Purification and characterization of a membrane-bound enzyme
complex from the sulfate-reducing archaeon
Archaeoglobus fulgidus
related to heterodisulfide reductase from methanogenic archaea
Gerd J. Mander
1
, Evert C. Duin
1
, Dietmar Linder
2
, Karl O. Stetter
3
and Reiner Hedderich
1
1
Max-Planck-Institut fu
¨
r terrestrische Mikrobiologie, Marburg, Germany;
2
Biochemisches Institut, Fachbereich Humanmedizin,
Justus-Liebig-Universita
¨
t Giessen, Germany;
3
Lehrstuhl fu
¨
r Mikrobiologie und Archaeenzentrum, Universita
¨
t Regensburg, Germany
Heterodisulfide reductase (Hdr) is a unique disulfide reduc-
tase that plays a key role in the energy metabolism of

enzyme. In addition to EPR signals due to [4Fe-4S]
+
clus-
ters, signals of an unusual p aramagnetic species with g values
of 2.031, 1.994, and 1.951 were obtained. The paramagnetic
species could be reduced in a one-electron transfer reaction,
but could not be further oxidized, and shows EPR properties
similar to t hose of a paramagnetic species r ecently identified
in Hdr. In Hdr this paramagnetic species is specifically
induced b y the substrates of the e nzyme a nd is thought to be
an inte rmediate of the catalytic cycle. Hence, Hdr and the
A. fulgidus enzyme not only share sequence similarity, but
may also have a similar active site and a similar catalytic
function.
Keywords: Archaeoglobus f ulgidus; heterodisulfide reductase;
Hmc complex; iron-sulfur proteins; sulfate-reducing
bacteria.
Heterodisulfide reductase (Hdr) is a key enzyme in the
energy metabolism of methanogenic archaea. In the final
step of methanogenesis, the mixed disulfide of the metha-
nogenic thiol coenzymes coenzyme M and coenzyme B is
generated in a reaction catalyzed by methyl-coenz yme M
reductase [1]. This disulfide is reduced by a unique disulfide
reductase, designated heterodisulfide reductase (Hdr). Two
types of Hdr from phylo genetically distantly related meth-
anogens have been identified a nd characterized. Neither
type of enzyme belongs to t he family of pyridine nucleotide
disulfide oxidoreductases [2].
Hdr from Methanothermobacter marburgensis is an
iron-sulfur flavoprotein composed of the subunits HdrA,

February 2002)
Eur. J. Biochem. 269, 1895–1904 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02839.x
[4]. A homologue of the M. marburge nsis HdrA subunit is
lacking in H dr from Methanosarcina species. It has
therefore been suggested that the conserved subunits
HdrD and HdrCB must harbor the catalytic site for the
reduction of the disulfide substrate. The active site of Hdr
was recently shown to contain a [4Fe-4S] cluster that is
directly involved in mediating heterodisulfide reduction
[6,7]. This extra iron-sulfur cluster has been proposed to
be co-ordinated by cysteine residues of the highly
conserved sequence motif CX
31)38
CCX
33)34
CXXC found
in subunits HdrD and HdrB. The nonconserved subunits
HdrE and HdrA are thought to interact with the physio-
logical electron donor, which differs in t he two types of
Hdr. The physiological electron donor of Hdr from
Methanosarcina species is thought to be the membrane-
soluble electron c arrier methanophenazine [8]. Hdr from
M. marburgensis forms a functional complex with the
MvhAGD hydrogenase [9]. This complex catalyzes the
reduction of CoM-S-S-CoB by H
2
.
Hdr w as originally thought to be u nique to methanogenic
archaea. However, in recent years, genes encoding pro-
teins r elated to the catalytic subunit of Hdr have been identi-

NADH–quinone oxidoreductases [10,14]. It is assumed to
function as a proton or sodium ion pu mp as well. In
addition, the m embrane fraction o f A. fulgidus catalyzes the
reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by
L
-lactate, which indicates that lactate dehydrogenase direct-
ly channels the reducing equivalents generated in the
oxidation of lactate to pyruvate into the menaquinone pool
[12]. A. fulgidus has been shown t o contain a modified
menaquinone as a m embrane-soluble e lectron c arrier [15]. It
is, however, not yet known how the reduced menaquinone
pool is electrically connected to the enzymes of sulfate
reduction, namely adenosine 5 ¢-phosphosulfate reductase
and sulfite reductase.
Here we report on the isolation and characterization of a
heme-containing membrane protein from A. fulgidus related
to Hdrfrom M. barkeri. Afunction ofthis enzymeas reduced
menaquinone–acceptor oxidoreductase is discussed.
MATERIALS AND METHODS
Materials
Redox dyes were obtained from Aldrich–Sigma. DMN w as
from Sigma. Potassium trithionate was a gift from Peter
M. H. Kroneck (Universita
¨
t K onstanz). All other chemicals
were from Merck. The chromatographic materials were
from Amersham Pharmacia Biotech.
Growth of the organism
A. fulgidus (VC16, DSMZ 304) was grown in a 300-L
fermenter at 83 °C on lactate/sulfate medium as described

M
Mops/KOH (pH 7.0) containing 2 m
M
dodecyl-b-
D
-malto-
side (buffer A). Protein was eluted in a s tepwise NaCl
gradient (80 mL each in buffer A): 0 m
M
, 300 m
M
,
400 m
M
,500m
M
,600m
M
,and1
M
. The majority of the
heme-containing protein(s) were eluted at 600 m
M
NaCl.
These f ractions were applied to a Superdex 200 gel-filtration
column (2.6 · 60 cm) equilibrated w ith buffer A containing
100 m
M
NaCl. Protein was eluted using the same buffer.
Heme-containing protein(s) were eluted after 120 mL (peak

tometrically. DMN or DMNH
2
was added to the enzyme
solution [1 mgproteinÆmL
)1
in 50 m
M
Mops/KOH (pH 7.0)]
to a final concentration of 150 l
M
, and spectra were
recorded every 5 s. DMNH
2
was prepared a s described
previously [16].
Analytical methods
Non-heme iron was quantified colorimetrically with neo-
cuproin (2,9-dimethyl-1,10-phenanthroline) and ferrozine
1896 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
[3-(2-pyridyl)-5,6-bis-(4-phenylsulfonate)-1,2,4-triazine] as
described by Fish [17]. Acid-labile sulfur was analyzed as
methyleneblue[18].
The protein concentration was routinely determined by
the method of Bradford (Rotinanoquant; Roth Karlsruhe,
Germany) using BSA as standard.
Heme was extracted with acetone/HCl and the pyridine
hemochrome derivate was formed as described. Reduced
minus oxidize d differe nce spectra were recorded at room
temperature [19]. The spectra obtained were compared with
the pyridine hemochrome spectra obtained with heme

lated using nonco mmercial programs based on formulas
described previously [20].
Redox titrations
Redox titrations were carried out at 18 °Cinananaerobic
chamber under N
2
/H
2
(95 : 5, v/v). Potentials w ere a djusted
with small amounts of freshly prepared sodium dithionite
(20 m
M
stock solution) or freshly prepared potassium
ferricyanide (20 m
M
stock solution). All redox potentials
quoted here are relative t o the standard hydrogen electr ode.
In these titrations, a selection of the following mediators
(final concentration 20 l
M
) were added individually to the
enzyme solution: 1,2-naphthoquinone (E°¢ ¼ +134 mV),
duroquinone (E ¢ ¼ +86 mV), 1,4-naphthoquinone
(E ¢ ¼+69 mV), thionine (E ¢ ¼+64 mV), methylene blue
(E ¢ ¼ +11 mV), indigodisulfonate (E ¢ ¼ )125 mV),
2-hydroxy-1,4-naphthoquinone (E ¢ ¼ )145 mV), anthra-
quinone-1,4-disulfonate (E ¢ ¼ )170 mV), phenosafranin
(E ¢ ¼ )252 mV), anthraquinone-2-sulfonate ( E°¢¼
)255 mV), safranin O (E ¢ ¼ )289 mV), and neutral red
(E ¢ ¼ )325 mV). The final concentration of Hdr-like

ments were generated using the
FASTA
3server(http://
www.ebi.ac.uk/fasta3/).
RESULTS
Purification of a heme-containing enzyme complex
from the membrane fraction of
A. fulgidus
The genome of A. fulgidus encodes several membrane-
bound oxidoreductases that share sequence similarity with
subunits of Hdr from methanogenic archaea, in particular
with the membrane-bound enzyme from M. barkeri [4,10],
which is anchored in the cytoplasmic membrane via a b-
type cytochrome [3]. We used this knowledge to identify
and purify heme-containing membrane-bound enzymes
from A. fulgidus cells cultivated on lactate/sulfate medium
by following the characteristic absorption of heme proteins.
The membrane fraction was isolated, and proteins were
solubilized with the detergent dodecyl-b-
D
-maltoside. On
anion-exchange chromatography on Q-Sepharose, the
major heme-containing fraction was eluted at 600 m
M
NaCl. Approximately 70% of the heme presen t in solubi-
lized membranes was found in this fraction. A further
purification of the proteins in this heme-containing fraction
by gel fi ltration on Superdex 200 resulted again in only one
heme-containing fraction eluted after 120–150 mL. In the
final purification step, the sample was chromatographed on

upstream o f A F499 is AT-rich and contains typical a rchaeal
promoter elements. The sequence AAAGGTTAATATA
was f ound 64 bp upstream of the start codon of AF499; this
corresponds to the BRE element and the box A element of
archaeal promoters [21,22]. The A F499–AF503 gene cluster
can therefore be predicted to form a transcription unit
(Fig. 2). This transcription unit contains one gene (AF500)
for which no corresponding protein was found in the
purified enzyme preparation. The results of the sequence
analyses of the deduced proteins are given in Table 2.
The protein encoded by AF502 has a calculated mole-
cular mass of 64.4 kDa. The protein shows about 35%
sequence identity with the proposed catalytic s ubunit H drD
from M. barkeri. The closest relative of t he protein e ncoded
by AF502 (40% sequence identity) is the dissimilatory
sulfite reductase (Dsr)K protein from the sulfur-oxidizing
phototrophic bacterium Allochromatium vinosum. The DsrK
protein is encoded by the dsr locus, which also encodes the
subunits of the siroheme sulfite reductase [23]. Another
relative of the protein encoded b y AF502 is the high-
molecula r-ma ss c-type cytochrome (Hmc)F protein of
Desulfovibrio vulgaris (20% sequence identity) [24].
A characteristic of HdrD o f M. barkeri is the presence of
two t ypical [4Fe-4S] cluster binding motifs in th e N -terminal
part of the protein. HdrD contains 10 additional cysteine
residues found in two CX
31)38
CCX
33)34
CXXC sequence

preparation shown in (B) lacks this polypeptide. M, Low-molecular-
mass markers ( Amersham Pharmacia Biotech). The m olecular mass es
of the marker proteins are given o n the right side. Lane A1, 15 lgof
the A. fulgidus Hme c omplex denatured fo r 30 min at room t emper-
ature in SDS sample buffer; lane B1, 10 lg Hme complex denatured
for 30 min at room temperatu re i n SDS sam ple buffer; lane B2, 10 lg
Hme complex denatured for 5 min at 100 °C in SDS sample buffer.
The polypeptide with an apparent molecular mass of 34 kDa, identi-
fied as a b-type cytochro me-like protein by N-terminal sequencing,
shows a lower intensity in the b oiled sample; it probably forms
aggregates that do not run into the gel (lane B2). This behavior is
typical of integral membrane proteins. The polypeptide with an
apparent molecular mass of 53 kDa appears as a double band in
unboiled samples (lanes A1 and B1).
Table 1. N-Terminal sequences of the polypeptides of the purified enzyme. N-Terminal sequen ces were eit her obtained by Edman degradation
(column 1) or d er ived from the genome sequence of A. fulgidus (column 2). The c orresp onding genes are given in column 3. Amino acids p re sent in
both sequences are u nd erlined, a nd am ino acids th at co uld not be determined with ce rtainty i n the Ed man d egradation are g iven in parentheses.
Sequence derived by Edman
degradation
Sequence derived from the A. fulgidus
genome sequence Identified ORF
(
M)(E)RMRE(I)IEIKAKFP MEEMPERIEIKQKFP AF502
MIGVIFGVIVFYIAV MIGVIFGVIVFYIAV AF501
(
K)TQFIESPEEV(V)EK MMSRRKFLLLTGAAAAGAILTPQISA
KTQFIESPEEVREK
AF499
MYNK-YVIPLILVFL MSEMYNKKYVIPLILVFL AF503
1898 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002

purified enzyme, has a calculated molecular mass of
43 kDa. This protein shows highest sequence identity to
the DsrP protein from A. vinosum. It shows low sequence
similarity to the HmcC protein of D. vulgaris. Topological
analysis suggests that AF500, like DsrP and HmcC, has 10
membrane-spanning helices. These three proteins are also
related to the DmsC protein of dimethylsulfoxide reductase
[29]. The latter protein contains only eight predicted
transmembrane helices.
Catalytic properties of the Hme complex
and characterization by UV/Vis spectroscopy
To determine whether the cytochrome present in the Hme
complex is reduced by menaquinone, in vitro assays were
performed using the more hydrophilic analogue of men-
aquinone, DMN. The enzyme purified u nder anoxic condi-
tions generally contained the heme groups in the reduced
state. Any enzyme molecules that contained oxidized h eme
groups could be rapidly reduced by sodium dithionite.
Addition of DMN to the reduced enzyme resulted in rapid
oxidation of the heme present in the enzyme. The oxidized
heme groups could be rapidly reduced using DMNH
2
as
electron donor. The r ates of h eme reduction by DMNH
2
or
oxidation by D MN were too rapid to be resolved. Figure 3
shows t he dithionite-reduced minus air-oxidized absorbance
difference spectrum of an enzyme preparation containing
only minor amounts of th e 16-kDa c-type cytochrome. The

catalytic subunit
of Hdr
Cytochrome,
integral membrane
protein
Extracytoplasmic
iron-sulfur protein
Extracytoplasmic
c-type cytochrome
Integral membrane
protein
Ó FEBS 2002 Hdr-like enzyme complex from A. fulgidus (Eur. J. Biochem. 269) 1899
absorption maxima at 420 nm (c band), 530 nm (b band)
and 557 nm (a band) are characteristic of cytochrome b.
Heme was extracted from the protein with acidic acetone
and the pyridine hemochrome spectrum (reduced minus
oxidized) was recorded. T he spectrum contained maxima of
the a and b band at 553 and 521 nm, respectively. These
maxima are blue-shifted by a bout 4 nm relative t o published
values for protoheme IX [30]. In a control experiment the
pyridine hemochrome spectrum of hemoglobin was deter-
mined under identical conditions resulting in maxima
identical with published values (525 nm for the b band
and 557 nm for the a band). A similar blue shift was f ound
in heme o of cytochrome bo [30]. As pyridine hemochrome
spectra are very sensitive to substitutions on the porphyrin
ring, the results indicate that th e e xtractable heme of Hme is
not protoheme IX. Further studies are necessary to
elucidate the nature of the extractable heme present in this
enzyme.

Redox titrations were monitored by EPR to characterize
the different iron-sulfur clusters presen t in the enzyme. As
expected, t he enzyme showed broad unresolved EPR signals
at redox potentials u p to )100 mV that w ere only detectable
at temperatures below 1 0 K. These signals are most
probably due to the bulk of [4Fe-4S]
+
clusters present in
the enzyme. At potentials higher than 0 mV, an unusual
paramagnetic species was detected with g values at 2.031,
1.994, and 1.951. The resonance started to develop at
potentials ‡ 0 mV and was stable at potentials up to
+350 mV. The loss and formation of the resonance was
associated with a one-electron redox process with a
midpoint potential of +90 ± 10 mV (Fig. 4). The spin
concentration of the signals in the different titrations was
generally near 0.4 spinÆ(mol enzyme)
)1
. Because of overlap
with radical signals around g ¼ 2, the signal was simulated
(Fig. 4) and double integrated to obtain the spin intensity.
Temperature studies showed that the signal is readily power
saturated at 4.5–15 K. At 15–35 K, the signal could be
measured under nonsaturating conditions. At higher tem-
peratures, the signal started to broaden a nd was broadened
beyond detection at 60 K.
The EPR signal observed has EPR characteristics very
similar to a unique signal described for Hdr from metha-
nogens. The two paramagnetic species have similar g values,
show the same temperature behavior, and are only detect-

in 50 m
M
Tris/HCl
(pH 7 .6)] was reduced with so dium dithionite and su bsequently oxi-
dized by air. The oxidized spectrum was subt racted from the red uced
spectrum. When the enzyme was oxidized by DMN, the same differ-
ence spectrum was observed (not shown). The arrow indicates the
absorption maximum of the aband at 557 nm.
1900 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
could not be detected after SDS/PAGE in gels stained with
either Coomassie or silver (data not shown), which suggests
that this subunit does no t copurify with the other subunits
of the enzyme complex.
From the primary structure, the b-type cytochrome-like
protein AF501 and subunit AF500 are clearly predicted to
be integral membrane proteins. The iron-sulfur protein
AF499 contains a characteristic twin-arginine leader pep-
tide. This strongly suggests t hat this p rotein is located at t he
extracytoplasmic side of the membrane [27]. The c-type
cytochrome AF503 contains a typical Sec-dependent hydro-
phobic leader peptide [28] that is not cleaved off by a l eader
peptidase as i t was still present in the purified protein.
Therefore, this protein can also be predicted t o be located
on the extracytoplasmic side of the membrane and to have
an N-terminal membrane anchor. The AF502 protein,
which is related to the catalytic subunit of Hdr, is a
hydrophilic iron-sulfur protein. The protein does not
contain a leader sequence and therefore may be attached
to the integral membrane subunits on the cytoplasmic side.
It cannot, however, be excluded that this protein binds to

,[3Fe-4S]
+
, [4Fe-4S]
+
,or[4Fe-4S]
3+
cluster. CoM-
Hdr reacts with H-S-CoB to produce an EPR-silent form.
This indicates that only a half reaction is catalyzed when
only H -S-CoM is pr esent and that a reaction intermediate of
the catalytic cycle is trapped [6]. Variable-temperature
magnetic circular dichroism spectroscop y studies of CoM-
Hdr have provided compelling evidence f or the p resence of a
novel type of [4Fe-4S]
3+
cluster at t he active site of Hdr [6,7].
When oxidized Hdr is incubated with H-S-CoB, an EPR
signal with similar g values is obtained, but the midpoint
potential is shifted t o higher values ()30 mV for Hdr from
M. marburgensis and >0 mV for Hdr from M. barkeri).
From these data it has been concluded that H-S-CoB also
reacts with the active site of the enzyme. As this reaction is
only observed at nonphysiological redox potentials, it has
been proposed that this species could not be an intermediate
of the catalytic cycle, but rather is the product of a side
reaction that occurs at these high redox potentials. Similar
results have been obtained with other thiols, such as
dithiothreitol, which are not substrates of the enzyme [6].
In contrast with Hdr, the paramagnetic species in the
enzyme co mple x f rom A. fulgidus co uld a lready be observed

Ó FEBS 2002 Hdr-like enzyme complex from A. fulgidus (Eur. J. Biochem. 269) 1901
This value is more positive than the standard redox
potentials of the APS/sulfite couple ()60 mV) and the
sulfite/sulfide couple ()116 mV), which are thought to be
the final electron a cceptors (see b elow). It therefore has to be
considered that the signal in the A. fulgidus enzyme is
generated nonspecifically at high redox potentials. The
reaction of the e nzyme with its physiological substrate may
result in a shift of the midpoint potential of this species to
lower values, as has been observed w ith Hdr [6].
The sequence analysis of the A. fulgidus enzyme clearly
shows that t he AF502 protein is related to the catalytic
subunit HdrD of H dr from M. barkeri. I n particular, AF502
and HdrD share a common cysteine motif that in Hdr is
thought to co-ordinate the special [4Fe-4S] cluster in the
active site. I n the four Hdr sequences currently available, this
motif (CX
31)38
CCX
33)34
CXXC) is present in two copies in
each sequence. The Hdr-like p roteins contain either one or
two copies of this sequence motif. The three closely related
proteins AF502, DsrK, and HmcF contain only one copy,
and this may be sufficient for metal-cluster binding. Only in
the A F502 protein does an aspartate residue replace one of
the five cysteine residues. Aspartate can in principle also
function as a ligand of an iron-sulfur cluster [25].
Enzymes related to the Hme complex from A. fulgidus
described in this work are also encoded by the genomes

/sulfate medium, and expression is about fourfold lower
in cells cultivated on lactate/sulfate or pyruvate/sulfate
medium [33]. In addition, a mutant strain in which most of
the hmc operon is deleted has been constructed. This
deletion mutant grows normally when lactate or pyruvate
serve as e lectron donors for sulfate r eductio n. T he mutant is
still able to grow on H
2
/sulfate, although at a growth rate
lower t han that of the wild-type. The mutant is a lso d eficien t
in low-redox-potential niche establishment [34]. From these
various observations, it has been concluded that the Hmc
complex is involved in t he electron transfer from H
2
,which
is activated by a periplasmic hydrogenase, to an electron
acceptor on the cytoplasmic side o f the membrane. As
growth of the hmc deletion mutant on H
2
/sulfate is not
completely abolished, the organism may be able to synthe-
size an alternative enzyme complex with a function similar
to that of Hmc.
Proteins with the highest sequence similarity to the five
subunits of the A. fulgidus enzyme complex were found to
be encoded by the dsr locus of A. vinosum [23]. dsrA and
dsrB encode the a and b subunit of the dissimilatory sulfite
reductase of this organism. These two g enes are o rganized in
a cluster with genes encoding proteins highly related to the
AF499–AF503 proteins (Table 2) [23]. Polar insertion

b
2
structure and contains
siroheme, nonheme iron, and acid-labile sulfur [36]. An
additional protein with an apparent molecular mass of
11 k Da is associated with sulfite reductase from D. vulgaris
[38] and De sulfovibrio desulfuricans [39,40]. The function of
this so calle d c subunit is not yet known. In most of the
organisms t hat h ave been studied, t he enzyme has been
isolated from the soluble fraction. Sulfite reductase from
D. desulfuricans was found to be partially membrane-
associated after gentle disruption of t he cells [39,40].
On the basis of our results and comparisons with
published results for other organisms, we propose that the
Hme complex of A. fulgidus functions as a menaquinol
oxidoreductase. The sequence analysis of the enzyme
indicates that it is composed of two modules that may have
distinct functions. The first module is related to Hdr from
M. barkeri [3]. It is composed of the b-type cytochrome-like
protein AF501 and the AF502 protein, which has sequence
similarity to the catalytic subunit of Hdr. We propose that
this module of the enzyme complex mediates the electron
transfer from menaquinol to an unidentified electron
acceptor on the cytoplasmic side o f the membrane. This is
supported by the findin g that the heme g roups of the
1902 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
purified A. fulgidus enzyme were rapidly reduced by
DMNH
2
or were rapidly oxidized by DMN. Furthermore,

It has been proposed that the Hmc complex from
D. vulgaris mediates the electron t ransfer from a periplasmic
hydrogenase to t he cytoplasmic side where reduction of
sulfate occurs [24,44]. A. fulgidus is also able to grow with H
2
as electron donor for sulfate reduction [11]. The genome of
A. fulgidus, however, does not encode a s oluble periplasmic
hydrogenase as found in sulfate-reducing bacteria. Instead,
thegenomeofA. fulgidus encodes an extracytoplasmic
hydrogenase (AF1379 to AF1381), which is predicted to
contain a b-type cytochrome subunit (AF1379) as a
membrane anchor [10]. This hydrogenase is therefore
predicted to catalyze the hydrogen-dependent reduction of
menaquinone, as do oth er hydrogenases of this type [45].
Methanogenic archaea belonging to the family Meth-
anosarcinales contain two different energy-conserving elec-
tron-transport chains t hat catalyze the reduction of the
heterodisulfide. W hen the organism grows on methanol,
reduced coenzyme F
420
is generated during methanol
oxidation to CO
2
[46]. The organism contains a mem-
brane-bound electron-transport chain which mediates the
reduction of the heterodisulfide by F
420
H
2
. I t is c omposed of

acceptor on the extracytoplasmic side of the membrane or
to an acceptor in the cytoplasm. The latter electron
acceptor, which is still unknown, is thought to function in
its reduced form as electr on donor of the e nzymes of sulfate
reduction.
ACKNOWLEDGEMENTS
This work was supported by the Max-Planck-Gesellschaft,theDeutsch e
Forschungsgemeinsc haft,theFonds der Chemischen Industrie, and by a
fellowship f rom the Humboldt Stiftung to E. D. We thank P eter M. H.
Kroneck for the gift of potassium trithionate. We thank Karen A.
Brune for editing the manuscript.
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