The auxiliary [4Fe-4S] cluster of the Radical SAM heme synthase from: Methanosarcina barkeri is involved in electron transfer

Article abstract of DOI:10.1039/c6sc01140c

The heme synthase AhbD catalyzes the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme during the alternative heme biosynthesis pathway occurring in sulfate-reducing bacteria and archaea. AhbD belongs to the family of Radical SAM enzymes and contains two [4Fe-4S] clusters. Whereas one of these clusters is required for substrate radical formation, the role of the second iron-sulfur cluster is not known. In this study, the function of the auxiliary cluster during AhbD catalysis was investigated. Two single cluster variants of AhbD from M. barkeri carrying either one of the two clusters were created. Using these enzyme variants it was shown that the auxiliary cluster is not required for substrate binding and formation of the substrate radical. Instead, the auxiliary cluster is involved in a late step of AhbD catalysis most likely in electron transfer from the reaction intermediate to a final electron acceptor. Moreover, by using alternative substrates such as coproporphyrin III, Cu-coproporphyrin III and Zn-coproporphyrin III for the AhbD activity assay it was observed that the central iron ion of the porphyrin substrate also participates in the electron transfer from the reaction intermediate to the auxiliary [4Fe-4S] cluster. Altogether, new insights concerning the completely uncharacterized late steps of AhbD catalysis were obtained.

Full text of DOI:10.1039/c6sc01140c

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The auxiliary [4Fe–4S] cluster of the Radical SAM
heme synthase from Methanosarcina barkeri is
involved in electron transfer†
Cite this: DOI: 10.1039/c6sc01140c
Melanie Kuhner,a Peter Schweyen,b Martin Hoffmann,b Jose Vazquez Ramos,c
¨
´
d
d
b
ac
*
Edward J. Reijerse, Wolfgang Lubitz, Martin Broring and Gunhild Layer
¨
The heme synthase AhbD catalyzes the oxidative decarboxylation of two propionate side chains of iron-
coproporphyrin III to the corresponding vinyl groups of heme during the alternative heme biosynthesis
pathway occurring in sulfate-reducing bacteria and archaea. AhbD belongs to the family of Radical SAM
enzymes and contains two [4Fe–4S] clusters. Whereas one of these clusters is required for substrate
radical formation, the role of the second iron–sulfur cluster is not known. In this study, the function of
the auxiliary cluster during AhbD catalysis was investigated. Two single cluster variants of AhbD from
M. barkeri carrying either one of the two clusters were created. Using these enzyme variants it was
shown that the auxiliary cluster is not required for substrate binding and formation of the substrate
radical. Instead, the auxiliary cluster is involved in a late step of AhbD catalysis most likely in electron
transfer from the reaction intermediate to a final electron acceptor. Moreover, by using alternative
substrates such as coproporphyrin III, Cu-coproporphyrin III and Zn-coproporphyrin III for the AhbD
activity assay it was observed that the central iron ion of the porphyrin substrate also participates in the
electron transfer from the reaction intermediate to the auxiliary [4Fe–4S] cluster. Altogether, new
insights concerning the completely uncharacterized late steps of AhbD catalysis were obtained.
Received 11th March 2016
Accepted 22nd March 2016
DOI: 10.1039/c6sc01140c
www.rsc.org/chemicalscience
protoporphyrin IX (PROTO IX).8 In contrast, during the alternative
pathway UROGEN III is converted to precorrin-2 which is subse-
Introduction
quently oxidized to sirohydrochlorin. The insertion of iron leads
to the formation of siroheme which is then decarboxylated to
12,18-didecarboxysiroheme. This intermediate is transformed
into iron-coproporphyrin III (Fe-COPRO III) which is nally con-
verted to heme.6,7
The last reaction in the alternative heme biosynthesis
pathway, i.e. the conversion of Fe-COPRO III to heme, is cata-
lyzed by the heme synthase AhbD (alternative heme biosyn-
thesis enzyme D) which is, so far, scarcely characterized.9
During the reaction the two propionate side chains at positions
C3 and C8 of Fe-COPRO III are oxidatively decarboxylated to the
corresponding vinyl groups (Scheme 1).
In almost all living organisms heme serves as an essential cofactor
in enzymes, redox proteins involved in electron transport chains
or gas sensing regulators. Depending on the organism, heme is
either acquired from external sources or synthesized by the
organism itself. In eukaryotes and most bacteria, heme is formed
via the so-called classical biosynthesis pathway, whereas in
sulfate-reducing bacteria and archaea an alternative route is
operative.1–7 In both pathways heme is synthesized from the
common precursor uroporphyrinogen III (UROGEN III). During
the classical route, heme is then formed via the intermediates
coproporphyrinogen
III,
protoporphyrinogen
IX,
and
AhbD belongs to the large family of Radical SAM (RS)
enzymes. As such, the amino acid sequence of AhbD contains
a typical cysteine-rich motif at the N-terminus (CX3CX2C) which
provides three cysteine ligands for the coordination of a catalyti-
cally essential [4Fe–4S]2+/1+ cluster present in all RS enzymes (RS
cluster).10 The fourth, non-cysteine ligated iron atom of this
cluster is coordinated by one molecule of S-adenosyl-L-methio-
nine (SAM) which is used to initiate radical catalysis.11 Thus, the
common reaction steps employed by all members of the RS
family consist of the initial reduction of the RS cluster to the
[4Fe–4S]1+ state and subsequent electron transfer onto the bound
SAM which results in the cleavage of the latter to methionine and
a
¨
Institute of Microbiology, Technische Universitat Braunschweig, Spielmannstr. 7,
38106 Braunschweig, Germany
b
¨
Institute of Inorganic and Analytical Chemistry, Technische Universitat Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
cInstitute of Biochemistry, Leipzig University, Bruderstraße 34, 04103 Leipzig,
¨
Germany. E-mail: gunhild.layer@uni-leipzig.de
d
¨
Max Planck Institute for Chemical Energy Conversion, Stistr. 34-36, 45470 Mulheim
an der Ruhr, Germany
† Electronic supplementary information (ESI) available: The cyclic voltammetry
experiments, the binding assays with the substrate analogs, the determination
of Kd values, the SAM cleavage assays with the substrate analogs, the
decarboxylase activity assays with the substrate analogs and the synthesis and
characterization of Zn-COPRO III. See DOI: 10.1039/c6sc01140c
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AhbD sequence. As described above, the two enzymes catalyze
the same reaction but use different substrates: an iron-porphyrin
in the case of AhbD in contrast to a porphyrinogen in the case of
HemN. Moreover, in contrast to HemN, AhbD was shown to
contain a second [4Fe–4S] cluster in addition to the RS cluster.9
Based on amino acid sequence alignments, it was proposed that
this auxiliary cluster might be coordinated by the cysteine resi-
dues present in an additional cysteine-rich motif (CX2CX5CX2-
CX17C) at the C-terminal end of AhbD.9 However, the function of
the auxiliary cluster of AhbD during catalysis is not known. It
might be involved in the binding or positioning of the substrate
Fe-COPRO III. Alternatively, it might be required for a late step
during catalysis such as electron transfer to a terminal electron
acceptor. Therefore, the aims of this study were to obtain novel
insights into the unknown function of the auxiliary [4Fe–4S]
cluster of AhbD and to investigate the late steps of AhbD catalysis
taking place aer substrate radical formation.
Scheme 1 Reaction catalyzed by AhbD.
a 50-deoxyadenosyl radical (DOAc). This radical then abstracts
a hydrogen atom from the actual substrate which is unique for
each RS enzyme.12 In the case of AhbD, it is assumed that the
hydrogen atom abstraction occurs at the b-carbon of the propi-
onate side chain of Fe-COPRO III leading to substrate radical
formation in analogy to the formation of the substrate radical
during HemN catalysis (see below).6,13 The substrate radical is
then decarboxylated to form the vinyl group of the reaction
product. Overall, this reaction takes place twice in order to obtain
both vinyl groups at C-atoms C3 and C8 of the end product heme.
Assuming that the decarboxylation step yields CO2, a terminal
electron acceptor is required for the overall reaction (Scheme 2).
Interestingly, the coproporphyrinogen III dehydrogenase
HemN, which also belongs to the RS enzyme family,10 performs
exactly the same reaction during the classical heme biosynthesis
pathway. However, HemN uses coproporphyrinogen III as its
substrate which is converted to protoporphyrinogen IX.14 The
three-dimensional structure of HemN was determined and the
rst steps of its catalytic mechanism up to substrate radical
formation (as described above) were elucidated.11,13 In contrast,
the nal steps of HemN catalysis are still uncharacterized. AhbD
from M. barkeri and HemN from Escherichia coli share only 17%
amino acid sequence identity within the 349 amino acids of the
Results and discussion
Production of single cluster AhbD variants
The rst aim of this study was to analyze the properties of the two
[4Fe–4S] clusters within AhbD from M. barkeri individually. For
this purpose, two single cluster variants were created in which
either the N-terminal RS cluster or the C-terminal auxiliary
cluster was missing. For the deletion of the RS cluster the two
cysteine residues Cys19 and Cys23 of the typical RS cysteine motif
C19X3C23X2C26 (numbers corresponding to the AhbD sequence
from M. barkeri) were exchanged against alanine by site directed
mutagenesis. The resulting AhbD variant was named AhbD
C19A/C23A. The auxiliary [4Fe–4S] cluster of AhbD was proposed
to be ligated by three or four cysteine residues of the additional
cysteine-rich motif C312X2C315X5C321X2C324X17C342.7,9 In
order to delete this C-terminal cluster, the two cysteine residues
Cys321 and Cys324 were exchanged against alanine resulting in
the formation of the AhbD variant C321A/C324A. The recombi-
nant wild type (wt) AhbD and the two single cluster variants were
produced as fusion proteins carrying an N-terminal His6-tag and
puried aerobically via immobilized metal ion affinity chroma-
tography (IMAC). Aer the purication of the proteins, an in vitro
iron–sulfur cluster reconstitution was performed under anaer-
obic conditions. The puried, reconstituted wt AhbD contained
7.6 mol iron and 7.5 mol sulde per mol monomeric AhbD in
agreement with the presence of two [4Fe–4S] clusters. In contrast,
the iron and sulde contents of the puried and reconstituted
single cluster variant AhbD C19A/C23A were only 54% and 55%,
respectively, compared to the wt protein. Similarly, the enzyme
variant AhbD C321A/C324A exhibited iron and sulde contents of
43% and 51%, respectively, compared to the wt protein. These
results suggested that in each AhbD variant one [4Fe–4S] cluster
binding site was successfully eliminated.
Characterization of the individual [4Fe–4S] clusters by EPR
spectroscopy
In order to further characterize the two [4Fe–4S] clusters indi-
Scheme 2 Initial reaction steps of AhbD catalysis.
vidually and to verify their structural integrity in the single cluster
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variants, EPR spectroscopy was performed. Aer dithionite
reduction, EPR spectra were recorded for wt AhbD and the two
single cluster variants (Fig. 1). The measurement of each variant,
AhbD C19A/C23A and AhbD C321A/C324A, showed a typical
spectrum of a single [4Fe–4S]1+ cluster with g ¼ 2.0525(5),
1.928(1), 1.903(1) for AhbD C19A/C23A and g ¼ 2.039(1), 1.912(1),
1.896(1) for AhbD C321A/C324A. The sum of the two individual
spectra of the AhbD variants equals the spectrum of wt AhbD
conrming the presence of two distinct [4Fe–4S]2+/1+ clusters
within AhbD, one located at the N-terminus and the other at the
C-terminus of the enzyme.
Both [4Fe–4S] clusters of AhbD are essential for its heme
synthase activity
The ability of puried, reconstituted wt AhbD and the two single
cluster variants to convert Fe-COPRO III into heme was tested
using an established in vitro enzyme activity assay with sodium
dithionite (DT) as the reducing agent for the initial reduction of
the RS cluster.7 Aer incubation of the assay mixtures their
tetrapyrrole content was analyzed by HPLC. As shown in Fig. 2,
heme was formed in the presence of wt AhbD in the activity
assay. In this case, the substrate Fe-COPRO III was completely
ꢀ
consumed aer 24 h of incubation at 17 C. In addition to the
nal reaction product heme, another iron-porphyrin was
detected (retention time of about 30.1 min) most likely repre-
Fig. 2 Heme synthase activity of wt AhbD and the AhbD variants. The
tetrapyrrole content in the assay mixtures was analyzed after 24 h of
incubation at 17 ꢀC by HPLC. The elution of the tetrapyrroles was
monitored by measuring the absorption at 400 nm (solid line) and the
fluorescence (dotted line, excitation at 409 nm, emission at 630 nm).
Fe-COPRO III eluted at 20.9 min, the potential monovinyl intermediate
at 30.1 min and heme at 37.5 min.
senting
a
monovinyl reaction intermediate (Fe-harder-
oporphyrin or Fe-isoharderoporphyrin) as observed previously.9
In contrast to the wt enzyme, both AhbD variants, C19A/C23A
and C321A/C324A, were unable to convert Fe-COPRO III into
detectable amounts of heme under identical reaction condi-
tions (Fig. 2). Moreover, in both cases no monovinyl reaction
intermediate was observed. Therefore, the deletion of either one
of the two [4Fe–4S] clusters apparently abolished the ability of
AhbD to decarboxylate the two propionate side chains of Fe-
COPRO III to the corresponding vinyl groups. This result
showed that both [4Fe–4S] clusters are essential for the overall
heme synthase activity of AhbD from M. barkeri.
For the AhbD variant C19A/C23A this result was not
surprising. This variant lacked the N-terminal RS cluster and
was therefore unable to initiate SAM cleavage and radical
catalysis. The reason for the lack of heme synthase activity of
variant C321A/C324A was less obvious. Possibly, the C-terminal
cluster might also be involved indirectly in SAM cleavage.
Alternatively, it might be required for substrate binding or it
could play an essential role for a late step during catalysis.
These different hypotheses for the role of the auxiliary cluster of
AhbD were tested and the obtained results are described in the
following sections.
The auxiliary cluster of AhbD is not required for SAM cleavage
As described above, both [4Fe–4S] clusters of AhbD are required
for the overall heme synthase activity of the enzyme. However,
the reaction sequence of AhbD can be dissected into at least two
observable parts: (i) the reductive cleavage of SAM and (ii) the
overall formation of heme. The homolytic cleavage of SAM
results in the formation of methionine and the 50-
Fig. 1 EPR spectra of the [4Fe–4S]1+ clusters in AhbD. The spectra
were recorded at 10 K for (a) wt AhbD, (c) AhbD C321A/C324A and
(d) AhbD C19A/C23A. The trace in (b) represents the sum of (c) and (d).
Experimental parameters: field modulation
frequency ¼ 9.6428 GHz. Power 0.2 mW.
8 Gauss. Microwave
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deoxyadenosyl radical which is quenched by the abstraction of even without the substrate, but was enhanced about ten-fold in
a hydrogen atom from the substrate. This rst part of the AhbD the presence of Fe-COPRO III.9 Since for AhbD from M. barkeri
reaction sequence can be followed by the HPLC analysis of the the formation of DOA is dependent on the presence of the
SAM cleavage product 50-deoxyadenosine (DOA). In vitro, some substrate, the SAM cleavage assay can be used as an indirect
Radical SAM enzymes such as RlmN and HemN require the indicator for the binding of Fe-COPRO III to the enzyme.
presence of their substrate in order to be able to cleave SAM,15,16 Consequently, in the following, we assume that if DOA is
whereas for others, like HydE and HydG, the substrate is not formed, the substrate is bound in the active site of AhbD.
necessary for SAM cleavage.17 In the latter case, the 50-deoxy-
adenosyl radical is probably quenched by hydrogen abstraction their ability to cleave SAM in the presence of Fe-COPRO III. As
from the solvent or the enzyme itself. expected, the AhbD variant C19A/C23A was unable to cleave
Next, the two single cluster variants of AhbD were tested for
Here, we rst tested whether wt AhbD from M. barkeri SAM conrming the role of the N-terminal [4Fe–4S] cluster as
requires the presence of its substrate Fe-COPRO III for SAM the RS cluster of AhbD. In contrast, the AhbD variant C321A/
cleavage. As shown in Fig. 3, no DOA was formed in the SAM C324A lacking the C-terminal cluster was able to cleave SAM
cleavage assay conducted without Fe-COPRO III. In contrast, in comparable to the wt enzyme (Fig. 3). Therefore, the auxiliary
the presence of Fe-COPRO III in the reaction mixture the C-terminal [4Fe–4S] cluster is apparently not required for the
formation of DOA was observed by HPLC analysis. Thus, in the rst part of the AhbD reaction sequence up to the step of
case of AhbD from M. barkeri the substrate Fe-COPRO III is substrate radical formation. Instead, it must full an essential
required for SAM cleavage in vitro. This observation is in line function during the late steps of AhbD catalysis since it is
with the previous description of the SAM cleavage activity of the absolutely required for overall heme formation (see above).
homologous AhbD protein from Desulfovibrio vulgaris. In this Alternatively, the C-terminal cluster might also be required for
case, SAM cleavage was observed to take place to a small extent substrate binding. However, this possibility seems less likely
since our SAM cleavage assay which also serves as an indicator
for substrate binding was positive for the enzyme variant
C321A/C324A. Nevertheless, in order to further characterize the
ability of the two single cluster variants to bind Fe-COPRO III,
a substrate binding assay using UV-visible absorption spec-
troscopy was performed.
Neither of the iron–sulfur clusters is required for substrate
binding
For the substrate binding assay an Fe(III)-COPRO III solution
was mixed with either wt AhbD, AhbD C19A/C23A or AhbD
C321A/C324A, and UV-visible absorption spectra were recorded
immediately aer mixing and aer 270 s incubation time.
Additionally, the Fe(III)-COPRO III solution was also mixed with
either buffer alone or alcohol dehydrogenase (Sigma-Aldrich) as
a control, and UV-visible absorption spectra were recorded aer
the same time intervals as for the mixtures containing AhbD
(Fig. 4). The Soret band of monomeric Fe(III)-COPRO III in buffer
was recorded at 382 nm while the second peak at about 350 nm
indicated that the substrate solution also contained a fraction
of dimeric Fe(III)-COPRO III.18 When no protein was added to
this substrate solution (buffer control) the absorption spectrum
of Fe(III)-COPRO III remained unchanged within the time frame
of the measurements and even aer prolonged incubation for
24 h. In contrast, the addition of wt AhbD led to an immediate
shi of the Soret band of the monomeric form of the substrate
to 390 nm indicating the binding of Fe(III)-COPRO III to the
enzyme. Aer 270 s of incubation the peak was further shied to
392 nm. Finally, aer 24 h of incubation the Soret band of the
enzyme-bound substrate displayed its maximum at 396 nm.
Essentially the same spectral changes were observed aer
the addition of either AhbD C19A/C23A or AhbD C321A/C324A
to the substrate solution with only insignicant variations of the
Fig. 3 SAM cleavage activity of wt AhbD and the AhbD variants. The
formation of the SAM cleavage product DOA was followed by HPLC
initial wavelength shis. Additionally, the absorption spectra
measured aer 24 h of incubation clearly showed that both
analysis. The elution of SAM and DOA was monitored by measuring the
absorption at 254 nm. SAM eluted at 12.3 min and DOA at 18.6 min.
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The redox potential of the auxiliary [4Fe–4S] cluster of AhbD is
compatible with a role in electron transfer
The redox potentials of the two [4Fe–4S] clusters within AhbD
were determined for the wt protein and the two variants C19A/
C23A and C321A/C324A either in the absence or presence of
SAM and the substrate Fe-COPRO III. All electrochemical
measurements of the proteins showed a quasi-reversible
reduction of the [4Fe–4S]2+/1+ clusters with half-wave potentials
in the range of ꢂ340 to ꢂ400 mV (ESI Fig. C†), which is common
for proteins containing such clusters.19,20 The half-wave poten-
tials of the individual clusters were determined to be at
ꢂ344 mV for the RS cluster (AhbD C321A/C324A) and at
ꢂ359 mV for the auxiliary cluster (AhbD C19A/C23A) in the
absence of SAM. For both clusters these potentials were not
signicantly altered in the presence of SAM (ꢂ343 mV for the RS
cluster and ꢂ366 mV for the auxiliary cluster). The cyclic vol-
tammogram of the wt AhbD, which contains both iron–sulfur
clusters, showed one single reduction process at ꢂ371 mV in
the absence of SAM. This nding indicates that there is no
electronic interaction between the two iron–sulfur clusters and
such an electrochemical behavior was also described for other
proteins, which contain two cuboidal [4Fe–4S] units.21 More-
over, this observation is in agreement with the reported
reduction potential determined for AhbD from D. vulgaris, for
which also one single reduction potential at ꢂ390 mV was
measured.9 Further measurements of wt AhbD in the presence
of added SAM did not result in a signicantly shied potential
(ꢂ396 mV). Also, cyclic voltammograms of AhbD in the pres-
ence of Fe-COPRO III displayed traces representing the sum of
the single compounds without any noticeable shis in the
potentials of the iron–sulfur clusters (ESI Fig. C†). In summary,
these experiments showed that not only the RS cluster is redox
active, but also the C-terminal auxiliary cluster supporting its
putative function in electron transfer during AhbD catalysis.
One possible scenario for the electron transfer events during
the AhbD catalytic cycle is depicted in Scheme 3 and ESI Fig. D.†
Aer the formation of the substrate radical, the single electron
is delocalized over the iron-porphyrin ring system and the
central metal ion facilitating the decarboxylation of the propi-
onate side chain. Assuming that CO2 is the product of the
decarboxylation event, the vinyl group formation is accompa-
nied by the reduction of the central iron ion to the Fe(II) state.
Then, the Fe(II)-harderoporphyrin might transfer one electron
to the auxiliary iron–sulfur cluster which in turn would pass the
electron further on to an external, terminal electron acceptor.
The resulting Fe(III)-harderoporphyrin could then serve as the
substrate for the second decarboxylation reaction proceeding by
a mechanism identical to the one for the rst decarboxylation
aer release of methionine and DOA and rebinding of a new
SAM. Thus, the role of the C-terminal [4Fe–4S] cluster would be
to accept an electron from the reduced reaction intermediate
and to pass it on to a terminal, so far unknown electron
acceptor.
Fig. 4 Substrate binding assay for wt AhbD and the AhbD variants. The
binding of Fe-COPRO III to wt AhbD and the AhbD variants was fol-
lowed by UV-visible absorption spectroscopy. Fe-COPRO III was
mixed with either buffer or the indicated proteins and spectra were
recorded (a) directly after mixing (0–100 s), after 270 s (270–370 s) and
(b) after 24 h. The observed shift of the Soret band upon addition of
AhbD is indicated. An UV-visible absorption spectrum of purified wt
AhbD alone is shown in ESI Fig. A† for comparison.
AhbD variants were able to bind the substrate comparable to the
wt enzyme. In contrast, no shi of the Soret band of monomeric
Fe(III)-COPRO III was observed aer the addition of alcohol
dehydrogenase to the substrate solution even aer 24 h of
incubation.
In order to quantify the binding of the Fe(III)-COPRO III to wt
AhbD and the two AhbD cluster variants, the dissociation
constants (Kd) were determined by the measurement of the
quenched tryptophan uorescence of AhbD upon substrate
binding (ESI Fig. B†). Using this method, a Kd value of 14.2 ꢁ
1.9 mM was determined for the wt AhbD. Similar Kd values of
15.3 ꢁ 1.8 mM for AhbD C19A/C23A and 11.2 ꢁ 1.2 mM for AhbD
C321A/C324A were obtained. Thus, all three AhbD proteins bind
the substrate with similar affinity.
Altogether, these experiments demonstrate that Fe(III)-
COPRO III binds specically to AhbD from M. barkeri and that
both single cluster variants are still able to bind the substrate
with affinities comparable to that of the wt protein. Therefore,
concerning the role of the auxiliary C-terminal [4Fe–4S] cluster
we conclude that it is not required for substrate binding as
already suggested by the results of the SAM cleavage assay
described above. Since the auxiliary cluster of AhbD is not
required for substrate binding and SAM cleavage, it might be
involved in a late step of the AhbD reaction sequence such as
the transfer of an electron from the substrate radical onto
a terminal electron acceptor. In order to investigate whether
such a role in electron transfer is feasible, the redox potentials
of the individual iron–sulfur clusters within AhbD were
measured by cyclic voltammetry.
In order to test whether an electron transfer from a reduced
iron-porphyrin to the C-terminal cluster (ꢂ359 mV) is feasible,
we also determined the half-wave potential of Fe-COPRO III and
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Scheme 3 Proposed reaction mechanism for AhbD.
heme (haemin) by cyclic voltammetry. It can be assumed that substrate analogs. For the metal-free COPRO III the Soret band
the redox potential of Fe-harderoporphyrin, which was not was shied from 397 nm for the unbound form to 406 nm for
available for measurements, lies between those of Fe-COPRO III the AhbD-bound form. For the Cu-COPRO III the shi of the
and heme. In the absence or presence of AhbD half-wave Soret band upon AhbD addition was from 379 nm to 398 nm.
potentials in the range of ꢂ650 mV to ꢂ659 mV were deter- Finally, the Soret band of free Zn-COPRO III at 403 nm was
mined for Fe-COPRO III. The half-wave potential of haemin in shied to 410 nm in the presence of AhbD.
buffer (ESI Fig. E†) was determined at ꢂ609 mV. Therefore,
In addition to the binding assays using UV-visible absorption
assuming that the redox potential of Fe-harderoporphyrin lies spectroscopy, we also performed SAM-cleavage assays of AhbD
between ꢂ659 mV and ꢂ609 mV, the proposed electron transfer in the presence of the substrate analogs to test their binding to
from the monovinyl Fe(II)-porphyrin intermediate to the auxil- the enzyme. As described above, the cleavage of SAM only
iary cluster is possible.
occurs when the substrate is bound to AhbD. In the presence of
For the proposed reaction mechanism including the revers- all three substrate analogs SAM was cleaved and DOA was
ible reduction/oxidation of the central metal ion of the detected by HPLC analysis (ESI Fig. G†). However, for COPRO III
porphyrin substrate/intermediate, the central iron plays and Cu-COPRO III the SAM cleavage activity seemed to be a little
a crucial role. In order to further substantiate the proposed bit lower than with Fe-COPRO III and the SAM cleavage activity
mechanism and to investigate the role of the central iron ion, in the presence of Zn-COPRO III was even more impaired,
alternative substrates were used for AhbD activity assays. These although still detectable.
substrate analogs contained either no metal at all (cop-
In order to test, whether these slight differences in SAM
roporphyrin III (COPRO III)) or Cu (Cu-COPRO III) or Zn (Zn- cleavage in the presence of the substrate analogs as compared
COPRO III). Before testing the AhbD activity with these to the SAM cleavage in the presence of the true substrate were
substrates, we tested whether the enzyme was able to bind the due to differences in the binding affinities, the Kd values for the
substrate analogs.
substrate analogs and wt AhbD were determined by the
measurement of tryptophan uorescence quenching (ESI
Fig. H†). For COPRO III a Kd of 33.9 ꢁ 3.9 mM, for Cu-COPRO III
a Kd of 56 ꢁ 3.5 mM and for Zn-COPRO III a Kd of 38.8 ꢁ 2.4 mM
was determined. Thus, all three substrate analogs bind with
slightly lower affinity to the wt AhbD than the true substrate
according to the two-fold higher Kd values for COPRO III and
Zn-COPRO III and the four-fold higher Kd for Cu-COPRO III. The
somewhat higher Kd values might explain the slightly lower SAM
cleavage activity in the presence of COPRO III and Cu-COPRO
AhbD binds the substrate analogs COPRO III, Cu-COPRO III
and Zn-COPRO III
The binding of the substrate analogs to AhbD was tested under
identical conditions as for the true substrate Fe-COPRO III. A
shi of the Soret band to longer wavelengths upon addition of
the enzyme was taken as an indication for the formation of an
enzyme–substrate complex. As shown in Fig. 5 and ESI Fig. F,†
such a shi of the Soret band was observed for all three
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III, but do not explain the low SAM cleavage activity in the
presence of Zn-COPRO III. Here, a slightly different orientation
of the substrate analog within the active site might be respon-
sible for the inefficient SAM cleavage.
Nevertheless, all three methods, UV-visible absorption
spectroscopy, SAM-cleavage assays and uorescence quenching,
showed that AhbD was able to bind all three articial substrates
and, therefore, enzyme activity assays employing these mole-
cules were performed.
AhbD displays very low decarboxylase activity with the
substrate analogs COPRO III, Cu-COPRO III and Zn-COPRO III
To investigate the inuence of the central iron ion of Fe-COPRO III
on the activity of AhbD, an enzyme activity assay using COPRO III
instead of Fe-COPRO III was performed. For comparison, the
AhbD assay was performed under the same assay conditions
using the true substrate Fe-COPRO III. As already described
above, for the assay containing Fe-COPRO III the substrate was
completely consumed aer 24 h of incubation and converted to
heme or at least the monovinyl intermediate (Fig. 6 and 2). In
contrast, in the assay mixture containing COPRO III there was
still a high amount of COPRO III (retention time around
25.6 min) present aer the same incubation time. However,
minor amounts of PROTO IX (retention time 40.5 min) and
a potential monovinyl intermediate (retention time 34.5 min)
were also detected. These reaction products were not found in
a control assay mixture lacking AhbD. Therefore, AhbD was able
to convert COPRO III to the corresponding monovinyl inter-
mediate and the product PROTO IX; however, the activity of
AhbD with the articial substrate was very low compared to its
activity in the presence of the true substrate Fe-COPRO III.
Since the binding affinity of COPRO III to AhbD is not
drastically different from that of Fe-COPRO III (only two-fold
higher Kd) and SAM cleavage is clearly taking place in the
presence of COPRO III, this result indicates that the central iron
ion of the porphyrin substrate indeed plays a crucial role for the
efficient conversion of the propionate side chains to the corre-
sponding vinyl side chains by AhbD.
In order to further substantiate this idea, the same AhbD
activity assay was also performed using Cu-COPRO III and Zn-
COPRO III. As shown in ESI Fig. I† similar results were obtained
as in the assay with COPRO III. In both cases a very low decar-
boxylase activity of AhbD was observed since low amounts of
monovinyl intermediates and the reaction products Cu-PROTO IX
and Zn-PROTO IX (detected as PROTO IX due to extraction
conditions) were detected by HPLC analysis. These reaction
products were not found in the respective assay mixtures lack-
ing AhbD. However, as in the case for the assay with COPRO III
and in contrast to the assay with Fe-COPRO III, the major
amount of the two articial substrates was not consumed aer
24 h showing that they served only as very poor substrates for
AhbD. In the case of the Zn-COPRO III the product formation is
comparable to that of the assay with COPRO III despite the
seemingly lower SAM cleavage activity. For Cu-COPRO III
product formation is hardly detectable at all. Again, these very
low decarboxylase activities of AhbD in presence of the
Fig. 5 Binding assay for wt AhbD using different substrate analogs.
The binding of (a) Fe-COPRO III, (b) COPRO III, (c) Cu-COPRO III and
(d) Zn-COPRO III to wt AhbD was followed by UV-visible absorption
spectroscopy. The spectra were recorded after 24 h of incubation after
mixing the protein with the respective substrate analog. The shift of the
Soret bands upon AhbD addition is indicated.
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a 50-deoxyadenosyl radical and methionine thereby initiating
radical catalysis. In AhbD from M. barkeri a second, auxiliary
[4Fe–4S] cluster is present, coordinated via cysteine residues at
the C-terminal end of the protein. Like for several other RS
enzymes containing auxiliary [4Fe–4S] clusters, the overall
enzyme activity of AhbD is lost upon deletion of the auxiliary
cluster. The functions of the auxiliary clusters in the various RS
SAM enzymes are diverse and include roles in substrate binding
and orientation, sulfur donation and electron transfer.22 In the
case of AhbD, a role in sulfur donation is irrelevant and a role in
substrate binding could be excluded by the experiments in this
study. However, based on the results described here, we propose
a role of the auxiliary [4Fe–4S] cluster in electron transfer from
the reaction intermediate to a terminal, so far unknown elec-
tron acceptor. Furthermore, we observed that the central iron
ion of the Fe-COPRO III substrate of AhbD participates in this
electron transfer process. Therefore, we obtained new insights
into the reaction steps that follow the initial formation of the
substrate radical during AhbD catalysis. Obviously, these steps
differ from those (still uncharacterized) occurring during HemN
catalysis which catalyzes the identical reaction but uses
a different substrate (porphyrinogen without metal ion) and
does not contain an auxiliary iron–sulfur cluster. Nevertheless,
both enzymes require a terminal electron acceptor for the
oxidative decarboxylation of the propionate side chains which
in both cases also remains to be identied in the future.
Fig. 6 Decarboxylase activity of wt AhbD with COPRO III as the
substrate. The tetrapyrrole content in the assay mixtures was analyzed
after 24 h of incubation at 17 ꢀC by HPLC. The elution of the tetra-
pyrroles was monitored by measuring the absorption at 400 nm (solid
line) and the fluorescence (dotted line, excitation at 409 nm, emission
at 630 nm). Fe-COPRO III eluted at 20.9 min, the potential monovinyl
intermediate at 30.1 min and heme at 37.5 min (top panel). COPRO III
eluted at 25.7 min, the corresponding monovinyl intermediate at
34.5 min and PROTO IX at 40.5 min (lower panels). The respective
monovinyl intermediates are highlighted by an asterisk.
Experimental section
Chemicals
All chemicals and reagents were obtained from Sigma-Aldrich
(Tauirchen, Germany), Carl Roth (Karlsruhe, Germany),
Gerbu (Heidelberg, Germany), Merck (Darmstadt, Germany).
DNA polymerases, restriction enzymes, PCR requisites as well as
the Q5 Site-Directed Mutagenesis Kit were purchased from New
England Biolabs (Frankfurt a. M., Germany). Oligonucleotide
primers and plasmid-miniprep Kits were purchased from
Metabion (Martinsried, Germany). PCR purication and gel
extraction Kits were obtained from Qiagen (Hilden, Germany).
substrate analogs cannot be explained by their lower binding
affinity alone.
Overall, the experiments with the substrate analogs support
the hypothesis that the central iron ion of the substrate Fe-
COPRO III participates in catalysis most likely as an electron
relay ultimately transferring the electron from the reaction
intermediate to the auxiliary [4Fe–4S] cluster. In our current
activity assay, the reductant for the RS cluster in order to initiate
catalysis is sodium dithionite which is commonly used for
activity assays of Radical SAM enzymes. However, in the pres-
ence of sodium dithionite the auxiliary cluster as well as the
substrate itself are most likely also present in their reduced
forms. Considering our proposed reaction mechanism this
might be a reason why the reaction catalyzed by AhbD is quite
slow in vitro. Therefore, in the particular case of AhbD, sodium
dithionite should be replaced by a physiological electron donor
such as avodoxin or ferredoxin in future experiments.
¨
Ni-NTA agarose was purchased from Macherey-Nagel (Duren,
Germany). Porphyrins were all purchased from Frontier Scien-
tic (Logan, UT, USA) except of Zn-coproporphyrin III. The for
E. coli codon-optimized gene Mbar_A1458 from M. barkeri was
synthesized by Life Technologies GmbH (Darmstadt, Germany).
Construction of plasmids
For the construction of plasmid pETDuetahbD/HIS the codon-
optimized gene Mbar_A1458 (encoding AhbD) was PCR amplied
using the primers NirJ1_Duet_BamHI_fw (G GAT CCG ATG ATT
GCC ATG ACC) and NirJ1_Duet_HindIII_bw (GAA GCT TTT ATT
TTT TAC CCG GAC GGT) containing BamHI and HindIII restric-
tion sites (underlined) and rst cloned into the pJET1.2/blunt
cloning vector (Thermo Scientic) according to the manufactur-
Conclusions
All RS enzymes contain at least one [4Fe–4S] cluster coordinated er's instructions. Mbar_A1458 was then cut from this plasmid
by three cysteine residues which is able to cleave SAM into with BamHI and HindIII endonucleases and the resulting DNA
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fragment was ligated into the correspondingly digested vector
pETDuet-1 (Novagen) generating pETDuetahbD/HIS.
For the construction of plasmid pETDuetahbDp.C19A/C23A/
HIS plasmid pETDuetahbD/HIS was used as template for
mutagenesis with the Q5 Site-Directed Mutagenesis Kit (New
England Biolabs) using the primers nebN_NirJ1 (CAT GCT CGT
GGT GCA AGC ACC AG) and nebN_NirJ1_anti (AAC AGC ATT
CAG ATT ACA ACC TGC GGT C) (introduced mutations
underlined).
For the construction of plasmid pETDuetahbDp.C321A/
C324A/HIS plasmid pETDuetahbD/HIS was used as template for
mutagenesis with the Q5 Site-Directed Mutagenesis Kit (New
England Biolabs) using the primers nebC_NirJ1 (GGT GCT CGT
GCA CGT GCA TAT GC) and nebC_NirJ1_anti (TGC AGC AAC
TTT TTT ATA CTC GCA GAT GC) (introduced mutations
underlined).
In vitro iron–sulfur cluster reconstitution
The in vitro reconstitution of iron–sulfur clusters was performed
under anaerobic conditions in an anaerobic chamber (Coy
Laboratories, Grass Lake, MI, USA) as previously described.23 All
required solutions (1 M DTT, 20 mM ammonium iron citrate,
20 mM lithium sulde) were freshly prepared in buffer B.
10 equiv. (relative to the protein concentration) of DTT were
added to the protein solution and incubated for 1 h before the
addition of 10 equiv. of ammonium iron citrate. Aer incubation
for 5 min, 10 equiv. of lithium sulde were added slowly and the
mixture was incubated for 35 min. The excess of iron and sulde
was removed by centrifugation and subsequent passage of the
protein solution through a NAP-25 column (GE Healthcare)
which was used according to the manufacturer's instructions.
Determination of iron and sulde contents
The iron content of puried proteins was determined according
to Fish24 aer denaturation of the protein with 1 M perchloric
acid and bathophenanthroline as the chelating reagent. The
sulde content was determined as previously described.25
Bacterial strains and growth conditions
E. coli DH10B was used as the host for cloning. For production
of recombinant proteins, the E. coli strain BL21(DE3) was used.
For recombinant protein production the E. coli strains carrying
ꢀ
the corresponding vectors were grown at 37 C in LB-medium
Molecular mass determination
(aerobic growth conditions) supplemented with appropriate
antibiotics. Protein production was induced by adding 500 mM
IPTG (isopropyl-b-D-thiogalactopyranoside) to the cultures as
soon as an optical density at 600 nm of 0.6 was reached. Aer
induction the cells were further cultivated at 25 ꢀC overnight
and then harvested by centrifugation. Cell pellets were stored at
The native molecular mass of puried proteins was estimated
by gel permeation chromatography as previously described.5
In vitro enzyme activity assay for AhbD
ꢀ
ꢂ20 C.
The assay was performed under anaerobic conditions in a glove
box (Coy Laboratories). Stock solutions of 500 mM Fe-COPRO III,
COPRO III and Cu-COPRO III were generated according to Chim
et al.26 For Zn-COPRO III a stock solution of about 200 mM was
prepared. The enzyme activity assay mixture contained either
20 mM of substrate or 20 mM of substrate analog, 500 mM SAM,
1 mM sodium dithionite (DT) and 5 mM puried, reconstituted
AhbD in buffer B. The reaction mixtures were incubated for 24 h
at 17 ꢀC. To stop the enzymatic reaction 5% (v/v) of concentrated
HCl were added to the mixture which was then stored at ꢂ20 ꢀC
until further use.
Purication of enzymes
Recombinant wild type AhbD (wt AhbD) from M. barkeri and the
AhbD variants (AhbD C19A/C23A and AhbD C321A/C324A) were
puried by IMAC. The cell pellet was resuspended in buffer A
(50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole) and
the cells were disrupted using a French Press system (1000 psi).
The soluble protein fraction was obtained by ultracentrifuga-
tion (60 min, 175 000 ꢃ g, 4 ꢀC). The supernatant was sonicated
twice using a Sonopuls HD 2070 (Bandelin, Berlin, Germany)
equipped with a KE 76 tip (1 min, 4 ꢃ 10%) and aerwards
loaded on a Ni-NTA column by gravity ow. The column was
washed with 10 column volumes (CV) of buffer A before eluting
the bound proteins with 6 CV of buffer A containing 300 mM
imidazole. The protein content of the elution fractions was
analyzed by SDS-polyacrylamide gel electrophoresis and the
fractions containing recombinant AhbD were pooled. The nal
buffer exchange against buffer A without imidazole (¼ buffer B)
was performed with a NAP-25 Sephadex column (Illustra NAP-
25, GE Healthcare). The puried proteins were stored at 4 ꢀC.
SAM cleavage assay
The conditions of the SAM cleavage assay were identical to those
of the enzyme activity assay described above except the
concentration of SAM which was used at a nal concentration of
1 mM.
Preparation of HPLC samples
HPLC samples for the analysis of tetrapyrroles within enzyme
activity assay mixtures and the corresponding HPLC standards
were prepared as described previously.7 For the preparation of
HPLC samples of SAM cleavage assays, the assay mixture con-
Determination of protein concentration
For the determination of protein concentrations the Bradford taining 5% (v/v) of concentrated HCl was mixed using a Vortex-
Reagent (Sigma-Aldrich) was used according to the manu- Genie 2 mixer (Scientic Industries Inc., Bohemia, NY, USA) for
facturer's instructions with BSA as the protein standard.
1 min followed by centrifugation (10 min, 16 100 ꢃ g).
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mixture was stirred at room temperature for 30 min. The
precipitated brown Zn-COPRO carboxylate zinc salt was collected
by centrifugation and washed multiple times with water. The
residue was again solved in 0.1 M NaOH. During that process zinc
hydroxide precipitated out and was removed by centrifugation.
To prepare the protonated Zn-COPRO III, the solution was acid-
ied with 0.1 M HCl. The precipitate was collected by centrifu-
gation, dissolved in methanol, ltered over Na2SO4 and the
solvent was evaporated to yield Zn-COPRO III as a purple solid
(6.5 mg, 9 mmol, 74%). 1H NMR (600 MHz, (CD3)2SO): d 12.35 (br.
s, 4H), 10.17 (s, 1H), 10.10 (s, 2H), 10.08 (s, 1H), 4.36–4.29 (m,
8H), 3.62 (s, 12H), 3.18 (m, 8H). 13C NMR (150 MHz, (CD3)2SO):
d 174.4 (2C), 174.2 (2C), 147.7, 147.6, 147.6, 147.5, 146.9 (2C),
146.8, 146.7, 139.6, 139.3, 136.7, 136.6, 136.5, 136.5, 97.2, 97.1
(2C), 97.0, 37.6 (2C), 37.4 (2C), 21.7 (2C), 21.6 (2C), 11.5 (4C).
HRMS (ESI): m/z ¼ 716.1822 [M]+ calcd for C36H36N4O8Zn+:
716.1819 (error ¼ 0.4 ppm). UV-Vis (MeOH): lmax/nm (3[rel]) ¼
406 (1), 538 (0.1), 574 (0.1). See also ESI Fig. J–M.†
High performance liquid chromatography of tetrapyrroles and
SAM
Tetrapyrrole extracts were analyzed using a JASCO HPLC 2000
series system (Jasco, Gross-Umstadt, Germany) as previously
described.7 The chromatographic separation of samples (20 mL
injection volume) containing SAM and 50-deoxyadenosine
(DOA) was performed on a HYPERCARB column (Thermo
Scientic, Dreieich, Germany) with 5 mm particle size and
100 mm ꢃ 2.1 mm column dimensions at 25 ꢀC. The
substances were eluted at a ow rate of 0.2 mL minꢂ1 using
a gradient system. Solvent A consisted of 0.1% triuoroacetic
acid (for UV-spectroscopy, Fluka) and solvent B of acetonitrile
(HPLC grade, Sigma-Aldrich) containing 0.08% triuoroacetic
acid (HPLC grade, Sigma-Aldrich). At the time of sample injec-
tion the mobile phase consisted of 100% solvent A. Within
25 min the content of solvent B was increased to 100% with
concomitant decrease of solvent A. Then, the content of solvent
B was held for 5 min before returning to the initial conditions.
SAM and DOA were detected by photometric diode array anal-
ysis at 200–650 nm.
Binding assay of different substrate analogs
The assay was performed as described above. The resulting
solutions contained a total amount of either 5 mM Fe-COPRO III,
5 mM COPRO III, 5 mM Cu-COPRO III, or 5 mM Zn-COPRO III and
20 mM wt AhbD, respectively. Mixtures containing the respective
substrate analog but without wt AhbD served as a control. Stock
solutions of Cu-COPRO III and Zn-COPRO III were prepared
according to Chim et al.26
EPR-spectroscopy
EPR samples were prepared under anaerobic conditions in
a glove box (Coy Laboratories). 400 mM of puried AhbD in
buffer B was mixed with 10 mM sodium dithionite in a total
volume of 300 mL. This solution was transferred to a 5 mm od
quartz EPR tube and frozen in liquid nitrogen aer 15 min of
incubation. CW EPR spectra were recorded on a Bruker Elexsys
E 500 X-band EPR spectrometer equipped with an Oxford
ESR900 helium ow cryostat. The EPR samples were accom-
modated in a dual mode rectangular resonator operating in the
perpendicular excitation mode. The EPR spectra were processed
using home-written Matlab scripts and analyzed using the
Easyspin Matlab package.27 The g-tensor values of the single
cluster mutants were evaluated using the least-squares tting
procedure “est” including the line-shape and g-strain models
supported by Easyspin.
Quantication of substrate binding by uorescence
quenching
Dissociation constants (Kd) for the affinity of Fe-COPRO III to wt
AhbD and the AhbD cluster variants C19A/C23A and C321A/
C324A as well as for COPRO III, Zn-COPRO III and Cu-COPRO III
to wt AhbD were determined by uorescence spectroscopy. The
quenching of the intrinsic tryptophan uorescence of AhbD upon
substrate or substrate analog addition was followed using
a JASCO spectrouorimeter FP-8500 (Jasco) with excitation at 295
nm and emission at 337 nm. The samples contained 1 mM AhbD
and increasing amounts of the substrate or substrate analogs
(0–300 mM) in a total volume of 50 mL of buffer B. The protein and
the substrate or substrate analog were mixed and incubated at
room temperature overnight prior to the uorescence measure-
ments. The uorescence values at 337 nm for the samples with
substrate/substrate analog were corrected by the subtraction of
the uorescence values for the corresponding samples contain-
ing only substrate/substrate analog. The difference in uores-
cence between the sample containing no substrate and the
corrected values for the samples containing increasing amounts
of substrate was plotted against the substrate concentration (see
ESI Fig. B and H†). The Kd values were then determined using
equation29
Fe-COPRO III binding assay
The assay was performed under anaerobic conditions in a glove
box (Coy Laboratories). Either puried AhbD in buffer B or
buffer B alone as a control was directly added to a diluted
Fe-COPRO III solution in a quartz SUPRASIL precision cell with
¨
10 mm light path (Hellma Analytics, Mullheim, Germany) and
mixed by pipetting. The resulting solution contained a total
amount of 5 mM Fe-COPRO III and 10 mM AhbD or only 5 mM
Fe-COPRO III without AhbD as a control. UV-visible absorption
spectra from 250–700 nm were recorded using a V-650 spec-
trophotometer (Jasco, Gross-Umstadt, Germany) immediately
aer mixing (0–100 s), aer 270 s (270–370 s) and aer 24 h.
Synthesis of Zn-COPRO III (zincphyrin28)
COPRO III dihydrochloride (Frontier Scientic, 9.0 mg,
12.3 mmol, 1.0 equiv.) was solved in 0.1 M NaOH (5 mL). Zinc
acetate dihydrate (11 mg, 50 mmol, 4.0 equiv.) was added and the and the soware OriginPro 8G (Originlab Corporation,
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¨
Northampton, MA, USA). [E] ¼ AhbD concentration, [S] ¼
substrate concentration, DF ¼ difference in uorescence at [S],
DF0 ¼ difference in uorescence at [S] ¼ 0, DFmax ¼ difference in
uorescence at substrate saturation.
7 M. Kuhner, K. Haufschildt, A. Neumann, S. Storbeck, J. Streif
and G. Layer, Archaea, 2014, 2014, 327637.
8 G. Layer, J. Reichelt, D. Jahn and D. W. Heinz, Protein Sci.,
2010, 19, 1137–1161.
9 S. A. Lobo, A. D. Lawrence, C. V. Romao, M. J. Warren,
M. Teixeira and L. M. Saraiva, Biochim. Biophys. Acta,
Proteins Proteomics, 2014, 1844, 1238–1247.
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N. E. Miller, Nucleic Acids Res., 2001, 29, 1097–1106.
11 G. Layer, J. Moser, D. W. Heinz, D. Jahn and W. D. Schubert,
EMBO J., 2003, 22, 6214–6224.
12 P. A. Frey, A. D. Hegeman and F. J. Ruzicka, Crit. Rev.
Biochem. Mol. Biol., 2008, 43, 63–88.
13 G. Layer, A. J. Pierik, M. Trost, S. E. Rigby, H. K. Leech,
Cyclic voltammetry
Cyclic voltammetry measurements were performed using an
Ametek Versastat 3. The measurements were carried out in
a self-made anaerobic three-electrode electrochemical cell
ushed with nitrogen. As the reference, a silver/silver-chloride
electrode was used (3 mol Lꢂ1 KCl). All potentials in the text and
gures are given vs. NHE (+210 mV). A platinum wire was used
as the counter electrode, with glassy carbon as the working
electrode. Before each measurement the platinum wire was
annealed in a natural gas ame and the glassy carbon electrode
was pretreated in nitric acid, neutralized, polished with 0.05 mm
alumina and annealed in a natural gas ame as described
before.30,31 For each experiment 20 cycles were recorded. The
potential slightly dried only over the rst 10 cycles and stabi-
lized thereaer. In this work, only the stabilized potential is
¨
K. Grage, D. Breckau, I. Astner, L. Jansch, P. Heathcote,
M. J. Warren, D. W. Heinz and D. Jahn, J. Biol. Chem.,
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¨
14 G. Layer, K. Verfurth, E. Mahlitz and D. Jahn, J. Biol. Chem.,
2002, 277, 34136–34142.
¨
15 F. Yan, J. M. LaMarre, R. Rohrich, J. Wiesner, H. Jomaa,
A. S. Mankin and D. G. Fujimori, J. Am. Chem. Soc., 2010,
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discussed. The cycles were recorded with a scan rate of 1 V sꢂ1
.
All electrochemical experiments were carried out at ambient
temperature in a 100 mL drop. Samples were prepared under
anaerobic conditions in a glove box (Coy Laboratories) and
transferred into HPLC vials before injecting into the CV
chamber directly on the glassy carbon electrode. Samples con-
tained 120 mM AhbD, 500 mM SAM, 40 mM Fe-COPRO III, 40 mM
Zn-COPRO III, Cu-COPRO III or 40 mM heme in diverse
combinations in buffer B.
¨
16 G. Layer, K. Grage, T. Teschner, V. Schunemann, D. Breckau,
A. Masoumi, M. Jahn, P. Heathcote, A. X. Trautwein and
D. Jahn, J. Biol. Chem., 2005, 280, 29038–29046.
17 J. K. Rubach, X. Brazzolotto, J. Gaillard and M. Fontecave,
FEBS Lett., 2005, 579, 5055–5060.
18 E. Tipping, B. Ketterer and P. Koskelo, Biochem. J., 1978, 169,
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20 J. M. Blamey, M. Chiong, C. Lopez and E. T. Smith, Anaerobe,
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Acknowledgements
The authors would like to thank Prof. Dr Dieter Jahn and Dr
21 F. A. Armstrong, H. A. O. Hill and N. J. Walton, Acc. Chem.
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22 N. D. Lanz and S. J. Booker, Biochim. Biophys. Acta, Proteins
Proteomics, 2012, 1824, 1196–1212.
¨
¨
Jurgen Moser (Technische Universitat Braunschweig, Germany)
for helpful discussions. This work was supported by grants from
the Deutsche Forschungsgemeinscha to MB (BR 2010/12-1)
and GL (LA 2412/5-1).
¨
¨
23 L. Fluhe, T. A. Knappe, M. J. Gattner, A. Schafer,
O. Burghaus, U. Linne and M. A. Marahiel, Nat. Chem.
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