Báo cáo khoa học: Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli pdf

Heavy metal ions inhibit molybdoenzyme activity by
binding to the dithiolene moiety of molybdopterin in
Escherichia coli
Meina Neumann and Silke Leimku
¨
hler
Department of Proteinanalytics, Institute of Biochemistry and Biology, University of Potsdam, Germany
Molybdenum is the only second-row transition metal
that is required by most living organisms, and the few
species that do not require molybdenum use tungsten,
which lies immediately below molybdenum in the Peri-
odic Table [1,2]. Molybdenum- and tungsten-contain-
ing enzymes often catalyse the same types of reaction;
while molybdenum-containing enzymes are found in
all aerobic organisms, including humans, tungsten-
containing enzymes are found only in obligate, typi-
cally thermophilic, anaerobic bacteria and archaea
[3,4]. The molybdenum cofactor (Moco) is the essential
component of a group of redox enzymes which cata-
lyse a variety of transformations at carbon, sulfur and
nitrogen atoms. More than 40 molybdenum and tung-
sten enzymes have been identified in bacteria, archaea,
plants and animals to date [5,6]. Some of the better
known Moco-containing enzymes include sulfite oxi-
dase, xanthine dehydrogenase and aldehyde oxi-
dase in humans [6], assimilatory nitrate reductase in
plants [7] and dissimilatory nitrate reductase,
Keywords
copper; dithiolene; metal toxicity; Moco;
molybdenum
Correspondence

S. Leimku
¨
hler, Institute of Biochemistry and
Biology, University of Potsdam, D-14476
Potsdam, Germany
Fax: +49 331 977 5128
Tel: +49 331 977 5603
E-mail:
(Received 15 July 2008, revised
3 September 2008, accepted
19 September 2008)
doi:10.1111/j.1742-4658.2008.06694.x
Molybdenum insertion into the dithiolene group on the 6-alkyl side-chain
of molybdopterin is a highly specific process that is catalysed by the MoeA
and MogA proteins in Escherichia coli. Ligation of molybdate to
molybdopterin generates the molybdenum cofactor, which can be inserted
directly into molybdoenzymes binding the molybdopterin form of the
molybdenum cofactor, or is further modified in bacteria to form the dinu-
cleotide form of the molybdenum cofactor. The ability of various metals to
bind tightly to sulfur-rich sites raised the question of whether other metal
ions could be inserted in place of molybdenum at the dithiolene moiety of
molybdopterin in molybdoenzymes. We used the heterologous expression
systems of human sulfite oxidase and Rhodobacter sphaeroides dimethylsulf-
oxide reductase in E. coli to study the incorporation of different metal ions
into the molybdopterin site of these enzymes. From the added metal-con-
taining compounds Na
2
MoO
4
,Na

2
WO
4
, NaVO
3
, Cu(NO
3
)
2
, CdSO
4
and
NaAsO
2
during the growth of E. coli, only molybdate and tungstate were
specifically inserted into sulfite oxidase and dimethylsulfoxide reductase.
Other metals, such as copper, cadmium and arsenite, were nonspecifically
inserted into sulfite oxidase, but not into dimethylsulfoxide reductase. We
showed that metal insertion into molybdopterin occurs beyond the step of
molybdopterin synthase and is independent of MoeA and MogA proteins.
Our study shows that the activity of molybdoenzymes, such as sulfite
oxidase, is inhibited by high concentrations of heavy metals in the cell,
which will help to further the understanding of metal toxicity in E. coli.
Abbreviations
FeVco, iron–vanadium cofactor; hSO, human sulfite oxidase; hSO-MD, human sulfite oxidase Moco domain; ICP-OES, inductively coupled
plasma-optical emission spectrometry; MGD, molybdopterin guanine dinucleotide cofactor; Moco, molybdenum cofactor; MPT,
molybdopterin; Wco, tungsten cofactor.
5678 FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS
dimethylsulfoxide reductase and formate dehydroge-
nase in bacteria [8–10].

Moco biosynthesis has been studied extensively in
Escherichia coli using a combination of biochemical,
genetic and structural approaches [11,12], and addi-
tional insights have been provided by studies in
eukaryotes [13]. The biosynthesis of Moco can be
divided into three general steps in all organisms: (a)
formation of precursor Z from GTP; (b) formation of
molybdopterin (MPT) by insertion of two sulfur atoms
into precursor Z; (c) insertion of molybdenum into the
dithiolene group of MPT, thus forming Moco. In bac-
teria, such as E. coli, an additional modification of
Moco occurs with the attachment of GMP to the
phosphate group of Moco, forming the MPT guanine
dinucleotide cofactor (MGD) [14,15]. In total, more
than 10 genes are involved in the biosynthesis of Moco
in E. coli, and highly conserved proteins have been
identified in other organisms.
Molybdenum enters the cell as the soluble oxyanion
molybdate, for which high-affinity molybdate trans-
porters have been described in bacteria [11,16] and in
higher eukaryotes [17,18]. In the step of molybdenum
insertion into MPT, the gene products of moeA and
mogA are involved in E. coli. It has been observed that
MoeA mediates molybdenum ligation to newly synthe-
sized MPT at low concentrations of molybdate, and
MogA helps to facilitate this step in vivo in an ATP-
dependent manner via an MPT-adenylate intermediate
[19,20]. The crystal structure of the Arabidopsis thali-
ana Cnx1 G protein, a homologue of E. coli MogA,
shows that copper is bound to the MPT dithiolene

sulfurs of the MPT–AMP complex [21]. It has been
proposed that copper binding to MPT–AMP on Cnx1
is physiologically relevant and that, in vivo, copper
may serve to protect the dithiolene moiety prior to the
binding of molybdenum. However, as several metal
ions are known to bind tightly to sulfur-rich sites, a
more recent report by Morrison et al. [22] investigated
the effect of copper-limiting reaction conditions on
molybdoenzymes in E. coli and Rhodobacter sphaero-
ides. Their results demonstrated that the activities of
dimethylsulfoxide reductase and nitrate reductase were
not repressed under copper starvation, showing that
copper is not strictly required for the biosynthesis of
Moco in bacteria [22].
In addition to copper, various metals are known to
bind tightly to sulfur-rich sites, leading to the question
of whether other metal ions can bind to the dithiolene
moiety of Moco and be inserted into molybdoenzymes.
Some of the most common environmental toxins are
cadmium and arsenic. Arsenic is ubiquitous in the
environment and is most commonly found in an insol-
uble form associated with rocks and minerals [23,24].
In soluble form, arsenic occurs as trivalent arsenite
[As(III)] and pentavalent arsenate [As(V)]. Arsenate, a
phosphate analogue, can enter cells via the phosphate
transport system, and is toxic because it can interfere
with normal phosphorylation processes by replacing
phosphate [25]. The competitive substitution of arse-
nate for phosphate can lead to rapid hydrolysis of the
high-energy bonds in compounds such as ATP. Arse-

nite has recently been demonstrated to enter cells at
neutral pH by aqua-glyceroporins (glycerol transport
proteins) in bacteria, yeast and mammals [26], and its
toxicity lies in its ability to bind sulfhydryl groups of
cysteine residues in proteins, thereby inactivating them.
Arsenite is considered to be more toxic than arsenate
and can be oxidized to arsenate chemically or microbi-
ally [27,28].
In contrast, cadmium is soluble as its bivalent cation
Cd
2+
, and Cd
2+
ions are readily taken up by bacterial
and eukaryotic cells, presumably by the Mn
2+
uptake
system [29]. Cadmium toxicity may be caused by bind-
ing to zinc binding proteins, e.g. proteins that contain
zinc finger protein structures [30]. Zinc and cadmium
are in the same group in the Periodic Table, contain
the same common oxidation state (+2) and, when
ionized, are almost the same size. As a result of these
similarities, cadmium can replace zinc in many biologi-
cal systems, in particular systems that contain sulfur
ligands [31]. Cadmium can be bound up to 10 times
more strongly than zinc to certain biological systems,
and is thus difficult to remove [31]. In addition, cad-
mium can replace magnesium and calcium in certain
proteins [30].

Vanadium is chemically similar to molybdenum, and
can replace molybdenum in its role in nitrogenase, form-
ing the iron–vanadium cofactor (FeVco) [32]. Like
molybdenum, vanadium is available in anionic and cat-
ionic forms, the most common being, under physiologi-
cal conditions, vanadate (H
2
VO
4
)
) and vanadyl (VO
2+
)
[33]. Vanadate can act as a competitor to phosphate
(HPO
4
2)
), or as a transition metal ion that competes
with other metal ions in coordination with biogenic
compounds. Because of the low molecular weight of
VO
3
)
, like phosphate, the VO
3
)
ion is able to permeate
plasma membranes and the intestinal wall in humans
with relative ease [34–36]. Vanadate ions also mimic
most of the rapid actions of insulin in the cell [37].

In this report, we examined the in vivo and in vitro
incorporation of metal ions of molybdenum, tungsten,
vanadium, copper, cadmium and arsenic into the MPT
cofactor. For in vivo studies, we heterologously pro-
duced human sulfite oxidase (hSO) and R. sphaeroides
dimethylsulfoxide reductase in the presence of metal
M. Neumann and S. Leimku
¨
hler Metal ion insertion into E. coli molybdoenzymes
FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS 5679
ions in E. coli and studied the incorporation of
metal-bound MPT into these enzymes. Our results
demonstrate that only molybdate and tungstate are
specifically inserted into MPT and MGD in E. coli.
Bivalent copper and cadmium ions and trivalent arse-
nite can be inserted nonspecifically into MPT, which is
inserted into hSO, thus inhibiting enzyme activity.
However, copper, cadmium and arsenite are not
inserted into bis-MGD containing dimethylsulfoxide
reductase in E. coli. Our results suggest that enzymes
containing the MPT form of Moco can easily be inhib-
ited by copper, cadmium, arsenic and other metal ions
binding to sulfur-rich sites, whereas bis-MGD-contain-
ing enzymes are rather protected from nonspecific
metal insertion.
Results
Investigation of metal ion insertion into hSO
during heterologous expression in E. coli
The expression of hSO in an E. coli modC
)

strain [38]
results in an MPT-containing form of hSO, which is
free of metal ions at the MPT site (data not shown). It
has been shown previously that the addition of 100 lm
molybdate is sufficient to complement the modC
)
phe-
notype [39,40]. Thus, the production of hSO in the
E. coli modC
)
strain is an ideal system to study the
nonspecific insertion of metal ions, other than molyb-
date, into hSO. In addition, both the E. coli moeA
)
and mogA
)
strains [38,41] were used for comparative
studies, as MoeA and MogA have been shown to be
involved in the specific insertion of molybdate into
MPT [19,20]. Comparative studies of the metal
contents of hSO after production in E. coli modC
)
,
mogA
)
and moeA
)
strains should make it possible to
distinguish whether the metal ions are inserted specifi-
cally or nonspecifically into hSO. For metal insertion

into hSO, the protein was produced in the E. coli
modC
)
, moeA
)
and mogA
)
strains in the presence of
100 lm of Na
2
MoO
4
,Na
2
WO
4
, NaVO
3
, Cu(NO
3
)
2
,
CdSO
4
or NaAsO
2
. For metal analysis, hSO was puri-
fied after expression. In addition, the uptake of metal
ions was analysed in E. coli cell extracts. As shown in

Fig. 1A, the addition of 100 lm molybdate during
expression in the modC
)
strain resulted in a 65%
molybdenum-saturated hSO. Surprisingly, with the
exception of vanadate, all other metal ions were also
readily detectable in hSO. Although the saturation lev-
els of hSO with copper and arsenite were only 27%
and 23%, respectively, the saturation level for tung-
state was 44% and, for cadmium, 36% (Fig. 1A). In
contrast, the levels of MPT saturation in hSO were in
A
B
C
Fig. 1. Metal and MPT saturation of purified hSO. Purified hSO
was analysed after expression from plasmid pTG818 in E. coli
strain RK5202 (modC
)
) (A), E. coli strain AH69 (moeA
)
) (B) and
E. coli strain RK5206 (mogA
)
) (C). The following metals were
added at a concentration of 100 l
M to the growth medium: I,
Na
2
MoO
4

; II, Cu(NO
3
)
2
; III, Na
2
WO
4
; IV, NaAsO
2
; V, CdSO
4
; VI,
NaVO
3
. Dark grey bars, metal contents (lM metalÆlM
)1
hSO) were
determined by ICP-OES (see Experimental procedures) using multi-
element standards. Light grey bars, the MPT content of hSO was
quantified after its conversion to Form A. 100% metal or MPT satu-
ration is related to a fully active hSO in a 1 : 1 ratio. White bars,
hSO activity (unitsÆmg
)1
) defined as an absorbance change of
1.0 AUÆmin
)1
Æmg
)1
protein monitoring the reduction of cyto-

chrome c at 550 nm. ND, none detectable.
Metal ion insertion into E. coli molybdoenzymes M. Neumann and S. Leimku
¨
hler
5680 FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS
the range 55–74% when cells were grown in the pres-
ence of Na
2
MoO
4
, Cu(NO
3
)
2
,Na
2
WO
4
or NaAsO
2
(Fig. 1A). Cells grown in the presence of CdSO
4
or
NaVO
3
showed lower levels of MPT saturation of
37% and 39%, respectively. However, this underlines
the fact that metal-free MPT is inserted into hSO inde-
pendent of the availability of metals.
In comparison, the metal insertion pattern was

different in the E. coli moeA
)
strain (Fig. 1B). When
using the moeA
)
strain, neither molybdate nor tung-
state was detected in hSO, showing that both metals
are specifically inserted into MPT by the MoeA pro-
tein. In contrast, saturation levels of copper, arsenite
and cadmium were found to be in the region of 21%,
39% and 60%, respectively. Again, no vanadate was
detected in hSO. Analysis of the uptake of the metal
ions showed that all metals were present in the E. coli
extract (data not shown). Thus, E. coli is able to take
up vanadate, but vanadate is not inserted into MPT;
this was a rather surprising observation, as vanadium
is known to functionally replace the molybdenum
atom in nitrogenase. The MPT saturation of hSO after
expression in the moeA
)
strain in the presence of dif-
ferent metal ions was comparable, and varied between
71% and 88%.
Furthermore, we also examined metal insertion dur-
ing production in the E. coli mogA
)
strain (Fig. 1C). It
has been described previously by Miller and Amy [39]
that a molybdate concentration of 1 mm is sufficient to
reverse the mogA

)
phenotype, restoring nitrate reduc-
tase activity. Thus, at a concentration of 100 lm,
neither molybdate nor tungstate reconstituted the
cofactor of hSO. In contrast, saturation levels of
copper, arsenite and cadmium were found at 41%,
40% and 65%, respectively. As observed after expres-
sion in the modC
)
and moeA
)
strains, no vanadate
was detected in hSO. The MPT saturation of hSO
after production in the mogA
)
strain in the presence of
different metal ions was comparable, and varied
between 62% and 73%.
Competitive insertion of metal ions into hSO
during expression in E. coli modC
)
cells
To analyse the specificity of the insertion of different
metal ions into MPT, competition experiments were
performed. E. coli modC
)
cells were grown in the pres-
ence of 100 lm Na
2
MoO

4
and 100 lmNa
2
WO
4
,
NaAsO
2
, Cu(NO
3
)
2
or CdSO
4
. The same competition
experiment was performed with Cu(NO
3
)
2
and all
other metal ions. As shown in Table 1, the MPT satu-
ration of hSO was comparable, and varied between
69% and 92%. The results in Table 1 clearly show
that the presence of equal amounts of molybdate dur-
ing growth was sufficient for the specific insertion
of molybdenum into MPT. When Na
2
MoO
4
and

Na
2
WO
4
, Cu(NO
3
)
2
, CdSO
4
or NaAsO
2
were present
during growth, the molybdate saturation in hSO was
comparable, and varied between 84% and 70%,
whereas only about 5% of the competing metal was
inserted (Table 1). This result clearly shows the high
specificity of molybdate for insertion into MPT. A dif-
ferent pattern was obtained for the insertion of tung-
state, arsenite or cadmium in the presence of equal
amounts of copper during growth. When Na
2
WO
4
and
Cu(NO
3
)
2
were added to the medium, hSO was satu-

rated with 48% tungstate and 11% copper; equal
amounts of about 30% copper and arsenite were
inserted when NaAsO
2
and Cu(NO
3
)
2
were added, and
more cadmium (67.8%) than copper (22.2%) was
inserted in the presence of CdSO
4
and Cu(NO
3
)
2
in
the cell (Table 1). Thus, the affinity of MPT for
Table 1. Activity, metal content and MPT saturation of purified hSO heterologously produced in E. coli modC
)
cells. Metals were added dur-
ing growth at a concentration of 100 l
M each. hSO activity (unitsÆmg
)1
) is defined as an absorbance change of 1.0 AUÆmin
)1
Æmg
)1
protein
monitoring the reduction of cytochrome c at 550 nm. The MPT content of hSO was quantified after its conversion to Form A in comparison

with a fully active hSO. 100% MPT saturation is related to a fully active hSO in a 1 : 1 ratio. Metal contents (l
M metalÆlM
)1
hSO) were deter-
mined by ICP-OES (see Experimental procedures) using multielement standards. 100% metal saturation is related to a fully MPT-saturated
enzyme in a 1 : 1 ratio. ND, none detectable.
hSO activity and
metal ⁄ MPT saturation
Metals added to the growth medium
Mo ⁄ Cu Mo ⁄ WMo⁄ As Mo ⁄ Cd Cu ⁄ WCu⁄ As Cu ⁄ Cd
hSO activity (unitsÆmg
)1
) 621 ± 29 822 ± 54 719 ± 59 725 ± 25 ND ND ND
MPT (%) 83.7 ± 2.1 74.5 ± 2.4 82.3 ± 2.2 89.5 ± 1.7 81.7 ± 4.4 68.6 ± 7.2 91.6 ± 7.7
Mo (%) 78.7 ± 3.0 70.2 ± 6.6 77.2 ± 6.1 84.3 ± 1.2 ND ND ND
Cu (%) 4.5 ± 0.4 ND ND ND 11.3 ± 0.7 32.9 ± 0.7 22.2 ± 1.7
W (%) ND 5.4 ± 1.4 ND ND 48.2 ± 0.7 ND ND
As (%) ND ND 5.2 ± 0.7 ND ND 31.2 ± 1.7 ND
Cd (%) ND ND ND 4.1 ± 1.5 ND ND 67.8 ± 3.3
M. Neumann and S. Leimku
¨
hler Metal ion insertion into E. coli molybdoenzymes
FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS 5681
cadmium is higher than that for copper, but, in gen-
eral, there is no preference for the nonspecific insertion
of any other metal ion than molybdate.
Analysis of metal insertion into R. sphaeroides
dimethylsulfoxide reductase
Most molybdoenzymes in E. coli contain the bis-
MGD cofactor. For the biosynthesis of MGD, GMP

is attached to the phosphate group of MPT by the
MobA protein [42]. It has been shown previously that
MGD formation is catalysed by MobA only after the
coordination of a molybdenum atom to the dithiolene
moiety of MPT [19]. To determine whether molybdate
can be replaced by other metals for the biosynthesis
of MGD, R. sphaeroides dimethylsulfoxide reductase
was heterologously produced in the presence of differ-
ent metal ions at a concentration of 100 lm in the
E. coli modC
)
strain, and analysed for its metal con-
tent. Metal analysis of purified dimethylsulfoxide
reductase revealed that only tungstate and molybdate
were inserted into the enzyme (Fig. 2). Consistent with
a previous report [43], dimethylsulfoxide reductase
was active in the tungsten-bound form. Copper, cad-
mium, arsenite and vanadate were not present in the
purified dimethylsulfoxide reductase (Fig. 2, data not
shown). In addition, the dimethylsulfoxide reductase
proteins were devoid of bis-MGD, showing that metal
ions such as copper or cadmium cannot replace
molybdate in the biosynthesis of MGD, whereas tung-
state is able to substitute for this role. Thus, GMP is
only added to molybdenum- or tungsten-containing
MPT.
Metal insertion into purified hSO in vitro
It has been shown previously that Moco-free hSO can
be reconstituted with nascent MPT and molybdate
in vitro [44]. At the molybdate concentrations used in

the assay (> 1 mm), MoeA and MogA were not
required for the generation of the active form of
Moco, showing that the ATP-dependent activation of
MPT and molybdenum is not required for the in vitro
ligation of molybdate. It was of interest to examine
the affinity of free MPT for other metal ions, and
to determine whether MPT chelated with other non-
specific metals can also be inserted into hSO in vitro.
For these experiments, the molybdenum-free human
sulfite oxidase Moco domain (hSO-MD) was used as
MPT source [45], and extracted MPT was incubated
with 100 lm of Na
2
MoO
4
,Na
2
WO
4
, NaVO
3
,
Cu(NO
3
)
2
, CdSO
4
or NaAsO
2

, before purified apo-
hSO was added to the mixture. As shown in Fig. 3,
the amount of MPT inserted into hSO was about the
same in all incubation mixtures, independent of the
added metal, with an MPT saturation in the range
43–47%. Analysis of the metal content of hSO revealed
that, at a metal concentration of 100 lm, only the
bivalent copper and cadmium ions were inserted into
hSO (Fig. 3A). As shown previously, molybdate is not
inserted into MPT at a concentration of 100 lm [44].
As tungstate, arsenite and vanadate were not inserted
into MPT at a concentration of 100 lm, higher con-
centrations of added metals were analysed. However,
the addition of these metals at concentrations of up to
1mm to the reaction mixture did not result in their
insertion into MPT (data not shown). In addition, we
tested the insertion of metal ions into MPT-containing
hSO. None of the metals was inserted into hSO
(Fig. 3B), which is consistent with the previous data for
the insertion of molybdate into molybdenum-free
MPT-hSO [44], and makes nonspecific binding of the
metals to the protein surface unlikely. This shows that,
after MPT insertion, hSO adopts a conformation that
is not competent for the insertion of metals. Thus, any
nonspecific metal insertion into MPT has to occur
before the insertion of MPT into hSO.
Fig. 2. Activity, metal and MPT saturation of R. sphaeroides
dimethylsulfoxide reductase (DMSOR). Dimethylsulfoxide reductase
was purified after expression from plasmid pJH820 in E. coli
RK5202 (modC

)
) cells. The following metals were added during
growth at a concentration of 100 l
M:I,Na
2
MoO
4
; II, Cu(NO
3
)
2
; III,
Na
2
WO
4
; IV, CdSO
4
. Dark grey bars, metal contents (lM metalÆlM
)1
hSO) were determined by ICP-OES (see Experimental procedures)
using multielement standards. Light grey bars, the MPT content of
dimethylsulfoxide reductase was quantified after the conversion
of bis-MGD to Form A. 100% metal or MPT saturation was related
to a fully MPT-saturated dimethylsulfoxide reductase in a 1 : 1 or
1 : 2 ratio, respectively. White bars, dimethylsulfoxide reductase
activity (unitsÆmg
)1
) defined as the reduction of 1 lmol of dimethyl-
sulfoxideÆmin

)1
Æmg
)1
protein. ND, none detectable.
Metal ion insertion into E. coli molybdoenzymes M. Neumann and S. Leimku
¨
hler
5682 FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS
Release of MPT bound to MPT synthase by
metal ions
It has been suggested that, during the biosynthesis of
Moco, the cofactor and its intermediates remain
protein bound until insertion into the specific target
protein, as oxygen seems to be a major factor of free
Moco inactivation [46]. Our results showed that metal
ions, such as copper, cadmium or arsenite, might be
inserted nonspecifically into MPT, without the involve-
ment of the MogA or MoeA proteins. As MPT is pro-
duced by MPT synthase, it was of interest to analyse
the effect of metal ions on the release of MPT bound
to MPT synthase. For our studies, we chose to com-
pare the MPT release after metal addition to MPT-
containing R. capsulatus MPT synthase in comparison
with E. coli MPT synthase. The comparison of the two
MPT synthases from different sources was of particu-
lar interest, as the phenotype of the R. capsulatus
moeA
)
strain has been shown to be repairable with
1mm molybdate [47], which is not the case for the

E. coli moeA
)
strain [39]. Thus, we first analysed the
effect of high molybdate concentrations on MPT-satu-
rated MPT synthase from the two different sources.
The results in Fig. 4 show that, under the same assay
conditions, 85.5% of MPT remained bound to E. coli
MPT synthase when 1 mm molybdate was added,
whereas only 12.3% of MPT remained bound to
R. capsulatus MPT synthase. This result shows that
E. coli MPT synthase binds MPT more tightly; how-
ever, the rate of conversion of precursor Z to MPT
was the same in both MPT synthases (data not
shown).
To analyse the effect of other metal ions on MPT
release by MPT synthase, we chose the R. capsulatus
MPT synthase, which binds MPT less tightly; thus, an
effect of metal ions on MPT release should be detected
easily. R. capsulatus MPT synthase was saturated
with MPT before the addition of Na
2
WO
4
, NaVO
3
,
Cu(NO
3
)
2

, CdSO
4
or NaAsO
2
. The incubation
B
A
Fig. 3. In vitro reconstitution of hSO with MPT and metal ions. (A)
20 l
M MPT extracted from hSO-MD was incubated with 100 lM of
Na
2
MoO
4
(I), Cu(NO
3
)
2
(II), Na
2
WO
4
(III), NaAsO
2
(IV), CdSO
4
(V) or
NaVO
3
(VI) for 10 min at 4 °C. Subsequently, 10 lM of purified apo-

hSO was added to the mixture and incubated for 20 min at 4 °C,
before unbound MPT or metal ions were removed by gel filtration.
Dark grey bars, metal contents (l
M metalÆlM
)1
hSO) were deter-
mined by ICP-OES (see Experimental procedures) using multi-
element standards. Light grey bars, the MPT content of hSO was
quantified after its conversion to Form A. 100% metal or MPT satu-
ration is related to a fully active hSO in a 1 : 1 ratio. (B) 40 l
M
MPT-containing hSO was incubated with 100 lM of Na
2
MoO
4
(I),
Cu(NO
3
)
2
(II), Na
2
WO
4
(III), NaAsO
2
(IV), CdSO
4
(V) or NaVO
3

(VI)
for 30 min at room temperature, before unbound MPT or metal
ions were removed by gel filtration. Light grey bar, MPT content of
hSO. Metal content (l
M metalÆlM
)1
hSO) of MPT-hSO was below
detection limit. ND, none detectable.
Fig. 4. Comparison of the effect of 1 mM molybdate on MPT-satu-
rated MPT synthase from E. coli and R. capsulatus. 33.2 l
M of
MPT-saturated MPT synthase from either E. coli (I, II) or R. capsula-
tus (III, IV) was incubated in the presence (II, IV) or absence (I, III)
of 1 m
M molybdate. The MPT content of MPT synthase was quan-
tified after its conversion to Form A.
M. Neumann and S. Leimku
¨
hler Metal ion insertion into E. coli molybdoenzymes
FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS 5683
mixtures were subjected to gel filtration and the MPT
synthase fraction was analysed for its MPT content.
The results in Fig. 5A show that, under the assay con-
ditions, MPT remained bound to MPT synthase when
metals other than molybdate were added. As metal
analysis of MPT synthase revealed that none of the
metals was inserted into MPT (Fig. 5A), the dithiolene
group of MPT must be protected in MPT synthase,
and, apparently, only molybdate is able to trigger the
release of MPT. This result clearly shows that the non-

specific insertion of metal ions into MPT occurs in a
step after the release of MPT from MPT synthase. The
reverse experiment showed that MPT was only bound
to MPT synthase in its metal-free form, and not in the
presence of metal ions (Fig. 5B).
Discussion
Homeostasis of metal ions is a highly regulated com-
plex process in the cell [48]. As a defence against metal
toxicity, organisms have developed systems for metal
detoxification, including specific export systems, as
found in E. coli [49–51]. Our results show that, at high
concentrations of metal ions and in the absence of
molybdate ions, copper, cadmium and arsenite are
inserted into Moco found in hSO. In addition to the
metals investigated in this report, other metals ions
known to bind tightly to sulfur-rich sites, such as zinc
and cobalt, are inserted into MPT (data not shown).
However, only molybdate and tungstate are specifically
inserted into hSO, requiring the catalytic activity of
the MoeA protein. Although the synthesis of the dioxo
Moco found in hSO seems to be more susceptible to
nonspecific metal insertion, the biosynthesis of the bis-
MGD cofactor present in dimethylsulfoxide reductase
provides an additional control step. In dimethylsulf-
oxide reductase, only molybdate and tungstate are
found to be coordinated to bis-MGD, and the inser-
tion of either metal results in an active form of dimeth-
ylsulfoxide reductase. This result shows that MobA is
only able to add GMP to the molybdate- or tungstate-
substituted form of MPT, and not to other metal-

substituted forms of MPT. The chemical and physical
similarities of tungstate and molybdate are a result of
their equal atomic and ionic radii and similar electro-
negativity and coordination characteristics, contribut-
ing to the discrimination of these metals in biological
systems [2,52]. In general, tungstate can replace molyb-
denum in molybdoenzymes in selected organisms,
forming the tungsten cofactor (Wco) [3]. Although
molybdenum-containing enzymes are found in all aero-
bic organisms, tungsten-containing enzymes are gener-
ally found only in obligate, typically thermophilic,
anaerobes. Tungsten may have been the first of these
elements to be acquired by living organisms. However,
when the atmosphere became more aerobic, the oxygen
sensitivity of tungsten compounds made them less
available, and the water solubility of high-valent
molybdenum oxides may have become more advanta-
geous [3]. Because tungsten and molybdenum have
similar chemistry, it is possible that, initially, as the
transition to an oxygen-rich environment occurred, the
A
B
Fig. 5. Analysis of the effect of added metals on MPT binding or
MPT release in purified R. capsulatus MPT synthase. (A) 33.2 l
M
MPT-saturated MPT synthase was incubated with 1 mM of the indi-
cated metal ions, and unbound material was removed by gel filtra-
tion. The MPT content of MPT synthase was quantified after its
conversion to Form A. (B) 1 m
M of metal ions was incubated with

1m
M of MPT prior to the addition of 21.3 lM MPT synthase.
Unbound MPT and metal ions were removed from MPT synthase
by gel filtration. The MPT content of MPT synthase was quantified
after its conversion to Form A. (A, B) Metal contents were deter-
mined by ICP-OES (see Experimental procedures) using multi-
element standards. All metal contents were below the detection
limit. 1 m
M of Cu(NO
3
)
2
,Na
2
WO
4
, NaAsO
2
or CdSO
4
was added.
ND, none detectable.
Metal ion insertion into E. coli molybdoenzymes M. Neumann and S. Leimku
¨
hler
5684 FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS
latter substituted for the former in enzyme active sites.
The results on dimethylsulfoxide reductase show that
tungsten is still able to replace molybdenum at the
bis-MGD cofactor, resulting in an active enzyme.

However, in R. capsulatus, the expression of dimethyl-
sulfoxide reductase has been shown to be regulated by
the availability of molybdenum [53]; therefore, it is
unlikely that, during normal growth conditions, tung-
sten replaces molybdenum to produce an active
dimethylsulfoxide reductase in this organism.
Our studies also show that bivalent copper and cad-
mium ions and trivalent arsenite ions can be inserted
nonspecifically into MPT without the catalytic activity
of the MoeA or MogA protein. Copper and cadmium
also show a higher affinity than molybdate for the
dithiolene group of MPT, as revealed by the in vitro
insertion of metal-substituted MPT into hSO, as these
metals were already inserted into MPT at concentra-
tions of 100 lm. Our results show that the nonspecific
insertion of metal ions into MPT occurs at a step
beyond the MPT synthase reaction. In MPT synthase,
the dithiolene group seems to be protected in a manner
that makes it inaccessible for the insertion of nonspe-
cific metal ions. Only in the presence of high molyb-
date concentration is MPT released and molybdate
inserted. This result may imply that, under conditions
of high molybdate availability in the cell, the activa-
tion of MPT by adenylation may not be required, as
molybdate is directly inserted into released MPT after
its completion by MPT synthase, as shown by the
molybdate repairable phenotype of the E. coli mogA
)
strain [39]. The comparison of MPT synthase from
E. coli and R. capsulatus shows that MPT is more eas-

ily released from R. capsulatus MPT synthase when
1mm molybdate is added, implying that MPT is less
tightly bound in R. capsulatus MPT synthase in com-
parison with the protein from E. coli. This result also
explains the phenotype of the R. capsulatus moeA
)
strain, which can be complemented by the addition of
1mm molybdate [47], which is not the case for the
E. coli moeA
)
strain [39]. High molybdate concentra-
tions result in the release of MPT from MPT synthase,
and thus MogA or MoeA are not required under these
conditions.
Our investigations analysed the insertion of metal
ions into MPT beyond the reaction by the MogA and
MoeA proteins. The insertion of copper, cadmium and
arsenite is independent of the MogA and MoeA pro-
teins. Even if the copper-containing MPT–AMP inter-
mediate is formed in E. coli, copper can be replaced in
this intermediate by other metal ions without the cata-
lytic activity of the MoeA protein. Our results suggest
Fig. 6. Model for the specific and
nonspecific insertion of metal ions
during Moco biosynthesis in E. coli.
Specific reactions are marked by full
lines, nonspecific reactions by broken
lines. Details of the reactions are given
in the text.
M. Neumann and S. Leimku

¨
hler Metal ion insertion into E. coli molybdoenzymes
FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS 5685
that copper is not required as an intermediate in Moco
biosynthesis in E. coli. In addition to the known toxic
effects, the toxicity of copper, cadmium and arsenic in
the environment may also be caused by an inhibition
of molybdoenzyme activity in E. coli. We present a
model for nonspecific metal insertion during Moco
biosynthesis in E. coli (Fig. 6). Under physiological
molybdate concentrations (1–10 lm), the MogA and
MoeA proteins are required in E. coli to form an
MPT–AMP intermediate, facilitating molybdate inser-
tion and Moco formation in the cell (full lines). Using
the same pathway, tungstate can be specifically
inserted into MPT to form Wco. Under high molyb-
date concentrations (> 1 mm), MPT–AMP formed by
MogA is not required and molybdate can be directly
inserted into MPT by the aid of the MoeA protein. All
other metal ions, when present at high concentrations,
are inserted nonspecifically into MPT, not requiring
the MogA and MoeA proteins (broken lines). How-
ever, this nonspecific reaction can be outcompeted by
the presence of molybdate, showing that this is the
specific pathway in the cell. Nonspecifically formed
Cu–MPT, Cd–MPT or As–MPT can be inserted into
MPT-binding molybdoenzymes, such as sulfite oxidase,
but not into bis-MGD-containing enzymes, such as
dimethylsulfoxide reductase. Here, the E. coli provides
an additional ‘quality control step’ by the MobA pro-

tein, which only forms the bis-MGD cofactor when
molybdenum or tungsten is inserted into MPT. How-
ever, the nature of this quality control step and the
details of bis-MGD formation are not yet known. In
our experiments, we were unable to show a copper-
containing MPT–AMP intermediate. Thus, for Moco
biosynthesis in E. coli, copper is not required and is
rather an inhibitor of molybdoenzymes, inhibiting
enzyme activity when inserted nonspecifically.
Experimental procedures
Bacterial strains, plasmids, media and growth
conditions
E. coli BL21(DE3) cells were used for the heterologous
expression of the R. capsulatus moaE and moaD1 genes. For
metal incorporation experiments, E. coli RK5202
(chlD202::Mu cts [modC
)
]), RK5206 (chlG206::Mu cts
[mogA
)
]) [38] and AH69 (moeA113 DzbiK-Km [moeA
)
]) cells
[41] were used for the production of hSO from plasmid
pTG718 [54]. R. sphaeroides dimethylsulfoxide reductase was
expressed in E. coli RK5202 cells from plasmid pJH820 [42]
under the same conditions as hSO. Moco-free apo-hSO was
obtained after expression in E. coli RK5200 (chlA200::Mu
cts [moaA
)

]) [38]. MPT-containing hSO-MD and hSO were
expressed from pTG818 [54] or pTG718 in E. coli RK5202
cells. Dimethylsulfoxide reductase, hSO and hSO-MD were
purified as described by Temple et al. [54]. E. coli MoaE was
expressed from plasmid pGG110 in E. coli BL21(DE3) cells
(Novagen, La Jolla, CA, USA), cotransformed with plasmid
pREP4 (Qiagen, Hilden, Germany) and purified as described
previously [55]. E. coli MoaD was expressed from plasmid
pMW15aD in E. coli BL21(DE3) cells and purified as
described previously [56]. Cell strains containing expression
vectors were grown aerobically in Luria–Bertani medium at
30 °C in the presence of either 150 lgÆmL
)1
ampicillin
and ⁄ or 25 lgÆmL
)1
kanamycin. Metals were added as indi-
cated at a concentration of 100 lm.
Cloning, expression and purification of
R. capsulatus MoaE and MoaD1
DNA fragments containing the coding regions for R. cap-
sulatus moaE and moaD1 were amplified by PCR, and
flanking restriction sites were introduced. The moaE gene
was cloned into the NdeI-BamHI sites of expression vector
pET16b (Novagen) and moaD1 into the NdeI-XhoI sites of
pET28a (Novagen), resulting in plasmids pSL241 and
pMN67, respectively. For the production of His
6
-tagged
MoaE and MoaD1, E. coli BL21(DE3) cells were

transformed with plasmid pSL241 or pMN67. One litre of
Luria–Bertani medium was inoculated with 10 mL of an
overnight culture and incubated at 30 °C until an absor-
bance (A) at 600 nm of 0.3–0.5. The expression was
induced with 100 lm isopropyl thio-b-d-galactoside, and
cells were harvested after an additional growth of 5 h. The
cell pellet was resuspended in phosphate buffer (50 mm
NaH
2
PO
4
, 300 mm NaCl, pH 8.0). Cells were lysed by sev-
eral passages through a French pressure cell, and the
cleared lysate was applied to 0.5 mL nickel-nitrilotriacetate
resin (Qiagen) per litre of culture. The column was washed
with 20 column volumes of phosphate buffers, one contain-
ing 10 mm and the other 20 mm of imidazole. Proteins were
eluted with buffer containing 250 mm imidazole and, after
concentration, the proteins were applied to a PD10 column
(GE Healthcare, Munich, Germany) exchanging the buffer
for 100 mm Tris, pH 7.2.
Metal analysis by inductively coupled
plasma-optical emission spectrometry (ICP-OES)
Metal analysis was performed using a Perkin-Elmer Optima
2100DV inductively coupled plasma-optical emission spec-
trometer (Perkin-Elmer, Fremont, CA, USA). Protein sam-
ples were incubated overnight in a 1 : 1 mixture with 65%
nitric acid (Suprapur, Merck, Darmstadt, Germany) at
100 °C. Samples were filled to a 10-fold volume with water
prior to ICP-OES analysis. As reference, the multielement

standard solutions XII and XVI (Merck) were used.
Metal ion insertion into E. coli molybdoenzymes M. Neumann and S. Leimku
¨
hler
5686 FEBS Journal 275 (2008) 5678–5689 ª 2008 The Authors Journal compilation ª 2008 FEBS
Moco/MPT analysis
The Moco and MPT contents of purified hSO were quanti-
fied after conversion to Form A-dephospho, as described
previously [57]. To determine the MGD content of dimeth-
ylsulfoxide reductase, the protein was incubated at 95 °C
for 30 min in the presence of acidic iodine to convert
MGD to Form A. Released Form A was detected as
described previously [57].
Enzyme assays
The activity of hSO (unitsÆmg
)1
) was determined as
described previously [58] by monitoring the reduction of
cytochrome c at 550 nm, and is defined as an absorbance
change of 1.0 AUÆmin
)1
Æmg
)1
protein.
Dimethylsulfoxide reductase activity (unitsÆmg
)1
) was
assayed as described by McEwan et al. [59] with dithionite-
reduced benzyl viologen as the electron donor, and is
defined as the reduction of 1 lmol of dimethylsulfoxideÆ

min
)1
Æmg
)1
protein.
In vitro incorporation assays
Free MPT was obtained from MPT-containing hSO-MD as
described previously [57]. For in vitro metal incorporation
into apo-hSO, 20 lm of extracted MPT was incubated with
100 lm of Na
2
MoO
4
,Na
2
WO
4
, NaAsO
2
, Cu(NO
3
)
2
,
CdSO
4
or NaVO
3
. Subsequently, 10 lm of purified apo-
hSO was added to the mixture, incubated at 4 °C for

20 min, and free MPT and metal ions were subsequently
removed by gel filtration. For the determination of metal
incorporation into MPT after insertion into hSO, 40 lm of
MPT-containing hSO was incubated at 4 °C for 30 min
with 100 lm of Na
2
MoO
4
,Na
2
WO
4
, NaAsO
2
, Cu(NO
3
)
2
,
CdSO
4
or NaVO
3
. Unbound metals were removed by gel
filtration using a Nick column (GE Healthcare), and metal
incorporation into hSO was determined by ICP-OES
analysis.
MPT synthase in vitro assays
To determine the release of MPT from MPT synthase, sepa-
rately purified R. capsulatus or E. coli MoaE and MoaD1

were assembled and incubated with excess MPT prior to the
removal of unbound MPT by gel filtration. Subsequently,
1mm of Na
2
MoO
4
,Na
2
WO
4
, NaAsO
2
, Cu(NO
3
)
2
or
CdSO
4
was added to 33.2 lm MPT-loaded MPT synthase.
After a further incubation step of 10 min, released MPT
was removed by an additional gel filtration step and the
protein fraction was analysed for MPT and metal content.
To determine the binding of metal-containing MPT to
MPT synthase, 1 mm of Na
2
MoO
4
,Na
2

WO
4
, NaAsO
2
,
Cu(NO
3
)
2
or CdSO
4
was added to 60 lm MPT prior to the
addition of 21.3 l m MPT synthase. After a further incuba-
tion step of 10 min, free MPT and metal ions were removed
by gel filtration and the protein fraction was analysed for
MPT and metal content.
Acknowledgements
We thank K. V. Rajagopalan (Duke University,
Durham, NC, USA) for helpful discussions, and for
providing pTG718, pTG818, pMW15aD and pJH820.
This work was supported by Deutsche Forschungs-
gemeinschaft Grant LE1171 ⁄ 3-3 and the Fonds der
Chemischen Industrie (FCI).
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