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IUBMB

Life, 59(2): 51 59, February 2007

Critical Review
Common Themes and Variations in the Rhodanese Superfamily
Rita Cipollone1, Paolo Ascenzi1,2 and Paolo Visca1,2
1
2

Dipartimento di Biologia, Universita` Roma Tre, Roma, Italy


Istituto Nazionale per le Malattie Infettive IRCCS Lazzaro Spallanzani, Roma, Italy

Summary
The rhodanese homology domain is a ubiquitous fold found in
several phylogenetically related proteins encoded by eubacterial,
archeal, and eukaryotic genomes. Although rhodanese-like proteins
share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese
superfamily. The variability occurs at dierent levels including
sequence, active site loop length, presence of a critical catalytic Cys
residue, and domain arrangement. Even within the same genome,
multiple genes encode rhodanese-like proteins presenting with
variably arranged rhodanese domain(s): as single or tandem
domain(s), or combined with other protein domain(s). Given the
highly variable organization of the rhodanese domain(s) and the
context where it is found, here we review the structural organization
and function of the rhodanese-like proteins. The overview of the
most recent ndings about rhodanese allow us to depict a
superfamily of versatile proteins relying on persulde chemistry to
accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to
sulfur metabolism and progression of cell cycle.
IUBMB Life, 59: 5159, 2007
Keywords

Rhodanese; sulfurtransferase; sulfane sulfur; cyanide


scavenging.

Abbreviations MST, 3-mercaptopyruvate:cyanide sulfurtransferase;


Rhobov, mitochondrial bovine (Bos taurus) rhodanese;
ST, sulfurtransferase; TST, thiosulfate:cyanide sulfurtransferase.

INTRODUCTION
In 1933, Lang reported that certain tissues of mammals
contained an enzyme able to catalyze the reaction between
thiosulfate and cyanide leading to the formation of thiocyanate and sulte (1). The responsible enzyme was designed
Received 19 December 2006; accepted 8 January 2007
Address correspondence to: Rita Cipollone, Department of
Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146
Roma, Italy. Tel: 39 (0)6 5517 6346. Fax: 39 (0)6 5517 6321.
E-mail: mic.stud@uniroma3.it
ISSN 1521-6543 print/ISSN 1521-6551 online 2007 IUBMB
DOI: 10.1080/15216540701206859

rhodanese, from the German name for thiocyanate (rhodanid), with the ending ese indicating that this compound was
formed through the enzymatic reaction. According to the
ocial nomenclature rules, the proper name of the enzyme is
thiosulfate:cyanide sulfurtransferase (TST, EC 2.8.1.1), based
on the reaction catalyzed in vitro; however, the trivial name of
rhodanese is still commonly used.
The rst and best characterized rhodanese is the mitochondrial bovine (Bos taurus) TST, conventionally referred to as
Rhobov (2 4). Analysis of the Rhobov structure (293 amino
acids long), highlights that the protein consists of two equallysized globular domains, the inactive N-terminal and the
catalytic C-terminal rhodanese domain, showing identical a/b
topology. Each domain is about 120 amino acids long with a
low sequence similarity. Only the C-terminal domain carries
the six-amino acid active site loop hosting at the rst position
the Cys residue involved in the catalytic process. The counterpart of the Cys residue in the N-terminal domain is an Asp
residue which has no involvement in catalysis (5) (Fig. 1).
The peculiarity of rhodanese resides in two patterns of
amino acid sequences that can be recognized at the N-terminal
region ([F/Y]-X3-H-[L/I/V]-P-G-A-X2-[L/I/V/F]) and at the
C-terminal end of the protein ([A/V]-X2-[F/Y]-[D/E/A/P]-G[G/S/A]-[W/F]-X-E-[F/Y/W]) (Fig. 1). These sequences, called
rhodanese signatures, have a remarkable degree of conservation among rhodaneses and therefore are used to recognize
these proteins encoded by dierent genomes. Rhodaneses
are present in organisms belonging to dierent phyla, and
although these proteins can dier signicantly at the sequence
level (Fig. 1), the tandem domain three-dimensional structure
resembling that of Rhobov is highly constrained. For instance,
Pseudomonas aeruginosa rhodanese shows 79% and 22%
sequence identity with Azotobacter vinelandii rhodanese and
Rhobov respectively, but no striking dierences emerge by
comparing their three-dimensional structures (Fig. 2). Thus,
the highly conserved double domain architecture of Rhobov
has been assumed as the reference structure of the entire
family of evolutionary conserved tandem-domain TSTs, which
can be addressed as rhodaneses sensu stricto.

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CIPOLLONE ET AL.

Figure 1. Partial sequence alignment of the rhodanese domains from representative members of the rhodanese superfamily:
Rhobov (P00586), E. coli SseA (P31142), A. vinelandii RhdA (P52197), P. aeruginosa RhdA (Q9HUK9), human Cdc25A
(P30304), E. coli GlpE (P0A6V5), W. succinogenes Sud (Q56748), and E. coli ThiI (P77718). Rhodanese signatures are
highlighted in grey. Residues forming the six-amino acid active-site loop of the catalytic domain are underlined. Amino acid
sequences were retrieved from Swiss-Prot/TrEMBL database (www.expasy.org/sprot/) and aligned with the program
CLUSTAL_W with default parameters (55). Identical residues, conserved and semi-conserved substitutions are indicated by
asterisks, colons, and periods, respectively. Note that translation of Rhobov reading frame diers from chemically determined amino acid sequence by an N-terminal Met and the three residue C-terminal extension Gly-Lys-Ala (not shown in the
gure).

RHODANESE STRUCTURE AND FUNCTION

Figure 2. Three-dimensional structure of Rhobov (1RHD), A.


vinelandii RhdA (1E0C) and P. aeruginosa RhdA. The three
dimensional structure of Rhobov and A. vinelandii RhdA were
recovered from the Protein Data Bank (www.rcsb.org).
Homology modelling of P. aeruginosa RhdA has been
obtained using A. vinelandii RhdA as the template and the
SWISS-MODEL
facility
(http://swissmodel.expasy.org/
SWISS-MODEL.html).

Rhodanese catalytic process occurs via a double displacement (ping-pong) mechanism involving the stable formation of
a persulde-containing intermediate (ES) (6 8):

53

Values of catalytic parameters for prototypical TST action are


reported in Table 1.
The transferring sulfur is bound to an invariant catalytic
Cys residue and is transferred formally as S0 by nucleophilic
reaction with cyanide, to yield thiocyanate and regenerate the
active Cys residue for another round of catalysis. The chemical
species intervening in sulfur delivery is a sulfane sulfur, a
sulfur atom covalently bound to the sulfur atom of Cys, which
has an apparent oxidation state of 0 or -1 (6 8).
Several metabolic reactions are known to generate sulfane
sulfur in vitro but their relative importance in vivo is not yet
known. Current knowledge about sulfur donors assumes that
the primary source of sulfane sulfur in eukaryotes relies on
L-cysteine desulfuration pathway (9). It has been demonstrated
that rat liver cystathionase (EC 4.4.1.1) greatly enhances the
trans-sulfuration of cyanide to thiocyanate; thus, a product of
the cystathionase reaction, thiocystine, has been suggested to
serve as the sulfur donor substrate for rhodanese (10).
In prokaryotes, the information available is more vague.
An interesting hypothesis about the sulfur donor has been
entertained by Forlani and co-workers (11) on the basis of the
in vitro persulfuration of A. vinelandii TST by Escherichia coli
L-cysteine desulfurase (IscS). However, although this hypothesis provides a rst evidence for an enzymatic persulfuration
of rhodanese, it still lacks in vivo substantiation using the
homologous experimental system.
Reviews covering biochemical and functional properties of
STs have been published in the past years (3, 12, 13). Here we
will combine recent knowledge on dierent aspects of the
rhodanese structure and reaction mechanism to provide a
critical overview of the dierent functions performed by these
proteins. For this purpose, a novel classication of the
rhodanese-like proteins, possibly corresponding to functional
groups, is proposed.

THE RHODANESE SUPERFAMILY


Inspection of the SMART resource (http://smart.emblheidelberg.de/), using rhodanese as the keyword to identify the
rhodanese homology domain, reveals that rhodanese is widely
distributed in Eubacteria, Archea, and Eukarya. A substantial
proportion of the predicted gene products is functionally
uncharacterized or only tentatively classied. Analysis of their
sequences highlights that they are highly heterogeneous
despite the conservation of the rhodanese signatures (12).
Hence it seems necessary to properly address these proteins as

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CIPOLLONE ET AL.

Table 1
Values of catalytic parameters for TST actiona
Substrate, Km (mM)
Enzyme

Cyanide

Thiosulfate

Double domain TSTs


Rhobovb
Azotobacter vinelandii RhdAc
Pseudomonas aeruginosa RhdAd

0.063
8.7
16

7.0
1.0
7.4

Single domain TSTs


Escherichia coli GlpEe
Escherichia coli PspEf
Arabidopsis thaliana BAB10409g
Arabidopsis thaliana BAB10422g

17
4.6

78
27
7.4
1.1

Vmax
600 mmol min71 mg71
1250 mmol min71 mg71
815 mmol min71 mg71
230 s71
0.0067 s71
4 s71
10.7 s71

Values of catalytic parameters were obtained between pH 8.5 and 9.0, and between 20.08C and 25.08C.
From 56.
c
From 57.
d
From 8.
e
From 15.
f
From 16.
g
Values of catalytic parameters were obtained at [cyanide] 60 mM. From 58.
b

rhodanese-like (or rhodanese-related) proteins and introduce


a classication possibly based on their common traits.
Accordingly, here we present the rhodanese-like proteins
characterized so far grouped into four distinct classes, likely
corresponding to homogeneous structural groups. Interestingly, genome analysis of P. aeruginosa reveals the presence of
ten rhodanese-like proteins (14) belonging to each of the
rhodanese groups here proposed, with the only exception of
the members belonging to the elongated active-site loop
proteins group (i.e., group IV), a class of rhodanese-like
proteins that are apparently restricted to eukaryotic organisms. Thus, the coexistence of several rhodanese-like proteins
in the same organism suggests a plethora of dierent
physiological roles fullled by the rhodanese superfamily.

Group I: Single Domain Proteins


From an historical perspective, the importance of the
coexistence of two stably folded domains and the presence of
the N-terminal domain for rhodanese stability clearly
emerged. Thus, the idea that a functionally active rhodanese
could exist only for the presence of an intact double domain
structure was widely accepted. However, proteins containing
either a catalytic or an inactive rhodanese domain have been
characterized only in the past few years. Interestingly,
structural and functional studies performed on the E. coli
TST GlpE, containing a single 108-amino acid rhodanese
domain, demonstrated that a single domain can be selfsucient for thiocyanate formation from thiosulfate in the
presence of cyanide (15) (see Figs 1 and 2, and Table 1).

Single catalytic rhodanese domain proteins are encoded by


both eukaryotic and prokaryotic genomes. These proteins
have been associated with specic stress conditions as in the
case of the Drosophyla melanogaster heat shock protein 67-B2,
of the E. coli phage-shock protein E (PspE) (16), and of the
Vibrio cholerae shock protein Q9KN65 (17). Proteins containing single rhodanese domain have also been associated with
the process of leaf senescence in plants. These include Sen1 in
Arabidopsis thaliana (18), Ntdin in Nicotiana tabacum, and
Din1 in Raphanus sativus (19). Other recently identied
proteins showing TST activity in vitro are P15 and P16.2
from the bacterium Acidithiobacillus ferrooxidans, possibly
involved in sulfur oxidation (20), and the 121 amino acid
rhodanese domain At4g01050 from A. thaliana, whose
solution structure has been determined despite its still
unknown function (21).
The only reported case of a single domain protein endowed
with a dierent activity from TST is the polysulde:cyanide ST
Sud from the eubacterium Wolinella succinogenes, a periplasmic protein which uses the same Cys residue not only for
catalyzing the sulfur transfer but also for additional hydrolase
activity (22). The highest similarity to the Sud protein is
displayed by the A. ferrooxidans rhodanese-like protein P14.
However, this protein lacks the signal peptide for periplasmic
export, and a dierent function has been inferred from the
dierential cellular localization of Sud and P14 (20).
Proteins displaying only the inactive domain, i.e., lacking
the active Cys residue, are less common and restricted to the
eukaryotic dual-specic MAPK-phosphatases, and yeast

RHODANESE STRUCTURE AND FUNCTION

(Ubp4, Ubp7, Ubp8) and mammalian (Ubp-Y) ubiquitin


hydrolases (23, 24).

Group II: Tandem-Domain Proteins


Although catalytic and inactive domains seem to be phylogenetically distinct (12), proteins containing both domains
are very common and in most cases belong to the family of
STs (EC 2.8.1.x). In fact, tandem-domain rhodaneses include
rhodaneses sensu stricto (or double domain TSTs) and the
strictly related 3-mercaptopyruvate:cyanide sulfurtransferases
(MSTs, EC 2.8.1.2).
Double domain TSTs have been characterized from dierent
eukaryotic and prokaryotic sources including B. taurus (3),
A. vinelandii (7), and P. aeruginosa (8), all showing the salient
features of rhodaneses sensu stricto (see Figs 1 and 2).
MSTs share common structural and functional features with
TSTs, but utilize 3-mercaptopyruvate as the specic sulfur
donor to catalyze the sulfur transfer reaction. The catalytic
activity of MST subfamily members is centered on a reactive
invariant Cys residue as for TSTs, but little is known about the
molecular mechanism of sulfur transfer from 3-mercaptopyruvate (25). In this respect, kinetic studies performed on bovine
kidney and rat MSTs have indicated that, dierently from what
observed in double domain TSTs, transfer of a sulfur atom
seems to occur via a single displacement mechanism, without
the formation of a stable persulde intermediate (25).
Only Rattus norvegicus, A. thaliana, Leishmania major, and
E. coli MST have been biochemically characterized so far.
SseA, the product of the E. coli sseA gene endowed with MST
activity in vitro, represents the prototype of the MST
subfamily (25). MSTs display clear sequence homology with
rhodaneses, with which they can share up to 66% identical
residues (26). Note that it is possible to convert the sulfur
donor specicity of MST to TST and vice versa, by sitedirected mutagenesis, given the high structural similarity of
MST to TST (26). In fact, main dierences between TSTs and
MSTs occur at the level of active-site loop amino acid
sequences, being CRXGX[R/T] and CG[S/T]GVT, respectively. The amino acid properties of the active-site loop of
known TSTs and MSTs correlates with the distinct ionic
charge of their in vitro substrates, thiosulfate(2-) and 3mercaptopyruvate(1-), respectively (12).
An example of tandem-domain rhodanese that seems to
belong to none of the ST subfamilies is represented by the
RdlA protein from Halanaerobium congolense. This protein
was originally predicted as TST, but it does not display TST
activity in vitro, despite containing two domains with
potentially reactive Cys residues (27). The role of RdlA is
uncertain but it is deemed to be involved in reductive cleavage
of thiosulfate (27).
Group III: Multidomain Proteins
The catalytic rhodanese domain is often combined with
other characterized protein domains. In particular, the

55

involvement of a rhodanese domain in a given process seems


to be related to the functional properties of the accompanying
domain. Paradigmatic examples are ThiI- and ThiF/MoeBrelated proteins, which are involved in the metabolism of the
sulfur-containing biomolecules thiamin and molybdopterin,
respectively (28 30).
ThiI is common to the biosynthetic pathways leading to
both thiamin and 4-thiouridine, a modied base present in
certain bacterial tRNAs (28). Almost all the proteins referable
to the ThiI family have an N-terminal THUMP domain
involved in delivering a variety of RNA modication enzymes
to their targets, and a C-terminal rhodanese module. In the
proposed ThiI reaction mechanism, a partner desulfurase
(IscS) rst catalyzes the transfer of sulfur from a free Cys to
the Cys catalytic residue of the rhodanese homology domain
of ThiI which in turn becomes the sulfur source for thiamine
and 4-thiouridine biosynthesis (29). ThiI is also endowed with
TST activity in vitro, but at a very low rate (28).
The occurrence of rhodanese domains has also been
observed at the C-terminal end of ThiF/MoeB-related proteins, which are involved in molybdopterin biosynthesis;
however, MoeB-like proteins lacking the rhodanese domain
also exist (30). An example is provided by the human cytosolic
MOCS3 protein which contains an N-terminal MoeB-like
domain and a C-terminal module displaying similarities with
rhodanese (31). The MOCS3 rhodanese domain is believed to
catalyze the thiocarboxylation of MOCS2A, a subunit of the
molybdopterin synthase, via a reaction in which the MoeB-like
domain of MOCS3 activates the C-terminal Gly residue of
MOCS2A to form an acyl adenylate. Subsequently, the
rhodanese-like domain of MOCS3 acts as a direct sulfur
donor for the formation of the thiocarboxylate group in
MOCS2A (31):

Another protein is the E. coli YbbB, which consists of a


rhodanese catalytic domain and a second domain containing a
P-loop (Walker A) motif. This enzyme is the only member of
the rhodanese superfamily capable of catalyzing the last
biosynthetic step of 2-selenouridine, a modied base with
unknown function that is present at the wobble position of
Lys, Glu, and Gln tRNAs. Upon reaction with selenophosphate to generate a perselenide at the Cys catalytic residue of
the rhodanese domain, the transfer of selenium replaces sulfur

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CIPOLLONE ET AL.

in 2-thiouridine yielding 2-selenouridine (32). YbbB provides


evidence that substrate specicity of the rhodanese homology
domain is not restricted solely to the transfer of sulfur. A
conrmation of the capacity of rhodanese-like proteins to
function as components of the delivery system for reactive
selenium has also been provided by in vitro experiments with
RhdA from A. vinelandii, showing specic interaction between
selenodiglutathione and the catalytic Cys residue (33).
In A. thaliana a multi-domain protein containing an inactive rhodanese homology domain has been described as an
extracellular calcium-sensing receptor, thus suggesting a
signaling-like function (34).

Group IV: Elongated Active-site Loop Proteins


Cdc25A phosphatases are involved in the dephosphorylation of cyclin-CDK complexes for the progression of the cell
cycle. In their active-site loop, these enzymes contain the
CX5R motif, a typical sequence of the protein-tyrosine
phosphatase superfamily. However, the structure of the
human Cdc25A catalytic domain reveals an unexpected
structural similarity with each half of tandem domain
rhodaneses (12, 24). Dierently from any other rhodaneserelated protein, the active-site loop of Cdc25A (which is
coincident with the tyrosine phosphatase motif) is an
elongated stretch of the rhodanese active-site loop. In this
case, the catalytic Cys residue directly attacks the phosphate
atom of the phosphorylated cyclin-CDK complex forming a
phosphocysteine intermediate in the rhodanese homology
domain (35). The importance of the features of the catalytic
loop in the discernment of the proper substrate has been
conrmed by mutagenesis experiment performed on A.
vinelandii RhdA. The elongation of the native RhdA catalytic
loop, in fact, results in the impairment of productive
interaction with substrates involved in the sulfur transfer
reaction and in the productive interaction with an articial
phosphatase substrate. Taken together, these results clearly
indicate that ST and phosphatase activities do not exist
concurrently (12). Considering the very distant relationship
between eukaryotic Cdc25 proteins and the rest of the
rhodanese-like proteins, it is not surprising that even the
rhodanese signatures are not completely respected (Fig. 1).
The typical feature of an elongated active-site loop is
conserved also in the Arath;CDC25 protein from A. thaliana
(36). Based on the pattern of conserved residues in the activesite loop, other proteins presenting with similar amino acid
sequence are the Saccharomyces cerevisiae Acr2, a unique
eukaryotic arsenate resistance enzyme (note that arsenic is
chemically similar to phosphorus), and Yg4E whose function
is still unknown (12).

RHODANESE-LIKE PROTEIN FUNCTIONS


Apart from the elongated active-site loop proteins, which
recognize phosphate as the substrate, it can be argued from the

general features laid out above that rhodaneses provide


eukaryotic and prokaryotic cells with a labile reactive sulde
which is at the basis of dierent cellular processes involving
sulfur transfer reactions (37, 38). Only in case of ThiI- and
ThiF/MoeB-related proteins, an univocal function in the
metabolism of the sulfur-containing biomolecules, thiamine,
4-thiouridine, and molybdopterin has been reported (for
details, see 38).
About the other rhodanese-like proteins, the predominant
view is their involvement in distinct biological functions, as
suggested by the wide variability of amino acid residues in the
active-site loop. In fact, the amino acid side chains extending
from this structural element dene the ridge of the catalytic
pocket, and are therefore expected to play a key role in
substrate recognition and catalytic activity (12). In case of the
best known proteins, i.e., tandem-domain TSTs and MSTs,
the biological function is still largely debated and spans from
cyanide detoxication to sulfur metabolism. The extreme
indetermination in attributing a dened role to STs is likely
due to the fact that the identication of the in vivo substrates
has thus far proven inconclusive. It appears unlikely that
privileged natural substrates of rhodaneses are those identied
by in vitro reactions given that the thiosulfate/mercaptopyruvate and cyanide anity is in the millimolar range (Table 1),
thus apparently incompatible with the supposed physiological
role in enzymatic cyanide detoxication (8, 25, 39). Therefore,
alternative functions, including sulfur and selenium metabolism and biosynthesis of prosthetic groups in iron-sulfur
proteins, have been proposed (40 46).

Cyanide Detoxification
It has been understood for nearly a century that most of the
sub-lethal quantities of cyanide absorbed by either ingestion
or inhalation is detoxied by cyanide reaction with reactive
sulfur. In eukaryotes, rhodaneses and MSTs are supposed to
be involved in cyanide detoxication. As far as tandemdomain TSTs are concerned, the hypothesis is supported by
the high concentration of these enzymes in tissues and organs
exposed to cyanide (47). Although the distribution pattern of
tandem-domain TSTs in dierent tissues is species specic,
rhodanese levels are usually high in hepatocytes that are in
close proximity to the blood supply of the liver, in epithelial
cells surrounding the bronchioles (a major entry route for
gaseous cyanide) and in proximal tubule cells of the kidney
(serving to facilitate cyanide detoxication and elimination as
thiocyanate). In cattle, rhodanese is maximally expresses in the
stomach epithelium of rumen, omasum, and reticulum. In
these districts, where cyanide is released following ingestion of
plant cyanogenic glucosides, rhodanese activity is signicantly
higher than in liver, arguing for a role of rhodanese in cyanide
detoxication (48). Moreover, rhodanese has recently been
detected also in the mouse brain (49), and found to be
localized in areas which synthesize cyanide possibly acting as a
neuromodulator (50). Thus, a possible regulatory role of

RHODANESE STRUCTURE AND FUNCTION

rhodanese in tuning (through conversion to thiocyanate) the


neuromodulator properties of cyanide has been hypothesized
(51).
However, since tandem-domain TST activity is conned to
the mitochondrial matrix where thiosulfate enters with low
eciency (3), it has been suggested that rhodanese actually
adopts other unidentied sulfur source(s) for cyanide detoxication (52). Alternatively, on the basis of the local
distribution of STs, it has been proposed that rhodaneses
detoxify cyanide together with MSTs (52). In fact, MSTs are
distributed both in cytoplasm and in mitochondria (52). When
cyanide enters the cytoplasm, cytosolic MSTs could catalyze a
primary sulfuration reaction of cyanide and the poison
eventually escaping this rst detoxication step could further
be sulfurated by mitochondrial rhodanese, denitively avoiding the lethal inhibition of cellular respiration (i.e., of
cytochrome c oxidase activity) (52). It is noteworthy, however,
that the therapy for acute cyanide poisoning combines
intravenous administration of sodium nitrite and sodium
thiosulfate. Sodium nitrite oxidizes oxy-hemoglobin, resulting
in met-hemoglobin whose anity for cyanide is higher than
that of cytochrome c oxidase. Sodium thiosulfate is the
substrate of tandem-domain TSTs and poorly serves MSTs for
cyanide detoxication. Moreover, mercaptopyruvate administered intravenously is not eective for cyanide detoxication,
due to its rapid decomposition in the blood (52). Thus, the
specic contribution of tandem-domain TSTs and MSTs in
physiological cyanide detoxication remains controversial.
Interestingly, the function in protection against cyanide
toxicity, attributed as rst to Rhobov in eukaryotes (2, 3) has
also been established in prokaryotes (14, 53). The capacity of
Bacillus stearothermophilus to detoxify cyanide is greatly
increased in mutants showing 5- to 6-folds rhodanese activity
than wild-type (54). Moreover, RhdA from the cyanogenic
bacterium P. aeruginosa has recently been reported to contribute to cyanide detoxication (8, 14, 53). In fact, although
RhdA is endowed with low anity for both thiosulfate and
cyanide in vitro, it has been shown to provide protection
against cyanide toxicity when overexpressed in E. coli as
heterologous host (53). RhdA also promotes the viability of P.
aeruginosa during growth under cyanogenic conditions (14).
Thus, in vitro biochemical studies which predict a minor role
of RhdA in cyanide detoxication could underestimate the
actual role of the enzyme in vivo.

Sulfur and Selenium Metabolism


Rhodanese-like proteins may have a role in sulfur and
selenium metabolism. The expression of the P21 rhodaneselike protein from A. ferrooxidans seems to be induced during
growth on dierent oxidizable sulfur compounds to generate
thiosulfate, which is used as electron donor by this chemolithotrophic bacterium (40). Yet, disruption of the gene
encoding the rhodanese-like enzyme CysA from Saccharopolyspora erythraea results in cysteine auxotrophy (41). In both

57

cases there is no signicant eect on the cellular TST activity.


It should be noted that the pathway for cysteine biosynthesis
in S. erythraea diers from that established, for instance, in E.
coli, in so far as only the former includes thiosulfate as an
intermediate. Thus, it is possible that P21 as well as CysA have
the specic function to synthesize thiosulfate (40).
An alternative rhodanese function has been proposed by
Ogasawara and co-workers (42) who reported that the E form
of Rhobov can bind selenium at a 1:1 molar ratio in vitro,
leading to the formation of the stable perselenide form (E-Se)
of rhodanese. E-Se rhodanese was prepared in a reaction with
SeO327 and glutathione, both of which at physiologically
meaningful concentrations. Therefore, E-Se rhodanese has
been proposed to be necessary to generate the reactive form of
selenium for the synthesis of selenophosphate (SePO337)
which is the active selenium-donor compound required by
bacteria and eukaryotes for the specic synthesis of SeCystRNA, the precursor of selenocysteine in selenoenzymes.

Synthesis or Repair of Iron-sulfur Proteins


Rhodanese appears to contribute to the recovery of the
native architecture of reconstituted iron-sulfur protein(s) by
mobilizing sulfur for the formation or repair of iron-sulfur
clusters. The iron-sulfur clusters of ferrodoxins, succinate
dehydrogensase, and mitochondrial NADH dehydrogenase
can be reconstituted by incubation with Rhobov, a sulfur
donor, a sulfur acceptor (dihydrolipoate), and an iron source
(43 45). However, the participation of tandem-domain TSTs
in the formation of iron-sulfur clusters has been questioned,
since there is strong evidence that Nif/Isc-related proteins are
involved in the mobilization of sulfur from cysteine for the
biosynthesis of iron-sulfur clusters (46).

CONCLUDING REMARKS
No unique reaction can be written at present to completely
dene the biological function(s) of the rhodanese superfamily
members. The three-dimensional structure of some of the
representative members has been obtained from both prokaryotic and eukaryotic species, highlighting the underlying
invariant features as well as the range of possible structures
for other superfamily members. On the other hand, there are
still numerous unanswered questions regarding the physiological functions of individual proteins and their mechanisms of
action. The increasing number of novel rhodanese-like
proteins with diverse and highly specialized functions supports
the notion that rhodaneses are involved in dierent cellular
processes, and coexist within the same organism since they
accomplish essential cell functions.
However, the large body of work carried out in vitro and
in vivo on dierent rhodanese superfamily members indicates
that the determinants responsible for functional selectivity of
the rhodanese domain reside in the features of the active-site
loop and in the domain arrangement (see Figs 1 and 2).

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CIPOLLONE ET AL.

Both elements, in fact, concur to ensure the formation, interconversion, and transport of the highly reactive sulfane sulfur
species to the proper sulfur acceptor, whose involvement in
some cellular processes (e.g., biosynthesis of cofactors
and thionucleosides) and in environmental adaptation (e.g.,
cyanide detoxication) is preponderant.
Undoubtedly, much of research on rhodanese has been
focused on cyanide resistance, as rst suggested after the discovery of the Rhobov. However, increasing body of evidence
suggest that Nature has settled on protein persulde groups the
privileged sulfur source for several biochemical processes. The
identity of the primary sulfur source is still questionable,
but whatever it is, the unexpected occurrence of so many
rhodanese-like proteins suggests that cells use sulfane sulfur to
tune sulfur ux through pathways involving rhodanese
domains. The rhodanese domain seems to be the key enzymatic
withholder of the persulde group, which can be transferred
directly from the enzyme that incorporates it into a substrate,
or can be passed to another protein acceptor before nal
delivery.
Although Rhobov was discovered more than 70 years ago,
the list of open issues about the rhodanese-like protein superfamily is still long. The availability of large-scale genomic
databases holds out the prospect of discovering novel
rhodanese classes which can infer an even wider range of
functions than those presently known. Hopefully, the investigation of these new proteins will help to answer impelling
questions regarding the identication of possible sulfur
donor(s) and/or acceptor(s), and will oer fascinating possibilities for examining the not fully resolved regulatory role of
sulfur in cellular metabolism.

ACKNOWLEDGEMENTS
This work was supported by grants from Istituto Superiore per
la Prevenzione e la Sicurezza sul Lavoro (B1-39/DML/04),
Ministero dellUniversita` e della Ricerca (COFIN 2004), and
Ministero della Salute (Ricerca Corrente INMI Lazzaro
Spallanzani 2005) to P.A. and P.V.
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