate may have actually catalyzed the sluggish pyrite-forming
reaction. Alternatively, the nitrate may have promoted the oxidation of Fe
2+
in
solution, thus generating copious H
2
from water. Note, however, in the Drobner
et al. (1990) experiments the reactions were blanketed with CO
2
, whereas in the
Blo chl et al. (1992) experiments, Argon was used instead. As described later, a CO
2
atmosphere may not have been as inert as Drobner et al. (1990) had anticipated.
Blo chl et al.s second set of experiments explored whether the pyrite-forming
reaction could promote model organic reductions, e.g., the reduction of acetylene
(ethyne), acetaldehyde, 2-mecaptoethanol (2-thio-ethanol), ethylene glycol, and
1,2-dithioethane. In the case of acetylene and acetaldehyde, experiments run for
7, 14, and 21 days revealed reduction to both ethylene and ethane (with maximum
yields of 0.7% and 80 ppm, respectively, from acetylene and 950 ppm and 25 ppm,
respectively, from acetaldehyde). In the case of both thiolated ethane derivatives,
reduction to ethylene was observed with 1% yields.
The substantial yields associated with thioethyl derivatives was interpreted by
Blo chl et al. to reflect an intermediary role of H
2
S. A subsequent test of the

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acetylene reactions with 50 mol of H
2
and no H
2
S revealed only minimal reduc-
tion, again suggesting that H
2
S addition reactions preceded reduction as was pro-
posed in the Wachtershausers theoretical papers (Wachtershauser 1988a, 1990,
1992). However, Blo chl et al. do not report detecting any thioacetaldhyde (an
expected intermediate), nor is the reduction in yield with exchange with H
2
a par-
ticularly convincing result, as the solubility of H
2
in water is considerably less than
that of H
2
S.
Blo chl et al. performed additional experiments with thioglycolic acid, reporting
a 50% conversion to acetic acid. In the absence of H
2
S the yield dropped to 4%,
and in the presence of H
2
S alone the yield was 1%. Blo chl et al. (1992) interpreted
this result to suggest that H
2
S operated in a catalytic fashion by converting the
thioglycolic acid first to thiolated thioacetate, which then lost the 2-thio group via
a pyrite-pulled reaction with FeS to produce thioacetate and pyrite (e.g., similar to
Reaction 6, reaction class C). Thioacetate would rapidly react with water to produce
acetic acid. If these reactions did occur in the manner suggested by Blo chl et al.,
this would provide evidence for the key reactions invoked by Wachtershauser for
the pyrite-pulled metabolism hypothesis. However, the data presented by Blo chl
et al. (1992) do not provide for an unambiguous mechanistic determination of
reaction pathway.
The final experiment described by Blo chl et al. involved the reduction of an alpha
ketoacid (e.g., phenyl pyruvic acid) via the FeS to pyrite reaction to form cinnamic
acid and phenyl propionate. The formation of phenyl propionate demonstrates
the reductive power of the pyrite-forming reaction. Expected intermediates such
as phenyl lactic and phenyl thiolactic acid were not detected. Similar reactions
with oxaloacetic acid yielded fumaric and succinic acid, but no malic or thiomalic
acid.
Two years later, Kaschke et al. (1994) published the results of experiments
exploring the FeS to pyrite reaction for the reduction of cyclohexanone. Rather
than use aqueous solutions, Kaschke et al. preferred dimethyl foramide and, in
one case, methanol. Although such a solvent medium appears hardly relevant
to the early Earth, Kaschke et al. argued that the lower dielectric constant of the
solvent provided a valid comparison with similar reactions run in high temperature
water. FeS was synthesized prior to reaction from solutions of ferrous chloride by
sulfidization with H
2
S.
Kaschke et al. (1994) noted that in various reactions, run for 4 h and at ei-
ther 25 or 100
C, nearly complete conversion of cyclohexanone to various or-

ganic products was observed. In one experiment, the dominant compound was the
dithioketal of cyclohexanone, as would be expected. In all of the experiments, nu-
merous reduced compounds, including thiocyclohexane, dicyclohexane disulfide,
and dicyclohexane trisulfide, were detected. No cyclohexene or cyclohexane was
reported as a product in any of the reactions. Given the reducing power of the pyrite
forming reaction shown by Blo chl et al. (1992), a lack of cyclohexane suggests
that a kinetic barrier exists for dethiolation, possibly owing to the aprotic nature
of the solvent.

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The formation of dicyclohexane trisulfide is particularly interesting. Kaschke
et al. (1994) considered whether the pyrite forming reaction actually provided the
reducing power, i.e., they considered whether the redox reaction

H
2
S H
2
+ S
o
(15)

might also have provided reducing potential. The presence of dicyclohexane trisul-
fide strongly suggests that this is the case. Notably, Kaschke sought, but did not
find, any evidence for pyrite formation in their reaction. It is certainly reasonable
to assume that the aprotic solvent medium used in this study promoted Reaction
15 over the pyrite-forming reaction. These results further suggest that the FeS to
pyrite reaction does not involve direct sulfidization of FeS (e.g., Schoonen et al.
1999), rather, it involves sulfidization of the Fe
2+
in solution.

Pyrite-Pulled Amide Formation

For transition metal sulfidemediated chemistry to have true significance to pre-
biotic chemistry, it is clearly critical to demonstrate that it can drive condensation
reactions as well as reductions. Keller et al. (1994) set out to test this aspect of
Wachtershausers hypothesis (Wachtershauser 1998a, 1990, 1992) by demonstrat-
ing the formation of amide bonds through a reaction that they argued must proceed
through a reactive thioacid intermediate. Note that the thioacid in Wachtershausers
hypothesis serves a similar function as de Duves thioesters (de Duve 1991). What
makes Keller et al.s reaction interesting is that one cannot start with a thioacid;
such compounds are unstable in hot water and will rapidly react to form carboxylic
acids, thus it must be formed as a reaction intermediate.
Keller et al. (1994) performed the following reaction. To an aqueous solu-
tion containing FeS (formed from the prior aqueous sulfidization of FeSO
4
with
Na
2
S), 50 mol thioglycolic acid, 100 mol of a given amino compound, and
2 mmol of H
2
S (in aqueous solution) was added. The reaction solution was blan-
keted with argon and heated to 100
C for two and four days. In the case where

aniline was chosen as the acetyl acceptor, Keller et al. (1994) reported a yield of
2% of the acetanalide. Reactions with other amino compounds, such as tyro-
sine and orthocarboxyl aniline, also yielded the corresponding amides. A small
amount of reaction evidently occurred in the absence of H
2
S, but was signifi-
cantly enhanced when H
2
S was added. Pyrite formation was suggested by the
presence of a floating glistening surface layer [see discussions regarding Heinen
& Lauwers (1996) experiment below]. In the absence of FeS, no reaction was
evident.
Blo chl et al. (1992) had proposed that the reduction of thioglycolic acid to
acetic acid, promoted by the FeS to pyrite reaction, proceeded through a thioacid
intermediate; however, specific evidence for such a pathway was absent. In the
present case, the formation of acetylated amino compounds virtually requires the
presence (albeit transient) of thioacids. Furthermore, Keller et al. (1994) noted that
when acetic acid was substituted for thioglycolic acid, no amide formation was

4
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observed. Thus, Keller et al. (1994) appear to have provided the first experimental
evidence for a pyrite-pulled condensation reaction.

A Fly in the Ointment?

It is perhaps not surprising to note that not all scientists with active interest in
prebiotic (i.e., protometabolic) chemistry were enamored with Wachtershausers
pyrite-pulled metabolism hypothesis (see, for example, de Duve & Miller 1991,
wherein valid criticisms are raised regarding the plausibility of the iron-sulfur
World thesis). In 1995, Keefe et al. designed an experiment to test Wachtershausers
FeS to pyrite reaction for the promotion of amino acid, purine, and/or pyrimi-
dine synthesis. Their initial reaction involved reaction of FeS (1 mmol) in 1 ml
of water with 0.5 mmol ammonia, 1 mmol H
2
S, and 1 mmol CO
2
heated for
122 days at 100
C. Subsequent analysis was performed with high performance

liquid chromatography after reaction with o-phthaldialdehyde (a molecule that
provides a fluorescent tag to amino bearing compounds). Keefe et al. detected no
amino acids, purines, or pyrimidines with a base line confidence level of greater
than 10%4% yield. What Keefe et al. (1995) did discover was that the trace amount
of amino acid contamination present prior to reaction was in fact reduced following
reaction, indicating that the contaminants were destroyed in the course of reaction.
Keefe et al. then chose to explore whether the FeS to pyrite reaction could
convert butyrate to norvaline:

CH
3
(CH
2
)
2
COO

+ CO
2
+ 2FeS + NH
+
+ 2H
2
S
CH
3
(CH
2
)
2
CH(NH
+
)COO

+ 2H
2
O + 2FeS
2
. (16)
This reaction ran for 26 days at 100
C, with the corresponding result that no

norvaline was detected as a product. Keefe et al. (1995) concluded that given the
broad range of steps required to form any of the target compounds, starting with
purely inorganic substrates, such a synthesis was kinetically improbable, and per-
haps impossible, given numerous expected energetic barriers to key intermediate
formation along the reaction path.
The question naturally arises as to whether the experiments of Keefe et al. (1995)
actually refute the hypothesis of Wachtershauser (1988a, 1990, 1992). First, recall
that in Nakajima et al.s (1975) synthesis of phenyl alanine, the final yield was
only 0.3%. Such a low yield was hardly surprising considering the numerous steps
required to form this compound even when starting with the thioester precursor
(see Reactions 914). Given the breadth in reaction branching expected starting
on a path initiated exclusively with CO
2
and NH
+
, it is clear that even if one could
synthesize a given amino acid, this product would exist in extreme dilution with
multitudes of other reduced organic compounds. An extremely complex product
suite will severely reduce detection limits.
A second issue involves the use of a single reaction interval being chosen at
122 days. If organo-synthesis reactions are driven by pyrite-forming reactions,
then the yield of such reactions may be balanced by other reactions that lead to

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their destruction in the hot aqueous solution. Given a closed system, such reactions
would proceed to yield a steady state concentration of organic compounds only so
long as the FeS to pyrite reaction continued. If this reaction ceased to operate within
10 or even 20 days, then after 122 days at 100
C any reactive compounds, such as

amino acids, purines, and pyrimidines, formed would have been destroyed. Clearly,
a more thorough experimental assessment would follow the reactions across over
short, intermediate, and long time intervals.
The lack of synthesis of substantial quantities of norvaline starting with bu-
tyrate provides a more valid test of Wachtershausers hypothesis. Mechanistically,
the first step in such a reaction would require the reaction of butyrate with H
2
S
to form thiobutyrate and H
2
O; Wachtershauser assumes in his hypothesis that an
equilibrium must exist between thioacids and carboxylic acids (Wachtershauser
1988a, 1990). The next step involves the reductive carboxylation of the thioacid
to form 2-oxo pentanoic acid [in essence, the reaction demonstrated by Nakajima
et al. (1975)]. Reductive amination of the alpha-keto acid would then yield norva-
line. Hafenbradl et al. (1995) showed that this later reaction can occur driven by
the reductive power of the FeS to FeS
2
reaction. Therefore, the lack of norvaline
synthesis must reflect the fact that any thio acid-carboxylic acid equilibrium in hot
aqueous solutions is shifted very strongly toward carboxylic acids.

CO
2
Reduction in the System FeS + H
2
S
In their review of the current state of Origins of Life inquires, Chyba & McDonald
(1995) noted that the central claim of Wachtershausers iron-sulfur World hypoth-
esis, i.e., that inorganic carbon could be reduced during pyrite formation, had not
yet been demonstrated as of 1995. Shortly thereafter, however, experiments by
Heinen & Lauwers (1996) appear to have closed this crucial experimental gap.
In a series of reactions using FeS, H
2
S, and water, blanketed by an atmosphere
of N
2
/CO
2
or pure CO
2
, Heinen & Lauwers demonstrated the apparently facile
reduction of CO
2
to form alkane thiols coincident with the formation of pyrite.
Heinen & Lauwers reaction utilized FeS obtained from three different chemical
suppliers and each certified as 99.9% pure. The amount of FeS added to 10 ml of
H
2
O ranged from 10 to 150 mg. H
2
S was formed via reaction of sodium sulfide with
sulfuric acid, yielding a starting pH of 4.6. Depending on the specific reaction, 1
10 ml of the H
2
S-bearing solution was added to the initial 10 ml aqueous solution.
All reactions were run in 60 ml serum bottles at between 75
C and 90
C for
multiple days. Reaction progress was monitored by sampling the headspace with a
gas tight syringe followed by analysis with GC and GC-mass spectrometry (MS).
H
2
and H
2
S were analyzed separately from the volatile organic phases.
The carbon-containing compounds detected in the gas phase included carbon
disulfide, dimethyl disulfide, and nine alkane thiols. The predominant product was
methane thiol, accompanied by a homologous series of alkane thiols detected out to
pentane thiol. No attempt was made to derive an absolute yield of organic sulfur-
containing species. Following the reaction, Heinen & Lauwer (1996) reported
the appearance of a silvery floating layer, which when recovered and analyzed

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with X-ray diffraction was shown to be mainly pyrite with minor amounts of
marcasite. No attempt was made to analyze the aqueous solution for any polar
organic molecules.
A very interesting result was obtained when HCl was substituted for H
2
S.
Heinen & Lauwers (1996) observed that H
2
and alkane thiol production occurred
in the case where H
2
S was derived directly from FeS. They observed, however,
that there was an optimum concentration of HCl, i.e., the yield of thiols reached
a maximum with increasing HCl and then dropped at higher HCl concentrations.
As HCl dissolves FeS, moving Fe
2+
and HS

into solution, evidently the bisulfide
(or H
2
S depending on the pH) thus generated rapidly reacts with the Fe
2+
to form
pyrite and H
2
. These results suggest that the pyrite-forming reaction occurs with
Fe
2+
in solution rather than any direct sulfidization of FeS. Similar conclusions
can be drawn based on the previously discussed results of Kascke et al. (1994).
A detailed discussion of all of the reactions performed in Heinen & Lauwers
(1996) is beyond the scope of this paper. It is sufficient to state that as a result of
these experiments, a clear correlation has been shown between the oxidation of
FeS to form pyrite and the reduction of CO
2
to form organic compounds. This is
as much as Wachtershausers hypothesis predicted; however, Heinen & Lauwers
reactions revealed some details that require some discussion.
The formation of a homologous series of alkane thiols strongly suggest a
surface-catalyzed, Fischer-Tropsch (FT)-type (Fischer 1935) reaction (see
Figure 3). The FT reaction proceeds through the sequential carbonylation of a

Figure 3 A schematic representation of the catalytic, Fischer-Tropsch reduction of
CO on FeS
2
, where iron atoms are black and sulfur atoms are cross-hatched. A sequence
of carbonyl insertion followed by reduction leads to progressive hydrocarbon chain
growth.

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catalyst surface and reduction of the carbonyl by hydrogen. If the probability of
reduction by hydrogen is high, extensive chain growth will not occur. Alterna-
tively, if a carbonyl is near to the partially reduced carbon (Figure 3), there is
a high probability that a carbonyl insertion will occur. Sequential insertions and
reductions lead to alkane chain growth. At low levels of polymerization, relative to
the pool of unreacted carbon and hydrogen, the C1 alkanes will dominate and the
distribution function of higher alkanes will follow a power law (Flory 1936). The
detection of isopropane thiol and sec butane thiol, in addition to n-alkane thiols,
suggests the production of unsaturated hydrocarbons, e.g., propene and butene,
which were subsequently thiolated by reaction with H
2
S.
The formation of n-alkane thiols (and iso-alkane thiols) clearly suggest a FT-
type reaction and, therefore, suggest that carbon monoxide, not CO
2
, was the form
of carbon being reduced to alkanes. The reduction of CO
2
to CO might very well
have occurred in the solution coupled to the pyrite-forming reaction. The site of
the FT reaction, however, would most likely have been on the pyrite surfaces,
accessible to CO and H
2
in the reactor head space by virtue of the fact that the
pyrite grains floated at the top of the solution.
These reactions differ considerably from those postulated to occur in
Wachtershausers iron-sulfur world (Wachtershauser 1988b, 1990, 1992); they are
not pyrite pulled, but may well utilize pyrite as an essential catalyst. The carbonyl
insertion reaction may be one of the most important classes of reaction promoted
by transition metal sulfides when considering their role in protometabolism.

The Synthesis of Activated Acetic Acid by Carbonyl
Insertion on (Ni,Fe)S

Recognizing the significance of the production of methane thiol, as demonstrated
by Heinen & Lauwers (1996), Huber & Wachtershauser (1997) conceived of the
following series of experiments designed to test whether transition metal sulfides
were capable of promoting a carbonyl insertion reaction. They sought to demon-
strate whether various transition metal sulfides could catalyze a reaction wherein
a metal-bound methyl group was converted to a metal-bound acetyl group. In this
regard, it is well known that Group VIII elements generally provide the best cat-
alytic performance for carbonyl insertion reactions (see discussions in Cotton &
Wilkinson 1988).
As an example of their typical protocol, the following reaction was performed
yielding 41% conversion of methane thiol to acetic acid. To a deareated reaction
vessel (120 ml serum vial) 1 mM of FeSO
4
7H
2
O, 1mM of NiSO
4
6H
2
O, and
an 2 ml aqueous solution of Na
2
S (2 mM) was added. Huber & Wachtershauser
anticipated the reaction,

FeSO
4
7H
2
O + NiSO
4
6H
2
O + 2Na
2
S
2(Fe, Ni)S + 2SO
2
+4Na
+
+13H
2
O, (17)

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would proceed and provide them with a mixed-metal monosulfide. Huber &
Wachtershauser did not characterize their precipitates, e.g., through X-ray diffrac-
tion or elemental analysis, although in Drobner et al. (1992), the products of the
same reaction with only ferrous sulfate were reported to be amorphous FeS.
Following the synthesis of the sulfide, the serum vial head space was then
charged with 1.05 bar (4 mM) carbon monoxide. The pH was then raised to 8
by the addition of 150 l of 4N NaOH solution. Oxygen-free water was then added
to bring the solution volume up to 10 ml. At this point, 2.5 ml of methane thiol
(presumably as an aqueous solution of sodium thiomethoxide, although this detail
is omitted in Huber & Wachtershauser 1997) was added, yielding 100 mol of
methane thiol to reaction. Reaction proceeded at 100
C for 7 days. After reaction,

the solution pH was reported to have dropped to 6.5. The final solution was analyzed
via GC, wherein 41 moles of acetic acid was detected.
Subsequent experiments were carried out spanning a pH range from 1.8 up
to 10. The salient point is that Huber & Wachtershauser explored the catalytic
effects of several metal sulfides, including FeS, NiS, and CoS. Using the yield of
acetate as a gauge of the quality of the catalysts, Huber & Wachtershauser reported
that FeS was incapable of promoting carbonyl insertion across the pH range; CoS
only gave a high yield (15% conversion) at pH >7, whereas NiS yielded acetate
across the pH range with relatively high yields (yields >30%) at low pH (<2.5)
and intermediate pH (>7). The particularly surprising result was that the (Ni,Fe)S
catalyst exhibited the highest performance, but over a very restricted pH range
(5.5 < pH < 7.5). The catalytic quality of Ni(OH)
2
, precipitated in the absence
of Na
2
S, was also tested and provided significant yields of acetate (>30%) at
pHs >7.0. In the course of this reaction, it was reported that the Ni(OH)
2
turned
black, suggesting that this phase reacted with sulfur derived from methane thiol to
produce NiS.
The unusual catalytic enhancement observed by the mixed (Ni,Fe)S phase was
interpreted by Huber & Wachtershauser to result from COs high affinity for iron
and the methyl cations affinity for nickel (Figure 4). The attraction of such a
scenario is that it appears to coarsely mimic the stoichiometry of the active center
for acetyl formation in the CoA synthase enzyme complex utilized by methanogens
and acetogens for primary carbon fixation (Gottschalk 1985, Qiu et al. 1994).
The acetyl-CoA synthase enzyme complex has been extensively studied in
a variety of organisms, and in particular in the acetogenic bacterium, Moorella
thermoaceticum [formerly known as Clostridia thermoaceticum (see for example,
Lindahl et al. 1990, Qiu et al. 1994)]. The reaction pathways start with CO
2
and H
2
and lead ultimately to the formation of a transferable acetyl group as diagramed in
Figure 5. The essential feature of the complex is a pair of reaction branches, one
leading to the progressive reduction of CO
2
, ultimately to a transferable methyl
group, whereas a second branch promotes the reduction of CO
2
to CO via a biolog-
ical equivalent of the water-gas-shift-reaction (WGSR). The two reaction branches
join at the point where a methyl group (CH
3
-) is transferred, first to a cobalt center
in a cofactor (cobalamin) and ultimately to a nickel atom in a Ni-X-Fe
4
S
4
cluster;

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Figure 4 A schematic representation of the reaction between methane thiol (CH
3
SH)
and carbon monoxide on a stoichiometric surface of (Ni,Fe)S as proposed by Huber &
Wachtershauser (1997). A methyl group derived from methane thiol is transferred to a
nickel atom. An adjacent iron atom is carbonylated with carbon monoxide. Carbonyl
insertion leads to the formation of the nickel-bound acetyl group. Nucleophilic attack
by either hydoxyl, bisulfide, or methane thiol yields acetic acid, thioacetic acid, or
methyl thioacetate, respectively.

where X maybe sulfur (Qiu et al. 1994). A key step in the reaction scheme involves
the carbonyl insertion reaction that occurs at the Ni-X-Fe
4
S
4
cluster and yields the
transferable acyl group on the nickel atom (Lindahl et al. 1990, Qiu et al. 1994).
It is the Ni-X-Fe stoichiometry in reaction center A (Figure 5) that led Huber
& Wachtershauser (1997) to suggest that perhaps their (Ni,Fe)S phase served, in
a prebiotic sense, as a precursor to acetyl CoA synthase. Note that subsequent
analysis via high-resolution protein X-ray crystallography (2.2 A

resolution) has
led to a reinterpretation of the configuration of the A site as being [Fe
4
S
4
]-X-M
1
-
X-M
2
(where M
1,2
are transition metals and X is likely to be sulfur) (Doukov et al.
2002). Doukov et al. (2002) further concluded that whereas the M
2
site is occupied
by nickel, the M
1
site is occupied by Cu(I) and may be the site of acetyl formation
rather than the nickel center.
Huber & Wachtershauser (1997) reported other intriguing reactions associated
with their (Ni,Fe)S phase. For example, upon addition of 200 mol of aniline
with 100 mole of methane thiol and after reaction over the (Ni,Fe)S phase,
4.7 moles of acetanilide were detected; thus, this catalyst can promote the for-
mation of peptide bonds through acetyl transfer to primary amines. Small amounts
of the thioester methyl thioacetate were also detected when the reactions were run

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.

Figure 5 A schematic diagram depicting the synthesis of acetyl CoA at reaction
center C in the enzyme complex acetyl CoA synthase. In the overall reaction, scheme
CO
2
and H
2
are reduced along one reaction branch to a methyl group. The methyl group
is transferred to a cobalt-containing cofactor, which in turn transfers the methyl group
to the nickel atom in reaction center C. Along a separate reaction branch, CO
2
and H
2
are catalytically reacted to form carbon monoxide, which then carbonylates an iron
atom in the ferredoxin cluster [Fe
4
S
4
], carbonyl insertion at the nickel atom leads to
the formation of the transferable acetyl group for the ultimate synthesis of acetyl CoA.

in the presence of pure NiS at a pH of 1.6. Methyl thioacetate is expected to be
extremely unstable in aqueous solution at 100
C. Huber & Wachtershauser (1997)

confirmed this expectation by showing that 100 mol of methyl thioacetate heated
at 100
C in an aqueous solution (pH = 1.6) hydrolyzes almost entirely in 20 h.

Finally, Huber & Wachtershauser (1997) replicated aspects of the experiment
performed by Heinen & Lauwers (1996) by showing that reactions with both NiS
and (Ni,Fe)S in the presence of excess H
2
S yield acetic acid using carbon monoxide
as a sole carbon source. In the course of this reaction, carbonyl sulfide (COS) and
methane thiol where detected. Clearly, such results at relatively low temperatures
and pressures demonstrated the catalytic potential of transition metal sulfides.

Peptide Synthesis Revisited: Activation of Amino Acids
with CO and (Ni,Fe)S

Recalling that the addition of aniline served as an acyl acceptor leading to the for-
mation of a simple peptide, Huber & Wachtershauser (1998) proceeded to explore
whether their (Ni,Fe)S catalyst in the presence of excess CO and amino acids, e.g.,

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phenylalanine, glycine, or tyrosine, might promote the formation of dipeptides.
The principal problem is that the formation of dipeptides from the condensation of
amino acids is endergonic by 3 Kcal/mol; therefore, any equilibrium between free
amino acids and dipeptides will be strongly shifted toward free amino acids (see
discussions by Shock 1992). The formation of dipeptides must be coupled, there-
fore, with some sufficiently exergonic reaction. The standard method for peptide
synthesis, employed both in bio- and abioprotein synthesis, involves activating
amino acids via acylation (see for example Liu & Orgel 1997). The condensation
of acylated amino acids is thermodynamically very favorable (Abeles et al. 1992).
Reactions (100
C, 14 days) in aqueous solutions containing (Ni,Fe)S, methane

thiol, H
2
S, and various enantiomerically pure (L) amino acids, when blanketed with
carbon monoxide, all yielded the corresponding dipeptides, e.g., glycylglycine. In
one case, tripeptides of tyrosine were detected. Subsequent experiments under
similar conditions but starting with dipeptides yielded the respective amino acids.
Huber & Wachtershauser note that these results indicate that the amount of peptides
detected correspond to a steady state of formation and destruction.
Huber & Wachtershauser also noted that promotion of peptide formation ex-
hibited a strong dependence on pH with optimum yield at pHs between 8 and 9.5;
above or below these pHs minimal peptide formation was observed. Furthermore,
it was also observed that both the phenylalanine and tyrosine dipeptides had un-
dergone extensive epimerization and existed as pure L, D, and mixed L and D
dipeptides.
The formation of dipeptides is obviously important, but surprising. The spon-
taneous condensation of amino acids to form peptides is both thermodynamically
and kinetically inhibited (Shock 1992), and clearly the reaction must be coupled
to another exergonic reaction. The obvious choice would involve coupling the
condensation reaction of amino acid with the hydrolysis of a thioester. Huber &
Wachtershauser tested this by the addition of an enormous excess of methyl thioac-
etate and thioacetate [previously detected by Huber & Wachtershauser (1997) in
similar reactions] in place of CO and observed no acylated amino acids or peptide
formation. Of course, to be most effective, the amino acid must be activated, and
acylation is one route (Liu & Orgel 1997), but this did not appear to be the reaction
operating in Huber & Wachtershausers experiment.
Huber & Wachtershauser (1998) proposed two hypothetical possibilities for
amino acid activation: one route involving reaction with COS, the other an N-
formylated amino acid; the products of either reaction could hypothetically react
with a free amino acid to form dipeptide. Huber & Wachtershauser (1998) con-
cluded that the thermodynamic driving force for amino acid activation appeared
coupled to either the oxidation of CO to COS or reaction of CO with H
2
O to
produce formic acid. It was noted that addition of COS in place of CO and H
2
S
promoted the formation of dipetides; however, only in the presence of (Ni,Fe)S.
Furthermore Huber & Wachtershauser (1998) reported detecting small quantities
of N-formylated amino acids. The precise reaction mechanism remains unknown,
although (Ni,Fe)S appears necessary as a catalyst.

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.

Carbonylated Iron-Sulfur Clusters and the Synthesis
of Alpha-Keto Acids

Nakajima et al. (1975) had previously shown that alpha-keto acids could be
formed via a reductive carboxylation reaction using solvent (THF, methanol,
water)-soluble iron sulfur clusters (Schrauzers complex; see discussions above)
and thioesters. Organo metallic, transition metal sulfide complexes can be easily
formed from monosulfides, e.g., NiS, CoS, and FeS (see Able & Crosse 1967).
Schrauzers complex, for example, is synthesized by reaction of FeS and dipheny-
lacetylene; alternatively, iron pentacarbonyl and sulfur can be substituted for FeS
(Schrauzer et al. 1966). This fact, and given the successful demonstration of
(Ni,Fe)S-catalyzed carbonyl insertion reactions (Huber & Wachtershauser 1997),
suggested another route to the formation of alpha-keto acids. Cody et al. (2000)
explored whether under high-pressure reactions of alkane thiols, carbon monoxide
and FeS might provide a source of alpha-keto acids without an initial abundance
of thioesters.
Experiments were run with pure FeS (synthesized from puratronic-grade iron
and sulfur), aqueous formic acid, and nonane thiol loaded into welded gold cap-
sules. The reaction conditions were 250
C and either 50, 100, or 200 MPa pressure

run for 6 h. After reaction, the product solutions were observed to be strongly col-
ored red. UV-visible light and Raman spectroscopy of the solutions revealed the
presence of carbonylated iron-sulfur species (Cody et al. 2000). The intensity of
absorption increased substantially with increased pressure. Analysis of the prod-
ucts revealed substantial quantities of methyl nonyl sulfide, decanoic acid, dinonyl
disulfide, and dinonyl trisulfide.
The synthesis of decanoic acid is analogous to the acetate-forming reaction
of Huber & Wachtershauser (1997); however, in their experiments, FeS did not
promote acetate synthesis. In the case of the Cody et al. (2000) experiment, it is
possible that the site of reaction was the carbonylated iron-sulfur clusters. Clearly,
considerable CO or CO
2
reduction had also occurred, as evident by the relatively
high yields of the methyl nonyl sulfide. The synthesis of dinonyl trisulfide indicates
the presence of S

in the reaction, for example,
2CH
3
(CH
2
)
8
SH + S
0
CH
3
(CH
2
)
8
S
3
(CH
2
)
8
+ H
2
. (18)
In the case of Kaschke et al. (1994), S

was derived from H
2
S disproportionation. In
the Cody et al. (2000) reaction, however, S
o
was inferred to be derived from the Red-
Ox disproportionation of FeS in the presence of CO, yielding iron pentacarbonyl
via Reaction 19,

FeS + 5CO Fe(CO)
5
+ S
0
. (19)

The source of the solution-phase carbonylated iron-sulfur species was inter-
preted to be derived from the reactions of iron pentacarbonyl with nonane thiol
or a concerted reaction involving CO, nonane thiol, and FeS (e.g., Abel & Crosse

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1967), for example,
2FeS + 6CO + 2nonyl SH Fe
2
(nonyl S)
2
(CO)
6
+ 2S
0
+ H
2
. (20)
Note, however, that both the UV-vis and Raman spectra indicated that there
likely existed many different carbonylated iron-sulfur compounds, conceivably
including also noncarbonylated iron-sulfur clusters similar to Schrauzers complex
(Schrauzer et al. 1966).
Of particular interest to protometabolic chemistry, Cody et al. (2000) identified
traces of pyruvic acid (conclusively identified) and 2-oxo undecanoic acid (inferred
from MS) in the product suite. The pyruvic acid must have formed from methane
thiol derived from CO reduction. Although it is likely that Nakajima et al.s ex-
periment yielded alpha-keto acids via a reductive carboxylation (e.g., Reactions
5 and 11), given the severe conditions of Cody et al.s experiment, it is possible
that the alpha-keto acids formed via double carbonylation followed by hydrolysis.
Such reactions have been reported and typically employ rhodium, palladium, and
cobalt catalysts and high CO pressures (see Tanaka et al. 1985 and discussions in
March 1991). Alpha-keto acid synthesis via a reductive carboxylation mechanism,
however, can not be excluded.

Up the Down Staircase

As discussed earlier, both Wachtershauser (1988a, 1990, 1992) and Russell & Hall
(1997) both favor the RCC (Figure 1) as the primordial carbon-fixation pathway.
Cody et al. (2001) set out to explore citric acid reactions in aqueous solutions at high
pressures and in the presence of transition metal sulfides in order to gain insight
into the viability of the RCC as an archaic carbon-fixation pathway. Citric acid
reactions in high temperature water had been previously studied by Carlsson et al.
(1994). It was shown that citric acid decomposes in hot aqueous media along two
different reaction pathways. In one pathway (, Figure 6) citric acid decomposes
to oxaloacetic acid and acetic acid via a retro aldol reaction (see Figures 1 and 2);
oxaloacetic acid undergoes a beta decarboxylation to form pyruvic acid. Pyruvic
acid can undergo either a reductive decarboxylation to acetic acid, CO
2
, and H
2
,
or a straight decarboxylation, yielding acetaldehyde and CO
2
. A second pathway
( , Figure 6) involves citric acid decomposing via elimination of water followed
by a cascade of decarboxylations, leading first to the dicarboxylic acids itaconic,
mesaconic, citraconic, and citramalic acids, and finally down to methacrylic acid.
Both pathways exhibit strong temperature dependence.
Cody et al. (2001) explored these reactions at elevated pressures (100 Mpa)
and as a function of solution pH. High pressures, as might be found at the depths
of Hadean oceans, had been invoked by Wachtershauser (1992) as favoring au-
totrophic anabolic metabolism. Cody et al. (2001) discovered that the degradative
pathways and were both accelerated by high pressures. Experiments that var-
ied the solution pH revealed a strong pH dependence on both the reaction rates
and branching ratios of paths and , with the fastest reactions occurring along

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Figure 6 The principal degradative pathways of citric acid in hot water. Reaction pathway
mimics the branch point in the RCC, i.e., the retro aldol reaction that leads to the formation
of oxaloacetate and acetate. Oxaloacetic acid undergoes a facile decarboxylation to yield
pyruvic acid and CO
2
. Pyruvic acid undergoes an oxidative decarboxylation yielding acetic
acid, CO
2
, and H
2
. A separate degradation pathway, , involves citric acid eliminating water to
form aconitic acid, which undergoes a decarboxylation to form itaconic acid and CO
2
(note
that itaconic acid exists in equilibrium with two other unsaturated isomers as well as the
hydroxylated dibasic acid, citramalic acid). Itaconic acid decarboxylates to yield methacrylic
acid and CO
2
, which in turn can decarboxylate to form propene. Abiotic reversal of the
pathway does not appear probable. However, under reducing conditions and in the presence
of effective catalysts (such as NiS), a pathway,
0
, exists that can plausibly lead from propene
up to citric acid.

path at moderately acidic conditions, and path being favored over path at
intermediate pH.
Approaching the problem of initiating a carbon fixation pathway via a reverse
synthesis of citric acid, Cody et al. (2001) considered the potential reversibil-
ity of either reaction path or . They also considered several other known

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synthetic routes, but discounted these as not relevant to natural environments. Now,
Wachtershauser (1998) explicitly favored a pathway similar to that of the acetyl
CoA pathway (e.g., Huber & Wachtershauser 1997). Starting with this pathway
requires the RCC initiation point to pass through pyruvate and up to oxaloacetate
(e.g., Figures 1 and 2). The problem is that whereas there is clear evidence that
pyruvate can be formed abiotically (e.g., Nakajima et al. 1975, Cody et al. 2000),
the beta carboxylation of pyruvate to form oxaloacetic acid (e.g., Wachtershausers
postulated Reaction class D, Reaction 7) has not been performed abiotically and
does not appear straightforward.
Cody et al. (2001) focused on whether the citric acid degradation path might
not be reversed and provide a viable geochemical route to citric acid synthesis. The
reaction proposed to do this is the Koch reaction (also known as hydrocarboxyla-
tion). The idea was that if the prebiotic world had the capacity to form simple olefins
such as propene [see discussion of Heinen & Lauwers (1996) experiment], then
in the presence of CO and appropriate catalysts, e.g., transition metal sulfides,
one should be able to form carboxylic acids. Reactions using 1-nonene (Cody
et al. 2001) over NiS in the presence of aqueous formic acid at 250
C and 200
MPa yielded 27.5% conversion to C
10
acids, where carboxylation occurred pre-
dominantly at the interior carbons. This reaction implies that a similar reaction with
propene would yield both butyric and isobutyric acid. Reactions under the same
conditions with the mono-carboxylic acid, methyl acrylic acid (isobutenoic acid),
and the dicarboxylic acid, itaconic acid, yielded the dicarboxylic acid, methyl
succinic acid, and the tricarboxylic acid, tricarballylic (hydroaconitic) acid, re-
spectively (Cody et al. 2001).
These reactions suggest that reversal of pathway may be a viable route to
citric acid. One more reaction, however, is required. The Koch reaction requires
(in this case) an olefinic carbon. Therefore, a demonstration of a partial oxidation
of isobutyric acid to methyl acrylic acid and methyl succinic acid to itaconic (or
citraconic or mesaconic acid) is required to prove viability, e.g., as shown for
isobutyric:
CH
3
CH(CH
3
)COOH CH
2
C(CH
3
)COOH + 2e

+ 2H
+
. (21)
Evidence for such a reaction was observed in the reaction of methyl acrylic acid
to methyl succinic acid, wherein thiocitramalic acid and thioisocitramalic acid were
both formed at near equivalent abundance with methylsuccinic acid (Cody et al.
2001). Both thiocitramalic and thioisocitramalic acids are equivalent in oxidation
state to itaconic acid and are capable of additional hydrocarboxylation reactions.
The identity of the electron acceptor in this reaction has not been determined (Cody
et al. 2001). It is certainly possible that the redox coupling reaction involved the
reduction of NiS to Ni
3
S
2
, although other reactions cannot be excluded. Regardless
of the details, it is clear from these experiments that a protometabolic path from
CO
2
up to citric acid catalyzed by transition metal sulfides is plausible (e.g.,
0
;
Figure 6).

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Special Phases or Catalytic Ubiquity

The theories of Wachtershauser (1988a, 1990) and Rusell & Hall (1997) place
a special emphasis on iron sulfide phases such as pyrrhotite and pyrite. Experi-
mentally, however, Huber & Wachtershauser (1997) demonstrated that it was the
mixed Fe,Ni sulfides that provided superior catalytic performance, at least for
carbonyl insertion reactions. Hall et al. (1998) explicitly favored Fe,Ni sulfides
such as violarite (FeNi
2
S
4
) as potential catalysts. Recently the catalytic qualities
of a broad range of transition metal (Fe, Ni, and Cu bearing) sulfides have been
assayed (Cody et al. 2004) under conditions that optimized reaction probability
and allowed for a phase-by-phase comparison of each minerals promoting activ-
ity for a test reaction (carbonyl insertion of CO to a metal-bound alkoyl group
leading to the formation of a fatty acid). This study revealed that all of the phases
studied (with the exception of covellite, CuS) provided some catalytic function for
carbonyl insertion. Furthermore, all of the mineral phases assayed promoted CO
reduction to metal-bound transferable methyl groups. These results reveal that of
the 11 Fe-, Ni-, and Cu-bearing sulfide phases studied, 10 were capable of promot-
ing the key catalytic reactions promoted by the acetyl CoA synthase complex. It is
clear from these results (Cody et al. 2004) that one need not resort to searching for,
or invoking, a special transition metal sulfide phase for lifes protometabolic roots;
most commonly occurring transition metal sulfides may well have been up to the
task.

CONCLUSIONS

The experiments described in this review constitute the proverbial tip of the
iceberg in terms of the potential reaction space that remains to be explored.
One interesting aspect of the experimental results described above is that if one
were to infer a primordial anabolic pathway arising from such natural chemistry,
the most similar extant pathway would be the acetyl CoA pathway utilized by
chemoautrophic organisms, e.g., methanogens and acetogens (Gottschalk 1986),
not the RCC. Methanogens, such as Methanococcous jannaschii, synthesize all
cellular material autotrophically (e.g., from CO
2
; H
2
; and inorganic sources of
nitrogen, phosphorous, and sulfur). Biosynthetic routes to the synthesis of var-
ious amino acids, sugars, and nucleobases radiate from acetyl CoA through an
incomplete version of the citrate cycle. Notwithstanding the superficial similarity
between the initial anabolic pathways utilized by methanogens and the chemistry
promoted by transition metal sulfides, there remain enormous gaps in the prebiotic
pathway toward Earths first life.
One of the most striking aspects of extant biochemistry is the homochiral nature
of the molecular constituents of biological macromolecules, e.g., proteins, RNA,
DNA, and polysaccharides. The transition metal sulfides are excellent hydrogena-
tion catalysts, thus should promote the reductive amination of alpha-keto acids
to form amino acids (e.g., Nakajima et al. 1975). Presuming that this reaction is

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surface catalyzed, there is the possibility that the surface topology of some transi-
tion metal sulfide may actually favor regioselective hydride transfer to the imine
intermediate perhaps favoring the L enantiomer over the D. Such an effect has
not yet been reported. A robust prebiotic pathway for the synthesis of diastere-
ometrically pure sugars using the reductive surface catalytic properties of pyrite
as predicted by Wachtershauser (1988a) has also not been demonstrated. Fur-
thermore, whether any of the precursor compounds for the initiation of an RNA
world (e.g., activated nucleotides) could form via reactions directly catalyzed by
transition metal sulfides appears doubtful.
In any origins of life scenario, however, catalysts were undoubtedly required
to exploit the chemical potential of the ancient environment. The abundance of
transition metal sulfides in the ancient environment, the demonstrations of their
capacity to catalyze biologically useful organic reactions, and the ubiquity of
transition metal sulfide clusters in key metabolic enzymes suggests that transition
metal sulfides likely supported the development of protometabolism and ultimately
the emergence of life.

ACKNOWLEDGMENTS

I gratefully acknowledge financial support from NASAs Astrobiology and Exo-
biology programs.

The Annual Review of Earth and Planetary Science is online at
http://earth.annualreviews.org

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Annual Review of Earth and Planetary Science
Volume 32, 2004

CONTENTS
GEOMORPHOLOGY: A Sliver Off the Corpus of Science, Luna B.

Leopold 1
EVOLUTION OF THE NORTH AMERICAN CORDILLERA, William
R. Dickinson

13

COMPUTER MODELS OF EARLY LAND PLANT EVOLUTION,
Karl J. Niklas

47

LATE CENOZOIC INCREASE IN ACCUMULATION RATES OF
TERRESTRIAL SEDIMENT: How Might Climate Change Have
Affected Erosion Rates? Peter Molnar

67
RECENT DEVELOPMENTS IN THE STUDY OF OCEAN
TURBULENCE, S.A. Thorpe

91
GLOBAL GLACIAL ISOSTASY AND THE SURFACE OF THE ICE-
AGE EARTH: The ICE-5G (VM2) Model and GRACE, W.R. Peltier

111
BEDROCK RIVERS AND THE GEOMORPHOLOGY OF ACTIVE
OROGENS, Kelin X. Whipple

151
QUANTITATIVE BIOSTRATIGRAPHYACHIEVING FINER
RESOLUTION IN GLOBAL CORRELATION, Peter M. Sadler

187
ROCK TO SEDIMENTSLOPE TO SEA WITH BERATES OF
LANDSCAPE CHANGE, Paul Robert Bierman, Kyle Keedy Nichols

215
RIVER AVULSIONS AND THEIR DEPOSITS, Rudy Slingerland,
Norman D. Smith

257
BIOGENIC MANGANESE OXIDES: Properties and Mechanisms of
Formation, Bradley M. Tebo, John R. Bargar, Brian G. Clement, Gregory
J. Dick, Karen J. Murray, Dorothy Parker, Rebecca Verity, Samuel M.
Webb

287
SPHERULE LAYERSRECORDS OF ANCIENT IMPACTS, Bruce M.
Simonson, Billy P. Glass

329

YUCCA MOUNTAIN: Earth-Science Issues at a Geologic Repository
for High-Level Nuclear Waste, Jane C.S. Long, Rodney C. Ewing

363
INFLUENCE OF THE MENDOCINO TRIPLE JUNCTION ON THE
TECTONICS OF COASTAL CALIFORNIA, Kevin P. Furlong, Susan Y.
Schwartz

403
COMPRESSIONAL STRUCTURES ON MARS, Karl Mueller, Matthew
Golombek

435
MULTISPECTRAL AND HYPERSPECTRAL REMOTE SENSING OF
ALPINE SNOW PROPERTIES, Jeff Dozier, Thomas H. Painter

465
MODERN ANALOGS IN QUATERNARY PALEOECOLOGY: Here
Today, Gone Yesterday, Gone Tomorrow? Stephen T. Jackson, John W.
Williams

495
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SPACE WEATHERING OF ASTEROID SURFACES, Clark R.
Chapman

539
TRANSITION METAL SULFIDES AND THE ORIGINS OF
METABOLISM, George D. Cody

569
GENES, DIVERSITY, AND GEOLOGIC PROCESS ON THE PACIFIC
COAST, David K. Jacobs, Todd A. Haney, Kristina D. Louie

601

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A

C), mildly acidic (pH 5.5 owing

C), rich in bisulfide (HS-), and reduced (H

C for 14 days under

C, nitrate was reduced to ammonia at nearly 42%

C, nearly complete conversion of cyclohexanone to various or-

C for two and four days. In the case where

C. Subsequent analysis was performed with high performance

C, with the corresponding result that no

C any reactive compounds, such as

C for 7 days. After reaction,

C. Huber & Wachtershauser (1997)

C in an aqueous solution (pH = 1.6) hydrolyzes almost entirely in 20 h.

C, 14 days) in aqueous solutions containing (Ni,Fe)S, methane

C and either 50, 100, or 200 MPa pressure

C. Comp. Geosci. 18:899947

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