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This paper is published as part of a Dalton Transactions themed issue on:
Bioorganometallic Chemistry
Guest Editor Charles Riordan
University of Delaware, USA
Published in issue 22, 2009 of Dalton Transactions
Images reproduced with permission of John Peters (left) and Toshikazu Hirao (right)
Papers published in this issue include:
Perspective: Hydrogenase cluster biosynthesis: organometallic chemistry nature's way
Shawn E. McGlynn, David W. Mulder, Eric M. Shepard, Joan B. Broderick and John W. Peters
Dalton Trans., 2009, 4274, DOI: 10.1039/b821432h
Role of aromatic substituents on the antiproliferative effects of diphenyl ferrocenyl butene
compounds
Ouardia Zekri, Elizabeth A. Hillard, Siden Top, Anne Vessières, Pascal Pigeon, Marie-Aude
Plamont, Michel Huché, Sultana Boutamine, Michael J. McGlinchey, Helge Müller-Bunz and
Gérard Jaouen, Dalton Trans., 2009, 4318, DOI: 10.1039/b819812h
Resin-bound models of the [FeFe]-hydrogenase enzyme active site and studies of their reactivity
Kayla N. Green, Jennifer L. Hess, Christine M. Thomas and Marcetta Y. Darensbourg
Dalton Trans., 2009, 4344, DOI: 10.1039/b823152d
Molecular structures of protonated and mercurated derivatives of thimerosal
Wesley Sattler, Kevin Yurkerwich and Gerard Parkin
Dalton Trans., 2009, 4327, DOI: 10.1039/b823467a
Critical aspects of [NiFe]hydrogenase ligand composition
Koji Ichikawa, Takahiro Matsumoto and Seiji Ogo
Dalton Trans., 2009, 4304, DOI: 10.1039/b819395a
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Dalton
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An international journal of inorganic chemistry
www.rsc.org/dalton
Number 22 | 14 June 2009 | Pages 4261–4384
Themed issue: Bioorganometallic chemistry
ISSN 1477-9226
PERSPECTIVE
Peters et al.
Hydrogenase cluster biosynthesis:
organometallic chemistry nature’s way
HOT ARTICLE
Schatzschneider et al.
Sonogashira and “Click” reactions
for the N-terminal and side-chain
functionalization of peptides with CO
releasing molecules
1477-9226(2009)22;1-2
www.rsc.org/dalton | Dalton Transactions
PERSPECTIVE
Hydrogenase cluster biosynthesis: organometallic chemistry nature’s way
Shawn E. McGlynn, David W. Mulder, Eric M. Shepard, Joan B. Broderick and John W. Peters*
Received 1st December 2008, Accepted 11th March 2009
First published as an Advance Article on the web 30th March 2009
DOI: 10.1039/b821432h
It has been over a decade now since it was revealed that the metal containing active sites of
hydrogenases possess carbonyl and cyanide ligands bound to iron. The presence of these ligands in
hydrogenases came as a surprise and to-date these ligands have not been observed to be associated with
any other enzymatic metallocenter. The elucidation of the structures of these unique metalloenzymes
and their associated metal clusters created opportunity for a number of different lines of research. For
synthetic chemists, the structures of hydrogenase active sites have provided attractive targets for
syntheses that advance our understanding of the electronic structure and reactivity of these unique
enzyme active sites. These efforts contribute to the synthesis of first row transition metal catalysts for
hydrogen oxidation and hydrogen production that could have significant impacts on alternative and
renewable energy solutions. Although effective synthetic approaches have been identified to generate
models with a high degree of similarity to these active sites, the details of how these metal clusters are
synthesized biochemically have not been resolved. Since hydrogen metabolism is presumed to be an
early feature in the energetics of life and hydrogen metabolizing organisms can be traced very early in
molecular phylogeny, the metal clusters at hydrogenase active sites are presumed to be among the
earliest of known co-factors. Comparison of mineral based precursors and synthetic cluster analog
chemistry to what is observed in contemporary biological systems may shed light on how
proto-metabolically relevant catalysts first arose prebiotically by the processes of adoption of
pre-existing functionality and ligand assisted catalysis.
Introduction
Hydrogenases occur in three forms [FeFe]-, [NiFe]-, and [Fe]hydrogenase so named to reflect their respective metal cluster
content. The [FeFe]- and [NiFe]-hydrogenases formally catalyze
reversible interconversion between dihydrogen gas and protons
and electrons where the [Fe]-hydrogenases which occur exclusively
Department of Chemistry and Biochemistry and the Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, Montana
59715, USA. E-mail: [email protected]; Fax: +1 406994-7470
in the methanogenic Archaea are given the systematic name,
H2 -forming methylene-H4 MPT dehydrogenase (Hmd), and catalyze the dehydrogenation of methylene-tetrahydromethanopterin
(methlylene-H4 MPT).
Hydrogenases are widely distributed in microorganisms.1,2 Hydrogen oxidation is used to couple the generation of reducing
equivalents to energy yielding reactions to support microbial
growth, while hydrogen production is utilized as a mechanism
to recycle reducing equivalents that accumulate during fermentative metabolism. In nature, hydrogen oxidizing and hydrogen
producing microorganisms frequently occupy the same or adjacent
David Mulder was born in 1983.
He studied chemistry at Calvin
College, receiving his B.S. degree in chemistry in 2005 and
is currently a Ph.D. candidate
in Prof. John Peters research
group at Montana State University where he received an INBRE
research fellowship. His scientific
interests are in iron–sulfur enzyme biochemical and spectroscopic characterization and hydrogenase biosynthesis.
Shawn McGlynn was born in
1983. He received a B.S. degree
in chemistry from Montana State
University in 2005 and is currently a Ph.D. candidate in Prof.
John Peters research group at
Montana State University and an
IGERT fellow in Geobiological
Systems. His research interests
include metal usage in biology,
hydrogenase active site assembly,
and the origins of life.
Shawn E. McGlynn
4274 | Dalton Trans., 2009, 4274–4285
David W. Mulder
This journal is © The Royal Society of Chemistry 2009
environmental niches and interspecies hydrogen transfer is of
paramount importance to the energetics and equilibrium processes
of microbial systems. So large is the role of hydrogen in microbial
ecosystems that interspecies hydrogen transfer and the associated
interdependence of hydrogen oxidizing and hydrogen producing
organisms has been suggested in two independent hypotheses as
having been the driving force of the evolution of the first eukaryotic
cell.3,4
The occurrence of hydrogenases in microbial hosts is an
interesting mystery of molecular evolution since to date [FeFe]hydrogenases have been found to be associated with bacteria and
lower eukaryotes such as algae and protists, but are not found
to be associated with archaea or cyanobacteria. In contrast, the
[NiFe]-hydrogenases, which are found to be associated with a wide
variety of bacteria, are also commonly found in many archaea
and cyanobacteria but are not found to be associated with any
lower eukaryotes. Finally, the occurrence of the [Fe]-hydrogenases
is observed exclusively in some hydrogenotrophic methanogenic
archaea. These discreet differences in the occurrence of the
different classes of hydrogenases is surprising given the common
features of their enzyme active site ligand architectures and
Eric Shepard was born in 1977.
He received a B.S. degree in
chemistry from Rocky Mountain
College. He studied copper and
TPQ-containing amine oxidases
under Dr David M. Dooley at
Montana State University, where
he was supported by a NSF
IGERT fellowship on complex
biological systems. He received
his Ph.D. degree in biochemistry
from MSU and is currently a
postdoctoral research associate
Eric M. Shepard
under Dr. Joan B. Broderick. His
research interests are metal cluster assembly in the [FeFe] hydrogenase system and [Fe–S] cluster spectroscopy and reactivity.
Joan Broderick was born in 1965.
She received a B.S. from Washington State University and a
Ph.D. from Northwestern University. She was an American Cancer Society postdoctoral fellow at
MIT before joining the faculty
at Amherst College as Assistant
Professor in 1993. She moved
to Michigan State University in
1998 and to Montana State University in 2005, where she is
currently Professor of Chemistry
and Biochemistry. Her research
Joan B. Broderick
interests are in mechanistic bioinorganic chemistry, with a particular
focus on enzymes utilizing iron–sulfur clusters to catalyze radical
reactions.
This journal is © The Royal Society of Chemistry 2009
indicate that these enzymes have evolved to common ligand
architectures and reactivity by convergent evolution.
Interest in hydrogen metabolism and hydrogenases has grown
exponentially over the past decade in part due to the growing
interest in alternative and renewable energy solutions. Hydrogen
production by microorganisms or the use of hydrogenase enzymes
or mimetic compounds as hydrogen oxidation or hydrogen
production catalysts has tremendous promise; however there
are significant barriers to surmount. Overcoming these barriers
will require an intimate understanding of the biosynthesis and
maturation of hydrogenase enzymes as well as their structure and
reactivity.
As thoughts concerning hydrogenase structure, function, and
reactivity continue to evolve, the relationships between the features
of hydrogenase active sites and those of other complex iron-sulfur
enzymes and iron-sulfur mineral structures and reactivity have
also been gaining interest. Given that complex iron-sulfur enzymes
are involved with reactions such as reversible hydrogen oxidation,
nitrogen fixation, and reversible carbon monoxide oxidation—
reactions that would have been important in the prebiotic earth
and necessary for the first stages of the emergence of life—it is
rational to think about these enzymes as having an ancient origin.
It is possible that the unique metal-containing prosthetic groups at
the active sites of these enzymes may represent the oldest of known
co-factors with what is observed today existing as highly evolved
versions of modified iron-sulfur mineral catalysts. Investigations
of hydrogenase structure, function, and biosynthesis may in fact
provide some keys as to the origin of life and perhaps even
contribute to the development of signatures for life beyond Earth.
Given the potential importance of hydrogenases at the origin of
life and the potential for hydrogenase based catalysis for renewable
energy and sustaining life on Earth, hydrogenase research is an
ideal combination of basic and applied research.
Active site metal cluster structure
In 1995, the first structure of a [NiFe]-hydrogenase was determined
by X-ray diffraction methods from the sulfate reducing bacteria
(SRB) Desulfovibrio gigas5 and with the structure came some
John Peters was born in 1965. He
received a B.S. in Microbiology
from the University of Oklahoma
and a Ph.D. from Virginia Tech.
He was a NIH postdoctoral fellow at Cal Tech before joining
Utah Sate University as Assistant Professor in 1997 where he
was the recipient of the Camille
Dreyfus Teacher-Scholar Award.
In 2002, he joined Montana State
University and is currently Professor of Chemistry and Biochemistry and Director of the NASA
John W. Peters
funded Astrobiology Biogeocatalysis Research Center and the MSU
Thermal Biology Institute. His research interests are in complex
iron-sulfur enzymes, specifically hydrogenase and nitrogenase structure, function and biosynthesis.
Dalton Trans., 2009, 4274–4285 | 4275
surprises (Fig. 1a). From this work, it was revealed that the
active site possessed an Fe atom that was previously missed by
biochemical and spectroscopic work. Even more intriguing was
that the Fe was coordinated by a number of non-protein ligands
that could not be resolved by X-ray crystallography. The nonprotein ligands were determined to be cyanide and carbonyl
ligands by Fourier transform infrared (FTIR) spectroscopy and
chemical analysis, which resolved long standing issues concerning
the interpretation of carbon monoxide inhibited states of the
hydrogenase.6 Through this work, the active site of the D. gigas
[NiFe]-hydrogenase was found to exist as a heterometallic cluster
with a Ni ion coordinated by four cysteine thiolates to an Fe ion by
two bridging thiolates. Additional coordination to Fe is supplied
by three diatomic ligands (two cyanide and one carbonyl) and a
bridging species for which its composition has been proposed to be
a m-oxo, sulfido, hydroxo, peroxide, or sulfoxide group7–9 (Fig. 1b).
Structural characterization of the [FeFe]-hydrogenase came
later from Clostridium pasteurianum (CpI) and Desulfovibrio
desulfuricans10,11 (Fig. 1c) and revealed a similar iron coordination environment yet a somewhat different overall active site
composition. Common to these hydrogenases are the presence
of several accessory iron–sulfur clusters, which presumably function to shuttle electrons to and/or from external physiological
electron donors and/or acceptors. For the [FeFe]-hydrogenase
from Clostridium pasteurianum the apparent bifurcated pathway
with two separate accessory clusters approaching the surface at
different positions may suggest that the enzyme can accommodate
alternative electron donors and/or acceptors. The six iron catalytic
“H-cluster” active site (Fig. 1d) is comprised of a [4Fe-4S] cubane
bridged via a single cysteinyl thiolate to a 2Fe subcluster in
which both irons are coordinated by a CO and CN- ligand,
and bridged by a m-CO ligand and a non-protein dithiolate of
still unknown composition having been proposed to exist as
propane dithiolate, dithiomethylamine12 , or dithiomethylether.13
Knowledge as to the stereochemical position of the CO and CNligands on the active site was based on CO and CN- ability for
hydrogen bonding.11 Subsequent FTIR studies revealed that these
enzymes are reversibly inhibited by CO12,14 and the structure of the
CO-inhibited enzyme was determined via X-ray crystallography
from crystals of the C. pasteurianum enzyme grown in the presence
of CO.14 The structure revealed reversible hydrogen oxidation
likely occurs at a ligand-exchangeable site at the distal iron atom
of the 2Fe subcluster in relation to the [4Fe-4S] cluster. In the
presumed oxidized state of the C. pasteurianum enzyme, a water
molecule occupies the ligand-exchangeable site. However, this
water molecule is not present in the D. desulfuricans [FeFe]hydrogenase structure as it is believed to represent a more reduced
state of the active site.
The [Fe]-hydrogenases were first shown to contain iron ligated by CO ligands in 200415 and very recently, the structural
description of the [Fe]-hydrogenase was revealed with bound cofactor allowing for the first time side by side comparison of
these unique active sites.16 The [Fe]-hydrogenase active site exists
with iron coordinated by two CO ligands, a cysteinyl sulfur, the
Fig. 1 Ribbon/space filling diagram of the (A) [NiFe]-hydrogenase from D. gigas (PDB code 1YQ98 ). (B) [FeFe]-hydrogenase from C. pasteurianum
(PDB code 3C8Y13 ). For both structures, the protein domains are represented with different colors. For D. gigas, a small and a large subunit exist, for which
they can be divided into two and five separate domains, respectively, according to protein folds. Atomic models of the [NiFe]- and [FeFe]-hydrogenase
active site metal clusters are shown for each (B and D), respectively, (Fe: dark red, S: orange, O: red, N: blue, C: dark grey, unknown atom of dithiolate
ligand: magenta). For the D. gigas model, a bridging peroxide ligand is shown and represents the oxidized “unready” state of the active site. The
C. pasteurianum active site depicted is presumed to represent the oxidized form as a water molecule is present at the ligand-exchangeable site of the distal
Fe atom. All ribbon diagrams and atomic models were generated in PyMOL.103
4276 | Dalton Trans., 2009, 4274–4285
This journal is © The Royal Society of Chemistry 2009
nitrogen of a 2-pyridinol compound, and a ligand of unknown
composition.
Collectively, these enzyme active sites and features presumably
allow for the stabilization of reduced iron in a form capable of
catalyzing the hydrogenase reaction, and in the case of the [Fe]hydrogenase center, the dehydrogenation of methylene-H4 MPT.
The nature of the evolutionary and biochemical origins of these
complexes, as well as the electronic and structural basis for their
reactivity, poses an array of intriguing questions, which are being
addressed by research groups from a diverse range of backgrounds
and expertise.
Active site cluster mimics
The syntheses of hydrogenase active site analogs began soon
after the first structural report of the [NiFe]-hydrogenase when
workers were motivated by the prospects of creating biomimetic
catalysts and gaining further insight into the reactivity of the
hydrogenase active site clusters. Significant progress has been
made in generating active site cluster mimics for both [NiFe]- and
[FeFe]-hydrogenases and this work has been the topic of recent
reviews.17–20 A more topical summary of the progress in the field is
described here.
The development of synthetic techniques suitable for the
formation of heterobimetallic sulfur bridged CO and CN- ligated
clusters posed unique challenges to the field of organometallic
chemistry, and in the case of the [NiFe] center, the events leading to
the generation of sulfur coordinated Ni species ligated in bidentate
fashion to iron coordinated by CO and CN- were major highlights.
Within a year of the first structural description of the [NiFe]hydrogenase, Darensbourg and co-workers21 reported a heterobimetallic structural analogue (Fig. 2a) obtained by the reaction
of a sulfur coordinated Ni2+ complex with an iron carbonyl.
The resultant structure consisted of a nickel moiety bound to
a Fe(CO)4 fragment via a single bridging sulfur. In 1997, a [NiFe]hydrogenases active site model compound in which nickel was
linked to iron by two sulfur atoms (Fig. 2b) was reported22
from the reaction of an Fe(CO)2 (NO)2 with a nickel complex
to give the formation of a heterometallic [NiFe] complex with
Fe ligated by NO. The formation of mimics with heterometallic
[NiFe] complexes coordinated to CO ligated Fe came later with
the complex synthesized by the reaction of an iron thiolate with
NiCl2 (dppe) under an atmosphere of CO23 (Fig. 2c). In 2002, the
formation of an analog with Ni ligated solely by thiolates was
achieved24 (Fig. 2d), marking a significant advance from other
syntheses, which utilized phosphines as soft donor ligands.23,25
An additional advancement in bimetallic heteroatomic [NiFe]
analogue synthesis came in 2005; when it was realized that reaction
of the pre-formed iron carbonyl [Fe(CO)3 (CN)2 Br]- with a nickel
complex gave a fully sulfur coordinated Ni moiety bridged to
iron ligated by two CO and two CN- molecules26 (Fig. 2e). This
development represented the culmination of many years of work
by a diverse group of chemists to develop the reaction specificity
to yield organometallic compounds with three main qualities
requisite for [NiFe]-hydrogenase active site resemblance: sulfur
coordinated Ni bound via two sulfur thiolates to an iron center
ligated by both CO and CN- . Synthetic efforts to mimic the [NiFe]
site continue to advance the understanding of H2 activation by the
active site, with a recent report suggesting that coordination of
This journal is © The Royal Society of Chemistry 2009
Fig. 2 Selected synthetic structures mimicking the active sites of the (left)
[NiFe]-hydrogenase active site and (right) [FeFe]-hydrogenase active site
(discussed in text).
hydrogen by the [NiFe] center stems from an ability of Ni in the
active site to shift from square pyramidal to octahedral geometry,
this being based upon observations of various [NiFe] complexes.27
In the midst of efforts to develop [NiFe] active site models,
structural descriptions of the [FeFe]-hydrogenases in 1998 and
199910,11 catalyzed the development of a parallel vein of research
aimed at mimicking the related but distinct different active site
structures. Similar challenges were faced in the case of [FeFe]hydrogenase active site analog synthesis as in the case of the
[NiFe]-hydrogenases but with some variations. Beginning work
was directed at the synthesis of the 2Fe-subcluster of the H-cluster.
The previous synthesis and characterization of various diiron carbonyl complexes, dating back to 192828,29 (Fig. 2f)
and furthered in the 1980’s, provided a starting point for the
synthesis of H-cluster mimics and thus allowed the community to hit the ground running. These syntheses demonstrated
the formation of the di-ironhexacarbonyldisulfide compound
[Fe2 (SCH2 CH2 CH2 S)(CO)6 ] present with a propane dithiolate30–32
starting from either Fe3 (CO)12 or (m-S2 )Fe2 (CO)6 (Fig. 2g left).
Following structural elucidation of the [FeFe]-hydrogenase, two
of the CO ligands on this compound were substituted with
CN- by three different groups33–35 via reaction of Fe2 (pdt)(CO)6
(pdt = propyl dithiolate) with two equivalents of CN- (Fig. 2g
right) to give a remarkable analog of the H-cluster 2Fe-subcluster
exhibiting a short Fe-Fe distance (2.6 Å), and the presence of
both CO and CN- ligands. The parent compound for this reaction
was again a remnant of non-intentional hydrogenase active site
mimicry carried out some thirty years prior to the knowledge of
Dalton Trans., 2009, 4274–4285 | 4277
the active site structure; King showed in 196136,37 the development
of chemistry suitable for the thiolation of iron carbonyls by
reaction with alkyl sulfides, allowing m-sulfur-iron bond formation
of di-iron centers. Subsequent work on the di-iron alkyl-sulfide
ligated compounds resulted in variations in which the dithiolate
linkage was varied with central atomic positions being occupied
by ether38,39 and amine40 (Fig. 2h) functional groups, as opposed to
the methylene group at this position in the initial propane dithiol
ligated compounds. These variations were elicited by the inability
to assign the central atom of the bridging dithiolate based on
crystallographic data since the likely possibilities (CH2 , O, NH,
NH2 + ) are isoelectronic.
A further challenge presented in the development of structural
analogs included adding the presence of a m-CO ligand. This goal
was first met by Pickett and co-workers41 with the formation
of a mixed valent Fe1+ Fe2+ compound with a m-CO ligand,
although this compound proved to be very unstable. Later work
by two groups gave stable m-CO containing di-iron compounds42,43
(Fig. 2i) and in addition to these CO bridged compounds, a model
with a bridging hydride capable of binding and activating H2 at
an Fe2+ site has been reported.44,45 However, these compounds
did not have CN- ligands and instead were coordinated by bulky
IMES, PMe3 , and dppv ligands, respectively, which presumably
functioned to stabilize the unique geometry that the m-CO ligand
demands. A major advancement in the synthesis of H-cluster
mimics came in 2005 with the synthesis of the complete 6Fe
framework of the H-cluster46 (Fig. 2j). The bridging of a di-iron
carbonyl subcluster to a [4Fe-4S] cubane was accomplished by
first capping Fe3 (CO)12 by reaction with a three sulfur containing
thioester with the subsequent product being reacted with a thiolate
coordinated [4Fe-4S] cubane. This general approach allows significant flexibility for the formation of additional analogue targets as
well as providing a means to probing cluster characteristics as has
been recently demonstrated for the investigation of CO binding at
a 2Fe center.47
Mimetic synthesis of hydrogenase active sites metal clusters
resulted in the development of a suite of chemical approaches.
From a synthetic perspective, the starting materials for a number
of these compounds are simple iron carbonyl complexes such as
Fe(CO)5 , Fe2 (CO)9 , and Fe3 (CO)12 , to which sulfur in the form of
sulfide or thiolates and CN- from compounds such as Et4 NCN,
and a variety of nickel complexes may be added. In terms of
catalysts for hydrogen energy technologies, platinum chemistry
is very effective at catalyzing reversible hydrogen oxidation at
little or no overpotential, however, with the limited availability
of platinum there is a real need for examining non-noble metal
based solutions to accomplishing this chemistry. Lessons learned
in synthetic efforts are thus valuable for the continued pursuit
of hydrogenase active site mimics that may well yet prove to be
catalytic solutions for the accumulation of H2 as a fuel currency,
as well as providing a vantage point from which to consider the
biochemical events that led to the formation of these interesting
catalytic co-factors.
Biosynthesis and maturation
Biological utilization of metal-sulfide protein bound co-factors,
which are ubiquitous in living systems, stems from the inherent
electronic and reactive properties of both iron and sulfur.48
4278 | Dalton Trans., 2009, 4274–4285
The utilization and assembly of these co-factors necessitates the
operation of a complex and specialized set of proteins to allow
for the coordinated and regulated delivery of atoms in a way that
avoids the inherent toxicity of free aqueous metal ions.49–52 The
formation of the hydrogenases provides additional biosynthetic
challenges and has long puzzled biochemists as to the molecular
mechanisms responsible for the harnessing of the unique ligand
set of the active sites. The collective features of the hydrogenase
co-factors require the operation of a specialized set of proteins to
biosynthesize the active site cluster. Given the similarity in overall
composition between the hydrogenase active sites, it is interesting
if not surprising that the generation of the hydrogenase active sites
appears to occur by non-redundant chemistry, with distinct sets
of maturation proteins being required for active site synthesis of
each of the hydrogenases. A summary of the proposed activities for
gene products involved in [NiFe]- and [FeFe]-hydrogenase active
site cluster biosynthetic pathways is provided in (Fig. 3) and is
discussed further below.
[NiFe]-hydrogenase active site synthesis has been studied in a
number of organisms including Ralstonia eutropha, Bradyrhizobium japonicum, Rhizobium leguminosarum, Azotobacter species,
and E. coli and proceeds by the action of at least six accessory
proteins, with seven being required in cases in which proteolytic
processing is required at the final step of maturation.53 The
proposed roles of individual proteins in the maturation process
for E. coli hydrogenase 3 is summarized in (Fig. 4A) and begins
with the generation of cyanide from carbamoylphosphate by
the action of the two proteins HypF and HypE. In a two
step process that requires two ATP hydrolyzing events, HypF
transfers the carboxamide group of carbamoylphosphate to HypE
as thiocarboxyamide where dehydration occurs to yield an enzyme
bound thiocyanate, which can later be donated to iron.54–57
Subsequent interaction of thiocyanate bound HypE with the
HypD/HypC complex results in the donation of the CN- ligand
to an iron bound to HypD/HypC.58 The CN- ligated iron bound
by the HypD/HypC complex is then transferred by HypC to
the large subunit of the hydrogenase enzyme.54 Still in question
is the metabolic source of the CO ligand and at which stage
it is incorporated. Carbamoylphosphate has been ruled out as
the source of CO by labelling studies59,60 and the utilization of a
pathway involving acetate has been suggested.61 With CO and CNcoordinated Fe now present on the large subunit, Ni is inserted
by the actions of the Ni binding GTPase HypB62,63 and the Ni
binding HypA.64 The final step in [NiFe]-hydrogenase maturation
involves proteolytic cleavage of a C-terminal extension, if present.
[FeFe]-hydrogenase active site biosynthesis shares many of the
chemical challenges as just outlined for the [NiFe]-hydrogenase
site, but from what is known thus far appears to require fewer
gene products perhaps due to the presence of only a single
type of metal ion in the active site1 . Indeed, [FeFe]-hydrogenase
maturation was found to be dependent on three gene products
(HydE, HydF and HydG) based on analysis of deletion mutants
of Chlamydomonas reinhardtii in which the gene products HydEF
and HydG were identified to be required for the accumulation of
active hydrogenase.65 Subsequent analysis revealed that the gene
products HydE, HydF, and HydG are conserved in organisms
harbouring an active [FeFe]-hydrogenase, with HydEF existing as
a fusion product in C. reinhardtii. Based on sequence comparison,
the two proteins HydE and HydG were predicted to belong to the
This journal is © The Royal Society of Chemistry 2009
Fig. 3 Overview of genes involved in the biosynthesis of the [NiFe]-hydrogenase and [FeFe]-hydrogenase active site and their proposed functions.
radical S-adenosylmethionine (AdoMet) family of enzymes, and
HydF was predicted to bind an iron-sulfur cluster in addition
to having the ability to hydrolyse nucleotide tri-phosphates.65
Following this discovery, an E. coli based heterologous expression
allowing the co-expression of the [FeFe]-hydrogenase structural
protein with the three accessory proteins was developed and
shown to be sufficient for heterologous maturation, paving the
way for future biochemical studies. In subsequent work, the gene
based predictions for the enzymatic activities of these enzymes
was corroborated by analysis of anaerobically reconstituted Hyd
accessory protein homologues from the organism T. maritima.66, 67
The AdoMet class of enzymes comprises a large family of ironsulfur cluster containing enzymes capable of catalyzing a wide
range of free radical initiated reactions including carbon-carbon
bond formation, isomerisations, and sulfur insertions68 ; thus a
number of chemistries may be envisaged as taking part in H-cluster
biosynthesis. Knowledge of the enzyme families to which the
[FeFe]-hydrogenase assembly factors belong to led to a hypothesis
based off pre-established reactivity of AdoMet enzymes in which
the activities of HydE and HydG, respectively, are proposed to
act to generate the CO and CN- ligands, as well as the bridging
dithiolate ligand.69 The three maturation proteins are presumed to
be responsible in total for the generation of the ligand set of the
[FeFe] subcluster with the possibility that CO could be generated
through the action of carbon monoxide dehydrogenase excluded as
[FeFe]-hydrogenases are found in organisms which do not contain
this enzyme. In our hypothesis for [FeFe]-hydrogenase maturation,
the three gene products HydE, HydF, and HydG are thought to
be directed solely at the synthesis of the 2Fe subunit of the [FeFe]
active site, as the [4Fe-4S] moiety presumably would not require the
action of a specific set of assembly proteins but instead be formed
via the action of the endogenous host iron-sulfur cluster assembly
machinery. HydE or HydG is envisioned as acting to generate
the bridging dithiolate linkage in a mechanism analogous to that
of the radical-AdoMet protein LipA, which catalyzes carbon–
This journal is © The Royal Society of Chemistry 2009
sulfur bond formation; such C–S bond formation, occurring at
a bridging sulfide of an iron–sulfur cluster, would be expected
to shift the reactivity from the sulfur atoms to the irons of the
cluster. Following alkylation of the sulfides, a radical-initiated
amino acid decomposition of glycine is proposed to occur by the
action of HydE or HydG, resulting in the formation of CO and
CN- . The first step of this latter proposed reaction is analogous to
the chemistry catalyzed by the radical-AdoMet enzyme pyruvate
formate-lyase activating enzyme, lysine amino mutase, or ThiH
involved in thiamine biosynthesis which involves the generation of
an amino acid radical intermediate. These ligand-forming events
are hypothesized to result in assembly of the 2Fe subcluster of
the H-cluster on one of the accessory proteins. At this stage
the role of GTP binding and hydrolysis by HydF is unknown,
however, our results concerning in vitro activation of the [FeFe]hydrogenase would suggest that these activities are not directed at
cluster insertion as originally proposed.
Definitive knowledge as to the roles of the accessory proteins
in active site formation has been advancing rapidly, due in large
part to development of an in vitro system for the activation of the
structural hydrogenase protein.70 This in vitro system has led to the
recent identification of HydF as a scaffold or carrier protein which
acts at the terminal step of H-cluster biosynthesis71 , presumably
delivering the 2Fe sub-cluster to the immature structural protein
(Fig. 4B). Currently unresolved issues relate to the enzymatic roles
and substrates of the two AdoMet proteins HydE and HydG, the
precise nature of the cluster precursor bound by HydF prior to
delivery, and the role of GTP hydrolysis by HydF in the maturation
process.
Very little is known regarding the biosynthetic route for the case
of the [Fe]-hydrogenases. As is the case between the [NiFe]- and
[FeFe]-hydrogenases, however, phylogenetic evidence indicates
that the structural protein is evolutionarily isolated and stems
from a separate lineage, suggesting a separate route for the
generation of CO ligands at the iron center. One possible and
Dalton Trans., 2009, 4274–4285 | 4279
Fig. 4 Overview of the metal cluster assembly processes for the (A) [NiFe]- and (B) [FeFe]-hydrogenases. The [NiFe]-hydrogenase maturation scheme
is based on hydrogenase-3 in E. coli and the [FeFe]-hydrogenase maturation model is thought to be general to organisms containing this enzyme.
(A) CN- is generated by the action of HypE and HypF utilizing carbamoylphosphate and ATP forming thiocyanate bound by HypE. The CN- group on
HypE is transferred to the iron coordinating HypD/HypC complex, which is then transferred to the large subunit of the structural hydrogenase protein.
The source of CO is unknown. HypA and HypB act to deliver nickel, and in most cases a C-terminal peptide of the hydrogenase is endopeptidically
cleaved completing the maturation process. (B) The two SAM radical enzymes HydE and HydG are proposed to impose non-protein cluster ligands on
an existing [2Fe-2S] rhomb in two separate steps: (i) the formation of the dithiolate linkage and (ii) CO and CN- ligands, possibly from an amino acid
precursor on the scaffold protein HydF. HydF subsequently delivers the ligand modified 2Fe unit to the immature structural protein, which is presumed
to exist with the presence of a preformed [4Fe-4S] cluster, accomplishing activation.
4280 | Dalton Trans., 2009, 4274–4285
This journal is © The Royal Society of Chemistry 2009
intriguing correlation does exist however, as the gene encoding
a radical-AdoMet protein has been observed to cluster with
the gene encoding the [Fe]-hydrogenase structural protein in a
number of cases.72 Given this observation, it is interesting to
speculate about the commonality of radical-AdoMet chemistry
in the generation of essential CO ligands for both the [Fe]and the [FeFe]- hydrogenases and that the CO of both these
hydrogenases may be formed through a radical initiated amino
acid decomposition event.
Despite the distinct differences in the maturation systems for
the [NiFe]-, [FeFe]-, and [Fe]-hydrogenases, similar chemical and
catalytic functionality can be found. In the case of the [NiFe]and [FeFe]-hydrogenases, a cluster precursor has been proposed
to be assembled on a scaffold protein prior to insertion into
the immature hydrogenase structural protein, and in both cases
GTPase activity has been implicated in having a role in the
maturation process. These two hydrogenase maturation systems
differ however in their respective routes to the generation of the
diatomic ligands CO and CN- . Insofar as organisms harbouring
hydrogenases are able to occupy niches unavailable to organisms
without these enzymes, the functional, catalytic, and maturation
pathway similarities between hydrogenases attests to the ability of
evolution to produce functionally similar yet distinct solutions to
environmental problems. This points to an apparent optimum of
hydrogenase catalytic activity being associated with CO and CNligated iron, presumably as a direct consequence of the ability of
these ligands to stabilize metals in low oxidation states. In short,
three distinct hydrogenase enzymatic systems have convergently
evolved to contain, in whole or in part, iron coordinated by small
inorganic p acid ligands (CO and/or CN- ), suggesting that the low
valent iron stabilized by such p acids has been a central driving
force in hydrogenase evolution.
Prebiotic mineral clusters, ligand accelerated catalysis and
ancient enzymes
Hydrogen occupies a central role as a mobile electron carrier in
biological systems. Given the wide diversity of organisms that
utilize hydrogen oxidation/reduction in metabolic processes, as
well as the potential to derive high concentrations of hydrogen
geochemically via processes such as serpentinization73,74 and the
high concentration of hydrogen present on the early earth75 , it is
reasonable to suppose that hydrogen as a mobile electron carrier
and potent reductant has been coupled to metabolic processes
from their inception.2,74,76 The wide distribution of hydrogenases
in organisms and the presence of hydrogen on the early earth thus
make the consideration of hydrogenases and the events which led
to the formation of hydrogen oxidation and reduction catalysts
especially relevant to the consideration of early energetics and the
origin of life.
In addition to reversible hydrogen oxidation, small molecule
interconversions and cycling of the major atomic constituents
of life play a critical role in contemporary living systems and
must have also participated in the processes accompanying the
transitions from an abiotic to a biotic world. Given that processes
including nitrogen fixation, reversible carbon monoxide oxidation,
and hydrogenase activity occur biologically via the utilization
of metal clusters, it is possible that these clusters in biological
systems are signatures and even reflections of ancient metabolic
This journal is © The Royal Society of Chemistry 2009
and prebiotic processes in which transition metal based catalysts
occupied salient roles. Metal sulfides and metallic transition metals
have been shown to function as catalysts for biologically relevant
reactions and these catalytic activities may have been requisite
for the accumulation of molecules later incorporated into living
systems. Activities associated with metallic, metal sulfide, and
metal oxide based catalysts include the reduction of N2 to NH3 77–79 ,
the synthesis of pyruvate80 and amino acids, and peptides.81,82
These activities strongly suggest that transition metals, which
feature so prominently in contemporary biology, were relevant
to chemistry associated with the development of life.
As well as occupying an important role in individual reactions,
a further important role of both prebiotic and contemporary
catalysts is that of the reaction network connector. In the sense
that catalysts bound the metabolic capacity and flexibility of
an organism, the formation of such catalysts prebiotically may
have been a major barrier in the origin of life. Catalysts in
metabolic networks function to kinetically channel products and
substrates at time scales allowing for sufficient interconnectivity
to result in the ability to couple energy yielding reactions to those
that are utilized by organisms to maintain function. With these
activities in mind the importance of catalysis in the origins of
biological complexity can perhaps not be overstated. Taking into
account the aforementioned chemistry underlying the creation of
hydrogenase active sites both biologically and synthetically results
in a unique perspective from which to consider the origins of
prebiotic catalysts. A greater understanding of (i) the extent to
which minerals present on the early Earth may have harboured preexisting catalytic functionality, (ii) the ability of metal clusters to
form and the role of this in the formation of biologically accessible
molecules, and (iii) the contribution of ligand modifying events to
the emergence of catalytic functionality, may together yield insight
as to the nature or the original catalysts and catalytic events which
led to the emergence of life.
The present day biological utilization of the metal sulfide cofactors observed in hydrogenases and other enzymes to accomplish
efficient reactivity hints at the possibility that these co-factors
themselves may have been derived from geologically related
mineral precursors and thus have been adopted, modified, and
refined through evolutionary time to become the extant clusters
observed today existing as products of geomimicry. Various ironsulfur minerals exhibit structures similar to those seen in biology,
and attention has been drawn to the resemblance of biological
clusters such as that observed in the enzyme CODH and that of
the mineral greigite83 , as well as a similarity in geometry between
the mineral mackinawite and the di-iron center of the [FeFe]hydrogenase (Fig. 5a).84 These “ready made” clusters have been
proposed to have provided a structural basis for the catalytic events
required in the origin of life84 , itself being a product at least in part
of inherent geological functionalities.
Along with the similarities existing between mineral and enzyme
active site structure, an emerging body of work has contributed
to the understanding of how ligand modifications, including
carbonylation, can occur in prebiotic conditions. Iron carbonyl
complexes, which as described above form the reactive starting
material of many active site analogs, have been synthesized
from ferrous iron upon the addition of CO2 85 , CO and ethane
thiol86 , and as well as from aqueous preparations in a simulated
hydrothermal reactor upon the reaction of iron sulfide with CO.80,87
Dalton Trans., 2009, 4274–4285 | 4281
Fig. 5 Possible functional roles of prebiotic metal sulfides existing as (A) “ready made” as described by Russell in which some metal sulfides exist with
lattice structures of striking similarity to clusters observed in biology. Here, the iron sulfide mackinawite is shown, highlighting resemblance of the mineral
lattice structure to the 2Fe unit of the [FeFe]-hydrogenase, albeit existing without ligand modifications (adapted from ref. 84). (B) Possible formation of
the amino acid glycine via the condensation of iron bound CO and CN- with H2 O in a reverse reaction to that proposed to occur in the biosynthesis
of CO and CN- from glycine. (C) An iron-sulfur mineral surface, shown to be capable of ligand-accelerated autocatalysis and feedback as described
by89 as a means of catalyst formation and diversification. The mineral surface is initially capable of carrying out catalytic formation of a molecule that
subsequently binds to the mineral surface, and via ligand accelerated catalysis enhances the formation of more of the same molecule. In this way, the
catalyst is selected for kinetically, propagating itself through ligand-assisted autocatalysis.
In addition to these carbonylated iron species, iron cyanide species
in the form of Prussian blue have been isolated from Miller–Urey
types of experiments showing that spark discharge experiments
are sufficient for the generation of CN- , which reacts with ferrous
iron.88 The observation of iron carbonyl and iron cyanide species
indicate that the unique ligand set of the hydrogenase enzymes may
be accessible in prebiotic conditions. Furthermore, iron cyanide
synthesis has been shown to be feasible with nucleophilic iron
and electrophilic CN- and in the reverse situation57 , validating the
proposed model for CN- transfer occurring by the action of HypE
in [NiFe]-hydrogenase active site biosynthesis and drawing an
interesting parallel between geochemical and biochemical ligand
addition. A further possible parallel between geochemical and
biochemical events exists in the possibility of small molecule
formation by the condensation of these modifiers. In an extension
of chemistry in which CO and CN- become bound to iron, the
reverse reaction could take part in the generation of the amino
acid glycine. In the [FeFe]-hydrogenase maturation hypothesis
outlined above69 , glycine is proposed to be the CO and CN- ligand
precursor; a reverse process in which CO and CN- bound to iron
condense at the mineral–solution interface with water would result
in its formation (Fig. 5b), illustrating the possibility that both the
formation as well as the degradation of prebiotic molecules at
mineral surfaces could have had important consequences at the
origin of life. CO and CN- addition to iron in possible prebiotic
conditions may have thus acted as a source for the accumulation
of biologically accessible molecules such as amino acids, and as
described further below, as a driving force for the formation of
diverse catalytic functionality acting as metal surface modifying
agents via ligand addition.
The challenge of the formation of a diverse repertoire of
catalysts has been addressed in part by a proposal based off of
ligand modification. Here, the chemical reactions that lead to
metal ligand modification are viewed as acting in a type of chemical
evolution where catalytically derived products may themselves add
to metal centers in chemistry analogous to that described above,
modifying their reactivity perhaps for their betterment and thus
4282 | Dalton Trans., 2009, 4274–4285
act to diversify and enhance catalytic attributes.89 This process, in
which the addition of a specific ligand leads to a quickening of a
particular reaction is termed “ligand accelerated catalysis” and is
applicable to both heterogeneous and homogeneous catalysts, being especially relevant to transition metal based catalysts that may
exist in environments in which dynamic ligand exchange events are
possible.90 The series of events in which ligand modification may
serve as a route to positive feed back, termed ligand-accelerated
autocatalysis, has been proposed to operate as a most basic type of
reproductive mechanism89 and may function in a manner enabling
catalytic mineral core derived products to modify clusters thereby
producing generations and families of catalysts (Fig. 5c). Indeed,
this type of phenomenon may occur in a system wide sense,
with neighbouring mineral sites of differing catalytic proclivity
contributing to the formation of proficient neighbouring catalysts,
thus forming a network basis for catalytic fecundity.
Also relevant to the discussion of what events could comprise
catalyst formation is the notion of “defect” sites or areas of
discontinuity of bulk structure on mineral surfaces. At these
locations, which represent approximately 1% of total surface
area,91,92 defects in lattice structure result in unique electronic as
well as spatial localities, where molecules may transiently bind
in orientations and environments amenable to reaction progress.
Heterogeneous catalysis by mineral surfaces has indeed been
observed to occur at these sites93,94 and highlights the observation
of biological clusters existing as structurally, stoichiometrically,
and molecularly tuned derivatives of minerals.
In addition to the possibility of pre-existing functionality of
minerals and ligand modification reactions as having played
a role in the synthesis of relevant biological constituents, the
possibility of individual iron sulfur clusters in solution exists.
Iron–sulfur clusters of the nature [Fe4 S4 (SR)]2- have been shown
to assemble in aqueous solvent from iron, sulfide, and thiols
(RSH).95 Together with ligand modification schemes and reported
experiments showing the generation of iron carbonyls and reactive
precursors obtained through hydrothermal reaction conditions80 ,
these experiments show that a suite of chemistries and possible
This journal is © The Royal Society of Chemistry 2009
catalyst generating events closely related to those observed in
biology are possible in putative prebiotic conditions and indicate
that relevant transition metal based chemistry can occur outside
of proteins. Given this, it is interesting to speculate on the catalytic
properties of these modified metal species and the possibility that
cluster assembly events similar to those that occur biologically to
create the active site architecture of the hydrogenases and other
metalloproteins may at least be partly mimicked by naturally
occurring geochemical processes.
Based on the above discussion of synthetic, biosynthetic, and
prebiotic reactions, a putative route to biological catalyst creation
and the steps associated with this are figuratively portrayed in
Fig. 6 starting from an assortment of FeS mineral surfaces. Broadly
divided into stages these transitions include the concepts of (i) a
mineral basis for early catalysis, (ii) the formation of individual
catalytic entities; ligand modification and cluster formation, and
(iii) the encapsulation of the nascent metal sulfide clusters by
peptides. Specifically, the proposed process of catalyst evolution is
portrayed to include the inherent activities of mineral surfaces in
which the surface itself or its transformation (oxidation in the case
of pyrite formation) results in relevant reactivity (e.g. ref. 78,96–
98). Also at this stage is represented the aforementioned concept
of “ready made”84 clusters and defect sites which may catalyze
reactions contributing to the formation of biologically accessible
molecules. With these roles in mind, a next step in catalyst
evolution may have been the formation of individual clusters and
cluster components such as the di-iron hexacarbonyl species as
obtained under hydrothermal conditions upon the reaction with
CO and alkyl thiols80,99 , the formation of iron sulfur cubanes, and
the formation of Fe–CN bonds as observed in spark discharge
experiments. The integration of these concepts and chemistries
may lead to the generation of proto-metallo-co-factors which bear
similarity to those observed in biology today. These intermediary
clusters may, in a similar way as described by Milner-White and
Russell, be bound by peptide “nests” comprised of simple amino
acids.100,101 Finally, the incorporation of these clusters into more
complex peptide chains leads to protein bound catalysts capable
of supporting catalytic activity from a robust mobile peptide
platform and the formation of an early proto-metabolism replete
with catalysts of sufficient diversity by which to harness chemical
energy.
Prospectus
The structural and spectroscopic characterization of the hydrogenase enzymes created new lines of research that, in an
interesting complementarity, are applicable both to future energy
solutions and to understanding the past evolution of hydrogen
redox catalysts and their role in the formation of life. With the
rapid pace of research in this area, the field is certain to yield
additional new insights in the coming years. For example, much
is yet to be revealed regarding biosynthesis of the metal centers
of the hydrogenases, particularly the [FeFe] and [Fe] classes. In
addition, while synthetic studies have resulted in the development
of creative and often elegant chemistries, an effective first row
transition metal based catalyst for hydrogen production based
on the structure of hydrogenase active sites has not emerged,
perhaps hinting that the proteinaceous environments surrounding
the active sites of the hydrogenases are critical for fine-tuning
This journal is © The Royal Society of Chemistry 2009
Fig. 6 Possible set of major transition stages in the emergence of
prebiotically relevant catalysts from metal sulfide minerals. Evolutionary
routes to protein bound transition metal sulfide catalysts are postulated
as having originated from the basis of iron and sulfur bearing minerals.
As a first illustrative stage stemming from the inherent properties of
metal-sulfides, the lattice structure of pyrite104 is shown as being derived
from FeS by oxidation which can serve as a driver for prebiotic catalysis.
In addition, the structure of a nickel–iron sulfide is shown which may
harbour pre-existing functionality. Also depicted at this stage is the
portrayal of a mineral surface defect site where atomic irregularities
in the lattice structure may lead to relevant catalytic activity through
modified electronic and small molecule binding characteristics. Continuing
in catalyst evolution, the next stage involves individual cluster formation
and possible ligand modifications in the forms of CO and CN- addition and
the creation of individual metal centers resulting in the iron hexacyanide,
[4Fe-4S], and di-iron hexacarbonyl species (Fe: dark red, S: orange,
O: red, C: dark gray, R group: magenta, N: blue) resulting in an
intermediary step prior to the protein encapsulation of metal co-factors.
In the final stage (top), the combination of these chemistries leads to
similar metal clusters to those seen in biology today, being bound by
relatively simple peptides which serve to contribute the unique solvent
micro-environment observed in contemporary metalloenzymes and exist
as ligand modifying agents themselves. Shown as examples are the metal
cluster active sites of acetyl-CoA synthase105 , the [FeFe]-hydrogenase,
and carbon monoxide dehydrogenase.106 Together, these representations
illustrate only a few of the myriad possibilities and routes available in the
formation of transition metal based catalytic species.
the catalytic potential of the metal centers, making studies in
which synthetic metal co-factors are linked to designed peptides
particularly attractive.102 Given their high catalytic efficiencies,
hydrogenase enzymes themselves remain interesting prospects for
biotechnology for non-noble metal based hydrogen oxidation and
hydrogen production catalysis in a hydrogen fuel economy and a
thorough understanding of the structural determinants of catalysis
and the biosynthesis of these enzymes is required for the full
potential to be realized. Finally, the ideas of preformed geologically derived catalytic motifs, ligand accelerated autocatalysis,
Dalton Trans., 2009, 4274–4285 | 4283
and the chemical ability of catalysts derived as such, may together
prove to yield deep insight into the origins of modern biological
complexity. The brief account of possibilities concerning prebiotic
catalysis described herein indicates that much is possible in relevant
primordial conditions with concern to cluster assembly and ligand
modification. The observations presented demonstrate a diverse
array of modifications that minerals and individual metal ions may
undergo and indicate that studies into transition metal minerals
and the possible transformations possible from these may be
a highly insightful area of research with concern to prebiotic
catalysis. Continued work directed at the areas described herein
will shed new and fascinating light on hydrogenases; biology’s
response to the hydrogen redox couple.
Acknowledgements
The work was supported by an Air Force Office of Scientific
Research Multidisciplinary University Research Initiative Award
(FA9550–05-01–0365) to J. P. and the NASA Astrobiology
Institute-Montana State University Astrobiology Biogeocatalysis
Research Center (NNA08CN85A) award to J. B. and J. P. S. M. is
supported by an NSF IGERT Fellowship by the MSU Program
in Geobiological Systems (DGE 0654336).
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