Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate
In Vitro [FeFe] Hydrogenase Activation
Jon M. Kuchenreuther
1
, James A. Stapleton
1
, James R. Swartz
1,2
*
1 Department of Chemical Engineering, Stanford University, Stanford, California, United States of America, 2 Department of Bioengineering, Stanford University, Stanford,
California, United States of America
Abstract
Background: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H
2
. These proteins are
distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein
moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster
biosynthesis have yet to be elucidated.
Principal Findings: We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe]
hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified
hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases;
and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase
with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity
increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition
of guanosine triphosphate (GTP) improved hydrogenase maturation.
Significance: The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a
foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical
additives.
Citation: Kuchenreuther JM, Stapleton JA, Swartz JR (2009) Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation. PLoS
ONE 4(10): e7565. doi:10.1371/journal.pone.0007565
Editor: Anna Maria Delprato, Centre National de la Recherche Scientifique, France
Received July 31, 2009; Accepted October 4, 2009; Published October 26, 2009
Copyright:
2009 Kuchenreuther et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the Global Climate and Energy Project (GCEP) at Stanford University (http://gcep.stanford.edu/) as well as a National Defense
Science and Engineering (NDSEG) Fellowship (http://ndseg.asee.org/) to JAS. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jswartz@stanford.edu
Introduction
Hydrogenases are subdivided into three classes: [NiFe]
hydrogenases, [FeFe] hydrogenases, and [Fe] hydrogenases, each
characterized by a unique active site cofactor [1–5]. [NiFe] and
[FeFe] hydrogenases catalyze the reversible oxidation of dihydro-
gen: H
2
O2H
+
+2e
2
. Of these, [FeFe] hydrogenases have intrin-
sically higher in vitro H
2
evolution rates [6], making them more
attractive candidates for production of H
2
as a sustainable biofuel.
The [FeFe] hydrogenase active site cofactor, known as the H-
cluster, is composed of a conventional [4Fe–4S] cubane cluster
joined by a cysteinyl sulfur to a unique [2Fe] sub-cluster that
includes multiple non-protein ligands covalently attached to the
sub-cluster iron atoms [6]. These non-protein moieties have been
identified as carbon monoxide (CO), cyanide (CN) [2], and a
putative dithiopropane or dithiomethylamine bridge [3,7].
Three proteins required for active [FeFe] hydrogenase produc-
tion – HydE, HydF (fused as HydEF in eukaryotes), and HydG –
were first identified by analyzing C. reinhardtii mutants incapable of
H
2
photoproduction. Subsequent recombinant co-expression of
the C. reinhardtii [FeFe] hydrogenase with C. reinhardtii HydEF and
HydG in E. coli enabled production of active hydrogenase [8].
Following this discovery, in vitro work with the individual maturases
has shed light on their respective roles in the synthesis of the H-
cluster cofactor and its insertion into the hydrogenase active site.
HydE and HydG, which both contain [Fe–S] clusters and
sequence motifs generally attributed to radical SAM enzymes
[8], have been shown to reductively cleave SAM to form 59-
deoxyadenosine [9]. Recently, SAM-dependent HydG activity was
shown to increase in the presence of tyrosine, leading to a
hypothesis that a tyrosine-derived dehydroglycine intermediate is
the source for the H-cluster dithiol bridge [10]. HydF has been
identified as a GTPase based on sequence alignment analysis
[8,11] and its ability to hydrolyze GTP to GDP [11]. In earlier
efforts to reproduce apohydrogenase maturation, HydF was
isolated after recombinant co-expression with HydE and HydG.
The purified HydF partially activated apohydrogenase, suggesting
that this maturase is a scaffold protein for H-cluster cofactor
assembly and transfer to the hydrogenase [12].
Various recombinant systems have demonstrated active [FeFe]
hydrogenase synthesis, both in vivo [8,13–15] and in vitro
[12,14,16], and other in vitro metalloenzyme systems have shown
improved post-translational activation following incubation of the
apoproteins with their respective maturases along with exogenous
small molecules [17,18]. Despite these advancements, [FeFe]
hydrogenase studies have thus far failed to demonstrate enhanced
PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7565

hydrogenase maturation following small molecule addition,
limiting our ability to elucidate the specific biochemistry required
for H-cluster cofactor synthesis and installation.
In this work, we describe the first in vitro system in which
chemical additives stimulate activation of [FeFe] hydrogenase. We
recently reported a cell-free system for the production of active
hydrogenases [14]. Here, we separate translation and activation
into two distinct steps, allowing us to isolate the maturation process
and explore it in detail. In agreement with a previous study [16],
we noticed that hydrogenase apoprotein was partially activated
when added to a crude cell lysate containing the three maturases.
However, we observed significantly higher hydrogenase activities
when the small molecule components of the cell-free protein
synthesis system were also included. This discovery provided a
unique opportunity to identify which small molecules play a role in
hydrogenase activation and H-cluster biosynthesis.
Results and Discussion
The In Vitro System for Enhanced Activation of [FeFe]
Hydrogenase
The hydrogenase maturation system contains purified C.
reinhardtii HydA1 apohydrogenase; dialyzed E. coli cell extract
containing recombinant HydE, HydF, and HydG hydrogenase
maturases from Shewanella oneidensis (hereafter referred to as
maturase extract); and exogenous small molecules. The eukaryotic
C. reinhardtii hydrogenase HydA1 was chosen as our model protein
given its simplified structure and high degree of in vivo solubility.
Unlike prokaryotic hydrogenases, algal hydrogenases such as
HydA1 have only the C-terminal H-domain and lack N-terminal
[4Fe–4S] F-clusters [19]. The S. oneidensis maturases HydE, HydF,
and HydG were used since previous work established that these
proteins are effective in activating HydA1 both in vivo [14,15] and
in vitro [14].
HydA1 apohydrogenase was heterologously produced in E. coli
in the absence of the maturases and purified using immobilized
metal-affinity chromatography (IMAC). Pooled fractions con-
tained high purity HydA1 based on SDS-polyacrylamide gels
visualized with Coomassie stain (Fig. 1A). Purified apohydrogenase
(22.064.4 mg HydA1?L
21
of culture, n = 3) had 0.360.2 mol
Fe?mol
21
HydA1, which was measured using established methods
[20]. For some activation studies, as-isolated apoprotein (apo-
HydA1) was anaerobically incubated with 1 mM DTT, 0.5 mM
Fe(NH
4
)
2
(SO
4
)
2
, and 0.5 mM Na
2
S to reconstitute the [4Fe–4S]
cluster. Reconstituted apoprotein (apoHydA1
recon
) preparations
are yellow/brown. The UV-visible spectrum for desalted apoHy-
dA1
recon
(Fig. 1B) shows a broad peak at 400 nm with an A
400
:A
280
ratio of 0.5, in contrast to the spectrum for apoHydA1. This result
indicates the apohydrogenase is properly folded and incorporates
the H-domain [4Fe–4S] cluster prior to activation, similar to a
previous report [21].
Maturase extracts were produced from E. coli cells co-expressing
HydE, HydF, and HydG in the absence of an [FeFe] hydrogenase.
The extracts were dialyzed immediately before use to establish
reaction conditions well defined with respect to small molecules.
Following anaerobic incubation of apohydrogenase with dialyzed
maturase extract, hydrogen uptake activity from activated
hydrogenase was determined by measuring methyl viologen
reduction rates. Dialyzed maturase extracts were capable of
partially activating HydA1 without addition of exogenous
molecules (Fig. 2A), as observed with previously described systems
[16,21]. The partial activation may be attributed to [2Fe] sub-
clusters produced in vivo prior to cell lysis, which are associated
with the maturases. No methyl viologen-reducing activity was
observed from reaction mixtures when using cell extracts without
the maturases or when HydA1 apoprotein was not added.
Ferrous iron (Fe
+2
), inorganic sulfide (S
22
), SAM, and a mixture
of the standard 20 L-amino acids (20 aa) were initially identified as
chemical additives contributing to hydrogenase activation. Com-
plementing maturase extracts with 1 mM Fe
+2
, 1 mM S
22
,2mM
SAM, and 2 mM of each 20 aa increased hydrogenase activities 4-
fold (Fig. 2A). The comparable activities of matured as-isolated
and reconstituted apohydrogenase indicate that HydA1 does not
require an intact H-domain [4Fe–4S] cluster prior to addition to
this system. Exogenous Fe
+2
and S
22
were critical: enhanced
HydA1 activation did not occur without both ions despite the
presence of SAM and 20 aa. Moreover, Fe
+2
and S
22
were not
sufficient to increase hydrogenase activities without SAM and 20
aa. Partial and similar activation of as-isolated and reconstituted
apohydrogenase when only SAM and 20 aa were included suggest
the Fe
+2
and S
22
are involved in more than just reconstitution of
the hydrogenase [4Fe–4S] cluster. Iron and sulfide likely facilitate
reconstitution of the maturases’ [Fe–S] clusters, which may have
been oxidized during aerobic preparation of the cell extracts.
Chemical reconstitution of radical SAM proteins using Fe
+2
/Fe
+3
and S
22
has previously been shown to benefit enzyme activity
[22,23]. Additionally, iron and sulfide may be required substrates
for in situ synthesis of the [2Fe] sub-cluster.
We observed that incubating maturase extracts with Fe
+2
and
S
22
before addition of other small molecules and HydA1
apoprotein (termed extract reconstitution) provided more consistent
data for characterizing the effects of other exogenous substrates.
Incubating extracts with SAM and 20 aa following extract
reconstitution and before apohydrogenase addition (termed extract
pre-treatment) led to the immediate onset of maturation as well as
Figure 1. Characterization of purified C. reinhardtii HydA1
apohydrogenase. (Fig. 1A) SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) analysis of pooled elution fractions containing N-his
6
-
HydA1 apoprotein (48.4 kDa) following aerobic expression in E. coli and
subsequent Ni
+2
-affinity chromatography. The molecular weight marker
(MWM) is the Mark 12
TM
protein ladder (Invitrogen). Intermediate lanes
of the SDS-polyacrylamide gels were removed, maintaining alignment
between the MWM and Eluate lanes. (Fig. 1B) UV-visible spectra for
8 mM of as-isolated (black line) and reconstituted (red line) HydA1
apohydrogenase.
doi:10.1371/journal.pone.0007565.g001
[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7565

maximally enhanced activation (Fig. 2B). When SAM, 20 aa, and
HydA1 apoprotein were added to extracts concurrently, hydrog-
enase maturation was partially compromised, suggesting that such
experiments might be useful in exploring the maturation reaction
sequence. Extract reconstitution and extract pre-treatment were
implemented in all subsequent experiments.
Additional putative small molecule precursors were assessed,
although no conditions were identified in which addition of these
molecules influenced hydrogenase maturation. While GTPase
activity has been attributed to HydF from Thermotoga maritima [11],
we could not identify any reaction conditions in which exogenous
GTP benefited HydA1 activation. Moreover, addition of guano-
sine diphosphate (GDP) neither enhanced nor inhibited hydrog-
enase maturation. GTP may play a role in hydrogenase
maturation in a biochemical process upstream of those occurring
within our system. Carbamoyl phosphate has been identified as a
precursor for the cyano ligands associated with the [NiFe]
hydrogenase active site [24,25]. We speculated the H-cluster
CN
2
moieties may also derive from this compound. However,
addition of carbamoyl phosphate with exogenous Mg-ATP had no
effect on HydA1 activation. Moreover, SDS-PAGE and autora-
diography imaging following in vitro incubations with [
14
C]-
carbamoyl phosphate, Mg-ATP, and maturase extract suggest
that carbamoyl phosphate does not covalently associate with
HydE, HydF, or HydG. This result could be expected as none of
the maturases has a sequence motif characteristic of acyl
phosphatases or O-carbamoyltransferases like that of the [NiFe]
hydrogenase maturase HypF [26,27]. While thiocyanate report-
edly has a strong affinity to an anion-binding cavity of HydE from
T. maritima [28], conditions were not identified in which
thiocyanate or cyanide improved HydA1 maturation. Other in
vitro studies with [Fe–S] proteins have included reducing agents
such as sodium dithionite [17,18] and dithiothreitol (DTT)
[22,23,29], though neither compound improved hydrogenase
activation in our system. Use of ferrous iron and sulfide ions may
obviate the necessity for such reducing agents.
Exogenous SAM Stimulates In Vitro Hydrogenase
Activation
As shown in Fig. 3, reaction mixtures containing Fe
+2
,S
22
,20
aa, and SAM had 5-fold higher HydA1 activities compared to
mixtures without SAM. However, neither SAM nor the 20 aa
mixture individually enhanced hydrogenase activation (compare
Fig. 2B, Fig. 3). The results shown in Fig. 3 suggest that SAM is
utilized for in vitro HydA1 activation, likely by the maturases HydE
and HydG for synthesis of the H-cluster [2Fe] sub-cluster. Several
studies have shown that exogenous SAM stimulates in vitro
biosynthetic reactions catalyzed by radical SAM enzymes. In
some cases, a 59-deoxyadenosyl radical derived from homolytic
cleavage of SAM is thought to facilitate abstraction of protons
from organic substrates [30,31]. SAM is also required for the
synthesis of NifB-co, a precursor for the nitrogenase FeMo active
site cofactor; it has been suggested that radical SAM chemistry also
functions to build the FeMo-co [Fe–S] cage [17,18]. However, no
previously reported studies have demonstrated that exogenous
SAM improves post-translational [FeFe] hydrogenase activation.
To further explore the stimulatory effect of SAM on
hydrogenase activation, the SAM analog S-adenosyl homocysteine
(SAH) was tested. This analog was not an effective substitute for
SAM, likely because SAH contains a less electrophilic sulfur atom.
Reduction of a radical SAM enzyme’s [4Fe–4S] cluster to its
active state is required for radical chemistry [32]. In some in vitro
systems, this activation has been shown to further benefit from
exogenous NADPH along with SAM addition [29,31]. Nonethe-
less, NADPH had no significant effect on final HydA1 activities in
our system. Extract reconstitution with Fe
+2
and S
22
may generate
reduced [4Fe–4S] clusters associated with the maturases and thus
avoid the need for an additional reducing agent.
Figure 2. In vitro activation of C. reinhardtii HydA1 and the effects of exogenous small molecules. 2 mM of HydA1 apoprotein was
anaerobically incubated with 50–60% vol?vol
21
maturase extract. Exogenous substrates assessed included Fe
+2
(1 mM), S
22
(1 mM), SAM (2 mM),
and 20 aa (2 mM of each amino acid). (Fig. 2A) When included in reaction mixtures, Fe
+2
and S
22
were added to maturase extracts 2 hr before
addition of apoHydA1 (black bars) or apoHydA1
recon
(red bars). When SAM and 20 aa were included, maturase extracts were incubated with these
chemical additives for 1 hr prior to HydA1 addition. Final hydrogenase activities determined after 9 hr of incubation are from n = 2 to 5 independent
determinations 6 SEM. (Fig. 2B) Maturase extracts were reconstituted with Fe
+2
and S
22
for 2 hr (
N
,&,m) or 0 hr (+) before apoHydA1 addition;
extracts were also pre-treated with SAM and 20 aa for 1 hr (m) or 0 hr (&,
N
,+) before adding HydA1 apoprotein (as-isolated:
N
,m,+; reconstituted: &).
Data are from n = 2 independent measurements, and standard errors were less than 10% for all data.
doi:10.1371/journal.pone.0007565.g002
[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7565

We have yet to determine how many molecules of SAM are
required for activation of one hydrogenase polypeptide or for
synthesis of a single H-cluster cofactor. Consumption of multiple
SAM molecules per small molecule product for radical SAM-
based biochemistry has been reported [33,34]. Direct detection of
the highly reactive 59-deoxyadenosyl radical has proven difficult,
and detection of the allylic analog 59-deoxyadenosine is generally
used to characterize SAM radical chemistry [32]. Our efforts to
measure 59-deoxyadenosine accumulation using reverse-phase
HPLC did not show detectable levels in reaction mixtures
following HydA1 activation. Future work using purified maturases
and higher enzyme concentrations may be more effective for
characterizing the radical SAM biochemistry.
Tyrosine and Cysteine Enhance In Vitro Maturation of
HydA1 Hydrogenase
The requirement for the 20 aa mixture along with Fe
+2
,S
22
,
and SAM (Fig. 3) indicates that one or multiple amino acids may
be substrates for hydrogenase activation. A statistical design of
experiment approach was adopted to identify amino acids
positively or negatively influencing in vitro HydA1 activation.
Design Expert 7.1 software (Stat-Ease, Inc.) was used to create a 2-
level fractional factorial model and for statistical analysis of the
data. A 2
20–15
factorial design with resolution III was selected. 32
combinations of the 20 canonical L-amino acids were constructed
using the Design Expert software (Figure 4). Hydrogenase
activation reactions contained Fe
+2
,S
22
, SAM, and one of the
32 amino acid mixtures. Early hydrogenase activities at t = 10 min
(Response 1) as well as final hydrogenase activities at t = 9 hr
(Response 2) were measured.
Analysis of variance (ANOVA) was used to identify amino acids
with statistically significant effects on hydrogenase maturation. All
20 amino acids were included in the regression models. Tyrosine,
cysteine, and methionine were identified as having significant
positive contributions when analyzing each response, with p-values
,0.0001 for each amino acid. Cysteine had the most significant
effect on early HydA1 activities (Response 1). Tyrosine had the
most significant effect on overall HydA1 activities (Response 2),
which were 7- to 12-fold higher than the minimum activity. No
amino acid had a significant negative effect on HydA1 maturation.
With resolution III factorial models, single factor effects are
aliased with two-factor interactions. Therefore, our factorial model
did not have the ability to assess the independent significance of
cysteine, tyrosine, and methionine if two of these molecules have
interactive effects on hydrogenase activation. To complete the
evaluation, subsequent experiments were done with Fe
+2
,S
22
,
SAM, and the three amino acids. While cysteine and tyrosine
individually benefited hydrogenase activation, reaction mixtures
with both amino acids had the most effective maturation capability
for both maturation kinetics and final activities (Fig. 5A–B). These
data suggest that tyrosine and cysteine may have a cooperative
interaction for the in vitro activation of [FeFe] hydrogenase.
However, no conditions were identified in which addition of
methionine improved HydA1 activation (data not shown).
Examination of the 32 aa mixtures in Figure 4 shows that all 8
mixtures with both tyrosine and cysteine also contained methio-
nine. Thus, the apparent significant effect of methionine indicated
by the design of experiment data appears to be a product of the
limited discrimination provided by the resolution III factorial
model.
We have yet to characterize the biochemical role(s) of cysteine
for in vitro hydrogenase maturation. We speculate cysteine may be
a substrate for synthesis of the H-cluster [2Fe] sub-cluster,
specifically as a precursor for the dithiol bridging ligand sulfur
atoms. In the absence of cysteine, S
22
may substitute for synthesis
of the [2Fe] sub-cluster, which could explain why slower and
partial HydA1 activation occurred in mixtures containing Fe
+2
,
S
22
, SAM, and tyrosine (Fig. 5B). Alternatively, cysteine could be
involved in reconstitution of the maturase [Fe–S] clusters or the
hydrogenase [4Fe–4S] cluster; however, apoHydA1
recon
was
matured similarly to apoHydA1 in the absence of exogenous
cysteine (Fig. 5A).
3,4-Dihydroxy-L-phenylalanine Substitutes for Tyrosine
to Stimulate In Vitro HydA1 Activation
The effects of tyrosine analogs were examined to further
investigate the role of tyrosine as a substrate for [FeFe]
hydrogenase activation (Figure 6). All reaction mixtures contained
Fe
+2
,S
22
, SAM, and cysteine. Addition of the analog 3,4-
dihydroxy-L-phenylalanine partially substituted for tyrosine and
improved HydA1 maturation 4-fold. Other tyrosine analogs were
ineffective in stimulating hydrogenase activation. Recent in vitro
work has indicated that thiamine biosynthesis in E. coli may
require radical SAM chemistry, with tyrosine as a co-substrate
[29]. The authors proposed a mechanism by which the 59-
deoxyadenosyl radical generated from SAM abstracts the phenolic
hydrogen atom from tyrosine. Subsequent C
a
–C
b
bond cleavage
along with further oxidation of the glycinyl radical results in the
formation of dehydroglycine. Thiamine phosphate synthesis using
purified enzymes in conjunction with exogenous SAM, tyrosine,
and 1-deoxyxylulose-5-phosphate has also been shown [31].
Considering this proposed mechanism, the positive effect of 3,4-
dihydroxy-L-phenylalanine on hydrogenase activation could be
expected since the molecule has a para-hydroxyl group like that of
tyrosine. These results support the suggested role of tyrosine as a
substrate for SAM-based radical chemistry to produce intermedi-
ates required for synthesis of the H-cluster cofactor [10]. The
authors hypothesize that dehydroglycine is a precursor for the
Figure 3. Effects of SAM on in vitro HydA1 maturation. Maturase
extracts were reconstituted with Fe
+2
and S
22
for 60 min, and then pre-
treated for 60 min with the indicated small molecules prior to
apoHydA1 addition (3.6–4.6 mM). Reactions mixtures contained 50–
70% vol?vol
21
maturase extract. Final concentrations of chemical
additives were 1 mM Fe
+2
, 1 mM S
22
, 2 mM of each amino acid (20 aa),
2 mM SAM, 1 mM NADPH, and 2 mM SAH. Hydrogenase activities were
measured after 8–9 hr of anaerobic incubation. Data are the average for
n = 2 to 4 independent determinations 6 SEM.
doi:10.1371/journal.pone.0007565.g003
[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7565

[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7565

[2Fe] sub-cluster dithiomethylamine bridge based on the obser-
vation that p-cresol accumulated in HydG-catalyzed reactions
between SAM and tyrosine. However, we have yet to distinguish
which H-cluster non-protein moieties, if any, are derived from
tyrosine. Considering the structure of dehydroglycine (Figure 6)
and its carbonyl and imine groups, it is also possible the CO and
CN moieties may derive from a dehydroglycine precursor, in
addition to the dithiomethylamine bridge as proposed. The
observations that Fe
+2
,S
22
, SAM, cysteine, and tyrosine are
sufficient for HydA1 activation further supports this hypothesis as
no alternative reaction mechanisms for CO and CN synthesis from
these small molecules are apparent.
Conclusions
In this work, we demonstrate a platform for post-translational
activation of an [FeFe] hydrogenase. Utilizing this in vitro system,
we have shown for the first time the involvement of exogenous
small molecules, including ferrous iron, inorganic sulfide, SAM,
cysteine, and tyrosine in the activation of an [FeFe] hydrogenase.
These results now enable further investigation of H-cluster
cofactor biosynthesis using a defined system containing exogenous
substrates and purified enzymes.
Materials and Methods
Materials and Solution Compositions
Except for isopropyl b-D-1-thiogalactopyranoside (IPTG,
Invitrogen), SAM (New England Biolabs), and [
14
C]-carbamoyl
phosphate (American Radiolabeled Chemicals, Inc.), all chemicals
Figure 4. Experimental design for elucidation of amino acids enhancing in vitro hydrogenase activation. Amino acid mixtures (aa Mix)were
added to hydrogenase maturation reactions to a final concentration of 2 mM for each amino acid. The maturation reaction mixtures contained 60%
vol?vol
21
maturase extract reconstituted with Fe
+2
and S
22
for 60 min, and then pre-treated with SAM and one of the 32 aa mixtures for 60 min prior to
apoHydA1 addition (4.1 mM). HydA1 specific activities were determined at t = 10 min (Response R1)andt=9 hr(Response R2), and values are expressed
as pmol H
2
consumed?min
21
?ng
21
for n = 1 experiment. ANOVA was performed for both responses to determine F-statistics and p-values. The 20 individual
amino acid effects were selected for analysis by the regression models. Each F-statistic equals the ratio of mean squares for that particular amino acid (1
degree of freedom) to that of the residuals (11 degrees of freedom). P-values represent the statistical significance of the F-statistics.
doi:10.1371/journal.pone.0007565.g004
Figure 5. Effects of cysteine and tyrosine on in vitro HydA1
activation. Maturase extracts (final concentrations of 50–60%
vol?vol
21
) were reconstituted with Fe
+2
and S
22
for 60 min, and then
pre-treated with SAM and amino acids for 60 min prior to addition of
apoHydA1 (black bars) or apoHydA1
recon
(red bars). No additional
molecules were added with HydA1 apoprotein (3.6–4.6 mM). Final
concentrations of exogenous molecules were as follows: 1 mM Fe
+2
,
1mMS
22
, 2 mM SAM, 2 mM cysteine, 2 mM tyrosine, and 2 mM
methionine. (Fig. 5A) Hydrogenase activities were measured after 8–9 hr
of incubation. Data are the average for n = 2 to 5 independent
determinations 6 SEM. ApoHydA1
recon
was only tested for mixtures
with tyrosine and with cysteine plus tyrosine. Addition of methionine
did not enhance hydrogenase activities for all four conditions (data not
shown). (Fig. 5B) Reaction mixtures included as-isolated apoHydA1,
Fe
+2
,S
22
, SAM, and the following amino acids added as described
above: cysteine (&); tyrosine (6); cysteine and tyrosine (#). Data are
the average for n = 2 independent determinations. Standard errors were
less than 11% for all data.
doi:10.1371/journal.pone.0007565.g005
Figure 6. Assessment of tyrosine and tyrosine analogs for in
vitro hydrogenase activation. Maturase extracts were reconstituted
with 1.4 mM Fe
+2
and 1.4 mM S
22
for 60 min and subsequently pre-
treated with 2 mM SAM, 2 mM cysteine, and 2 mM of the following
amino acid(s) for 60 min prior to apoHydA1 addition (4.6 mM): (1) none,
(2) L-phenylalanine, (3) 3-hydroxy-DL-phenylalanine, (4) 3,4-dihydroxy-
L-phenylalanine, (5) 4-amino-L-phenylalanine, (6) L-tyrosine (4-hydroxy-
L-phenylalanine), (7) 20 aa. Reaction mixtures contained 60% vol?vol
21
maturase extract. Hydrogenase activities were measured after 9 hr, and
specific activities (*) are expressed as pmol H
2
consumed?min
21
?ng
21
HydA1. Data are the average for n = 2 independent determinations 6
SEM. The chemical structure for dehydroglycine (2-iminoacetic acid) is
provided (8). {ND: not determined.
doi:10.1371/journal.pone.0007565.g006
[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7565

were obtained from Sigma (Sigma-Aldrich). Commercial aqueous
SAM (32 mM stock concentration) contained 10% vol?vol
21
ethanol and 5 mM sulfuric acid, pH 2.7. Defined growth medium
for fermentations was prepared as previously described [14]. S30
buffer contained 10 mM Tris-acetate, pH 8.0; 14 mM magnesium
acetate; and 60 mM potassium acetate. 50 mM sodium phosphate
buffer, pH 7.4 with 100 mM NaCl (SP buffer) was used for Ni
+2
-
affinity chromatography. 100 mM HEPES/KOH, pH 7.2 with
100 mM NaCl (HP buffer) was used for maturase extract dialysis.
[FeFe] Hydrogenase and Maturase Expression Vectors
The hydrogenase gene hydA1 from C. reinhardtii as well as the
nucleotide sequences hydGx and hydEF encoding the S. oneidensis
hydrogenase maturases were PCR amplified from the pK7 shydA1
and pACYCDuet-1 hydGxEF plasmids [14] using Platinum H Taq
DNA Polymerase High Fidelity (Invitrogen). PCR products were
digested with restriction enzymes and inserted into expression
vectors between a T7 RNA polymerase promoter and terminator
using T4 DNA ligase (New England Biolabs). The pY71 vector
was used as the parent plasmid for construction of C. reinhardtii
shydA1 expression vectors. The plasmid pY71 cat encoding the
chloramphenicol acetyl transferase enzyme was synthesized by first
PCR amplifying the replication origin from the pUC19 plasmid
(Invitrogen), the kanamycin resistance gene from pK7 cat [35], and
the nucleotide fragment from pK7 cat containing the cat gene
flanked by the T7 RNA polymerase promoter and terminator
sequences. These three fragments were ligated using overlapping
PCR. The linear PCR product (<2.5 kb) was digested with
BamHI and ligated to form pY71 cat. Next, the shydA1 gene was
cloned into the pY71 vector, from which the cat gene had been
removed. The first 8 codons of shydA1 were conservatively
changed to ATG GCA GCA CCA GCA GCA GAA GCG for
reduced secondary structure as predicted using Mfold software to
improve in vitro translation (shydA1*). pY71 shydA1* was used for
addition of an N-terminal 6x-histidine tag, and the N-his
6
-shydA1*
insert was cloned back into the pY71 vector. Synthesis of the
expression vector containing the S. oneidensis maturase genes was
carried out in two parts. First, the hydGx gene segment was cloned
into multiple cloning site I of the pACYCDuet-1
TM
expression
vector (Novagen). Next, the hydEF gene segment was cloned into
multiple cloning site II of pACYCDuet-1–hydGx. All expression
vectors were confirmed by DNA sequencing and transformed into
E. coli strain BL21(DE3) (Invitrogen). Transformed cells were
selected against kanamycin resistance (40 mg L
21
) for pK7 and
pY71 plasmids, and against chloramphenicol resistance
(25 mg L
21
) for pACYCDuet-1 plasmids.
Apohydrogenase Expression, Purification, and
Characterization
In vivo apohydrogenase expression was carried out in the
absence of [FeFe] hydrogenase maturases using E. coli strain
BL21(DE3) pY71 N-his
6
-shydA1*. Cells were initially grown at
30uC in 2 L baffled flasks containing 1 L of LB Miller medium,
40 mg L
21
kanamycin, and 250 mg L
21
ferric ammonium
citrate. Shake flasks were transferred to 20uC shakers at an
OD
600
of 0.2, and L-cysteine was added to 1 mM. After 1 hr
(OD
600
<0.5), IPTG was added to 0.5 mM to induce hydrogenase
expression, and cultures were incubated for 12–15 hr at 20uC.
Following recombinant hydrogenase expression, cells were pellet-
ed and resuspended in 3 mL of Bug Buster Master Mix lysis
solution (Novagen) per gram wet cell mass. Cell suspensions were
incubated at 23uC for 30 min and then diluted with 56SP buffer
(10 mM imidazole final concentration). Cell lysates were clarified
by centrifugation at 30,0006g and 4uC for 30 min before being
loaded onto equilibrated 1 mL HisTrap
TM
HP Ni
+2
-affinity
columns (GE Healthcare). Columns were washed using 5 mL of
SP buffer with 40 mM imidazole. Apohydrogenase was eluted
using 5 mL of SP buffer with 250 mM imidazole. Eluate fractions
containing apoprotein were identified following SDS-PAGE and
Coomassie staining. Pooled fractions were dialyzed twice for 3 hr
each time against SP buffer with 10% vol?vol
21
sucrose.
Apohydrogenase aliquots were sealed and stored at 220uC.
Protein concentrations were determined with a Qubit fluorometer
according to manufacturer’s instructions (Invitrogen).
Reconstituted apoprotein solutions were prepared under
anaerobic conditions. Solutions were reduced with 1 mM DTT
for 15 min, incubated with 0.5 mM Fe(NH
4
)
2
(SO
4
)
2
for 15 min,
and then incubated with 0.5 mM Na
2
S for 2 hr. Reconstituted
protein solutions were centrifuged for 15 min at 8,0006g and
passed through PD–10 desalting columns (GE Healthcare)
equilibrated with HP buffer. Solutions analyzed spectrophotomet-
rically were sealed in quartz cuvettes within the anaerobic
chamber. UV-visible spectroscopy was performed using an HP
8425A Diode Array Spectrophotometer (Hewlett Packard). Iron
content was measured as previously described [20].
Production of Maturase Extract for In Vitro Hydrogenase
Activation
Recombinant expression of the S. oneidensis HydE, HydF, and
HydG maturases, and cell-free extract preparation were similar to
previously described methods [14]. E. coli strain BL21(DE3)
pACYCDuet-1–hydGx–hydEF was cultivated in a 5 L BioFlo 3000
fermentor (New Brunswick Scientific) in 4 L of defined growth
medium under oxic conditions at 30uC. The culture pH was
maintained at 7.0 using 1 N ammonium hydroxide. Growth
medium was supplemented with 25 mg L
21
chloramphenicol and
250 mg L
21
ferric ammonium citrate. At an OD
600
<2.0, 1 mM
L-cysteine was added, and recombinant maturase expression was
induced with 0.5 mM IPTG. After 45 min of induction, the
temperature set point was changed to 20uC. When cultures
reached 20uC, airflow was switched to 100% nitrogen at 1.5
SLPM to establish strict anoxic conditions. Agitation was reduced
from 500 rpm to 75 rpm. Cultures were anaerobically incubated
for 12–15 hr at 20uC before cell extract preparation.
All maturase extract preparation steps were carried out under
aerobic conditions. Cells were pelleted, resuspended in 1 mL of
S30 buffer per gram of wet cell mass, and lysed using a high-
pressure EmulsiFlex-C50 homogenizer (Avestin) operated at
15,000–20,000 psi. Cell lysates were clarified by centrifugation
at 30,0006g and 4uC for 30 min. Supernatant was collected,
frozen with liquid nitrogen, and stored at 280uC until used as
maturase extract for in vitro hydrogenase activation studies.
In Vitro Activation of [FeFe] Hydrogenase
Hydrogenase activation reaction mixtures were 25–50 mLin
volume and were incubated in 200 mL 8-well PCR strips (E&K
Scientific, Inc.). Mixtures contained 50–70% vol?vol
21
dialyzed
maturase extract, 1–5 mM HydA1 hydrogenase, and exogenous
substrates. When included, final concentrations of chemical
additives were as follows: 1 mM ferrous ammonium sulfate
(Fe
+2
), 1 1 mM sodium sulfide (S
22
), 2 mM SAM, a mixture 20
standard L-amino acids at 2 mM each, 2 mM SAH, 1 mM
NADPH, 2–20 mM magnesium chloride, 1–10 mM ATP, 2 mM
cysteine, 2 mM tyrosine, 2 mM methionine, 2 mM phenylalanine,
2 mM 4-amino-L-phenylalanine, 2 mM 3-hydroxy-DL-phenylal-
anine, 2 mM 3,4-dihydroxy-L-phenylalanine, 2 mM GTP, 2 mM
GDP, 1–5 mM carbamoyl phosphate, 2 mM sodium thiocyanate,
[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7565

2 mM sodium cyanide, 1 mM DTT, and 1 mM sodium
dithionite.
Generally, hydrogenase maturation reactions consisted of four
phases, with all procedures carried out in an anaerobic chamber
(Coy Laboratory Products) containing 98% N
2
and 2% H
2
. Phase
1, dialysis: 0.5–2.0 mL of maturase extract was buffer exchanged
three times (3 hr, 3 hr, overnight) against 0.75 L of HP buffer at
6uC using 6–8 kD MWCO RC dialysis tubing (Spectrum
Laboratories, Inc.). Dialyzed maturase extracts were used
immediately to avoid variability from freezing and thawing. Phase
2, extract reconstitution:Fe
+2
and S
22
were incubated with dialyzed
extracts for 60 min at 26uC before pre-treatment with small
molecules. Phase 3, extract pre-treatment: reconstituted extracts were
incubated with defined sets of exogenous substrates for 60 min at
26uC before apohydrogenase addition. Phase 4, hydrogenase activation:
either as-isolated or reconstituted apohydrogenase was added to
pre-treated maturase extracts, and reaction mixtures were
incubated at 26uC until assayed for hydrogenase activity.
Hydrogenase activity was determined with a H
2
consumption
and methyl viologen reduction assay as previously described [14],
with the modification that spectrophotometric measurements were
performed at 26uC instead of 37uC.
Acknowledgments
The authors wish to thank Marcus Boyer for contributions to the work.
Author Contributions
Conceived and designed the experiments: JMK JAS JRS. Performed the
experiments: JMK. Analyzed the data: JMK JRS. Contributed reagents/
materials/analysis tools: JMK. Wrote the paper: JMK JAS JRS.
References
1. Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, et al. (1995) Crystal
structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373:
580–587.
2. Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC (1998) X-ray crystal structure
of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8
angstrom resolution. Science 282: 1853–1858.
3. Nicolet Y, Piras C, Legrand P, Hatchikian CE, Fontecilla-Camps JC (1999)
Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual
coordination to an active site Fe binuclear center. Structure 7: 13–23.
4. Pilak O, Mamat B, Vogt S, Hagemeier CH, Thauer RK, et al. (2006) The
crystal structure of the apoenzyme of the iron-sulphur cluster-free hydrogenase.
J Mol Biol 358: 798–809.
5. Shima S, Pilak O, Vogt S, Schick M, Stagni MS, et al. (2008) The crystal
structure of [Fe]-hydrogenase reveals the geometry of the active site. Science
321: 572–575.
6. Adams MW (1990) The structure and mechanism of iron-hydrogenases.
Biochim Biophys Acta 1020: 115–145.
7. Nicolet Y, Cavazza C, Fontecilla-Camps JC (2002) Fe-only hydrogenases:
structure, function and evolution. J Inorg Biochem 91: 1–8.
8. Posewitz MC, King PW, Smolinski SL, Zhang L, Seibert M, et al. (2004)
Discovery of two novel radical S-adenosylmethionine proteins required for the
assembly of an active [Fe] hydrogenase. J Biol Chem 279: 25711–25720.
9. Rubach JK, Brazzolotto X, Gaillard J, Fontecave M (2005) Biochemical
characterization of the HydE and HydG iron-only hydrogenase maturation
enzymes from Thermatoga maritima. FEBS Lett 579: 5055–5060.
10. Pilet E, Nicolet Y, Mathevon C, Douki T, Fontecilla-Camps JC, et al. (2009)
The role of the maturase HydG in [FeFe]-hydrogenase active site synthesis and
assembly. FEBS Lett.
11. Brazzolotto X, Rubach JK, Gaillard J, Gambarelli S, Atta M, et al. (2006) The
[Fe-Fe]-hydrogenase maturation protein HydF from Thermotoga maritima is a
GTPase with an iron-sulfur cluster. J Biol Chem 281: 769–774.
12. McGlynn SE, Shepard EM, Winslow MA, Naumov AV, Duschene KS, et al.
(2008) HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis.
FEBS Lett 582: 2183–2187.
13. King PW, Posewitz MC, Ghirardi ML, Seibert M (2006) Functional studies of
[FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system.
J Bacteriol 188: 2163–2172.
14. Boyer ME, Stapleton JA, Kuchenreuther JM, Wang CW, Swartz JR (2008) Cell-
free synthesis and maturation of [FeFe] hydrogenases. Biotechnol Bioeng 99:
59–67.
15. Sybirna K, Antoine T, Lindberg P, Fourmond V, Rousset M, et al. (2008)
Shewanella oneidensis: a new and efficient system for expression and maturation
of heterologous [Fe-Fe] hydrogenase from Chlamydomonas reinhardtii. BMC
Biotechnol 8: 73.
16. McGlynn SE, Ruebush SS, Naumov A, Nagy LE, Dubini A, et al. (2007) In vitro
activation of [FeFe] hydrogenase: new insights into hydrogenase maturation.
J Biol Inorg Chem 12: 443–447.
17. Curatti L, Ludden PW, Rubio LM (2006) NifB-dependent in vitro synthesis of
the iron-molybdenum cofactor of nitrogenase. Proc Natl Acad Sci U S A 103:
5297–5301.
18. Curatti L, Hernandez JA, Igarashi RY, Soboh B, Zhao D, et al. (2007) In vitro
synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur,
molybdenum, and homocitrate using purified proteins. Proc Natl Acad Sci U S A
104: 17626–17631.
19. Meyer J (2007) [FeFe] hydrogenases and their evolution: a genomic perspective.
Cell Mol Life Sci 64: 1063–1084.
20. Fish WW (1988) Rapid colorimetric micromethod for the quantitation of
complexed iron in biological samples. Methods Enzymol 158: 357–364.
21. Mulder DW, Ortillo DO, Gardenghi DJ, Naumov AV, Ruebush SS, et al. (2009)
Activation of HydA,sup.EFG,/sup. requires a preformed [4Fe-4S] cluster.
Biochemistry.
22. Tse Sum Bui B, Mattioli TA, Florentin D, Bolbach G, Marquet A (2006)
Escherichia coli biotin synthase produces selenobiotin. Further evidence of the
involvement of the [2Fe-2S]2+ cluster in the sulfur insertion step. Biochemistry
45: 3824–3834.
23. Hernandez HL, Pierrel F, Elleingand E, Garcia-Serres R, Huynh BH, et al.
(2007) MiaB, a bifunctional radical-S-adenosylmethionine enzyme involved in
the thiolation and methylation of tRNA, contains two essential [4Fe-4S] clusters.
Biochemistry 46: 5140–5147.
24. Reissmann S, Hochleitner E, Wang H, Paschos A, Lottspeich F, et al. (2003)
Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands.
Science 299: 1067–1070.
25. Blokesch M, Albracht SP, Matzanke BF, Drapal NM, Jacobi A, et al. (2004) The
complex between hydrogenase-maturation proteins HypC and HypD is an
intermediate in the supply of cyanide to the active site iron of [NiFe]-
hydrogenases. J Mol Biol 344: 155–167.
26. Wolf I, Buhrke T, Dernedde J, Pohlmann A, Friedrich B (1998) Duplication of
hyp genes involved in maturation of [NiFe] hydrogenases in Alcaligenes
eutrophus H16. Arch Microbiol 170: 451–459.
27. Paschos A, Bauer A, Zimmermann A, Zehelein E, Bock A (2002) HypF, a
carbamoyl phosphate-converting enzyme involved in [NiFe] hydrogenase
maturation. J Biol Chem 277: 49945–49951.
28. Nicolet Y, Rubach JK, Posewitz MC, Amara P, Mathevon C, et al. (2008) X-ray
structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga
maritima. J Biol Chem 283: 18861–18872.
29. Leonardi R, Roach PL (2004) Thiamine biosynthesis in Escherichia coli: in vitro
reconstitution of the thiazole synthase activity. J Biol Chem 279: 17054–17062.
30. Guianvarc’h D, Florentin D, Tse Sum Bui B, Nunzi F, Marquet A (1997) Biotin
synthase, a new member of the family of enzymes which uses S-adenosylme-
thionine as a source of deoxyadenosyl radical. Biochem Biophys Res Commun
236: 402–406.
31. Kriek M, Martins F, Leonardi R, Fairhurst SA, Lowe DJ, et al. (2007) Thiazole
synthase from Escherichia coli: an investigation of the substrates and purified
proteins required for activity in vitro. J Biol Chem 282: 17413–17423.
32. Layer G, Heinz DW, Jahn D, Schubert WD (2004) Structure and function of
radical SAM enzymes. Curr Opin Chem Biol 8: 468–476.
33. Escalettes F, Florentin D, Tse Sum Bui B, Lesage D, Marquet A (1999) Biotin
Synthase Mechanism: Evidence for Hydrogen Transfer from the Substrate into
Deoxyadenosine. Journal of the American Chemical Society 121: 3571–3578.
34. Cicchillo RM, Iwig DF, Jones AD, Nesbitt NM, Baleanu-Gogonea C, et al.
(2004) Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to
synthesize one equivalent of lipoic acid. Biochemistry 43: 6378–6386.
35. Kim DM, Kigawa T, Choi CY, Yokoyama S (1996) A highly efficient cell-free
protein synthesis system from Escherichia coli. Eur J Biochem 239: 881–886.
[FeFe] Hydrogenase Maturation
PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7565