:Tungsten

As a micro-nutritional supplement the trace mineral tungsten belongs to the same family as chromium and molybdenum. Both chromium and molybdenum are known as essential minerals for very important biological functions. Chromium is essential for carbohydrate metabolism. Correcting chromium deficiencies helps with obesity and weight management. Dr. Henry Schroeder has very convincing data linking chromium deficiency and heart disease.

Trace Elements in Human and Animal Nutrition (fifth edition):

"Clinical symptoms, totally eliminated by supplementation [of molybdenum compound], included irritability leading to coma, tachycardia, tachypnea, and night blidness."

Studies show tungsten has biological effects similar to vanadium. In animal studies, tungsten mimics insulin. Diabetic rats given tungsten have lowering of both sugar and insulin-levels in a comparison with a control-group. The animals given tungsten stay healthy in both short and long term studies and they are free of any side-effects. Tungsten supplementation prevents cataracts in rats and mice. Tungsten is effective against an otherwise lethal dosage of radiation.

High levels of tungsten, as with other heavier trace elements, in human drinking water protects against schizophrenia and other mental disorders. Tungsten may be adaptogenic for the thyroid gland, balancing both hypothyroid and hyperthyroid states.

Tungsten is free of accumulation in the body and is free of any known affinity for a specific organ or tissue. Tungsten elimination is efficient. Tungsten and molybdenum compete for absorption.

:Study:

In most of eukaryotes and prokaryotes tungsten is an
antagonist of molybdenum and during growth of organ

isms the latter is easily replaced by tungsten due to their
chemical similarity. Inactive analogs of molybdenum

containing enzymes (Mo
enzymes) are thus formed. The
“true tungsten
containing enzymes” (W
enzymes”)—
formate dehydrogenase, aldehyde:ferredoxin
oxidore

ductase, formaldehyde:ferredoxin
oxidoreductase, etc.—
in which tungsten cannot be replaced by molybdenum
were isolated from hyperthermophilic archaea cells dis

covered in the last decade. Pterin
containing tungsten
cofactor, an analog of molybdenum cofactor of Mo
con

taining enzymes, is the active site of these enzymes.
However, there are some enzymes that exhibit catalytic
activity with both molybdenum and tungsten (formyl

methanofurane dehydrogenase, trimethylamine
N

oxide
reductase, etc.). Molybdopterin able to coordinate
both molybdenum and tungsten is an active site of these
enzymes.
CHEMICAL NATURE
OF TUNGSTEN COMPARED TO MOLYBDENUM
Together with chromium and molybdenum, tungsten
is an element of group VI of the periodic table of elements
and is similar to molybdenum in its chemical properties;
the important role of the latter in biological processes is
well known [1].
Tungsten and molybdenum have equal atomic
(1.40 Å) and ionic (0.68 Å) radii and similar electronega

tivity (1.4 and 1.3 for W and Mo, respectively); some
other coordination characteristics are also very close [2].
Both metals can be in various oxidation states (from +2
to +6) and are able to form polynucleotide complexes,
but only the oxidation states +4, +5, and +6 and
mononucleotide systems are biologically important [3,
4].
Tungsten and molybdenum are rare in occurrence
(the Clark of both elements is about 1.2 µg/ml) and they
rank 54th and 53rd in natural abundance, respectively [5].
The solubility of tungsten salts is less than that of molyb

denum salts, thus its concentration in fresh water rarely
exceeds 20 nM and is usually less than 0.5 nM, whereas
the concentration of molybdenum is two or more orders
of magnitude higher than this value. The concentration of
tungsten is sea water is extremely low—5·105 times lower
than that of molybdenum [2].
BIOLOGICAL ACTION OF TUNGSTEN
For many years tungsten was considered to be a bio

logical antagonist of molybdenum and was used for study
of the properties and functions of molybdenum in Mo

enzymes [6]. This was due to the fact that tungsten is able
Biochemistry (Moscow), Vol. 67, No. 2, 2002, pp. 196
200. Translated from Biokhimiya, Vol. 67, No. 2, 2002, pp. 234
239.
Original Russian Text Copyright © 2002 by L’vov , Nosikov, Antipov.
ACCELERATED PUBLICATION
0006
2979/02/6702
0196$27.00 ©2002 MAIK “Nauka/Interperiodica”
* To whom correspondence should be addressed.
Tungsten
Containing Enzymes
N. P. L’vov , A. N. Nosikov, and A. N. Antipov*
Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia;
fax: (095) 954
2732; E
mail: [email protected]
Received July 12, 2001
Revision received October 30, 2001
Abstract—The biological importance of tungsten has been fully proved in the last decade due to isolation of a number of tung

sten
containing enzymes (W
enzymes) from hyperthermophilic archaea. Tungsten was previously considered only as an
antagonist of molybdenum, because the replacement of molybdenum by tungsten (due to their chemical similarity) leads to
inactivation of molybdenum
containing enzymes (Mo
enzymes). In addition to the “true W
enzymes” in which tungsten
cannot be replaced by molybdenum, recently some enzymes have been isolated which can use either molybdenum or tung

sten in the catalytic process. This review briefly summarizes data on the participation of tungsten in catalysis by some enzymes
and the structure of the active sites of W
enzymes.
Key words: tungsten, molybdenum, enzymatic catalysis, pterin cofactor
TUNGSTEN
CONTAINING ENZYMES 197
BIOCHEMISTRY (Moscow) Vol. 67 No. 2 2002
to replace molybdenum in Mo
enzymes, forming catalyt

ically inactive (or possessing very low activity) analogs.
Together with the antagonistic action of tungsten on
Mo
enzymes, a positive effect of tungsten on nitrate
reductase activity of plants and bacteria has long been
known. Addition of small quantities of tungsten (0.25

1.0 µg/liter) to the growth medium stimulates the nitrate
reductase activity of plants [7].
More than 35% of nitrate reductase (NR) of plant
tissue is in the form of holoenzyme containing metal

lacking molybdenum cofactor retaining, however, the
ability to coordinate metals. It is precisely this molybde

num
lacking part of NR that can be “stabilized” (i.e.,
protected from proteolysis and other enzyme
inactivating
processes) by tungsten. Although this part of NR remains
inactive after inclusion of tungsten, it is able to replace
tungsten by molybdenum when the trace quantities of the
latter appear in the medium, as studies on spinach and
cauliflower demonstrated [7]. Thus, the “tungsten
stabi

lized” enzyme is activated. This stabilization effect of
tungsten can be significant: for example, growth of cauli

flower on medium containing equimolar concentrations
of tungsten and molybdenum (0.005 µg/liter) caused 20%
increase in NR activity, and plant growth was significant

ly accelerated. Hypersynthesis of NR apoprotein of
tobacco (immunochemically detected) is another possi

ble reason for the increase in NR activity of plants grow

ing on medium with equimolar concentrations of tung

sten and molybdenum [8].
Growth of the salt
tolerant yeast Rhodotorula glutinis
on medium containing molybdenum and tungsten in
equimolar concentrations (1 mM) caused stimulation of
synthesis of molybdenum cofactor and decrease in NR
activity. It is interesting to note that tungsten was not
included in NR composition and its action manifested
itself only on the level of molybdenum cofactor expres

sion [9].
In recent years, tungsten was reported to be able to
cause hyperexpression of the structural gene of some
other enzymes; for example, tungsten inhibited anaerobic
growth of Escherichia coli on glycerol–dimethylsulfoxide
medium, and this inhibition was partly compensated by
hypersynthesis of dimethylsulfoxide reductase [10].
Formate dehydrogenase (FDH) from Methylobacterium
sp. RXM is another example of an analogous effect of
tungsten [11]. Addition of tungsten (to 0.6 µM) to Mo

containing medium (0.6
0.9 µM) caused increase in
FDH activity by stimulation of enzyme synthesis.
TUNGSTEN
CONTAINING ENZYMES
In relation to molybdenum and tungsten, all organ

isms can be divided into at least three groups.
1. Organisms preferring molybdenum to tungsten. In
most prokaryotes and eukaryotes in vivo, tungsten easily
replaces molybdenum in Mo
enzymes; inactive tungsten
analogs of Mo
enzymes are thus formed [6, 12
14]. For
NR, this was shown for plants [15
17], fungi [18], algae
[19], and bacteria [20]. For sulfite oxidase and xanthene
oxidase, this was found for animal cells [21
23].
Ability of a microorganism to grow on medium with
nitrate and 1 mM sodium tungstate in the absence of
molybdenum indicates that NR of this microorganism
does not contain molybdenum. Inactivation of Mo

enzymes by tungsten is reversible; for example, on trans

fer of bacterial W
cells on a molybdenum
containing
medium, NR is converted into an active Mo
form, and
inactive tungsten nitrate reductase isolated from E. coli
W
cells can restore activity of NR defective in molybde

num cofactor of the fungus Neurospora crassa nit
1
mutant in the presence of 1 mM sodium molybdate [24].
In all the above
mentioned studies, tungsten could
be incorporated into Mo
enzymes only during growth.
Only in the case of animal sulfite oxidase was it possible
to replace tungsten by molybdenum in vitro and thus acti

vate the W
containing enzyme [21].
2. Organisms preferring tungsten to molybdenum.
The properties of the main “true W
enzymes” so far
described in the literature, in which tungsten cannot be
replaced by molybdenum or vanadium, are presented in
the table.
Interest in a probable biological function was first
raised in the early 70s when it was found that FDH of
clostridia can use tungsten along with molybdenum, the
former even being preferred [14]. Then tungsten incorpo

ration into FDH was found in the anaerobic methanogen
Methanococcus vannielii [33]. During growth of this
microorganism on tungsten
free medium two isoforms of
FDH were synthesized: one having molecular mass
105 kD and containing molybdenum and iron
sulfur
clusters and another, high
molecular
weight and con

taining selenium, molybdenum, and iron
sulfur clusters.
The latter isoform prevailed in the case of tungsten
con

taining medium.
However, spectacular progress in studies of biological
function of tungsten was achieved after the discovery of
hyperthermophilic archaea in the early 90s. Tungsten
enzymes isolated from hyperthermophilic archaea cells—
aldehyde:ferredoxin
oxidoreductase, formaldehyde: fer

redoxin
oxidoreductase, acetylene hydratase, etc. (see
table)—are exceptions from the group of W
enzymes
because the dependence of their catalytic activity on
tungsten is obligatory, this metal not being replaceable by
molybdenum or vanadium [34]. Some hyperthermophilic
archaea, for example, Pyrococcus furiosus, synthesize
three W
enzymes—aldehyde:ferredoxin
oxidoreductase,
formaldehyde:ferredoxin
oxidoreductase, and glycer

aldehyde
3
phosphate dehydrogenase [34]. An essential
difference between W
enzymes and their Mo
analogs is
that the former are expressed constitutively in most stud

ied microorganisms, whereas Mo
analogs are induced
198 L’VOV et al.
BIOCHEMISTRY (Moscow) Vol. 67 No. 2 2002
only in the presence of molybdenum [35, 36]. Besides
this, W
enzymes catalyze oxidation–reduction reactions
at much lower redox potentials than their Mo
analogs.
Thus, one can conclude that mesophilic microor

ganisms mainly synthesize Mo
enzymes, whereas
hyperthermophilic bacteria and archaea with anaerobic
metabolism synthesize W
enzymes. Since hyperther

mophilic archaea are considered to be the earliest
microorganisms [1, 37], cofactor (the active site) of
these enzymes consisting of molybdopterin and tungsten
but nucleotide
lacking is supposed to be a precursor of
the modern molybdenum cofactors of oligonucleotide
nature. Moreover, the presence of the same labile com

plex—molybdenum cofactor able to function both with
molybdenum and tungsten—in various organisms from
the earliest hyperthermophilic archaea to humans indi

cates the importance of Mo
and W
containing enzymes
in the evolutionary process. It is supposed that before
the appearance of molecular oxygen formed during pho

tosynthesis, molybdenum and tungsten were present on
the Earth as sulfides (MoS2 and WS2) rather than oxyan

ions (MoO4 and WO4). Since tungsten sulfide is better
soluble in water than molybdenum sulfide, it is supposed
that in the pre
oxygen epoch tungsten could be more
available to organisms than molybdenum. This sugges

tion agrees with the fact that “true W
enzymes” are
found in the strict anaerobic microorganisms, although
there is an exception—FDH of methylotrophic bacteria
[38].
FDH of sulfate
reducing bacteria Desulfovibrio gigas
(see table) could be also related to the “true W
enzymes”
[25]. This enzyme includes tungsten even during growth
on Mo
containing medium. It is interesting to note that
under the same conditions D. gigas cells synthesize Mo

aldehyde oxidase along with W
FDH. The authors [25]
suppose that in early evolution sulfate reducers of
Desulfovibrio genus were able to use both molybdenum
and tungsten, but now they use mainly molybdenum.
THE ACTIVE SITE
OF TUNGSTEN
CONTAINING ENZYMES
Study of the “true W
enzymes” (see table) mainly
isolated from the cells of hyperthermophilic archaea
proved that tungsten is bound similarly to molybdenum in
Mo
enzymes with 6
substituted pterin (molybdopterin)
[1, 37, 39, 40]. In contrast to Moco in Mo
enzymes,
pterin in W
cofactor is not bound with oligonucleotides
(GMP, CMP, AMP, or IMP). The main feature of tung

sten coordination in the active site of W
enzymes proba

bly providing their thermal stability is that every subunit
contains two molecules of molybdopterin, which are
coordinated by four sulfur ligands [41]. The similarity of
pterin components of W
cofactor and Moco is also
proved by the fact that W
FDH from Clostridium ther

moaceticum restored activity of NR defective in Moco of
the fungus Neurospora crassa nit
1 mutant [42]. Such
Enzyme
Formate dehydrogenase
Formate dehydrogenase
Aldehyde:ferredoxin
oxidoreduc

tase
Aldehyde:ferredoxin
oxidoreduc

tase
Formaldehyde:ferredoxin
oxidore

ductase
Acetylene hydratase
Carboxylic acid reductase
Reference
[2]
[25]
[26]
[27]
[28, 29]
[30]
[2]
[31, 32]
Tungsten
containing enzymes
Metal
and cofactor content,
mol/mol protein
W (2) Se (2)
Fe/S (20
40)
W (0.9) Fe (7)
W (2) FeS (4)
W (0.68) FeS (4)
W (4) FeS (4)
W (0.5) Fe (3) S (4)
W (1) Fe (29) S (25)
W (3) Fe (82) S (54)
FAD (2)
Enzyme
structure
?2?2 (96 and 76)*
?? (96 and 76)
?2 (67)
?2 (62)
?4 (69)
form I:
?? (64 and 14)
form II:
?3?3? (64, 14 and 43)
Microorganism
Clostridium
thermoaceticum
Desulfovibrio gigas
Pyrococcus furiosus
Desulfovibrio gigas
Thermococcus litoralis
Pelobacter acetylenicus
Clostridium
thermoaceticum
* Molecular mass of subunits (kD) is given in brackets.
TUNGSTEN
CONTAINING ENZYMES 199
BIOCHEMISTRY (Moscow) Vol. 67 No. 2 2002
reconstruction was possible only in the presence of 1 mM
sodium molybdate. The possibility to use the E. coli W

substituted NR as a donor of Moco for defective NR of N.
crassa nit
1 mutant in the presence of molybdenum was
demonstrated earlier [24]. These studies allow the con

clusion that Moco activity of NR of nit
1 mutant is exhib

ited only with molybdenum but not with tungsten.
3. Organisms to some extent able to use both metals.
In these organisms molybdenum can be replaced by tung

sten in vivo, and in this case the enzyme is not complete

ly inactivated but only decreases its catalytic activity.
Thus, in these organisms Mo
enzymes can probably use
molybdenum and less efficiently tungsten for catalysis.
For these organisms, tungsten seems to be a secondary
element in bacterial metabolism because most W

enzymes are analogs of Mo
enzymes with the same func

tions in the same organisms.
Formylmethanofurane dehydrogenase (FMFDH),
the first enzyme in the chain of enzymes participating in
the synthesis of methane, is the most studied enzyme
exhibiting activity both with molybdenum and tungsten
[43, 44]. It was noted that whereas the dependence of
growth of hyperthermophilic methanogenic archaea on
the presence of tungsten in the medium is obligatory,
thermophilic methanogenic archaea can use both tung

sten and molybdenum for growth and FMFDH synthesis.
For example, the thermophilic methanogen
Methanobacterium wolfei contains W
and Mo
isoen

zymes of FMFDH, and the W
isoenzyme is synthesized
during growth on W
containing medium, whereas the
Mo
analog is synthesized during growth on Mo
contain

ing medium [45
47]. In spite of a great similarity in the
structure and amino acid sequence of the N
end of two of
three subunits, these isoforms differ in some properties
(chromatographic behavior, substrate affinity and speci

ficity, sensitivity to O2 and cyanide).
For another thermophilic methanogen, Methano

bacterium thermoautotrophicum, tungsten is used prefer

entially to molybdenum for FMFDH synthesis [43].
During growth on molybdenum
containing medium one
Mo
isoenzyme is synthesized, and during growth on
tungsten
containing medium two isoenzymes are synthe

sized: one with molybdenum and another with tungsten;
in the latter case one of the FMFDH isoenzymes was
identical to the molybdenum isoenzyme and thus could
use both metals.
The Mo
enzyme trimethylamine
N
oxide reductase
(TMAOR) (EC 1.6.6.9) isolated from E. coli cells is
another example of an enzyme that can use both molyb

denum and tungsten for catalysis [48]. Growing E. coli on
media containing tungsten instead of molybdenum, the
authors obtained an active W
TMAOR which had two
times higher catalytic activity than Mo
TMAOR, higher
sensitivity to high pH, and increased thermal stability.
A usual Mo
cofactor or, more precisely, its molybde

num
free precursor, molybdopterin (MPT), which
although it can coordinate both metals seems to be also
synthesized by enzymes able to use both molybdenum
and tungsten.
There is little or no data on the possible participation
of tungsten in NR catalysis in the literature. It is only
known that replacement of molybdenum by tungsten in
E. coli NR resulted in decrease of Km value against nitrate
[49]. It is interesting that a strong inhibiting effect of
15 mM tungstate on E. coli nitrate reduction could be
overcome in the presence of only 10 µM molybdate [50].
It was also shown that for hyperthermophilic archaea, a
higher tungsten concentration in the medium is needed
for NR synthesis, and molybdenum does not replace
tungsten [51].
Thus, data obtained so far indicate a very important
biological role of tungsten in various metabolic processes
and the participation of this microelement in enzymes
catalyzing these processes.
This work was financially supported by the Russian
Foundation for Basic Research (grant Nos. 01
04
48553
and 01
04
48217).
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