Báo cáo khoa học: Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies potx

Azotobacter vinelandii
rhodanese
Selenium loading and ion interaction studies
Sonia Melino
1
, Daniel O. Cicero
1,2
, Maria Orsale
1
, Fabio Forlani
3
, Silvia Pagani
3
and Maurizio Paci
1,2
1
Dipartimento di Scienze e Tecnologie Chimiche and
2
INFM, Sez. B, University of Rome ‘Tor Vergata’, Italy;
3
Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy
Rhodanese is a sulfurtransferase which in vitro catalyzes the
transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic
interactions of the prokaryotic rhodanese-like protein from
Azotobacter vinelandii were studied by fluorescence and
NMR spectroscopy. The catalytic Cys230 residue of the
enzyme was selectively labelled using [
15
N]Cys, and changes
in
1

Hand
15
N NMR resonances on addition of different ions
were monitored. The results clearly indicate that the sulfur
transfer is due to a specific reaction of the persulfurated Cys
residue with a sulfur acceptor such as cyanide and not to the
presence of the anions. Moreover, the
1
H-NMR spectrum of
a defined spectral region is indicative of the status of the
enzyme and can be used to directly monitor sulfur loading
even at low concentrations. Selenium loading by the addition
of selenodiglutathione was monitored by fluorescence and
NMR spectroscopy. It was found to involve a specific
interaction between the selenodiglutathione and the catalytic
cysteine residue of the enzyme. These results indicate that
rhodanese-like proteins may function in the delivery of
reactive selenium in vivo.
Keywords:
15
N-NMR; Azotobacter vinelandii; rhodanese;
selenodiglutathione; sulfurtransferase.
The rhodanese from Azotobacter vinelandii (RhdA) is a
sulfurtransferase which catalyzes in vitro the production of
thiocyanate, transferring the sulfane sulfur atom from
thiosulfate to cyanide, by a double displacement mechanism
(thiosulfate–cyanide sulfurtransferase, EC 2.8.1.1) [1–3].
The best studied rhodanese is that from bovine liver
(Rhobov). Studies on its catalytic mechanism in vitro have
shown that, during the transfer of sulfane sulfur from

thiosulfate to cyanide, this enzyme cycles between two stable
intermediates, a sulfur-loaded (ES) and a sulfur-free form
(E). Physical properties of these intermediates have been
demonstrated to be different by a variety of solution
methods [4–6], but crystallographic data do not appear to
show appreciable flexibility in the rhodanese when ES
crystals are soaked with cyanide [7,8]. Thermodynamic
calculations [9] on the two forms of Rhobov reveal that the
ES form is about 8 calÆmol
)1
more stable than the E form. It
has been suggested that the conformational changes in rho-
danese may form the basis of its activity. The physiological
role of this class of enzyme is still unclear, but its wide
distribution among eukaryotes and prokaryotes suggests
that it is involved in essential metabolic pathways. The
proposed roles include cyanide detoxification [3], restoration
of iron-sulfur centres in Fe-S proteins such as ferredoxin
[10,11], and sulfur metabolism [3,12]. It has recently been
found to be involved in selenium trafficking [13]; selenium
uptake in the persulfide position of the bovine enzyme is
achieved by reaction with selenodiglutathione (SDG), the
primary metabolite of selenite. The selenium-loaded enzyme
(ESe) has been proposed to be the carrier of selenium for
selenophosphate synthase. It has been hypothesized that a
rhodanese-like enzyme may behave as a transferase for the
regulation of selenium concentration in vivo [13].
Recombinant RhdA is one of the most recently expressed
prokaryotic enzymes [14], and its 3D structure has been
elucidated [8]. In contrast with Rhobov, which has four

cysteine residues, RhdA has only one (Cys230), which is the
residue involved in the catalytic mechanism. This is a
fundamental advantage in the study of rhodanese-like
proteins.
The interconversion between the ES and E form has been
studied by NMR spectroscopy in parallel with fluorescence
methods [4]. Selective [
15
N]Cys labelling of RhdA was
performed in order to investigate, by NMR spectroscopy
changes in the status of the active site when the enzyme
cycles between the two forms. Analysis of high-resolution
1
H-NMR spectra of the ES and E form has revealed some
differences that are diagnostic of the two forms. In this
work, we propose the use of an alternative method, 1D
NMR spectroscopy, to investigate the interconversion
between the ES and E form in solution and to monitor
the state of the enzyme by addition of substrates or
inhibitors.
Correspondence to M. Paci, Dipartimento di Scienze e Tecnologie
Chimiche, Universita
`
di Roma ‘Tor Vergata’, Via della Ricerca
Scientifica, 00133-Rome, Italy.
Fax: + 39 0672594328, Tel.: + 39 0672594446,
E-mail:
Abbreviations: RhdA, rhodanese of Azotobacter vinelandii;E,sulfur-
free form of rhodanese; ES, sulfur-loaded form of rhodanese;
ESe, selenium-loaded form of rhodanese; HSQC, heteronuclear

single quantum coherence; Rhobov, bovine rhodanese;
SDG, selenodiglutathione.
Enzyme: rhodanese; thiosulfate–cyanide sulfurtransferase (EC 2.8.1.1).
(Received 9 July 2003, revised 25 August 2003,
accepted 5 September 2003)
Eur. J. Biochem. 270, 4208–4215 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03818.x
Materials and methods
Preparation of the protein
The plasmid pQER1 [14], containing the gene coding for
RhdA with an N-terminal His-tag, was used to transform
the BL-21(DE3)[pREP4] Escherichia coli strain [14], and
overexpression of the recombinant protein was induced by
addition of isopropyl thio-b-
D
-galactoside to a mid-expo-
nential culture. RhdA was purified by chromatography on
a Ni/nitrilotriacetate/agarose column (Qiagen). The His-
tagged protein was eluted by addition of 200 m
M
imidazole
and precipitated in 75% saturated ammonium sulfate. The
protein concentration was determined using A
0.1%
280
¼ 1.3
[2], and the molecular mass of 31 kDa was estimated by
SDS/PAGE. Rhodanese activity was measured by the
discontinuous colorimetric assay described by So
¨
rbo [15].

The presence of the His-tag did not affect enzymatic
activity. The sulfur-free form (E) was prepared by adding a
10-fold molar excess of cyanide to ES rhodanese in 50 m
M
Tris/HCl (pH 7.4)/0.3
M
NaCl followed by a 10-min
incubation at room temperature. Excess cyanide and
thiocyanate were removed by loading the protein solution
on to a Centricon-3 (3000 molecular mass cut-off; Amicon).
As a control, ES (to which no cyanide was added) was
analogously treated. The conversion of ES into E was
monitored by measuring the increase in fluorescence
quantum yield that accompanies the removal of the
persulfide sulfur [4].
Production of [
15
N]Cys-containing RhdA
His-tagged RhdA protein labelled with [
15
N]Cys was
expressed by growing the transformed BL21[pREP4] E. coli
strain in medium containing: 2 mgÆmL
)1
succinic
acid; 0.9 mgÆmL
)1
magnesium acetate tetrahydrate;
10 mgÆmL
)1

K
2
HPO
4
;2mgÆmL
)1
sodium acetate trihy-
drate; 1 mgÆmL
)1
ammonium chloride; 0.01 mgÆmL
)1
CaCl
2
; 0.004 mgÆmL
)1
FeCl
2
;0.05mgÆmL
)1
nicotinic acid;
0.05 mgÆmL
)1
thiamin; 0.1 lgÆmL
)1
biotin; 0.125 mgÆmL
)1
guanosine, cytosine and uracil; 0.08 mgÆmL
)1
thymine;
0.4 mgÆmL

)1
L
-alanine,
L
-glutamic acid,
L
-glutamine,
L
-arginine and glycine; 0.25 mgÆmL
)1
L
-aspartic acid;
0.1 mgÆmL
)1
L
-asparagine,
L
-histidine,
L
-isoleucine,
L
-lysine,
L
-proline,
L
-threonine,
L
-tyrosine and
L
-valine;

0.25 mgÆmL
)1
L
-methionine; 1.6 mgÆmL
)1
L
-serine;
1mgÆmL
)1
L
-leucine; 0.05 mgÆmL
)1
L
-tryptophan,
L
-cys-
tine,
L
-phenylalanine and
L
-cysteine; 1% glycerol;
0.1 mgÆmL
)1
ampicillin; 0.025 mgÆmL
)1
kanamycin. A
10-mL volume of BL21[pREP4]/pQER1 culture grown
overnight in Luria–Bertani medium was added to 500 mL
expression medium (filtered through 0.2-lm nylon filter),
and incubated (for 6 h) at 37 °Cinanorbitalshakerto

A
600
¼ 1. The culture was then induced with 1 m
M
isopropyl thio-b-
D
-galactoside, followed by a 10-min incu-
bation. The cell suspension was harvested by centrifugation,
washed twice with 500 mL 0.9 mgÆmL
)1
NaCl, and resus-
pended in the presence of 1 m
M
isopropyl thio-b-
D
-
galactoside, in 500 mL of the expression medium in which
both cystine and cysteine were replaced by
15
N-labelled
cystine (0.2 mgÆmL
)1
) (Isotec, Sigma-Aldrich, UK). After
2.5 h of induction at 37 °C, cells were harvested by
centrifugation and stored at )80 °C. The procedure used
for purification of the labelled protein was as described
above. After purification, the enzyme was assayed as
previously described [14]. The procedure used for the
expression and purification of the uniformly
15

N-labelled
protein will be reported elsewhere. The sample obtained
gives well-resolved and intense
15
N-NMR spectra; the
results will be reported elsewhere.
Preparation of the selenium-substituted rhodanese
RhdA in the E form was prepared by adding KCN to the
enzyme solution, with a molar ratio of E to KCN of 1 : 10,
in 50 m
M
Tris/HCl buffer, pH 7.4. After the reaction,
the protein solution was dialyzed at 4 °C for 12 h. The
selenium-loaded RhdA, E–Se, was prepared from the
persulfide-free enzyme by reaction with a solution of selenite
and glutathione (GSH), in a molar ratio of 1 : 4, respect-
ively, in 50 m
M
Tris/HCl, pH 7.4, containing 1 m
M
EDTA,
as described previously [13].
NMR spectroscopy
NMR measurements were performed at an RhdA concen-
tration of % 0.1–0.4 m
M
in 50 m
M
Tris/HCl (pH 7.25)/
0.3

M
NaCl and at 20 °C on a Bruker AVANCE instru-
ment, operating at a proton frequency of 700 MHz
equipped with a z-gradient triple resonance probe.
Data were processed and analysed on an IRIS O2 work
station (Silicon Graphics) using
NMRPIPE
[16] and
NMRVIEW
[17].
Fluorescence measurements
All fluorescence measurements were made using an LS50
Perkin–Elmer spectrofluorimeter equipped with a thermo-
statically controlled stirrer cell holder. The temperature was
maintained at 23 °C, and the protein concentration was
kept constant at 6 l
M
. The excitation and emission
bandwidths were 5 and 3 nm, respectively. The excitation
wavelength was set at 286 nm, and the spectra were
recorded from 300 to 400 nm. The changes in fluorescence
intensity at 336 nm (F
obs
) are given as DF %:
DF% ¼ abs½ðF
obs
À F
0
Þ=F
0

Â100
where F
0
is the original fluorescence intensity of RhdA.
ES and E were used as references. Fluorescence meas-
urements were made in the presence of different fixed ion
concentrations with an enzyme concentration of 6 l
M
in
50 m
M
Tris/HCl, pH 7.2.
Results and Discussion
15
N labelling of Cys230 of RhdA and
15
N-NMR
spectroscopy
Incorporation of [
15
N]Cys in the expression of RhdA
protein was estimated to be % 10% from
1
H-
15
N hetero-
nuclear single quantum coherence (HSQC) spectra, com-
paring the
15
N-filtered spectrum of [

15
N]Cys-RhdA with the
unfiltered enzyme at a fixed delay and also with the
1
H-
15
N
HSQC spectra of the uniformly
15
N-labelled RhdA
Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4209
(unpublished). The low level of [
15
N]Cys incorporation is
probably due to transamination reactions and to the use of a
nonauxotrophic strain of E. coli. In previous fluorescence
experiments and crystallographic investigations, native
RhdA was prepared as the persulfurated form at Cys230
[8,18]. We observed that RhdA is obtained as a mixture of
ES and E. In fact, two different cross-peaks were observed
in the
1
H-
15
N HSQC spectrum (Fig. 1A) of the over-
expressed [
15
N]Cys230–RhdA when protein purification
was performed without addition of thiosulfate. We evalu-
ated that, after purification, % 30% of the overexpressed

RhdA was in sulfur-loaded form and 70% in the sulfur-free
form (Fig. 1A). The fluorescence and MALDI-TOF data
from the same NMR samples confirm the estimate of the
ratio between the two forms of RhdA made from NMR
(data not shown). Addition of a thiosulfate and cyanide
excess, respectively, allowed identification of the
1
H-
15
N
correlation peaks corresponding, respectively, to the ES and
E form (Fig. 1B,C). In both cases, the samples were dialyzed
before the NMR experiments to remove the excess reagents.
The observed changes in chemical shift were % 0.26 and
1.0 p.p.m. for
1
Hand
15
N, respectively. Additional analysis
of
1
H-
15
N HSQC spectra from a uniformly labelled sample
of RhdA showed that about 20 peaks out of the 230
observed signals show different chemical shifts between the
two forms of the protein (unpublished). These changes may
reflect the conformational changes associated with sulfur
loading, probably of residues located near the active site.
1

H-NMR spectroscopy
1
H-NMR spectra were obtained for the ES and E forms.
They show a characteristic
1
H resonance and, in particular,
differences can be seen in the region typical of the indolyl
protons of tryptophan and imidazolyl protons of histidine.
Water presaturation was performed before data acquisition,
making it difficult to detect the fast exchanging NHs of
histidines. Moreover, all resonances in this region showed
15
N resonances at 128–131 p.p.m. (data not shown), typical
of the NH group of the side chain of tryptophans. These
two observations led us to tentatively assign those reso-
nances as belonging to tryptophans. Figure 2 shows the
selected regions that are diagnostic of the different state of
Fig. 1.
1
H-
15
NNMRHSQCspectraof[
15
N]Cys230-labelled RhdA.
(A)
1
H-
15
NHSQCspectrumof0.2m
M

[
15
N]Cys230-labelled RhdA in
50 m
M
Tris/HCl (pH 7.2)/0.3
M
NaCl, after purification at 20 °C. (B)
Spectrum of [
15
N]Cys230-labelled RhdA after treatment with 2 m
M
thiosulfate (ES form). (C) Spectrum of [
15
N]Cys230-labelled RhdA
after treatment with 2 m
M
KCN (E form).
Fig. 2. Selected regions of
1
H-NMR spectrum of RhdA. The two characteristic
1
H spectra of the ES and E forms of the enzyme are shown in (A) and
(B), respectively. The transition between the two forms can be observed as a shift in the peaks (the NH of the indole ring of a Trp, from 11.6 to
12.1 ppm) on conversion of the enzyme from the E to the ES form on addition of thiosulfate ions (E/thiosulfate, 1 : 10) (C).
4210 S. Melino et al.(Eur. J. Biochem. 270) Ó FEBS 2003
the enzyme. The shift in resonance from 11.6 to 12.1 p.p.m.
is a sensitive check of the transition from the ES to the E
form (Fig. 2A,B). Moreover, during this transition, a high-
field shift of the resonance of a methyl group was also

observed, i.e. the peak at 0.6 p.p.m. disappeared and a new
peak appeared at 1.25 p.p.m. Figure 2C shows the beha-
viour of the same methyl resonance upon reconversion of
E into ES by addition of thiosulfate. Identification of these
resonances requires extensive assignment work, but some
educated guesses may be made before the assignment is
complete. It has been reported that addition of cyanide in
soaking experiments on RhdA crystal results in the removal
of the persulfide S atom bound to Cys230, and this reaction
induces conformational changes in the Cys230 and Trp195
side chains, which disrupts the Arg235 side chain [8]. Close
inspection of the crystal structure reveals that the methyl
groups of Leu238 and Leu180 face Arg235 and Trp195, the
residues affected by the conformational changes around the
active site, thus these are likely candidates to be affected by
the change in the persulfurated state of the protein.
Previous studies have shown that a number of residues
surrounding the catalytic Cys230 are able to generate a
strong positive electrostatic field which reaches an estimated
value of 18 kTÆe
)1
under standard physiological conditions
(pH 7.5, ionic strength 0.15
M
) [19]. Therefore we studied
the interaction of the active site of RhdA with negative ions
by
1
H-NMR spectroscopy, monitoring whether there is
transition between the forms of the enzyme after addition of

these ions. Figure 3C,D shows the spectrum of the ES form
after addition of phosphate and hypophosphite ions. No
changes in chemical shift wereobserved up to amolar ratio of
RhdA (ES) to ion of 1 : 10 at pH 7.2. The results indicate
that, in solution, the catalytic Cys230 residue was not affected
by the presence of these ions, up to the concentrations used.
Fluorescence experiments
As also observed for Rhobov, RhdA shows an intrinsic
fluorescence with a maximum at 336 nm, resulting from
six tryptophan residues present in the polypeptide chain
[4,5,20]. Fluorescence spectroscopy is particularly useful in
the study of rhodanese as it can report on modifications of
the active site cysteine. In fact, formation of a persulfide
group in the active site quenches the intrinsic fluorescence
of the protein without affecting its shape (Fig. 4A). This
has been attributed to local perturbation or long-range
energy transfer [20]. Therefore we carried out a fluores-
cence quenching study to monitor the change in confor-
mation of RhdA. The results before and after addition of
hypophosphite ions are shown in Fig. 4B,C. There was a
small effect of quenching on the E form after addition of a
very high concentration of hypophosphite ions (Fig. 4C).
It is probable that these anions are electrostatically
attracted by the positively charged side chains of the
residues around the active site and bind in their proximity,
influencing the intensity of the fluorescence of the trypto-
phan residues and resulting in a fluorescence quenching
effect.
Fig. 3. Selected regions of the
1

H-NMR spectrum of RhdA of the two forms of the enzyme, ES (A) and E (B), with
1
H-NMR spectra of RhdA (ES)
after the addition of phosphate (C) and hypophosphite ions (D). A molar ratio of RhdA (ES) to ion of 1 : 10 in 50 m
M
Tris/HCl, pH 7.2 was used for
(C,D).
Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4211
Previous studies also showed that fluorescence changes in
RhdA seem to be modulated by phosphate anions, when the
protein was purified in phosphate buffer at pH 6.0 [19]. In
phosphate buffer, recovery of the intrinsic fluorescence after
the addition of KCN, to produce sulfur-free RhdA, was
significantly lower than in the presence of Tris/HCl (18% vs.
46%) [19]. The strong positive electrostatic field of the active
site may be decreased in intensity by a large excess of
phosphate ions, resulting in a decrease in the stability of the
persulfide bond. Crystallographic studies of the ES form of
RhdA after the addition of 5 m
M
phosphate or hypophos-
phite anions reported that these compounds completely
remove the persulfide sulfur atom from Cys230 and in
particular the hypophosphite anion was observed in the
catalytic pocket. In contrast, no phosphate anions were
observed near the active site [19]. An explanation for these
different results may be found in the different behaviour of
the protein in solution. We used these different experimental
conditions, i.e. the molar ratio of ions, pH and incubation
times, because our goal was to determine the behaviour of

different anions compared with cyanide to evaluate the
different affinities for the protein. A large excess of, and long
exposure to, phosphate, as used in the previous study [19],
may have a different effect on the stability of the S-S bond.
Previously characterized rhodaneses, including the bovine
liver enzyme [9] and the enzyme from E. coli [21], are
typically inhibited reversibly and competitively with respect
to thiosulfate by most anions (acetate, sulfate and phos-
phate anions) at very high concentration. Our results clearly
indicate that the removal of the persulfide group from
Cys230 is due to a selective reaction with a sulfane sulfur
acceptor, such as cyanide, in conditions close in pH and
ionic strength to physiological, and not to the simple
presence of anions. However, the limited survey of anions
performed in the present study does not allow us to rule out
the possibility that low molecular mass mimics of active site
groups of normal protein acceptors may also be able to
replace cyanide.
Reaction of RhdA with SDG
Selenium uptake in the persulfide position of RhdA was
monitored by fluorescence and NMR spectroscopy after
reaction with SDG. This compound was prepared by the
reaction of GSH with selenite as previously reported [13]
and based on earlier studies [22,23], suggesting that SDG
and its subsequent reduction to glutathionyl selenide anion
[24,25] are key intermediates in the selenium metabolic
pathway. It has been observed that in vitro the labile SDG
may react with Rhobov at neutral pH to generate an ESe
form [13]. The intrinsic fluorescence of RhdA before and
after addition of the GSH/selenite solution leads to the

selenium-loaded form of RhdA (ESe) (Fig. 5). In fact,
quenching of the intrinsic fluorescence corresponding to DF
of 22% at 336 nm was observed after incubation of RhdA
(E) with SDG solution [RhdA (E)/SeO3-2/GSH, 1 : 5 : 20]
at 37 °C for 10 min, whereas a DF of 11% was observed on
addition of a 10-fold molar excess of thiosulfate with the
same E form (Fig. 5). No further changes in intrinsic
fluorescence of the ESe form were observed after addition of
an excess of thiosulfate, confirming the presence of the
loaded form of the enzyme (data not shown). On the other
hand, fluorescence experiments with the ES form of RhdA
showed no changes after addition of SDG. Intrinsic
fluorescence was measured by adding selenite or GSH to
RhdA [at a molar ratio of RhdA (E) to SeO
3
2–
of 1 : 5 and
Fig. 4. Fluorescence spectra of RhdA in the presence of anions. (A) The
sulfur-loaded state of 6 l
M
RhdA in 50 m
M
Tris/HCl, pH 7.2, (solid
line), and the sulfur-free form of RhdA (dashed line) with thiosulfate
(E/thiosulfate, 1 : 100) (dotted line); (B) ES form (solid line) in the
presence of hypophosphite ions with a molar ratio of RhdA to ion of
1:1; 1:10; 1:20;1:50;1:100; (C) E form inthe presence of
hypophosphite ions at the molar ratio used in (B).
4212 S. Melino et al.(Eur. J. Biochem. 270) Ó FEBS 2003
RhdA (E) to GSH of 1 : 20], and the quenching (DF)

observed was 4% for both (data not shown). This indicates
that the labile SDG compound, produced by reaction
between selenite and GSH, reacts with RhdA at neutral pH
to generate an ESe rhodanese. The quenching of intrinsic
fluorescence for selenium loading of RhdA is higher than
that observed after treatment with thiosulfate because of the
higher quenching properties of selenium than of the
persulfide bond. These results are in agreement with those
of Cannella et al. [20], who prepared a selenium derivative
of Rhobov by using the synthetic substrate selenosulfate
and examined its spectroscopic properties. Selenium binding
to the protein was also detected by NMR spectroscopy.
Experiments on
15
N-Cys-labelled RhdA were performed
under the same conditions as for the fluorescence experi-
ments. Figure 6 shows the HSQC spectra of the [
15
N]Cys-
labelled RhdA (E) in the presence of GSH/selenite. The
spectrum indicates the formation of a new form of the Cys
residue. The
1
H-
15
N cross-peak of the catalytic cysteine was
shifted (
15
N, 118.5 p.p.m.;
1

H, 8.56 p.p.m.) upon reaction
with the SDG. Moreover, no changes in the
1
Hand
15
N
resonances of the [
15
N]Cys230 were observed after the
addition of the selenite/GSH mixture to the sulfur-loaded
RhdA (data not shown).
Figure 7 shows the
1
H-NMR spectral regions highly
indicative of the persulfurated and sulfur-free states of the
enzyme after addition of GSH alone, selenite alone, or
selenite/GSH equivalent to adding SDG. The
1
H-NMR
spectra of RhdA show that no loading occurred on addition
of either GSH or selenite alone to the E form (Fig. 7B,C). In
contrast, the addition of selenite/GSH to the E form induces
changes in the
1
H-NMR resonance pattern similar to those
observed by addition of thiosulfate.
No specific interactions with the catalytic site of the
enzyme were found in presence of the GSH or selenite ions
alone, indicating that the loading of selenium to the ESe
form occurs by a specific reaction with SDG. These results

confirm the hypothesis that rhodanese-like proteins may
function as components of the delivery system for reactive
selenium in vivo.
Prospective studies over the last few years have suggested
that Se intake may protect against cancer [26,27]. Several
mechanisms have been proposed to explain the anticarci-
nogenic effects of Se compounds [26]. One hypothesis is that
Se compounds induce apoptosis in initiated premalignant
cells, i.e. SDG induces p53 [28]. Furthermore, Ghose et al.
[29] recently reported that SDG induces apoptosis in oral
cell cultures. Induction of apoptosis has been attributed to
SDG because of the observation that it alters the redox
status of the cell by manipulating the level of a cellular
reducing agent, such as thioredoxin, which has been
implicated in growth control in other contexts and is
overexpressed in many tumours [30]. In fact, SDG has been
shown to be a specific oxidant of reduced thioredoxin and
inhibitor of thioredoxin reductase in a cell-free system
[31,32]. It has been shown that Rhobov has an affinity that
is 1000-fold higher for the reduced form of thioredoxin than
Fig. 6.
1
H-
15
N-NMR HSQC spectrum of the selectively [
15
N]Cys230-
labelled RhdA spectrum after addition of SDG. The [
15
N]Cys ESe was

obtained by addition of the SDG solution at 0.1 m
M
[
15
N]Cys230-
RhdA (E) [RhdA (E)/SeO
3
2–
/GSH, 1 : 10 : 40) in 20 m
M
Tris/HCl/
0.3
M
NaCl, pH 7.4.
Fig. 5. Fluorescence changes induced by SDG on RhdA. Sulfur-free
form (E) of 5 l
M
RhdA in 50 m
M
Tris/HCl (pH 7.4)/1 m
M
EDTA
(solid line); E form after addition of thiosulfate 50 l
M
(dotted line) or
SDG solution (to a final concentration of 25 l
M
SeO
3
2–

and 100 l
M
GSH) (dashed line).
Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4213
for cyanide [33], so it seems reasonable to suppose that SDG
may have an indirect effect in vivo on the thioredoxin system
through the rhodanese system. Moreover, the rhodanese-
like proteins may participate in detoxification of molecules
such as thiosulfate, selenite and SDG, raising interest about
the biological role of these proteins.
Our results show that a simple
1
H-NMR spectrum can be
used as a sensitive and fast monitor of sulfur or selenium
loading of RhdA. Although a protein of the size of RhdA
(31 kDa) gives hundreds of proton signals, the
1
H-NMR
spectrum shows two regions that are sufficiently well
resolved to follow the change in chemical shift induced by
the protein conversion of protons attached to nitrogen
(probably an indolyl proton of a tryptophan) and methyl
protons. Thus this NMR experiment can be performed
without the need of isotope labelling, and, given the
sensitivity of modern high-field spectrometers, protein
concentrations of 10–50 l
M
and experimental times of a
few minutes are sufficient to obtain information on the state
of the enzyme.

Acknowledgements
The technical assistance of Fabio Bertocchi is gratefully acknowledged.
This research was supported by MURST PRIN project ‘Sulfotrans-
ferasi procariotiche’ (1999–2001 and 2002–03) and the target Project of
Italian CNR ‘Biotecnologie’ and the project FIRB of Italian MIUR.
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