Download Anaerobic respiration with elemental sulfur and with disulfides

FEMS Microbiology Reviews 22 (1999) 353^381
Anaerobic respiration with elemental sulfur and with disul¢des
Reiner Hedderich a; *, Oliver Klimmek b , Achim Kroëger b , Reinhard Dirmeier c ,
Martin Keller c , Karl O. Stetter c
a
b
Max-Planck-Institut fuër terrestrische Mikrobiologie and Laboratorium fuër Mikrobiologie des Fachbereichs Biologie der Philipps-Universitaët,
Karl-von-Frisch-StraMe, D-35043 Marburg, Germany
Institut fuër Mikrobiologie der Johann-Wolfgang-Goethe-Universitaët Frankfurt am Main, Marie-Curie-StraMe 9, D-60439 Frankfurt am Main,
Germany
c
Lehrstuhl fuër Mikrobiologie, Universitaët Regensburg, UniversitaëtsstraMe 31, D-93053 Regensburg, Germany
Received 24 June 1998; received in revised form 21 October 1998 ; accepted 21 October 1998
Abstract
Anaerobic respiration with elemental sulfur/polysulfide or organic disulfides is performed by several bacteria and archaea,
but has only been investigated in a few organisms in detail. The electron transport chain that catalyzes polysulfide reduction in
the Gram-negative bacterium Wolinella succinogenes consists of a dehydrogenase (formate dehydrogenase or hydrogenase) and
polysulfide reductase. The enzymes are integrated in the cytoplasmic membrane with the catalytic subunits exposed to the
periplasm. The mechanism of electron transfer from formate dehydrogenase or hydrogenase to polysulfide reductase is
discussed. The catalytic subunit of polysulfide reductase belongs to the family of molybdopterin-dinucleotide-containing
oxidoreductases. From the hyperthermophilic archaeon Pyrodictium abyssi isolate TAG11 an integral membrane complex has
been isolated which catalyzes the reduction of sulfur with H2 as electron donor. This enzyme complex, which is composed of a
hydrogenase and a sulfur reductase, contains heme groups and several iron-sulfur clusters, but does not contain molybdenum
or tungsten. In methanogenic archaea, the heterodisulfide of coenzyme M and coenzyme B is the terminal electron acceptor of
the respiratory chain. In methanogens belonging to the order Methanosarcinales, this respiratory chain is composed of a
dehydrogenase, the membrane-soluble electron carrier methanophenazine, and heterodisulfide reductase. The catalytic subunit
of heterodisulfide reductase contains only iron-sulfur clusters. An iron-sulfur cluster may directly be involved in the reduction
of the disulfide substrate. z 1999 Published by Elsevier Science B.V. All rights reserved.
Keywords : Sulfur respiration; Disul¢de respiration; Polysul¢de reductase; Heterodisul¢de reductase; Wolinella succinogenes; Pyrodictium
abyssi ; Methanogenic archaea
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . .
2. Biology of sulfur and disul¢de respiration
2.1. Biology of sulfur respiration . . . . . . .
2.2. Biology of disul¢de respiration . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
* Corresponding author. Tel.: +49 (6421) 178230; Fax: +49 (6421) 178299; E-mail: [email protected]
0168-6445 / 99 / $19.00 ß 1999 Published by Elsevier Science B.V.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 3 5 - 7
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
.
.
.
.
.
.
.
.
.
.
.
.
354
354
354
356
354
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
3. Chemistry of elemental sulfur, polysul¢de, and organic disul¢des . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Polysul¢de as a possible intermediate of sulfur respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Sulfur respiration of Wolinella succinogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Bioenergetic data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Electron transport enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Mechanism of electron transfer from hydrogenase to polysul¢de reductase . . . . . . . . . . . . . . . . . . . . .
5.4. The function of the Sud protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. Mechanism of vp generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Sulfur respiration in hyperthermophilic archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Sulfur reduction in fermentative hyperthermophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Sulfur respiration in species of Pyrodictium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Disul¢de respiration in methanogenic archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Disul¢de respiration in Methanosarcina species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.1. Heterodisul¢de reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.2. Hydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.3. F420 H2 dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.4. Methanophenazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.5. Composition of the di¡erent respiratory chains and mechanisms of vp generation . . . . . . . . . .
7.2. Disul¢de respiration in Methanobacteriales, Methanococcales, Methanopyrales, and Methanomicrobiales
7.3. Other heterodisul¢de-generating reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4. Heterodisul¢de reductase ^ mechanistic considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5. Heterodisul¢de-reductase-related proteins in non-methanogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1. Introduction
2. Biology of sulfur and disul¢de respiration
Many microorganisms can utilize a variety of organic and inorganic compounds as terminal electron
acceptors of anaerobic respiration. Among these
electron acceptors, sulfur compounds (sulfate, sul¢te,
thiosulfate, organic sulfoxides, elemental sulfur,
polysul¢de, and organic disul¢des) may play important roles [1,2]. This article will focus on anaerobic
respiration with elemental sulfur, with polysul¢de,
and with organic disul¢des. In Section 2 the biology
of some relevant organisms will be brie£y discussed,
while Sections 3 and 4 will deal with the chemistry of
elemental sulfur, polysul¢de and organic disul¢des.
In Sections 5 and 6, a bacterial (Wolinella succinogenes) and an archael system (Pyrodictium) of sulfur
respiration will be described in detail. Section 7 covers the disul¢de respiration involved in catabolism by
methanogenic archaea. Sulfur respiration has been
reviewed previously in [3^5] and methanogenesis in
[6^11].
2.1. Biology of sulfur respiration
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
356
357
358
358
358
360
361
362
363
363
363
364
365
365
366
367
367
368
370
372
372
373
373
375
375
The ability to reduce sulfur using H2 or organic
substrates as electron donors is widespread among
bacteria and archaea (Table 1). Most of these organisms are hyperthermophilic and belong to the archaeal domain. Water-containing volcanic areas such
as terrestrial solfataric ¢elds and hot springs, and
shallow and abyssal submarine hydrothermal systems harbor hyperthermophilic archaea and bacteria,
which grow optimally above 80³C [55]. Recently, hyperthermophiles have also been discovered in oilbearing, deep-subterranean rocks, about 4000 m below the Earth's surface [56]. Within volcanic environments, sulfur may be formed in variable concentrations at the surface by oxidation of H2 S escaping
from the depths. In their hot biotopes, hyperthermophiles form complex ecosystems consisting of a variety of primary producers and decomposers of organ-
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
355
Table 1
Archaeal and bacterial genera harboring members able to reduce elemental sulfur to H2 S
Topt (³C)
pHopt
Electron donors
Reference
Archaea
Crenarchaeota:
Acidianus
Stygiolobus
Pyrobaculum
70^90
80
102
1.5^2.0
2.5^3.0
6.0
[12]
[13]
[14]
Thermo¢lum
Thermoproteus
85^90
85^90
5.0^6.0
5.0^6.5
Desulfurococcus
85^90
6.0^6.4
Igneococcus
90
5.5^6.0
H2
H2
H2 , peptone, extracts of meat and yeast,
bacterial and archaeal cell homogenates
Peptides
H2 , peptides, maltose, formate, fumarate,
ethanol, malate, methanol, glycogen, starch,
amylopectin, formamide
Peptides, starch, pectin, glycogen, yeast extract,
casein hydrolysate
H2
Pyrodictium
Stetteria
Thermodiscus
Thermosphaera
Staphylothermus
Hyperthermus
105
95
88
85
92
95^107
5.5^6.0
6.0
5.5
6.5
6.5
7.0
H2
H2
H2 , yeast extract
Yeast extract, peptone
Peptone, extracts of meat and yeast
Tryptone, peptone
Euryarchaeota:
Pyrococcus
96^100
6.8^7.0
Thermococcus
75^88
5.8^9.0
Caldococcus
Thermoplasma
Methanopyrus
Methanobacterium
Methanothermus
Methanococcus
88
59
98
37^65
88
85^90
6.4
1.0^2.0
6.5
7.0
6.5
6.0
Complex substrates, amino acids, starch,
maltose, pyruvate
Peptides, amino acids, sugars, starch, chitin,
pyruvate
Peptides
Extracts of yeast, meat, and bacteria
H2
H2
H2
H2 , formate
Bacteria
Aquifex
Ammonifex
Desulfurobacterium
Desulfuromonas
Desulfuromusa
Desulfurella
Desulfovibrio
Fervidobacterium
Geobacter
Pelobacter
Shewanella
Sulfospirillum
Thermotoga
85
70
70
37
35
55
37
65^70
35
37
30
37
66^80
6.8
7.5
6.0
7.5
6.5^7.0
7.0
7.2
6.5^7.0
6.5^7.0
6.5^7.0
6.5^7.0
6.5^7.5
6.5^7.5
Thermosipho
70^75
6.5^7.5
Wolinella
37
8.5
H2 , sulfur, thiosulfate
H2
H2
Acetate, pyruvate, ethanol
Acetate, propionate
Acetate
Organic acids, alcohols
Sugars, pyruvate, yeast extract
Acetate
H2 , ethanol
Lactate
H2 , formate
Sugars, peptone, yeast extract, bacterial and
archaeal cell homogenates
Yeast extract, brain heart infusion, peptone,
tryptone
H2 , formate
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
[15,16]
[17,18]
[19,20]
Huber et al.,
unpublished results
[21,22]
[23]
[17,24]
[25]
[26]
[27]
[28,29]
[30,31]
[32]
[33]
[34]
[34]
[34]
[34]
[35]
[36]
[37]
[38]
[39]
[40,41]
[42]
[43,44]
[45]
[46]
[47]
[48,49]
[50,51]
[52,53]
[54]
356
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
ic matter [55,57,58]. Mesophilic and thermophilic
sulfur reducers, mostly from the bacterial domain
[59], have been isolated from environments such as
anoxic marine or brackish sediments, fresh water
sediments, bovine rumen, hot water pools from solfataric ¢elds, and volcanic hot springs.
Among sulfur-reducing archaea and bacteria,
members of the genera Acidianus, Stygiolobus, Thermoproteus, Pyrobaculum, Igneococcus, Pyrodictium,
Wolinella, Desulfuromonas, Ammonifex, and Desulfurobacterium are able to gain ATP by lithotrophic
sulfur respiration. In contrast, members of the archaeal genera Desulfurococcus, Staphylothermus, Hyperthermus, Thermococcus, and Pyrococcus and of
the bacterial genera Thermotoga, Thermosipho, and
Fervidobacterium are strictly fermentative sulfur reducers [55,57^59]. The hyperthermophilic bacterium
Aquifex pyrophilus, although an aerobic chemolithoautotroph that uses sulfur in addition to hydrogen and thiosulfate as electron donor to reduce oxygen and nitrate, forms high levels of H2 S from S0
and H2 in the late exponential growth phase [35]. In
the presence of sulfur, also methanogenic archaea,
especially thermophilic and hyperthermophilic members of the genera Methanopyrus, Methanobacterium,
Methanothermus, and Methanococcus, produce substantial amounts of H2 S, while methanogenesis is
signi¢cantly reduced [34]. In some heterotrophs,
such as Pyrococcus furiosus and Thermotoga maritima, sulfur is thought to serve as an additional electron sink, but in many organisms, e.g., Aquifex pyrophilus and the methanogens, the metabolic
function of sulfur reduction is still uncertain.
2.2. Biology of disul¢de respiration
The ability to use a disul¢de substrate as an electron acceptor for organotrophic or lithotrophic
growth has been reported only for a small number
of microorganisms, all of which are sulfur-reducing
bacteria or archaea. Desulfuromonas acetoxidans
grows not only with sulfur, but also with cystine
and oxidized glutathione as electron acceptor and
acetate as electron donor [38]. Pyrobaculum islandicum can grow with cystine or oxidized glutathione as
electron acceptor and complex media as electron donor [14]. The sulfur-reducing bacteria W. succinogenes and Sulfospirillum deleyianum cannot grow
with these disul¢des as electron acceptors [3,60].
The enzymes that catalyze disul¢de reduction in D.
acetoxidans and P. islandicum have not been investigated. The enzyme responsible for disul¢de reduction in D. acetoxidans apparently di¡ers from the
enzyme that reacts with polysul¢de or sulfur, as suggested by the observation that the membrane fraction of D. acetoxidans catalyzes the reduction of sulfur with NADH, but not the reduction of disul¢des
[61].
In contrast to these organisms, which use an external disul¢de as electron acceptor for respiration,
methanogenic archaea generate a disul¢de in the ¢nal step of methanogenesis. This disul¢de is then
used as the terminal electron acceptor of the respiratory chain.
3. Chemistry of elemental sulfur, polysul¢de, and
organic disul¢des
The solubility of elemental sulfur in water at 25³C
is very low (5 Wg l31 ) [62]. The solubility at higher
temperatures is not known. Polysul¢de is formed by
dissolving sulfur £ower in an aqueous sul¢de solution (Reaction (a)) [63].
‡
nS0 ‡ HS3 ! S23
n‡1 ‡ H :
…a†
The S8 -ring is cleaved by nucleophilic attack of
HS3 . The amount of sulfur that can maximally be
dissolved in a sul¢de solution at pH 8 and 37³C is
nearly equivalent to the sul¢de content [63,64]. Much
less polysul¢de is formed at pH values below the pK
of H2 S (Table 2). Tetrasul¢de (S23
4 ) and pentasul¢de
(S23
5 ) are the predominant species of polysul¢de at
pH s 6. The pK of proton dissociation of HS3
4 and
HS3
5 are well below 7. Tetrasul¢de and pentasul¢de
dismutate rapidly according to Reaction (b) [63].
Table 2
Proton dissociation constants of compounds involved in polysul¢de reduction
Reaction
3
‡
H2 SCHS +H
HS3 CS23 +H‡
23
‡
HS3
4 !S4 ‡ H
23
‡
HS3
!S
‡
H
5
5
Temperature (³C)
pK
Reference
25
25
20
20
7.0
s 17
6.3
5.7
[65]
[65]
[66]
[66]
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
3
23
‡
3S23
5 ‡ HS 3 4S4 ‡ H :
…b†
As a consequence of the velocity of Reaction (b),
23
it is not known whether S23
4 or S5 is the preferred
substrate of polysul¢de reductase. For the same reason, the product of polysul¢de reduction is not
known. It is assumed that only one sulfur atom is
cleaved from the polysul¢de chain during catalysis
(Reaction (c)).
3
23
‡
H2 ‡ S23
n ! HS ‡ Sn31 ‡ H
vG0 ˆ 331 kJ=mol H2 :
…c†
The redox potential of polysul¢de can be estimated from that of elemental sulfur and the equilibrium constant of Reaction (a) assuming that only
one species of polysul¢de is formed in this reaction.
The value given in Table 3 refers to the reduction of
to HS3 and is only 15 mV more positive than
S23
4
that of elemental sulfur reduction to HS3 .
Similar to polysul¢de reduction, an S-S bond is
cleaved in disul¢de reduction (reaction (d)).
H2 ‡ R-S-S-R ! 2R-SH:
…d†
The redox potentials of the disul¢des reduced in
methanogenic archaea (CoM-S-S-CoB) is not
known. In Table 3, the redox potential for the cysteine/cystine couple (R-S-S-R/2R-SH) is given and is
approximately 50 mV more positive than that of
elemental sulfur. Hence, from an energetic standpoint, disul¢des should be the better electron acceptors.
4. Polysul¢de as a possible intermediate of sulfur
respiration
Elemental sulfur is not well suited as a substrate of
bacterial sulfur respiration because of its low solubility in water. However, `hydrophilic' or `colloidal'
sulfur has been reported to be reduced with considerable velocities in the presence of enzyme preparations obtained from sulfur-reducing bacteria [4,60].
Since elemental sulfur is readily converted to polysul¢de in aqueous solutions of sul¢de (Reaction (a)),
a product of sulfur respiration, it may be speculated
that polysul¢de is an intermediate of sulfur respira-
357
Table 3
Redox potentials pertinent to sulfur and disul¢de respiration
Redox couple
‡
H /H2
3
HCO3
3 =HCO2
0
3
S /HS
3
S23
4 =HS
R-S-S-R/2 R-SH
Menaquinone in ethanol
E0 0 (mV)
Reference
3420
3413
3275
3260
3220
374
[67]
[67]
[67]
See text
[68]
[69]
tion in general. To test this hypothesis, Schauder and
Muëller [70] measured the maximum amount of sulfur
dissolved according to Reaction (a) as a function of
pH and temperature. The authors found that the
concentration of polysul¢de sulfur should be well
above 10 WM in the growth medium of sulfur-reducing microorganisms growing in the presence of 1 mM
HS3 +H2 S at pH s 6. This concentration of polysul¢de sulfur (10 WM) is close to the apparent Km
measured with polysul¢de respiration of W. succinogenes [71] (see Table 6). With the assumption that
10 WM polysul¢de sulfur is also required for polysul¢de respiration to occur in the other bacteria, it
follows that polysul¢de may be an intermediate of
sulfur respiration in most of the known sulfur reducers (see Table 1).
The acidophilic archaea grow at temperatures
close to 90³C, where polysul¢de sulfur concentrations above 10 WM would require a pH s 5 [70].
However, these bacteria have their pH optimum at
about 2 (see Table 1). Hence, the environment of
these archaea should not contain enough polysul¢de
to allow polysul¢de reduction to occur outside of the
cytoplasmic membrane. In W. succinogenes, polysul¢de reduction occurs in the periplasm, as shown by
the orientation of the polysul¢de reductase towards
the outside of the cytoplasmic membrane [72]. The
orientation of the corresponding enzyme in other
sulfur-reducing bacteria is not known. A soluble cytoplasmic enzyme that catalyzes polysul¢de reduction by reduced ferredoxin or H2 has been discovered in P. furiosus [73], and therefore it is feasible
that polysul¢de reduction also occurs in the cytoplasm of the acidophilic archaea. For this to occur,
it has to be postulated that elemental sulfur di¡uses
across the cytoplasmic membrane and forms polysul¢de in the cytoplasm according to Reaction (a). Un-
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
358
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
fortunately, the di¡usion velocity of elemental sulfur
across the cytoplasmic membrane of growing acidophilic archaea is not known. The concentration of
elemental sulfur dissolved in the media at the growth
temperature of these archaea is probably considerably higher than at 25³C (5 Wg l31 ).
5. Sulfur respiration of Wolinella succinogenes
The actual electron acceptor of sulfur respiration
in W. succinogenes is polysul¢de [3,5,64,71]. This
anaerobic proteobacterium (O-subgroup) grows by
polysul¢de respiration with either H2 (Reaction (c))
or formate (Reaction (e)).
23
3
3
23
‡
HCO3
2 ‡ Sn ‡ H2 O!HCO3 ‡ HS ‡ Sn31 ‡ H
vG0 ˆ 330 kJ=mol H2 :
…e†
W. succinogenes has been reported to grow with
elemental sulfur as terminal electron acceptor under
conditions that were thought not to allow polysul¢de
formation [74]. In these experiments, the culture medium contained Fe2‡ to precipitate as FeS all the
sul¢de formed by the bacteria, and polysul¢de
should not be formed from elemental sulfur in the
absence of sul¢de (Reaction (a)). Recently, a soluble
sulfur compound was detected in the Fe2‡ -containing culture medium at a concentration corresponding
to 0.15 mM polysul¢de sulfur. The compound was
converted to SCN3 upon the addition of CN3 and
the Sud protein (see Reaction (f) in Section 5.4), and
may serve as the actual substrate in sulfur respiration. Although the nature of the compound is not yet
known, the result argues against a direct conversion
of elemental sulfur to sul¢de by W. succinogenes.
5.1. Bioenergetic data
The ATP gain (ATP/e) of polysul¢de respiration
has not been measured directly. The value given in
Table 4 (0.33 mol ATP per mol formate or ATP/
e = 1/6) was estimated from the growth yield (Y)
with polysul¢de using the known ATP gain and
the growth yield of fumarate respiration. With this
ATP gain, the free energy used for ATP synthesis
would be 116 kJ mol ATP31 in polysul¢de respiration, while that used in fumarate respiration is 127
kJ mol ATP31 . Both values are consistent with the
general observation that phosphorylation requires
about 100 kJ mol ATP31 in growing bacteria in
most instances [67]. In spite of the large di¡erence
between the free energy (or vE) available from respiration with polysul¢de and from respiration with
fumarate, nearly the same electrochemical proton
potential across the membrane (vp) has been measured during the respiration steady state with the two
electron acceptors. As a consequence, the H‡ /e ratio
with polysul¢de (H‡ /e = 1/2) should be maximally
half that measured with fumarate (H‡ /e = 1). The
two ratios correspond to the ATP/e ratios with an
H‡ /ATP ratio of 3, which has been measured with
the ATP synthase of W. succinogenes [79]. The values
of Y and vp measured with H2 instead of formate
are close to those given in Table 4. Therefore, the
remaining data of Table 4 are likely to apply also for
respiration with H2 (Reaction (c)).
5.2. Electron transport enzymes
The electron transport chain catalyzing polysul¢de
reduction by H2 or formate consists of polysul¢de
reductase (Psr) and hydrogenase (Fig. 1) or formate
dehydrogenase [80]. The enzymes are integrated in
Table 4
Bioenergetic data of the polysul¢de respiration with formate of W. succinogenes
Electron acceptor
pH
Y
(g cells/mol formate)
ATP/e
3vE
(V)
vEWF
ATP=e
(kJ/mol ATP)
vp
(V)
H‡ /e
Polysul¢de
Fumarate
8.4
7.9
3.2 [64]
7.0 [76]
(1/6)
1/3 [77,78]
0.20
0.44
116
127
0.17 [75]
0.18 [77]
(1/2)
1
The data are compared to those of fumarate respiration. The values of pH, Y and vE refer to the middle of the exponential growth phase at
3
37³C. vE was calculated from the vE0 0 given in Table 3 with the given values of pH and equal concentrations of HCO3
2 and HCO3 ,
3
polysul¢de sulfur and HS , and fumarate and succinate. The numbers in parentheses were estimated as described in the text.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
359
drogenase and Rhodobacter sphaeroides dimethylsulfoxide reductase [81]. The crystal structures of these
single-subunit enzymes are known [85,86]. At the
catalytic site of each enzyme, a molybdenum ion is
coordinated by two molybdopterin guanine dinucleotide molecules. PsrA is likely the catalytic subunit of polysul¢de reductase, and likely carries the
molybdenum ion coordinated by two molybdopterin
guanine dinucleotide molecules, although a lower
molybdopterin guanine dinucleotide content has
been determined experimentally (Table 5).
A mutant (vpsrABC) lacking the psrABC operon
does not catalyze polysul¢de reduction by H2 or formate when grown with fumarate as terminal electron
acceptor, in contrast to the wild-type strain [72]. Surprisingly, the mutant grows with polysul¢de. When
grown with polysul¢de, the mutant forms a membrane-integrated enzyme that replaces polysul¢de reductase (Psr). The enzyme formed by the vpsrABC
Fig. 1. Composition of the electron transport chain catalyzing
polysul¢de respiration with H2 in W. succinogenes. The sul¢de
dehydrogenase (Sud) protein is described in the text (Section
5.4). Mo, molybdenum linked to molybdopterin guanine dinucleotide ; Ni, nickel ion; Fe/S, iron-sulfur centers; Cyt b, diheme
cytochrome b; MKb , menaquinone bound to polysul¢de reductase; PsrA, B, C, polysul¢de reductase subunits; HydA, B, C,
hydrogenase subunits [84].
Table 5
Properties of polysul¢de reductase [82,83]
Turnover number:
3
[S]+BH3
4 CHS +BH3
Apparent Km
1700 s31
50 WM [S]
the cytoplasmic membrane with the catalytic subunits exposed to the periplasm [3,72]. The isolated
polysul¢de reductase, which consists of the three
subunits (PsrA, B, C) predicted from the nucleotide
sequence of the polysul¢de reductase operon
(psrABC) [81], catalyzes polysul¢de reduction by
BH3
4 to sul¢de, and sul¢de oxidation to polysul¢de
by 2,3-dimethyl-1,4-naphthoquinone (Table 5). The
enzyme contains molybdenum and molybdopterin
guanine dinucleotide. The amounts of iron and sul¢de in the enzyme are consistent with the presence of
¢ve tetranuclear iron-sulfur centers. The amino acid
sequence derived from the psrA nucleotide sequence
suggests that the catalytic subunit carries one tetranuclear iron-sulfur center, and PsrB is predicted to
carry four iron-sulfur centers [81].
The amino acid sequence of PsrA is similar to that
of the catalytic subunits of several molybdo-oxidoreductases, including Escherichia coli formate dehy-
Turnover number:
HS3 +DMN+H‡ C[S]+DMNH2
Apparent Km
1100 s31
25 mM HS3
Subunits:
Contents (mol/mol PsrABC) :
Molybdenum
MGD
Iron
Sul¢de
Menaquinone
Heme
Flavin
Other heavy metals
PsrA (81 kDa)
PsrB (21 kDa)
PsrC (34 kDa)
1
1
21
22
0.6^1.6
90.1
90.1
90.1
MGD, molybdopterin guanine dinucleotide ; [S], polysul¢de;
DMN, 2,3-dimethyl-1,4-naphthoquinone; Psr, polysul¢de reductase. The turnover numbers and the Km were determined with
the enzyme in an anoxic bu¡er (pH 8.3, 37³C) containing either
50 mM Tris-HCl and 10 mM KBH4 (polysul¢de reduction), or 0.2
M triethanolamine (pH 7.9) and 0.2 mM DMN (sul¢de oxidation).
The menaquinone content given was corrected for the amount of
menaquinone associated with the phospholipid present in the preparation (50^200 Wmol g protein31 ).
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
360
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
mutant grown with polysul¢de has not yet been isolated. Its properties appear to di¡er considerably
from those of the polysul¢de reductase enzyme.
The enzyme does not cross-react with antiserum
raised against PsrA. Like polysul¢de reductase, the
enzyme catalyzes sul¢de oxidation by dimethylnaphthoquinone. However, the apparent Km values for
sul¢de di¡er drastically. The value measured with
polysul¢de reductase is 25 mM (Table 5) and that
of the enzyme induced in the vpsrABC mutant is
approximately 1 mM [83]. The enzyme present in
the mutant is apparently absent from the wild-type
strain, as suggested by this di¡erence in the apparent
Km values.
5.3. Mechanism of electron transfer from hydrogenase
to polysul¢de reductase
A mutant of W. succinogenes lacking the hydrogenase structural genes (hydABC) does not grow
with H2 and either polysul¢de or fumarate [87].
The mutant grown with formate and fumarate does
not catalyze the reduction of polysul¢de or fumarate
by H2 , in contrast to the wild-type strain. Growth
and electron transport activities are restored upon
insertion of hydABC into the genome of the deletion
mutant. Hence, the same hydrogenase appears to
serve in the electron transport with polysul¢de and
with fumarate. The same holds true for formate dehydrogenase [80].
In fumarate respiration, electron transfer from the
dehydrogenases to fumarate reductase is mediated by
menaquinone, which is present in the bacterial membrane in more than 10-fold molar excess over the
electron transport enzymes [88,89]. Most of the menaquinone is thought to be dissolved in the lipid
phase of the membrane and to serve in transferring
electrons from the dehydrogenases to fumarate reductase by di¡usion. The mechanism of electron
transfer from the dehydrogenases to polysul¢de reductase is not known. Electron transfer by menaquinone di¡usion appears to be unlikely because the
standard redox potential of menaquinone at pH 7
is more than 200 mV more electropositive than
that of polysul¢de (Table 3).
The experiments illustrated in Fig. 2 suggest that
the electron transfer from the dehydrogenases to
polysul¢de reductase may require di¡usion and col-
Fig. 2. Electron transport activity with polysul¢de (A and C) or
fumarate (B and D) as a function of the phospholipid/membrane
protein ratio. The experiments were performed as described previously [90]. In C and D, the liposomes fused to the membrane
fraction of W. succinogenes contained menaquinone (20 Wmol/g
phospholipid) isolated from the membrane fraction. In A^D, formate (R) or H2 (b) were applied as electron donor of electron
transport. One unit of activity is equivalent to the oxidation of
1 Wmol formate or H2 per min at 37³C.
lision of the enzymes within the membrane. The
membrane fraction of W. succinogenes was fused
with increasing amounts of liposomes containing
menaquinone, and the electron transport activity
with polysul¢de (Fig. 2C) and that with fumarate
(Fig. 2D) was measured as a function of the amount
of phospholipid present in each preparation. The
speci¢c activities given are based on the amount of
membrane protein and are proportional to the turnover number of the enzymes in electron transport.
The activities of polysul¢de reductase, fumarate reductase, hydrogenase, and formate dehydrogenase
were hardly a¡ected by the dilution of the membrane
fraction with phospholipid (data not shown). In contrast, the electron transport activities with polysul¢de decreased by 70^80% upon maximal dilution
of the membrane fraction with phospholipid, while
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
those with fumarate increased slightly with the lower
amounts of phospholipid and were similar to the
activities of the original membrane fraction with
the highest amount of phospholipids. The di¡erent
e¡ects of membrane dilution can be explained on the
basis of the assumption that polysul¢de respiration is
limited by the di¡usion of the electron transport enzymes within the membrane, while fumarate respiration is not. The data of Fig. 2C ¢t to the Hardt
equation, which relates the collision frequency of
two protein molecules to their di¡usion coe¤cients
(1038 cm2 s31 ) within the membrane and their surface densities [90^92].
The experiments shown in Fig. 2A and B were
performed like those shown in Fig. 2C and D, except
that the liposomes applied did not contain menaquinone. Under these conditions, the electron transport
activities with either polysul¢de or fumarate decreased with the amount of phospholipid fused to
the membrane fraction. The e¡ect of membrane dilution on fumarate respiration can be explained on
the basis of the relatively high apparent Km of fumarate reductase for menaquinone, which is in the millimolar range [93]. The e¡ect of membrane dilution on
polysul¢de respiration is more pronounced in the
absence of menaquinone (Fig. 2A) than in its presence (Fig. 2C), suggesting that menaquinone is involved also in polysul¢de respiration. This view is
supported by the ¢nding that the isolated polysul¢de
reductase contains approximately 1 mol menaquinone per mol enzyme (Table 5). The menaquinone
involved in polysul¢de reduction is probably bound
to polysul¢de reductase and dissociates from its
binding site upon dilution of the membrane with
phospholipid. In contrast, the menaquinone involved
in fumarate respiration freely di¡uses within the
membrane.
The view that the function of menaquinone in the
respiration with polysul¢de di¡ers from that with
fumarate is supported by the following result [90].
When experiments similar to those shown in Fig.
2C and D were performed with vitamin K1 instead
of menaquinone, fumarate respiration did not decrease upon membrane dilution, and the data were
similar to those of Fig. 2D. In contrast, the e¡ect on
polysul¢de respiration was similar to that upon
membrane dilution in the absence of menaquinone
(Fig. 2A). Hence, vitamin K1 can replace menaqui-
361
none in the pathway of fumarate respiration, but
cannot replace menaquinone in the pathway with
polysul¢de. Vitamin K1 (four isoprene residues) differs from menaquinone (seven isoprene residues)
only in the length of the isoprenoid side chain and
its degree of saturation.
Hydrogenase catalyzes the reduction of menaquinone by H2 [84]. The quinone site is located on the
diheme cytochrome b subunit of the enzyme (HydC).
HydC mutants with one of the heme-B-ligating histidine residues substituted by another amino acid do
not catalyze quinone reduction or polysul¢de reduction by H2 [94]. This demonstrates that the intact
HydC is required for electron transfer from hydrogenase to polysul¢de reductase. The result also supports the view that the membrane anchor of polysul¢de reductase (PsrC) is involved in the electron
transfer from hydrogenase to polysul¢de reductase.
Bound menaquinone (Table 5) possibly serves as the
prosthetic group of PsrC and as the primary acceptor of the electrons delivered by HydC (Fig. 1). This
would explain why mutants that do not catalyze quinone reduction by H2 also lack electron transport
activity from H2 to polysul¢de.
Formate dehydrogenase catalyzes menaquinone
reduction by formate [93]. The quinone reactive site
of formate dehydrogenase is located on the diheme
cytochrome b subunit of the enzyme. The amino acid
sequence of this subunit is similar to that of hydrogenase cytochrome b [95]. Especially the four histidine residues coordinating the heme B groups are
predicted to be located at similar places on three
homologous membrane helices. Therefore, it is likely
that the mechanism of electron transfer from formate dehydrogenase to polysul¢de reductase is the
same as that with hydrogenase.
5.4. The function of the Sud protein
In the presence of sulfur, fumarate, and nitrate,
W. succinogenes grows by polysul¢de respiration,
while fumarate and nitrate are not reduced [96].
This preference suggests that W. succinogenes is primarily a sulfur (polysul¢de) reducer. Its ecological
role may be to supply sul¢de as a biosynthetic substrate to the methanogens in the rumen of cattle, the
habitat of W. succinogenes. The polysul¢de concentration in the rumen is estimated to be maximally
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
362
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
6 WM polysul¢de sulfur at pH 9 7 and 0.1 mM total
sul¢de (HS3 +H2 S) [97]. The apparent Km of polysul¢de reductase for polysul¢de has been measured
as 50 WM (Table 5). The same value has been obtained by measuring electron transport from H2 to
polysul¢de with the membrane fraction of W. succinogenes (Table 6). The apparent Km for polysul¢de
measured with intact bacteria grown with fumarate
is 70 WM. Hence, the Km for polysul¢de reduction is
about an order of magnitude above the actual polysul¢de concentration in the rumen. However, the
apparent Km measured with W. succinogenes grown
on polysul¢de (10 WM) is close to the ruminal polysul¢de concentration.
The lower Km for polysul¢de measured with cells
grown on polysul¢de is due to the induction of the
soluble periplasmic Sud protein under these conditions [71,98]. Sud was originally isolated as a sul¢de
dehydrogenase. The e¡ect of Sud on the Km for polysul¢de has been demonstrated using the activity of
electron transport from H2 to polysul¢de catalyzed
by the membrane fraction of W. succinogenes (Table
6). The electron transport activity is considerably
increased by the presence of the isolated Sud protein
at polysul¢de concentrations below 0.1 mM. The
apparent Km for polysul¢de decreases from 50 WM
in the absence of Sud to 7 WM in its presence. The
stimulating e¡ect of Sud is not observed at higher
polysul¢de concentrations [71] and is maximal with
0.8 WM Sud dimer added. Higher amounts of Sud do
not further increase electron transport activity. Polysul¢de-grown W. succinogenes cells contain nearly
equimolar amounts of Sud and Psr, whereas the molar ratio is 0.2 in fumarate-grown cells (Table 6). The
concentration of Sud in the periplasm of W. succinogenes grown with polysul¢de is more than two orders
of magnitude higher than that required for saturation of the electron transport activity of the mem-
brane fraction (0.8 WM). These results suggest that
the periplasmic Sud is bound to polysul¢de reductase
(Fig. 1).
Sud consists of two identical subunits (14.3 kDa)
and does not contain prosthetic groups or heavy
metal ions. Sud binds up to 10 mol polysul¢de sulfur
per subunit when incubated in a polysul¢de solution
[71]. Furthermore, Sud catalyzes sulfur transfer from
polysul¢de to cyanide according to Reaction (f)
3
3
23
S23
n ‡ CN !SCN ‡ Sn31
with a turnover number of about 104 s31 at 37³C.
These results suggest that Sud serves as a polysul¢de
sulfur transferase from aqueous polysul¢de to the
active site of polysul¢de reductase. Sud appears to
raise the a¤nity of polysul¢de reductase for polysul¢de. The function of Sud probably is to allow polysul¢de respiration to occur at a su¤cient speed even
at very low polysul¢de concentrations.
5.5. Mechanism of vp generation
The mechanism of vp generation in the polysul¢de
respiration of W. succinogenes is not known. Two
types of mechanism are feasible. Polysul¢de reductase may operate as a proton pump during electron
transport from H2 or formate to polysul¢de. Alternatively, the redox reactions of the menaquinone
that is probably bound to PsrC may be coupled to
proton translocation across the membrane. A schematic view of the stationary complex formed by hydrogenase and polysul¢de reductase in the cytoplasmic membrane during electron transfer is given in
Fig. 3. The bound menaquinone (MKb ) is assumed
to form the hydroquinone anion (MKb H3 ) upon
reduction by hydrogenase to account for the H‡ /e
ratio of 1/2 (Table 4). The site of quinone reduction
Table 6
Km values for polysul¢de in the electron transport from H2 to polysul¢de as a function of the amount of Sud protein present [71]
Membrane fraction
Membrane fraction
Cells grown with polysul¢de
Cells grown with fumarate
…f †
Sud concentration (WM)
Molar ratio (Sud/Psr)
Km for polysul¢de (WM)
0
0.8
160
15
0
30
1.1
0.2
50
7
10
70
Psr, polysul¢de reductase.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
363
tabolism in which sulfur acts as an additional electron sink. From this organism, two enzymes have
been isolated that catalyze the reduction of sulfur
or polysul¢de to H2 S. An NAD(P)H-dependent sul¢de dehydrogenase and a hydrogenase (termed sulfhydrogenase or sulfur:reduced ferredoxin oxidoreductase) couple the oxidation of reduced ferredoxin
to the reduction of either protons to H2 or sulfur to
H2 S [100,101]. However, the location of these two
enzymes in the cytoplasm, plus the ¢nding that products of maltose fermentation are virtually identical
during growth with or without sulfur, argue against
a conventional membrane-bound respiratory type of
metabolism in the presence of sulfur [102].
In these sulfur-reducing heterotrophs, the reduction of sulfur to H2 S is proposed to be a mechanism
for the disposal of excess reductant generated by
fermentation and of toxic H2 [28,51]. As shown for
other H2 S-producing organisms, sulfur reduction
could also lead to the formation of metal sul¢des,
thus allowing the removal of toxic metals [103].
Fig. 3. Hypothetical mechanism of vp generation by electron
transport from H2 to polysul¢de in W. succinogenes. The electron
transfer from hydrogenase (upper part) to polysul¢de reductase
(lower part) requires collision of the two enzymes within the
membrane. Ni, nickel ion; Mo, molybdenum ion linked to molybdopterin guanine dinucleotide; MKb , menaquinone bound to
polysul¢de reductase.
is envisaged to be located in a lipophilic environment. This would require the existence of proton
paths for proton uptake during quinone reduction
and for proton release during quinol oxidation. It
is assumed that the former path is provided by hydrogenase and the latter by polysul¢de reductase.
Consistent with the model shown in Fig. 3, the protons required for menaquinone reduction by H2 in
the membrane of W. succinogenes have been shown
to be taken up from the cytoplasm [99].
6. Sulfur respiration in hyperthermophilic archaea
6.1. Sulfur reduction in fermentative
hyperthermophiles
Several heterotrophic sulfur reducers, such as Pyrococcus furiosus, exhibit a fermentative type of me-
6.2. Sulfur respiration in species of Pyrodictium
In hyperthermophilic sulfur-respiring archaea, the
reduction of sulfur to H2 S is catalyzed by membrane-bound respiratory chains. Lithotrophs such
as Pyrodictium brockii and Stygiolobus azoricus use
molecular hydrogen as electron donor for this reaction [21,13], while organotrophic organisms such as
Thermodiscus maritimus and Thermo¢lum pendens
use peptides or carbohydrates [16,104]. Evidently,
the lithotrophic sulfur-respiring archaea must couple
electron transport to sulfur with phosphorylation of
ADP.
In the membranes of P. brockii, a hydrogenase, a
quinone, and a cytochrome c have been identi¢ed as
part of a proposed respiratory electron transport
chain [105^107]. The hydrogenase has been puri¢ed
and found to be of the Ni/Fe-type and to consist of
two subunits (66^68 kDa and 45 kDa) [106]. TLC
analysis of the quinone has shown migration characteristics similar to that of ubiquinone-6 (Q-6), but
NMR analysis has revealed evidence for a quinone
di¡erent from all quinones compared. Cytochrome c
(13^14 kDa) is the only cytochrome detected in the
membranes of P. brockii. Inhibition experiments
with the quinone analogue HQNO have suggested
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
364
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
the electron transfer sequence: hydrogenaseCquinoneCcytochrome c. After inactivation of the electron
transport activity by UV light, addition of ubiquinones Q-6, Q-10 or puri¢ed P. brockii quinone restores activity. Cytochrome c is thought to serve as
electron donor to the sulfur reductase, which has not
yet been identi¢ed [105^107].
The electron transport chain catalyzing sulfur reduction by H2 in P. abyssi isolate TAG11 di¡ers
from that of P. brockii with respect to composition
and organization of the components [22]. A
H2 :sulfur oxidoreductase complex, which catalyzes
the H2 -dependent reduction of sulfur to H2 S, has
been recently puri¢ed from the membranes of P.
abyssi isolate TAG11. The catalytic properties of
the enzyme complex suggest that it represents the
entire respiratory chain of the organism, with hydrogenase, electron transport components, and sulfur
reductase arranged in one stable multi-enzyme complex. The puri¢ed H2 :sulfur oxidoreductase consists
of at least nine subunits, two of which are b-type
cytochromes, and one a cytochrome c. The cytochrome c (30 kDa) is approximately twice as large
as that of P. brockii. It should be pointed out that
among hyperthermophiles, c-type cytochromes have
been detected only in species of Pyrodictium [22,105].
No quinone has been detected in the H2 :sulfur oxidoreductase or in the membrane fraction of P. abyssi
isolate TAG11. Although the respiratory chains of
P. brockii and P. abyssi isolate TAG11 di¡er in electron transport components, the hydrogenases appear
to be similar. The H2 :sulfur oxidoreductase consists
of two subunits (66 and 45 kDa), similar in size to
the P. brockii hydrogenase subunits. The N-terminal
amino acid sequence of the 66-kDa subunit is similar
to the N-terminal sequence of the catalytic subunits
of Ni/Fe-hydrogenases. The content of 1.6 mol nickel/mol H2 :sulfur oxidoreductase suggests that its hydrogenase, like the P. brockii enzyme, is of the Ni/
Fe-type. Both hydrogenases are insensitive to oxygen
and function as `H2 -uptake' hydrogenases, indicating
the respiratory role of these enzymes. During puri¢cation, the activity of the hydrogenase present in the
H2 :sulfur oxidoreductase complex, as measured with
viologen dyes, increases parallel with H2 :sulfur oxidoreductase activity.
Present data suggest that energy conservation via
respiration in hyperthermophiles appears to be sim-
ilar to that of mesophiles. A membrane-bound respiratory chain generates a chemiosmotic potential,
which is utilized by a membrane-bound ATP synthase to form ATP. Yet, due to their extreme habitats, hyperthermophiles have adapted their system
to high temperatures. For example, in P. abyssi isolate TAG11, not only the H2 :sulfur oxidoreductase
complex, but also a membrane-bound ATPase, likely
to function as ATP-synthase, show temperature optima around 100³C [22] (R. Dirmeier, unpublished
results). Thus far, the electron transport chain of
P. brockii and the H2 :sulfur oxidoreductase complex
from P. abyssi isolate TAG11 are the only described
examples of enzymes involved in the membranebound sulfur respiration of hyperthermophilic organisms. The stable organization of the di¡erent
components of the H2 :sulfur oxidoreductase complex from P. abyssi isolate TAG11 implies that further investigations will yield a better understanding
of sulfur respiration in hyperthermophiles.
7. Disul¢de respiration in methanogenic archaea
Methanogenic archaea derive their metabolic energy from the conversion of a restricted number of
substrates to methane. Most methanogens can reduce CO2 to CH4 with H2 as electron donor. A
few methanogens can utilize formate, ethanol, or isopropanol as an electron donor for CO2 reduction.
Some methanogens can convert methanol, methylamines, and methylmercaptans to CH4 and CO2 .
Acetate is the only C2 -compound utilized by some
Fig. 4. Structures of coenzyme M (H-S-CoM ; 2-mercaptoethanesulfonate), coenzyme B (H-S-CoB ; 7-mercaptoheptanoylthreonine
phosphate), and the heterodisul¢de (CoM-S-S-CoB) of coenzyme
M and coenzyme B.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
methanogens as sole energy substrate. It is converted
to CH4 and CO2 . (For a historical overview on
methanogenesis, see [6,7]; for recent reviews see [8^
11].)
In these di¡erent pathways of energy metabolism,
two unique thiol-containing coenzymes play a central role: coenzyme M (H-S-CoM; 2-mercaptoethanesulfonate) and coenzyme B (H-S-CoB; 7-mercaptoheptanoylthreonine phosphate) (Fig. 4). Coenzyme
M is converted to its methylthioether (CH3 -S-CoM),
which is the central intermediate of methanogenesis
(Fig. 5) (see [6,7]). This methylthioether reacts with
coenzyme B to yield methane and the heterodisul¢de
(CoM-S-S-CoB) of the two methanogenic thiol-containing coenzymes. This reaction is catalyzed by the
soluble methyl-coenzyme M reductase (for a review
see [11]). The heterodisul¢de thus generated plays a
central role in energy conservation; the reduction of
CoM-S-S-CoB is coupled with the generation of a
proton motive force [108^111]. Hence, CoM-S-SCoB is the terminal electron acceptor of a respiratory chain in these organisms. The enzyme reducing
the heterodisul¢de to the thiols H-S-CoM and H-SCoB, heterodisul¢de reductase, is membrane bound
and functions as a terminal respiratory reductase.
The electron donor for this disul¢de respiration
varies with the growth substrate. In the following,
the composition of the various respiratory chains
involved in the reduction of CoM-S-S-CoB will be
discussed in more detail.
Taxonomically, methanogens belong to the archaeal kingdom of euryarchaeota. They are classi¢ed in
¢ve orders, each of which is as distantly phylogenetically related to the other as the cyanobacteriales to
the proteobacteriales. The ¢ve orders are Methanobacteriales, Methanococcales, Methanomicrobiales,
Methanopyrales, and Methanosarcinales [112]. Of
these, only the Methanosarcinales can ferment acetate to CO2 and CH4 and can grow on methanol,
methylamines, and methylthiols as sole energy
source. In addition, the Methanosarcinales contain
cytochromes, whereas cytochromes have not been
found in organisms belonging to the other orders
of methanogens (see [8]). The ability to use a variety
of substrates and the presence of cytochromes have
an important in£uence on the composition of the
respiratory chains involved in disul¢de respiration.
Therefore disul¢de respiration in Methanosarcina
365
species will be discussed separately from disul¢de
respiration in organisms belonging to other phylogenetic groups of methanogens.
7.1. Disul¢de respiration in Methanosarcina species
Since Methanosarcina species can use several
growth substrates, these organisms contain di¡erent
respiratory chains for the reduction of CoM-S-SCoB. As will be shown below, these respiratory
chains are composed of a substrate-speci¢c dehydrogenase, a membrane-bound electron carrier, and heterodisul¢de reductase. The following enzymes and
electron carriers have been shown to participate in
these respiratory chains.
7.1.1. Heterodisul¢de reductase
Heterodisul¢de reductase (Hdr) was puri¢ed from
the membrane fraction of Methanosarcina barkeri
using detergents for solubilization [113^115]. The puri¢ed enzyme is composed of two di¡erent subunits,
a 23-kDa polypeptide designated HdrE and a 46kDa polypeptide designated HdrD. The enzyme contains approximately 0.6 mol heme b/mol enzyme and
about 20 mol non-heme iron and acid-labile sulfur/
mol enzyme. In contrast to most other disul¢de reductases, this disul¢de reductase does not contain a
£avin [115]. The 23-kDa polypeptide shows peroxidase activity, which indicates that this polypeptide
contains heme. Spectroscopic studies have shown it
to be a heme b [113,114].
The genes encoding the two subunits HdrD and
HdrE form the transcription unit hdrED. From the
deduced amino acid sequence, it can be predicted
that HdrE is an integral membrane protein with
¢ve transmembrane-spanning helices. Sequence analysis con¢rmed that HdrE is a b-type cytochrome as it
shows sequence similarity to other b-type cytochromes [115]. Analysis of the deduced amino acid
sequence of HdrD indicates that it is a hydrophilic
polypeptide that contains two classical binding motifs for [4Fe-4S] clusters close to its N-terminus. The
C-terminal domain of the polypeptide contains several cysteine residues, which could ligate an additional iron-sulfur cluster, as will be discussed below. In
Northern blot experiments with RNA isolated from
cells grown on methanol, H2 /CO2 , or acetate, probes
derived from hdrE or hdrD each hybridized to a 2.3-
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
366
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
Fig. 5. Scheme of methanogenesis from H2 /CO2 , methanol, and acetate. As a central intermediate of the various pathways, methyl-coenzyme M (CH3 -S-CoM) is formed and is converted to methane and the heterodisul¢de of coenzyme M and coenzyme B (CoM-S-S-CoB).
CoM-S-S-CoB thus generated functions as the terminal electron acceptor of the various respiratory chains. H2 and reduced coenzyme
F420 (F420 H2 ) are the electron donors for the reduction of CoM-S-S-CoB. The unknown mechanism of electron transfer from the reduced
ferredoxin (Fdred ) to CoM-S-S-CoB in acetate metabolism is symbolized by a question mark. The role of H2 as an intermediate of this reaction is discussed in the text. CH3 -H4 MPT, methyl-tetrahydromethanopterin; F420 H2 , reduced form of coenzyme F420 ; Fd, ferredoxin;
pFd, polyferredoxin.
kb mRNA, indicating that this operon is expressed
during growth on each of these substrates [115]. In
Southern blot hybridizations with total DNA from
M. barkeri, only one copy of hdrE and hdrD could
be detected. This indicates that the same heterodisul¢de reductase operates in H2 /CO2 , methanol, and
acetate metabolism (A. Kuënkel and R. Hedderich,
unpublished results).
Recently the puri¢cation and characterization of
heterodisul¢de reductase from Methanosarcina thermophila has been reported [116]. The enzyme exhibits
properties very similar to those of the M. barkeri
enzyme.
7.1.2. Hydrogenases
A membrane-bound hydrogenase has been puri¢ed from Methanosarcina mazei [117] and Methanosarcina barkeri [118] using detergents for solubilization. The puri¢ed enzyme contains Ni, non-heme
iron, and acid-labile sulfur. It is composed of two
di¡erent subunits with apparent molecular masses
of 60 kDa and 40 kDa. In the genome of M. mazei,
the structural genes for two closely related membrane-bound hydrogenases have been identi¢ed
[119]. They are encoded in two separate transcriptional units: vhoGAC (viologen-reactive hydrogenase
one) and vhtGAC (viologen-reducing hydrogenase
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
two). The genes vhoA and vhtA each encode a 60kDa subunit, which harbors the binding motifs for
the Ni-Fe active site. The genes vhoG and vhtG each
encode a 40-kDa subunit, which contains 10 conserved cysteine residues. Similar conserved residues
have been shown to ligate three iron-sulfur clusters
in the Desulfovibrio gigas hydrogenase [120]. Each
transcriptional unit contains one additional gene,
vhoC or vhtC, whose gene product is not present in
the puri¢ed enzyme. These genes encode b-type cytochromes. It is assumed that the hydrophobic VhoC
and VhtC subunits were separated from their two
hydrophilic subunits during puri¢cation of the enzymes. The vht operon contains a fourth gene
(vhtD), whose gene product is also not present in
the puri¢ed enzyme. The function of this gene is
not known. The ¢nding that the 5P-ends of the
vhoG/vhtG genes code for a long signal peptide supports the topology of the hydrogenase shown in Fig.
6.
Northern blot experiments have shown that the
expression of the genes encoding the two membrane-bound hydrogenases in M. mazei is substrate-dependent [121]. The vhoGAC operon is expressed during growth on H2 /CO2 , methanol, or
acetate, while the vhtGAC operon is only expressed
during growth on H2 /CO2 and methanol but not
during growth on acetate. Obviously the two hydrogenases have di¡erent functions in the metabolism of
Methanosarcina mazei. The amino acid sequences of
the homologous structural subunits (VhoG/VhtG
and VhoA/VhtA) are almost identical. The C-termini
of VhoC and VhtC are not homologous. This might
indicate that the two hydrogenases interact with different electron acceptors via this subunit. Since the
VhoGAC hydrogenase is synthesized during growth
on all substrates, it could be part of the respiratory
chain from H2 to CoM-S-S-CoB, while the VhtGAC
hydrogenase could be involved in reactions speci¢c
for H2 /CO2 and methanol metabolism [8].
7.1.3. F420 H2 dehydrogenase
The deaza£avin coenzyme F420 (E0 0 = 3360 mV) is
a two-electron redox carrier in methanogenic archaea. It functions as the physiological electron acceptor or donor of several oxidoreductases of the
pathways of energy metabolism [6]. The reduced
form of coenzyme F420 (F420 H2 ) functions as the
367
physiological electron donor for CoM-S-S-CoB reduction in Methanosarcina species. F420 H2 dehydrogenase, which catalyzes the oxidation of F420 H2 , is
an integral membrane protein and has been puri¢ed
from Methanolobus tindarius [122] and Methanosarcina mazei [123]. The enzyme from M. tindarius is
composed of ¢ve di¡erent subunits (45, 40, 22, 18,
and 17 kDa) and contains Fe/S centers but no £avin.
The enzyme from M. mazei is composed of ¢ve different subunits (40, 37, 22, 20, and 16 kDa) and
contains approximately 7 mol non-heme iron and
7 mol acid-labile sulfur. In addition, the enzyme contains FAD as prosthetic group. The puri¢ed enzyme
catalyzes the reduction of methanophenazine analogues, such as 2-hydroxyphenazine, with a speci¢c
activity of 9 U/mg protein and an apparent Km for
2-hydroxyphenazine of 35 WM. In vivo the lipophilic
methanophenazine present in the membrane is assumed to be the physiological electron acceptor of
this enzyme [111,124,125] as will be discussed below.
An F420 H2 :quinone oxidoreductase has been characterized from the sulfate-reducing archaeon Archaeoglobus fulgidus [126]. The genes encoding this enzyme have been identi¢ed in the totally sequenced
genome of A. fulgidus [127]. The subunits of this
enzyme show high sequence similarity to subunits
of energy-conserving NADH:quinone oxidoreductase.
7.1.4. Methanophenazine
Methanogenic archaea do not contain quinones.
Recently a compound was isolated from membranes
of M. mazei that could have a function in the respiratory chains of Methanosarcina species similar to
that of quinones in the respiratory chains of other
organisms [111]. This novel electron carrier is called
methanophenazine and is a 2-hydroxyphenazine derivative connected to a polyisoprenoid side chain via
an ether bridge. Since this component is almost insoluble in water, water-soluble analogues of methanophenazine, such as 2-hydroxyphenazine and 2-bromophenazine, have been used for in vitro enzyme
assays. These water-soluble analogues have been
shown to function as electron acceptors of the puri¢ed F420 H2 dehydrogenase [111,125]. In addition,
washed membranes of M. mazei catalyze the reduction of these methanophenazine analogues by H2 ,
suggesting that the methanophenazine functions as
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
368
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
electron acceptor of one of the membrane-bound
hydrogenases. Furthermore, the membrane-bound
heterodisul¢de reductase uses reduced 2-hydroxyphenazine as an electron donor for the reduction of
CoM-S-S-CoB [111,125]. From these data, it is reasonable to assume that methanophenazine plays an
important role in membrane-bound electron transport in vivo.
7.1.5. Composition of the di¡erent respiratory chains
and mechanisms of vp generation
When Methanosarcina species grow on H2 /CO2 ,
the electron donor for the reduction of CoM-S-SCoB is H2 (Fig. 5). A subcellular system from
Methanosarcina mazei consisting of washed inverted
vesicles catalyzes the reduction of CoM-S-S-CoB
with H2 [108,110].
H2 ‡ CoM-S-S-CoB
! H-S-CoM ‡ H-S-CoB
vG0 ˆ 340 kJ=mol:
…g†
This reaction is coupled with proton translocation
across the cytoplasmic membrane into the lumen of
the inverted vesicles. Two H‡ are translocated per
molecule of CoM-S-S-CoB reduced in this in vitro
system. Results of experiments with intact cells and
CH3 OH/H2 as substrate indicate a stoichiometry of
3^4 H‡ translocated per CoM-S-S-CoB reduced. The
discrepancy can be explained by the fact that only
about 50% of the vesicles in the in vitro system are
intact and thus couple CoM-S-S-CoB reduction with
H‡ translocation. A stoichiometry of 3^4 H‡ translocated per CoM-S-S-CoB reduced indicates that the
proton motive force is not generated solely by transmembrane electron transport, with H2 being oxidized
at the extracellular site of the cytoplasmic membrane. A di¡erent or additional mechanism for proton translocation must operate. The vp generated
drives the phosphorylation of ADP with inorganic
phosphate. From the present data, it is assumed
that the respiratory chain is composed of one of
the membrane-bound hydrogenases ^ most probably
VhoGAC ^methanophenazine, and heterodisul¢de
reductase (Fig. 6) [111,124].
During growth on methanol or methylamines, part
of the reducing equivalents are transferred to F420 to
generate F420 H2 (Fig. 5). Washed inverted vesicles of
M. mazei catalyze the reduction of CoM-S-S-CoB by
F420 H2 [128].
F420 H2 ‡ CoM-S-S-CoB ! F420
‡H-S-CoM ‡ H-S-CoB
vG 0 ˆ 329 kJ=mol:
Fig. 6. Putative scheme of the respiratory chain from H2 to
CoM-S-S-CoB and F420 H2 to CoM-S-S-CoB in Methanosarcina
species. MPox , methanophenazine in the oxidized form; MPred ,
methanophenazine in the reduced form. For other abbreviations,
see Fig. 3.
…h†
The reaction is coupled with proton translocation
across the cytoplasmic membrane with a stoichiometry of 2 H‡ translocated [109]. Recent data indicate
that this respiratory chain is composed of F420 H2
dehydrogenase, methanophenazine, and heterodisul¢de reductase (Fig. 6) [111,125]. Using washed
everted vesicles of M. mazei, it has been shown
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
that both the reduction of 2-hydroxyphenazine with
F420 H2 and the reoxidation of reduced hydroxyphenazine by CoM-S-S-CoB are coupled to proton
translocation across the cytoplasmic membrane
[124]. The mechanism of proton translocation is unknown. Since oxidation of F420 H2 and reduction of
CoM-S-S-CoB both occur on the cytoplasmic side,
transmembrane electron transport without proton
translocation can be excluded as the mechanism of
vp generation. Protons are translocated either by a
redox-driven proton pump or by the redox reactions
of methanophenazine (Fig. 6).
During growth on acetate, cleavage of the acetate
molecule is catalyzed by CO dehydrogenase/acetyl
CoA synthase. This reaction generates enzymebound CO and an enzyme-bound methyl group.
The methyl group is transferred to coenzyme M via
tetrahydromethanopterin (H4 MPT). The methyl
group of methyl-coenzyme M is subsequently reduced by H-S-CoB to CH4 , thereby forming CoMS-S-CoB. The CO bound to CO dehydrogenase/acetyl-CoA synthase is oxidized to CO2 , and the reducing equivalents are used for the reduction of CoM-SS-CoB. A ferredoxin has been shown to be the direct
electron acceptor of CO dehydrogenase/acetyl CoA
synthase (Fig. 5) (for a recent review see [9]). It is not
yet known how the electrons are transferred from the
ferredoxin to CoM-S-S-CoB. Based on studies with
whole cells [129,130] and cell extracts [131,132], H2
has been proposed to be an intermediate of this electron transfer reaction. Cell suspensions of M. barkeri
catalyze the conversion of external CO to CO2 and
H2 when methane formation is inhibited [133,134].
CO conversion to CO2 and H2 is coupled with the
generation of a proton motive force [133,134]. However, the molecular basis for the generation of H2 is
not known. Recently a novel hydrogenase was puri¢ed and characterized from acetate-grown cells of
M. barkeri, which could catalyze H2 formation via
this metabolic pathway [135]. The hydrogenase was
designated Ech (E for E. coli, c for the third (c)
hydrogenase, and h for hydrogenase) because its
properties are similar to those of the E. coli hydrogenase 3. The M. barkeri enzyme is an integral membrane protein composed of six di¡erent subunits. In
Northern blot experiments, the transcript of the ech
operon was detected in cells of M. barkeri grown
369
with acetate, methanol, or H2 /CO2 . The enzyme
shares the highest sequence similarity with the COinduced hydrogenase from Rhodospirillum rubrum
[136] and also has signi¢cant sequence similarity to
the Escherichia coli hydrogenases 3 and 4 [137,138].
R. rubrum can grow in the dark on CO as sole energy
source, forming H2 and CO2 . This reaction is
coupled with the formation of a proton motive force
in this organism. Since the CO dehydrogenase in R.
rubrum is a soluble enzyme, the membrane-bound
CO-induced hydrogenase is most likely the site of
energy conservation [136].
CO ‡ H2 O ! CO2 ‡ H2
vG 0 ˆ 320 kJ=mol:
…i†
Likewise, in the acetate metabolism of Methanosarcina, bound CO, generated via decarbonylation of
acetyl-CoA, might be converted to CO2 and H2 , catalyzed by CO dehydrogenase/acetyl-CoA synthase
and Ech hydrogenase. If H2 is an intermediate of
this electron transport chain, a second membranebound hydrogenase (an H2 uptake hydrogenase)
must be present in acetate-grown cells that together
with heterodisul¢de reductase catalyzes CoM-S-SCoB reduction by H2 . This is indeed the case. Acetate-grown cells of Methanosarcina species synthesize
the same membrane-bound hydrogenase as H2 /CO2 grown cells (VhoGAC in M. mazei) [118,121]. Thus,
the reduction of CoM-S-S-CoB by H2 in acetate metabolism could involve the same electron transport
chain as in H2 /CO2 metabolism. In summary, an
`intraspecies' hydrogen cycling is proposed which includes two di¡erent coupling sites for energy conservation: (i) the site for the conversion of bound CO
to CO2 and H2 and (ii) the site for the reduction of
CoM-S-S-CoB by H2 (Fig. 7).
Alternatively, Methanosarcina species could contain an electron transport chain that directly channels electrons from CO dehydrogenase/acetyl-CoA
synthase via a ferredoxin to heterodisul¢de reductase. Such a CO:heterodisul¢de oxidoreductase activity has been reconstituted with puri¢ed CO dehydrogenase/acetyl-CoA synthase, ferredoxin, washed
membranes, and partially puri¢ed heterodisul¢de reductase [140,141]. Since this in vitro system still contains the membrane fraction and thus both mem-
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
370
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
Fig. 7. Scheme of methanogenesis from acetate in Methanosarcina species. Recent data indicate that the 2 [4Fe-4S] ferredoxin (Fd) from
M. barkeri mediates electron transfer between CO dehydrogenase/acetyl-CoA synthase and Ech hydrogenase [139]. CH3 -H4 MPT, methyltetrahydromethanopterin ; MPox , methanophenazine in the oxidized form; MPred , methanophenazine in the reduced form ; Hdr, heterodisul¢de reductase ; Vho, viologen-reactive hydrogenase one.
brane-bound hydrogenases (VhoGAC and Ech), it
cannot be excluded that H2 is an intermediate in
this system.
7.2. Disul¢de respiration in Methanobacteriales,
Methanococcales, Methanopyrales, and
Methanomicrobiales
Most of the organisms belonging to these phylogenetic groups are restricted to H2 /CO2 as energy
substrates. These organisms do not contain cytochromes and thus b-type cytochromes can be excluded as membrane anchors and electron carriers
of membrane-bound dehydrogenases and reductases.
Reduction of CoM-S-S-CoB has been investigated
mainly with Methanobacterium thermoautotrophicum,
which belongs to the order Methanobacteriales.
Upon puri¢cation, heterodisul¢de reductase of this
organism was obtained in a tight complex with one
of the [Ni-Fe] hydrogenases, the so-called F420 -nonreducing hydrogenase [142,143]. This complex catalyzes the reduction of CoM-S-S-CoB with H2 at signi¢cant rates. At alkaline pH, the complex can be
dissociated into the two individual enzymes, heterodisul¢de reductase and hydrogenase. Heterodisul¢de
reductase is composed of three di¡erent subunits ^
HdrA, -B, and -C ^ encoded by the two separate
transcriptional units hdrA and hdrCB. The enzyme
contains FAD and iron-sulfur clusters. HdrA contains an FAD binding motif and four binding motifs
for [4Fe-4S] clusters. HdrC contains two binding
motifs for [4Fe-4S] clusters [144].
The F420 -non-reducing hydrogenase is also composed of three di¡erent subunits: a hydrogenase
large subunit containing the binuclear Ni-Fe active
site, a hydrogenase small subunit containing three
iron-sulfur clusters, and an additional small subunit
with unknown function. The operon encoding the
three subunits of this hydrogenase (mvhDGA, methylviologen-reducing hydrogenase) contains an additional open reading frame (mvhB), which encodes a
polyferredoxin [145]. The polyferredoxin has been
puri¢ed from M. thermoautotrophicum as an individual protein [146^148]. It is present in small amounts
in the puri¢ed H2 :heterodisul¢de oxidoreductase
complex, but a function as electron carrier in this
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
complex has not been clearly shown. After cell lysis,
the H2 :heterodisul¢de oxidoreductase complex is
present in the soluble fraction of M. thermoautotrophicum. The complex contains only hydrophilic polypeptides, as indicated by the deduced amino acid
sequence of the proteins. The three transcriptional
units encoding the di¡erent subunits of the complex
do not contain additional open reading frames encoding hydrophobic proteins, which in theory could
have been separated from the catalytic subunits during the puri¢cation. Hence, the major question is
how this non-integral membrane protein complex
can couple the reduction of CoM-S-S-CoB by H2
with the generation of the proton motive force. At
present there is no conclusive answer to this question, and the following ¢ndings should be considered.
Coupling of methanogenesis with ADP phosphorylation is not constant. During growth of methanogens on H2 /CO2 , the growth yield per mol CH4 increases with decreasing H2 concentrations [149,150]
indicating that at low H2 concentrations, energy coupling is tighter than at high H2 concentrations.
Hence, at di¡erent H2 concentrations, di¡erent electron transport chains could be involved in the reduction of CoM-S-S-CoB. The genome of M. thermoautotrophicum contains no additional gene cluster
encoding a second heterodisul¢de reductase [151].
The genome contains, however, two gene clusters
that presumably encode two additional hydrogenases, which have not yet been identi¢ed at the protein level [135,151]. The genes encoding the large and
small subunits of these postulated hydrogenases are
closely linked to genes encoding iron-sulfur proteins
and integral membrane proteins. Both the hydrophilic and the hydrophobic subunits of these putative
hydrogenases show signi¢cant sequence similarity to
subunits of the energy-conserving NADH:quinone
oxidoreductase from various organisms [152]. Similar
gene clusters are present in the genome of M. jannaschii [153]. These putative enzymes are interesting
candidates for proton pumps. It may be speculated
that under certain physiological conditions, such as
low H2 concentration, one of these hydrogenases interacts with heterodisul¢de reductase to couple the
reduction of CoM-S-S-CoB by H2 with the formation of a proton motive force. At high H2 concen-
371
trations, reduction of CoM-S-S-CoB might not be
coupled with energy conservation and might be catalyzed by the soluble H2 :heterodisul¢de oxidoreductase complex described above. This `uncoupling'
might allow a higher £ux through the metabolic
pathway and could compensate the lower energy
yield (see [154]).
There is only limited information about the
H2 :heterodisul¢de oxidoreductase reaction from organisms belonging to the orders Methanococcales,
Methanopyrales, and Methanomicrobiales. Heterodisul¢de reductase activity has been detected in organisms belonging to these phylogenetic groups [155].
As in M. thermoautotrophicum, most of the activity
is located in the soluble fraction.
The genome of Methanococcus jannaschii contains
two copies of hdrCB and one copy of hdrA [153]. No
data have been obtained with puri¢ed enzymes from
this organism. Heterodisul¢de reductase has been
puri¢ed from Methanopyros kandleri (R. Hedderich,
unpublished results). The enzyme has a subunit composition similar to that of heterodisul¢de reductase
from M. thermoautotrophicum. The N-terminal amino acid sequence of the 35-kDa subunit is highly
similar to that of HdrB from M. thermoautotrophicum. The gene encoding the subunit HdrA has been
cloned and sequenced, and the deduced amino acid
sequence shares high sequence similarity with HdrA
from M. thermoautotrophicum [115]. Hence, heterodisul¢de reductase in this organism seems to be quite
similar to the enzyme from M. thermoautotrophicum.
It is interesting to note that the sulfate-reducing archaeon A. fulgidus contains homologues of the genes
hdrA, hdrB, hdrC, mvhD, mvhG, and mvhA in a putative transcriptional unit hdrACBmvhDGA (genes
AF1377^AF1372) [127]. This ¢nding supports the
biochemical data obtained with the H2 :heterodisul¢de oxidoreductase complex from M. thermoautotrophicum that indicate that heterodisul¢de reductase
and methylviologen-reducing hydrogenase (Mvh)
form a functional complex.
An F420 -non-reducing hydrogenase, similar to the
M. thermoautotrophicum enzyme, is also present in
the Methanococcales. The enzyme from Methanococcus voltae has been characterized in detail [156]. The
enzyme from M. voltae does not form a tight complex with heterodisul¢de reductase in vitro.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
372
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
7.3. Other heterodisul¢de-generating reactions
Methyl-coenzyme M reduction with coenzyme B is
not the only reaction in which CoM-S-S-CoB is generated. Most methanogens contain a soluble fumarate reductase, which catalyzes the reduction of fumarate with H-S-CoM and H-S-CoB to succinate
and CoM-S-S-CoB [157,158].
Fumarate ‡ H-S-CoM ‡ H-S-CoB
! succinate ‡ CoM-S-S-CoB:
…j†
This reaction is part of a biosynthetic pathway for
the biosynthesis of 2-oxoglutarate. Since this anabolic reaction generates CoM-S-S-CoB, it also has
a link to energy conservation.
The reaction is catalyzed by thiol:fumarate reductase (Tfr). The enzyme is composed of two di¡erent
subunits, TfrA and TfrB [157,158]. TfrA contains
FAD and has high sequence similarity to the catalytic subunit of fumarate reductases and succinate
dehydrogenases. TfrB contains three binding motifs
for di¡erent Fe/S clusters and shows sequence similarity to the subunit HdrD of the M. barkeri heterodisul¢de reductase and to the subunits HdrC and
HdrB of the M. thermoautotrophicum heterodisul¢de
reductase. It is reasonable to assume that the subunit
TfrA harbors the catalytic site for fumarate reduction and TfrB the catalytic site for thiol oxidation.
barkeri is not present in M. thermoautotrophicum.
Instead, the M. thermoautotrophicum enzyme contains the FAD-containing subunit HdrA.
Since HdrD, HdrCB, and TfrB are conserved between both heterodisul¢de reductases and thiol:fumarate reductase, it is assumed that these polypeptides harbor the catalytic site for the reduction of the
disul¢de substrate. The heme-containing subunit
HdrE of the M. barkeri heterodisul¢de reductase is
clearly involved in electron transfer. The function of
the subunit HdrA of the M. thermoautotrophicum
enzyme is not known. Until recently it was thought
to harbor the catalytic site for the reduction of the
disul¢de substrate [144], but when the sequence of
the M. barkeri heterodisul¢de reductase became
available, it was obvious that subunit HdrA or a
related protein is not part of this enzyme. Therefore,
it is assumed that HdrA has a speci¢c function in
electron transfer in M. thermoautotrophicum heterodisul¢de reductase and does not harbor the catalytic
site for the reduction of the disul¢de substrate.
The proposed catalytic subunits HdrD and HdrCB
do not show any sequence similarity to other characterized disul¢de reductases. The M. barkeri enzyme and the proposed catalytic subunits HdrCB
7.4. Heterodisul¢de reductase ^ mechanistic
considerations
Heterodisul¢de reductase from M. barkeri and
heterodisul¢de reductase from M. thermoautotrophicum di¡er signi¢cantly in their subunit composition
and cofactor content. However, a sequence comparison of the enzymes indicates that they have homologous subunits. Subunit HdrD of the M. barkeri
enzyme is a homologue of a fusion protein consisting
of the M. thermoautotrophicum HdrC and HdrB subunits [115]. The N-terminal part of HdrD, which
contains two binding motifs for [4Fe-4S] clusters, is
similar to HdrC, while the C-terminal part of HdrD
is similar to HdrB. The subunit TfrB of thiol:fumarate reductase is highly similar to HdrD and HdrCB
(Fig. 8) [158]. The b-type cytochrome HdrE of M.
Fig. 8. Schematic alignment of heterodisul¢de reductase from M.
barkeri (Mb Hdr), heterodisul¢de reductase from M. thermoautotrophicum (Mt Hdr), and thiol :fumarate reductase from M. thermoautotrophicum (Mt Tfr). The subunits HdrD, HdrCB, and
TfrB, which show a high degree of sequence similarity, are
shown in blue. In addition to the 8 cysteine residues that ligate
the two [4Fe-4S] clusters, these subunits contain 10 conserved
cysteine residues (10 C) which might ligate an additional Fe/S
cluster and might form a redox-active disul¢de. The subunits
HdrE, HdrA, and TfrA have no sequence similarity and have
di¡erent functions in the di¡erent enzymes.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
of the M. thermoautotrophicum enzyme do not contain a £avin, but only Fe/S centers as prosthetic
groups. Hence, a £avin is not the direct electron
donor for the reduction of the disul¢de. This has
important mechanistic consequences. The central
problem that needs to be addressed in heterodisul¢de
reduction is how a one-electron donor, an iron-sulfur
cluster, can carry out a concerted reaction involving
reductive cleavage of the disul¢de substrate. A oneelectron-reduced intermediate seems likely to occur
in this catalytic cycle. Oxidation of the enzyme with
its substrate CoM-S-S-CoB induces an EPR spectrum not common to known iron-sulfur clusters
(Gxyz = 2.02, 2.00, 1.95; T = 6 50 K). Redox titrations indicate a midpoint potential of 3250 mV for
this paramagnetic center (R. Hedderich, S.P.J. Albracht, M.K. Johnson and E. Duin, unpublished results). The nature of this unusual paramagnetic center, which might represent an important intermediate
of the catalytic cycle, is currently under investigation.
HdrD, HdrCB, and TfrB contain two highly conserved binding motifs for [4Fe-4S] clusters and ten
additional conserved cysteine residues [158]. These
cysteine residues could ligate an additional iron-sulfur center and could form a redox-active disul¢de.
The extra iron-sulfur cluster and the redox-active
disul¢de could form the active site of the enzyme.
There is only one other disul¢de reductase known
to pose the same mechanistic problem as heterodisul¢de reductase, ferredoxin-thioredoxin reductase.
This enzyme is found in plants and cyanobacteria,
and catalyzes the reduction of thioredoxin with reduced ferredoxin, an iron-sulfur protein, as electron
donor. The active site of this enzyme contains a
[4Fe-4S] cluster and a redox-active disul¢de. Based
on spectroscopic data, a thiyl radical stabilized by an
iron-sulfur cluster has been postulated as an intermediate of the catalytic cycle for this enzyme
[159,160].
Although ferredoxin:thioredoxin reductase and
heterodisul¢de reductase do not share any sequence
similarity, a similar catalytic mechanism might operate in the enzymes. Their catalytic mechanism clearly
di¡ers from that of the enzymes belonging to the
family of pyridine nucleotide disul¢de oxidoreductases, such as glutathione reductase, NADPH-dependent thioredoxin reductase, and dihydrolipoamide dehydrogenase. In these enzymes, FAD
mediates a two-electron/hydride transfer
NAD(P)H to an active site disul¢de [161].
373
from
7.5. Heterodisul¢de-reductase-related proteins in
non-methanogens
In the DNA and protein databases, there is an
emerging group of proteins from non-methanogenic
organisms with high sequence similarity to HdrD,
HdrCB, and TfrB. This group includes proteins
from several bacteria and archaea (Table 7). The
genome of A. fulgidus, for example, contains 11 different genes coding for proteins with a high sequence
similarity to the proposed catalytic subunit of heterodisul¢de reductase [127]. A function has not been
assigned to any of these heterodisul¢de-reductase-related proteins from non-methanogens. The high sequence similarity to heterodisul¢de reductase indicates a role in disul¢de reduction or thiol
oxidation. It is therefore assumed that these enzymes
together with heterodisul¢de reductase form a family
of disul¢de oxidoreductases distinct from the enzymes belonging to the well-characterized family of
pyridine nucleotide disul¢de oxidoreductases and ferredoxin:thioredoxin reductase.
In methanogens, the disul¢de used as terminal
electron acceptor of the respiratory chain is not an
external substrate, but is generated in H2 /CO2 , methanol, or acetate metabolism (Fig. 5). Likewise, disul¢des could be generated in the energy metabolism of
other organisms and function as electron acceptor of
the respiratory chain. The presence of genes encoding proteins related to heterodisul¢de reductase in
several non-methanogenic organisms supports this
hypothesis. Many of these genes are closely linked
to genes encoding integral membrane proteins, such
as b-type cytochromes. This further supports the hypothesis of an involvement of these enzymes in respiration.
8. Conclusions
The respiratory systems described di¡er signi¢cantly in the properties of their terminal reductases.
Polysul¢de reductase, the key enzyme of sulfur respiration in the bacterium W. succinogenes, contains
molybdenum bound to a molybdopterin dinucleotide
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
374
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
Table 7
Gene products with signi¢cant sequence similarity to the postulated catalytic subunit HdrD of the M. barkeri heterodisul¢de reductase,
the postulated catalytic subunits HdrCB of the M. thermoautotrophicum heterodisul¢de reductase, and the subunit TfrB of thiol :fumarate
reductase
Gene
Organism
Comment/putative transcriptional unit
Sequence
identity to
Reference
AF 0506
Archaeoglobus fulgidus
^
[127]
AF 1773
Archaeoglobus fulgidus
^
AF 0755
Archaeoglobus fulgidus
Homologue of an hdrED fusion
AF 1998
Archaeoglobus fulgidus
^
AF 0547
Archaeoglobus fulgidus
AF 0867
Archaeoglobus fulgidus
Putative operon with a gene encoding
a b-type cytochrome
^
AF 0502
Archaeoglobus fulgidus
AF 0543
Archaeoglobus fulgidus
Putative operon with genes encoding Fe/S proteins
and b-type cytochromes
Most similar to hmc6 from D. vulgaris
AF 0544
Archaeoglobus fulgidus
Most similar to hmc6 from D. vulgaris
AF 1375
Archaeoglobus fulgidus
hdrABCmvhDGA operon
AF 0271
Archaeoglobus fulgidus
Similar to C-terminal part of HdrB
ywjF
Bacillus subtilis
MTCY 279.05c
hdrD
Mycobacterium
tuberculosis
Aquifex aeolicus
Putative operon with acdA encoding acyl-CoA
dehydrogenase
Similar to ywjF from B. subtilis
hdrB
Aquifex aeolicus
hdrABC operon
isp2
Thiocapsa roseopersicina
Hydrogenase operon
sdhC
Sulfolobus acidocaldarius
Succinate dehydrogenase operon
hdrB
Synechocystis sp.
^
hmc6
Desulfovibrio vulgaris
dsrK
Chromatium vinosum
hmc operon; encodes a multisubunit membrane
complex
dsr locus encoding sul¢te reductase
glpC
Escherichia coli
glcF
Escherichia coli
Subunit of anaerobic glycerol-3-phosphate
dehydrogenase
Subunit of glycolate oxidase
ysfD
Bacillus subtilis
Subunit of glycolate oxidase
glcF
Synechosystis sp.
Subunit of glycolate oxidase
HdrD (34%
from 385 aa)
HdrD (30%
from 390 aa)
HdrD (31%
from 380 aa)
HdrD (29%
from 305 aa)
HdrD (23%
from 431 aa)
HdrD (26%
from 220 aa)
HdrD (32%
from 149 aa)
HdrD (20%
from 354 aa)
HdrD (20%
from 338 aa)
HdrB (30%
from 300 aa)
HdrB (32%
from 128 aa)
HdrD (28%
from 390 aa)
HdrD (28%
from 288 aa)
HdrD (25%
from 409 aa)
HdrB (30%
from 300 aa)
HdrD (23%
from 398 aa)
HdrB (31%
(from 277 aa)
HdrB (33%
from 300 aa)
HdrD (33%
from 103 aa)
HdrD (23%
from 437 aa)
HdrD (27%
from 251 aa)
HdrD (23%
from 370 aa)
HdrD (22%
from 390 aa)
HdrD (22%
from 204 aa)
Hydrogenase operon
[127]
[127]
[127]
[127]
[127]
[127]
[127]
[127]
[127]
[127]
[162]
[163]
[164]
[164]
[165]
[166]
[167]
[168]
[169]
[170]
[171]
[162]
[167]
Note that most of the genes were identi¢ed from genome sequencing projects and that the function of these genes and their gene products is
unknown.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
cofactor, and the catalytic subunit is related to enzymes of the family of molybdopterin-dinucleotidecontaining oxidoreductases. Heterodisul¢de reductase of methanogenic archaea and the H2 :sulfur oxidoreductase from the hyperthermophilic archaeon
Pyrodictium abyssi isolate TAG11 do not contain
such a prosthetic group. Hence, the catalytic mechanism for disul¢de or sulfur reduction in these organisms should be di¡erent. It is presently not
known whether heterodisul¢de reductase and sulfur
reductase from P. abyssi are related enzymes. The
elucidation of the primary structure of sulfur reductase from P. abyssi might provide an answer to this
question.
In the respiratory chains described, di¡erent electron carriers mediate electron transfer from a dehydrogenase to the terminal reductase. In W. succinogenes a menaquinone tightly bound to polysul¢de
reductase seems to be the direct acceptor of electrons
delivered from hydrogenase or formate dehydrogenase. An unidenti¢ed quinone is postulated as electron
carrier in P. brockii, and the newly discovered methanophenazine is most likely the physiological electron carrier in methanogens of the order Methanosarcinales. Quinones have not been detected in the
membrane fraction of P. abysii. It remains to be
shown whether electron transfer in this organism is
by direct electron transfer from the hydrogenase to
sulfur reductase, which together form a tight complex. Alternatively, an unidenti¢ed electron carrier
might be involved in this process.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgments
This work was supported by grants from the
Deutsche Forschungsgemeinschaft to R.H. and
K.-O.S. (Schwerpunktprogramm: `Neuartige Reaktionen und Katalysemechanismen bei anaeroben
Mikroorganismen') and to A.K. (SFB 472). We
thank K. Brune for editing the manuscript.
References
[15]
[16]
[17]
[1] Moodie, A.D. and Ingledew, W.J. (1990) Microbial anaerobic
respiration. In: Advances in Microbiology and Physiology
[18]
375
(Rose, A.H. and Tempest, D.W., Eds.), pp. 225^269. Academic Press, London.
Fauque, G., LeGall, J. and Barton, L.L. (1991) Sulfate-reducing and sulfur-reducing bacteria. In: Variations in Autotrophic Life (Shively, J.M. and Barton, L.L., Eds.), pp. 271^
337. Academic Press, London.
Schauder, R. and Kroëger, A. (1993) Bacterial sulphur respiration. Arch. Microbiol. 159, 491^497.
Fauque, G.D. (1994) Sulfur reductase from thiophilic sulfatereducing bacteria. Methods Enzymol. 243, 353^367.
Fauque, G.D., Klimmek, O. and Kroëger, A. (1994) Sulfur
reductases from spirilloid mesophilic sulfur-reducing bacteria.
Methods Enzymol. 243, 367^383.
Wolfe, R.S. (1991) My kind of biology. Annu. Rev. Microbiol. 45, 1^35.
Wolfe, R.S. (1993) A historical overview of methanogenesis.
In: Methanogenesis (Ferry, J.G., Ed.), pp. 1^32. Chapman
and Hall, New York, NY.
Deppenmeier, U., Muëller, V. and Gottschalk, G. (1996) Pathways of energy conservation in methanogenic archaea. Arch.
Microbiol. 165, 149^163.
Ferry, J.G. (1997) Enzymology of the fermentation of acetate
to methane by Methanosarcina thermophila. BioFactors 6, 25^
35.
Reeve, J.N., Noëlling, J., Morgan, R.M. and Smith, D.R.
(1997) Methanogenesis: genes, genomes, and who's on ¢rst.
J. Bacteriol. 179, 5975^5986.
Thauer, R.K. (1998) Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiology 144, 2377^2406.
Segerer, A., Neuner, A., Kristjansson, J. and Stetter, K.O.
(1986) Acidianus infernus gen. nov., sp. nov., and Acidianus
brierleyi comb. nov.: facultatively aerobic, extremely acidophilic thermophilic sulfur-metabolizing archaebacteria. Int. J.
Syst. Bacteriol. 36, 559^564.
Segerer, A.H., Trincone, A., Gahrtz, M. and Stetter, K.O.
(1991) Stygiolobus azoricus gen. nov., sp. nov. represents a
novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. Int. J. Syst. Bacteriol. 41,
495^501.
Huber, R., Kristjansson, J.-K. and Stetter, K.O. (1987) Pyrobaculum gen. nov., a new genus of neutrophilic rod-shaped
archaebacteria from continental solfataras growing optimally
at 100³C. Arch. Microbiol. 149, 95^101.
Zillig, W., Gierl, A., Wunder, S., Janekovic, D., Stetter, K.O.
and Klenk, H.P. (1983) The archaebacterium Thermo¢lum
pendens represents a novel genus of the thermophilic, anaerobic sulfur respiring Thermoproteales. Syst. Appl. Microbiol. 4,
79^87.
Stetter, K.O., Segerer, A., Zillig, W., Huber, G., Fiala, G.,
Huber, R. and Koënig, H. (1986) Extremely thermophilic sulfur metabolizing archaebacteria. Syst. Appl. Microbiol. 7,
393^397.
Fischer, F., Zillig, W., Stetter, K.O. and Schreiber, G. (1983)
Chemolithoautotrophic metabolism of anaerobic extremely
thermophilic archaebacteria. Nature 301, 511^513.
Selig, M. and Schoënheit, P. (1994) Oxidation of organic com-
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
376
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
pounds to CO2 with sulfur or thiosulfate as electron acceptor
in the anaerobic extremely hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicun proceeds via
the citric acid cycle. Arch. Microbiol. 162, 286^294.
Bonch-Osmolovskaya, E.A., Slesarev, A.I., Miroshnichenko,
M.L., Svetlichnaya, T.P. and Alekseev, V.A. (1988) Characteristics of Desulfurococcus amylolyticus n. sp. ^ a new extremely thermophilic archaebacterium isolated from thermal
springs of Kamchatka and Kunashir Island. Mikrobiologiya
57, 94^101.
Zillig, W., Stetter, K.O., Prangishvilli, D., Schaëfer, W., Wunderl, S., Janekovic, D., Holz, I. and Palm, P. (1982) Desulfurococcaceae, the second family of the extremely thermophilic,
anaerobic, sulfur-respiring Thermoproteales. Zbl. Bakt. Hyg.
I. Abt. Orig. C3, 304^317.
Stetter, K.O., Koënig, H. and Stackebrandt, E. (1983) Pyrodictium gen. nov., a new genus of submarine disc-shaped sulfurreducing archaebacteria growing optimally at 105³C. Syst.
Appl. Microbiol. 4, 535^551.
Dirmeier, R., Keller, M., Frey, G., Huber, H. and Stetter,
K.O. (1998) Puri¢cation and properties of an extremely thermostable membrane-bound sulfur-reducing complex from hyperthermophilic Pyrodictium abyssi. Eur. J. Biochem. 252,
486^491.
Jochimsen, B., Peinemann-Simon, S., Voëlker, H., Stuëben, D.,
Botz, R., Sto¡ers, P., Dando, P.R. and Thomm, M. (1997)
Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur dependent crenarchaeote isolated from Milos,
Greece. Extremophiles 1, 67^73.
Stetter, K.O. (1986) Diversity of extremely thermophilic
archaebacteria. In: Thermophiles : General, Molecular and
Applied Microbiology (Brock, T.D., Ed.), pp. 39^74, John
Wiley, New York, NY.
Huber, R., Dyba, D., Huber, H., Burggraf, S. and Rachel, R.
(1998) Sulfur-inhibited Thermosphaera aggregans sp. nov., a
new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences.
Int. J. Syst. Bacteriol. 48, 31^38.
Fiala, G., Stetter, K.O., Jannasch, H., Langworthy, T. and
Madon, J. (1986) Staphylothermus marinus sp. nov. Represents a novel genus of extremely thermophilic submarine heterotrophic archaebacteria growing up to 98³C. Syst. Appl.
Microbiol. 8, 106^113.
Zillig, W., Holz, I., Janekovic, D., Klenk, H.-P., Imsel, E.,
Trent, J., Wunderl, S., Forjaz, V.H., Coutinho, R. and Ferreira, T. (1990) Hyperthermus buthylicus, a novel hyperthermophilic sulfur-reducing archaebacterium that ferments peptides. J. Bacteriol. 172, 3959^3965.
Fiala, G. and Stetter, K.O. (1986) Pyrococcus furiosus sp.
nov., represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100³C. Arch. Microbiol. 145,
56^61.
Gonzaèlez, J.M., Masuchi, Y., Robb, F.T., Ammerman, J.W.,
Maeder, D.L., Yanagibayashi, M., Tamaoka, J. and Kato, C.
(1998) Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa
Trough. Extremophiles 2, 123^130.
[30] Dirmeier, R., Keller, M., Hafenbradl, D., Braun, F.-J., Rachel, R., Burggraf, S. and Stetter, K.O. (1998) Thermococcus
acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids. Extremophiles 2,
109^114.
[31] Neuner, A., Jannasch, H., Belkin, S. and Stetter, K.O. (1990)
Thermococcus litoralis sp. nov.: a new species of extremely
thermophilic marine archaebacteria. Arch. Microbiol. 153,
205^207.
[32] Svetlichnyi, V.A., Slesarev, A.I., Svetlichnaya, T.P. and Zavarzin, G.A. (1987) Caldococcus litoralis, gen. nov. sp. nov. ^
a new marine, extremely thermophilic, sulfur-reducing archaebacterium. Mikrobiologiya 56, 831^838.
[33] Segerer, A., Langworthy, T. and Stetter, K.O. (1988) Thermoplasma acidophilum and Thermoplasma volcanicum sp. nov.
from solfatara ¢elds. System. Appl. Microbiol. 10, 161^
171.
[34] Stetter, K.O. and Gaag, G. (1983) Reduction of molecular
sulphur by methanogenic archaea. Nature 305, 309^311.
[35] Huber, R., Wilharm, T., Huber, D., Trincone, A., Burggraf,
S., Koënig, H., Rachel, R., Rockinger, I., Fricke, H. and Stetter, K.O. (1992) Aquifex pyrophilus gen. nov., represents a
novel group of marine hyperthermophilic hydrogen-oxidizing
bacteria. Syst. Appl. Microbiol. 15, 340^351.
[36] Huber, R., Rossnagel, P., Woese, C.R., Rachel, R., Langworthy, T. and Stetter, K.O. (1996) Formation of ammonium
from nitrate during chemolithoautotrophic growth of the extremely thermophilic bacterium Ammonifex degensii gen. nov.
sp. nov. Syst. Appl. Microbiol. 19, 40^49.
[37] L'Haridon, S., Cilia, V., Messner, P., Ragueènteés, G., Gambacorta, A., Sleytr, U.B., Prieur, D. and Jeanthon, C. (1998)
Desulfurobacterium thermolithotrophicum gen. nov., sp. nov.,
a novel autotrophic, sulphur-reducing bacterium isolated from
a deep-sea hydrothermal vent. Int. J. Syst. Bacteriol. 48, 707^
771.
[38] Pfennig, N. and Biebl, H. (1976) Desulfuromonas acetoxidans
gen. nov. and sp. nov., a new anaerobic, sulfur-reducing, acetate oxidizing bacterium. Arch. Microbiol. 110, 3^12.
[39] Liesack, W. and Finster, K. (1994) Phylogenetic analysis of
¢ve strains of Gram-negative, obligately anaerobic, sulfur-reducing bacteria and description of Desulfuromusa gen. nov.,
including Desulfuromusa kysingii sp. nov., Desulfuromusa bakii
sp. nov., and Desulfuromusa succinoxidans sp. nov. Int. J. Syst.
Bacteriol. 44, 753^758.
[40] Bonch-Osmolovskaya, E.A., Sokolova, T.G., Kostrikina,
N.A. and Zavarzin, G.A. (1990) Desulfurella acetivorans gen.
nov. and sp. nov. ^ a new thermophilic sulfur-reducing eubacterium. Arch. Microbiol. 153, 151^155.
[41] Schmitz, R.A., Bonch-Osmolovskaya, E.A. and Thauer, R.K.
(1990) Di¡erent mechanisms of acetate activation in Desulfurella acetivorans and Desulfuromonas acetoxidans. Arch.
Microbiol. 154, 274^279.
[42] Biebl, H. and Pfennig, N. (1977) Growth of sulfate-reducing
bacteria with sulfur as electron acceptor. Arch. Microbiol.
112, 115^117.
[43] Huber, R., Woese, C.R., Langworthy, T., Kristjansson, J. and
Stetter, K.O. (1990) Fervidobacterium islandicum sp. nov., a
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
new extremely thermophilic eubacterium belonging to the
`Thermotogales'. Arch. Microbiol. 154, 105^111.
Patel, B.K., Morgan, H.W. and Daniel, R.M. (1985) Fervidobacterium nodosum gen. nov. and spec. nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium. Arch. Microbiol. 141, 63^69.
Caccavo, F. Jr., Debra, J., Lonergan, D.J., Lovley, D.R.,
Davis, M., Stolz, J.F. and McInerney, M.J. (1994) Geobacter
sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol. 60, 3752^3759.
Lovley, D.R., Phillips, E.J.P., Lonergan, D.J. and Widman,
P.K. (1995) Fe(III) and S0 reduction by Pelobacter carbinolicus. Appl. Environ. Microbiol. 61, 2132^2138.
Moser, D.P. and Nealson, K.H. (1996) Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur
reduction. Appl. Environ. Microbiol. 62, 2100^2105.
Wolfe, R.S. and Pfennig, N. (1977) Reduction of sulfur by
Spirillum 5175 and syntrophism with Chlorobium. Appl. Environ. Microbiol. 33, 427^433.
Finster, K., Liesack, W. and Tindall, B.J. (1997) Sulfurospirillum arcachonense sp. nov., a new microaerophilic sulfurreducing bacterium. Int. J. Syst. Bacteriol. 47, 1212^1217.
Windberger, E., Huber, R., Trincone, A., Fricke, H. and Stetter, K.O. (1989) Thermotoga thermarum sp. nov. and Thermotoga neapolitana occurring in African continental solfataric
springs. Arch. Microbiol. 151, 506^512.
Huber, R., Langworthy, T., Koënig, H., Thomm, M., Woese,
C.R., Sleytr, U.B. and Stetter, K.O. (1986) Thermotoga maritima sp. nov. represents a new genus of unique extremely
thermophilic eubacteria growing up to 90³C. Arch. Microbiol.
144, 324^333.
Huber, R., Woese, C.R., Langworthy, T., Fricke, H. and
Stetter, K.O. (1989) Thermosipho africanus gen. nov., represents a new genus of thermophilic eubacteria within the `Thermotogales'. Syst. Appl. Microbiol. 12, 32^37.
Antoine, E., Cilia, V., Meunier, J.R., Guezennec, J., Lesogeur,
F. and Barbier, G. (1997) Thermosipho melanesiensis sp. nov.,
a new thermophilic anaerobic bacterium belonging to the order Thermotogales, isolated from deep-sea hydrothermal vents
in the Southwestern Paci¢c Ocean. Int. J. Syst. Bacteriol. 47,
1118^1123.
Macy, J.M., Schroëder, I., Thauer, R.K. and Kroëger, A. (1986)
Growth of Wolinella succinogenes on H2 S plus fumarate and
on formate plus sulfur as energy sources. Arch. Microbiol.
144, 147^150.
Stetter, K.O. (1998) Volcanoes, hydrothermal venting, and the
origin of life. In: Volcanoes and the Environment (Marti, J.
and Ernst, G.J., Eds.). Cambridge University Press, in press.
Stetter, K.O., Huber, R., Bloëchl, E., Kurr, M., Eden, R.D.,
Fielder, M., Cash, H. and Vance, I. (1993) Hyperthermophilic
archaea are thriving in deep North Sea and Alaskan oil reservoirs. Nature 365, 743^745.
Stetter, K.O. (1996) Hyperthermophilic procaryotes. FEMS
Microbiol. Rev. 18, 149^158.
Stetter, K.O. (1997) Primitive archaea and bacteria in the
cycles of sulfur and nitrogen near the temperature limit of
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
377
life. In: Progress in Microbial Ecology. Proceedings of Seventh International Symposium on Microbial Ecology, Santos,
Sao Paulo, Brazil, 1995 (Martins, M.T. et al., Eds.), pp. 55^
61. SBM/ICOME, Sao Paulo.
Widdel, F. and Pfennig, N. (1991) The genus Desulfuromonas
and other Gram-negative sulfur-reducing eubacteria. In: The
Prokaryotes (Balows, A., Truëper, H.G., Dwarkin, M., Harder,
W. and Schleifer, K.-H., Eds.), pp. 3379^3389.
Zoëphel, A., Kennedy, M.C., Beinert, Z.H. and Kroneck,
P.M.H. (1988) Investigations on microbial sulfur respiration.
1. Activation and reduction of elemental sulfur in several
strains of eubacteria. Arch. Microbiol. 150, 72^77.
Paulsen, J., Kroëger, A. and Thauer, R.K. (1986) ATP-driven
succinate oxidation in the catabolism of Desulfuromonas acetoxidans. Arch. Microbiol. 144, 78^83.
Bouleègue, J. (1978) Solubility of elemental sulfur in water at
298 K. Phosphorus Sulfur 5, 127^128.
Giggenbach, W. (1972) Optical spectra and equilibrium distribution of polysul¢de ions in aqueous solution at 20³. Inorg.
Chem. 11, 1201^1207.
Klimmek, O., Kroëger, A., Steudel, R. and Holdt, G. (1991)
Growth of Wolinella succinogenes with polysulphide as terminal acceptor of phosphorylative electron transport. Arch. Microbiol. 155, 177^182.
Ellis, A.J. and Giggenbach, W. (1971) Hydrogen sulphide ionization and sulphur hydrolysis in high temperature solution.
Geochim. Cosmochim. Acta 35, 247^260.
Schwarzenbach, G. and Fischer, A. (1960) Die Aciditaët der
Sulfane und die Zusammensetzung waësseriger Polysul¢dloësungen. Helv. Chim. Acta 43, 1365^1388.
Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy
conservation in chemotrophic anaerobic bacteria. Bacteriol.
Rev. 41, 100^180.
Jocelyn, P.C. (1967) The standard redox potential of cysteinecystine from the thiol-disulphide exchange reaction with glutathione and lipoic acid. Eur. J. Biochem. 2, 327^331.
Schnorf, U. (1966) Dissertation Nr 3871, ETH Zuërich.
Schauder, R. and Muëller E. (1993) Polysulphide as a possible
substrate for sulphur-reducing bacteria. Arch. Microbiol. 160,
377^382.
Klimmek, O., Kreis, V., Klein, C., Simon, J., Wittershagen, A.
and Kroëger, A. (1998) The function of the periplasmic Sud
protein in polysul¢de respiration of Wolinella succinogenes.
Eur. J. Biochem. 253, 263^269.
Kra¡t, T., GroM, R. and Kroëger, A. (1995) The function of
Wolinella succinogenes psr genes in electron transport with
polysulphide as the terminal electron acceptor. Eur. J. Biochem. 230, 601^606.
Adams, M.W.W. (1993) Enzymes and proteins from organisms that grow near and above 100³C. Annu. Rev. Microbiol.
47, 627^658.
Ringel, M., GroM, R., Kra¡t, T., Kroëger, A. and Schauder, R.
(1996) Growth of Wolinella succinogenes with elemental sulfur
in the absence of polysul¢de. Arch. Microbiol. 165, 62^
64.
Wloczyk, C., Kroëger, A., Goëbel, T., Holdt, G. and Steudel, R.
(1989) The electrochemical proton potential generated by the
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
378
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
sulphur respiration of Wolinella succinogenes. Arch. Microbiol. 152, 600^605.
Bronder, M., Mell, H., Stupperich, E. and Kroëger, A. (1982)
Biosynthetic pathways of Vibrio succinogenes growing with
fumarate as terminal electron acceptor and sole carbon
source. Arch. Microbiol. 131, 216^223.
Mell, H., Bronder, M. and Kroëger A. (1982) Cell yields of
Vibrio succinogenes growing with formate and fumarate as
sole carbon and energy sources in chemostat culture. Arch.
Microbiol. 131, 224^228.
Kroëger, A. and Winkler, E. (1981) Phosphorylative fumarate
reduction in Vibrio succinogenes: Stoichiometry of ATP synthesis. Arch. Microbiol. 129, 100^104.
Brune, A., Spillecke, J. and Kroëger, A. (1987) Correlation of
the turnover number of the ATP synthase in liposomes with
the proton £ux and the proton potential across the membrane.
Biochim. Biophys. Acta 893, 499^507.
Schroëder, I., Kroëger, A. and Macy, J.M. (1988) Isolation of
the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes. Arch. Microbiol. 149, 572^
579.
Kra¡t, T., Bokranz, M., Klimmek, O., Schroëder, I., Fahrenholz, F., Kojro, E. and Kroëger, A. (1992) Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase. Eur. J. Biochem. 206, 503^510.
Jankielewicz, A., Schmitz, R.A., Klimmek, O. and Kroëger, A.
(1994) Polysulphide reductase and formate dehydrogenase
from Wolinella succinogenes contain molybdopterin guanine
dinucleotide. Arch. Microbiol. 162, 238^242.
Klimmek, O. (1996) Dissertation, FB Biologie, University of
Frankfurt.
DroM, F., Geisler, V., Lenger, R., Theis, F., Kra¡t, T., Fahrenholz, F., Kojro, E., Ducheêne, A., Tripier, D., Juvenal, K.
and Kroëger, A. (1992) The quinone-reactive Ni/Fe-hydrogenase of Wolinella succinogenes. Eur. J. Biochem. 206, 93^102.
Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K.V. and
Rees, D.C. (1996) Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science
272, 1615^1621.
Boyington, J.C., Gladyshev, V.N., Khangulov, S.V., Stadtman, T.C. and Sun, P.D. (1997) Crystal structure of formate
dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster. Science 275, 1305^1308.
GroM, R., Simon, J., Theis, F. and Kroëger, A. (1998) Two
membrane anchors of Wolinella succinogenes hydrogenase
and their function in fumarate and polysul¢de respiration.
Arch. Microbiol. 170, 50^58.
Kroëger, A. and Unden, G. (1985) The function of menaquinone in bacterial electron transport. In: Coenzyme Q (Lenaz,
G., Ed.), pp. 285^300. John Wiley, Chichester.
Kroëger, A., Geisler, V., Lemma, E., Theis, F. and Lenger, R.
(1992) Bacterial fumarate respiration. Arch. Microbiol. 158,
311^314.
Jankielewicz, A., Klimmek, O. and Kroëger, A. (1995) The
electron transfer from hydrogenase and formate dehydrogenase to polysul¢de reductase in the membrane of Wolinella
succinogenes. Biochim. Biophys. Acta 1231, 157^162.
[91] Hardt, S.L. (1979) Rates of di¡usion controlled reactions in
one, two and three dimensions. Biophys. Chem. 10, 239^243.
[92] Chazotte, B. and Hackenbrock, C.R. (1988) The multicollisional, obstructed, long-range di¡usional nature of mitochondrial electron transport. Biol. Chem. 28, 14359^14367.
[93] Unden, G. and Kroëger, A. (1986) Reconstitution of a functional electron transport chain from puri¢ed formate dehydrogenase and fumarate reductase complex. Methods Enzymol. 126, 387^399.
[94] GroM, R., Simon, J., Lancaster, C.R.D. and Kroëger, A.
(1998) Identi¢cation of histidine residues in Wolinella succinogenes hydrogenase that are essential for menaquinone reduction by H2 . Mol. Microbiol., 30, 639^646.
[95] Berks, B.C., Dudley Page, M., Richardson, D.J., Reilly, A.,
Cavill, A., Outen, F. and Ferguson, S.J. (1995) Sequence
analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium : the integral membrane
subunit provides a prototype for the dihaem electron-carrying arm of a redox loop. Mol. Microbiol. 15, 319^331.
[96] Lorenzen, J.P., Kroëger, A. and Unden, G. (1993) Regulation
of anaerobic respiratory pathways in Wolinella succinogenes
by the presence of electron acceptors. Arch. Microbiol. 159,
477^483.
[97] Hungate, R.E. (1966) The Rumen and its Microbes. Academic Press, London.
[98] Kreis-Kleinschmidt, V., Fahrenholz, F., Kojro, E. and
Kroëger, A. (1995) Periplasmic sulphide dehydrogenase
(Sud) from Wolinella succinogenes: Isolation, nucleotide sequence of the sud gene and its expression in Escherichia coli.
Eur. J. Biochem. 227, 137^142.
[99] Geisler, V., Ullmann, R. and Kroëger, A. (1994) The direction
of the proton exchange associated with the redox reactions of
menaquinone during the electron transport in Wolinella succinogenes. Biochim. Biophys. Acta 1184, 219^226.
[100] Ma, K., Schicho, R.N., Kelly, R.M. and Adams, M.W.W.
(1993) Hydrogenase of the hyperthermophile Pyrococcus
furiosus is an elemental sulfur reductase or sulfhydrogenase:
evidence for a sulfur-reducing hydrogenase ancestor. Proc.
Natl. Acad. Sci. USA 90, 5341^5344.
[101] Ma, K. and Adams, M.W. (1994) Sul¢de dehydrogenase
from the hyperthermophilic archaeon Pyrococcus furiosus:
a new multifunctional enzyme involved in the reduction of
elemental sulfur. J. Bacteriol. 176, 6509^6517.
[102] Schoënheit, P. and Schaëfer, T. (1995) Metabolism of hyperthermophiles. W. J. Microbiol. Biotech. 11, 26^57.
[103] Means, J.L. and Hinchee, R.E. (1994) Emerging Technology
for the Bioremediation of Metals. CRC Press, Boca Raton,
FL.
[104] Stetter, K.O., Fiala, G., Huber, G., Huber, R. and Segerer,
A. (1990) Hyperthermophilic organisms. FEMS Microbiol.
Rev. 75, 117^124.
[105] Maier, R.J. (1996) Respiratory metabolism in hyperthermophilic organisms : hydrogenases, sulfur reductases, and electron transport factors that function at temperatures exceeding 100³C. Adv. Prot. Chem. 48, 35^73.
[106] Pihl, T.D. and Maier, R.J. (1991) Puri¢cation and characterization of the hydrogen uptake hydrogenase from the hyper-
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
[117]
[118]
[119]
thermophilic archaebacterium Pyrodictium brockii. J. Bacteriol. 173, 1839^1844.
Pihl, T.D., Black, L.K., Schulman, B.A. and Maier, R.J.
(1992) Hydrogen-oxidizing electron transport components
in the hyperthermophilic archaebacterium Pyrodictium brockii. J. Bacteriol. 174, 137^143.
Peinemann, S., Hedderich, R., Blaut, M., Thauer, R.K. and
Gottschalk, G. (1990) ATP synthesis coupled to electron
transfer from H2 to the heterodisul¢de of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate in
vesicle preparations of the methanogenic bacterium strain
Goë1. FEBS Lett. 263, 57^60.
Deppenmeier, U., Blaut, M., Mahlmann, A. and Gottschalk,
G. (1990) Reduced coenzyme F420 :heterodisul¢de oxidoreductase, a proton-translocating redox system in methanogenic bacteria. Proc. Natl. Acad. Sci. USA 87, 9449^9453.
Deppenmeier, U., Blaut, M. and Gottschalk, G. (1991)
H2 :heterodisul¢de oxidoreductase, a second energy-conserving system in the methanogenic strain Goë 1. Arch. Microbiol. 155, 272^277.
Abken, H.-J., Tietze, M., Brodersen, J., Baëumer, S., Beifuss,
U. and Deppenmeier, U. (1998) Isolation and characterization of methanophenazine and function of phenazines in
membrane-bound electron transport of Methanosarcina mazei Goë1. J. Bacteriol. 180, 2027^2032.
Boone, D.R., Whitman, W.B. and Rouvieére, P. (1993) Diversity and taxonomy of methanogens. In: Methanogenesis
(Ferry, J.G., Ed.), pp. 35^80. Chapman and Hall, New
York, NY.
Heiden, S., Hedderich, R., Setzke, E. and Thauer, R.K.
(1993) Puri¢cation of a cytochrome b containing
H2 :heterodisul¢de oxidoreductase complex from membranes
of Methanosarcina barkeri. Eur. J. Biochem. 213, 529^535.
Heiden, S., Hedderich, R., Setzke, E. and Thauer, R.K.
(1994) Puri¢cation of a two-subunit cytochrome-b-containing
heterodisul¢de reductase from methanol-grown Methanosarcina barkeri. Eur. J. Biochem. 221, 855^861.
Kuënkel, A., Vaupel, M., Heim, S., Thauer, R.K. and Hedderich, R. (1997) Heterodisul¢de reductase from methanol
grown cells of Methanosarcina barkeri is not a £avoenzyme.
Eur. J. Biochem. 244, 226^234.
Simianu, M., Murakami, E., Brewer, J.M. and Ragsdale,
S.W. (1998) Puri¢cation and properties of the heme- and
iron-sulfur-containing heterodisul¢de reductase from Methanosarcina thermophila. Biochemistry 37, 10027^10039.
Deppenmeier, U., Blaut, M., Schmidt, B. and Gottschalk, G.
(1992) Puri¢cation and properties of F420 -nonreactive membrane bound hydrogenase from Methanosarcina mazei strain
Goë1. Arch. Microbiol. 157, 505^511.
Kemner, J.M. and Zeikus, J.G. (1994) Puri¢cation and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS. Arch. Microbiol. 161, 47^54.
Deppenmeier, U., Blaut, M., Lentes, S., Herzberg, C. and
Gottschalk, G. (1995) Analysis of the vhoGAC and vhtGAC
operons from Methanosarcina mazei strain Goë1, both encoding a membrane-bound hydrogenase and a cytochrome b.
Eur. J. Biochem. 227, 261^269.
379
[120] Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E.C.,
Frey, M. and Fontecilla-Camps, J.C. (1995) Crystal structure
of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373, 580^587.
[121] Deppenmeier, U. (1995) Di¡erent structure and expression of
the operons encoding the membrane-bound hydrogenases
from Methanosarcina mazei Goë1. Arch. Microbiol. 164,
370^376.
[122] Haase, P., Deppenmeier, U., Blaut, M. and Gottschalk, G.
(1992) Puri¢cation and characterization of F420 H2 -dehydrogenase from Methanolobus tindarius. Eur. J. Biochem. 203,
527^531.
[123] Abken, H.-J. and Deppenmeier, U. (1997) Puri¢cation and
properties of an F420 H2 dehydrogenase from Methanosarcina
mazei Goë1. FEMS Microbiol. Lett. 154, 231^237.
[124] Abken, H.-J., Baëumer, S., Broderson, J., Murakami, E.,
Ragsdale, S.W., Gottschalk, G. and Deppenmeier, U.
(1998) Membrane-bound electron transport and H‡ -translocation in Methanosarcina mazei Goë1. BIOspectrum Sonderausgabe, 38.
[125] Baëumer, S., Murakami, E., Brodersen, J., Gottschalk, G.,
Ragsdale, S.W. and Deppenmeier, U. (1998) The
F420 H2 :heterodisul¢de oxidoreductase system from Methanosarcina species. 2-Hydroxyphenazine mediates electron transfer from F420 H2 dehydrogenase to heterodisul¢de reductase.
FEBS Lett. 428, 295^298.
[126] Kunow, J., Linder, D., Stetter, K.O. and Thauer, R.K.
(1994) F420 H2 :quinone oxidoreductase from Archaeoglobus
fulgidus: Characterization of a membrane-bound multisubunit complex containing FAD and iron-sulfur clusters. Eur.
J. Biochem. 223, 503^511.
[127] Klenk, H.P., Clayton, R.A., Tomb, J.F., White, O., Nelson,
K.E., Ketchum, K.A., Dodson, R.J., Gwinn, M., Hickey,
E.K., Peterson, J.D., Richardson, D.L., Kerlavage, A.R.,
Graham, D.E., Kyrpides, N.C., Fleischmann, R.D., Quackenbush, J., Lee, N.H., Sutton, G.G., Gill, S., Kirkness, E.F.,
Dougherty, B.A., McKenney, K., Adams, M.D., Loftus, B.
and Venter, J.C. (1997) The complete genome sequence of
the hyperthermophilic sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390, 364^370.
[128] Deppenmeier, U., Blaut, M., Mahlmann, A. and Gottschalk,
G. (1990). Membrane-bound F420 H2 -dependent heterodisul¢de reductase in methanogenic bacterium strain Goë1 and
Methanolobus tindarius. FEBS Lett. 261, 199^203.
[129] Lovley, D.R. and Ferry, J.G. (1985) Production and consumption of H2 during growth of Methanosarcina spp. on
acetate. Appl. Environ. Microbiol. 49, 247^249.
[130] Krzycki, J.A., Morgan, J.B., Conrad, R. and Zeikus, J.G.
(1987) Hydrogen metabolism during methanogenesis from
acetate by Methanosarcina barkeri. FEMS Microbiol. Lett.
40, 193^198.
[131] Terlesky, K.C. and Ferry, J.G. (1988) Ferredoxin requirement for electron transport from the carbon monoxide dehydrogenase complex to a membrane-bound hydrogenase in
acetate-grown Methanosarcina thermophila. J. Bacteriol. 263,
4075^4079.
[132] Fischer, R. and Thauer, R.K. (1990) Ferredoxin-dependent
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
380
[133]
[134]
[135]
[136]
[137]
[138]
[139]
[140]
[141]
[142]
[143]
[144]
[145]
[146]
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS). FEBS Lett. 269, 368^372.
Bott, M., Eikmanns, B. and Thauer, R.K. (1986) Coupling
of carbon monoxide oxidation to CO2 and H2 with the phosphorylation of ADP in acetate-grown Methanosarcina barkeri. Eur. J. Biochem. 159, 393^398.
Bott, M. and Thauer, R.K. (1989) Proton translocation
coupled to the oxidation of carbon monoxide to CO2 and
H2 in Methanosarcina barkeri. Eur. J. Biochem. 179, 469^
472.
Kuënkel, A., Vorholt, J.A., Thauer, R.K. and Hedderich, R.
(1998) An E. coli hydrogenase 3 type hydrogenase in methanogenic archaea. Eur. J. Biochem. 252, 467^476.
Fox, J.D., Kerby, R.L., Roberts, G.P. and Ludden, P.W.
(1996) Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. J. Bacteriol. 178, 1515^
1524.
Boëhm, R., Sauter, M. and Boëck, A. (1990) Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenylase components. Mol. Microbiol.
4, 231^243.
Andrews, S.C., Berks, B.C., McClay, J., Ambler, A., Quail,
M.A., Golby, P. and Guest, J.R. (1997) A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocation formate hydrogen lyase system. Microbiology 143,
3633^3647.
Meuer, J. (1998) Diploma thesis, Universitaët Marburg.
Peer, C.W., Painter, M.H., Rasche, M.E. and Ferry, J.G.
(1994) Characterization of a CO:heterodisul¢de oxidoreductase system from acetate-grown Methanosarcina thermophila.
J. Bacteriol. 176, 6974^6979.
Latimer, M.T., Painter, M.H. and Ferry, J.G. (1996) Characterization of an iron-sulfur £avoprotein from Methanosarcina thermophila. J. Biol. Chem. 271, 24023^24028.
Hedderich, R., Berkessel, A. and Thauer, R.K. (1990) Puri¢cation and properties of heterodisul¢de reductase from
Methanobacterium thermoautotrophicum (strain Marburg).
Eur. J. Biochem. 193, 255^261.
Setzke, E., Hedderich, R., Heiden, S. and Thauer, R.K.
(1994) H2 :heterodisul¢de oxidoreductase complex from
Methanobacterium thermoautotrophicum : composition and
properties. Eur. J. Biochem. 220, 139^148.
Hedderich, R., Koch, J., Linder, D. and Thauer, R.K. (1994)
The heterodisul¢de reductase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic of
pyridine nucleotide-dependent thioredoxin reductases. Eur.
J. Biochem. 225, 253^261.
Reeve, J.N., Beckler, G.S., Cram, D.S., Hamilton, P.T.,
Brown, J.W., Krzycki, J.A., Kolodziej, A.F., Alex, L.,
Orme-Johnson, W.H. and Walsh, C.T. (1989) A hydrogenase-linked gene in Methanobacterium thermoautotrophicum
strain vH encodes a polyferredoxin. Proc. Natl. Acad. Sci.
USA 86, 3031^3035.
Hedderich, R., Albracht, S.P.J., Linder, D., Koch, J. and
Thauer, R.K. (1992) Isolation and characterization of poly-
[147]
[148]
[149]
[150]
[151]
[152]
[153]
[154]
[155]
[156]
ferredoxin from Methanobacterium thermoautotrophicum.
The mvhB gene product of the methylviologen-reducing hydrogenase operon. FEBS Lett. 298, 65^68.
Steigerwald, V.J., Pihl, T.D. and Reeve, J.N. (1992) Identi¢cation and isolation of the polyferredoxin from Methanobacterium thermoautotrophicum strain vH. Proc. Natl. Acad.
Sci. USA 89, 6929^6933.
Noëlling, J., Ishii, M., Koch, J., Pihl, T.D., Reeve, J.N.,
Thauer, R.K. and Hedderich, R. (1995) Characterization of
a 45-kDa £avoprotein and evidence for a rubredoxin, two
proteins that could participate in electron transport from H2
to CO2 in methanogenesis in Methanobacterium thermoautotrophicum. Eur. J. Biochem. 231, 628^638.
Schoënheit, P., Moll, J. and Thauer, R.K. (1980) Growth
parameters (Ks , Wmax , YS ) of Methanobacterium thermoautotrophicum. Arch. Microbiol. 127, 59^65.
Morgan, R.M., Pihl, T.D., Noëlling, J. and Reeve, J.N. (1997)
Hydrogen regulation of growth, growth yields, and methane
gene transcription in Methanobacterium thermoautotrophicum
vH. J. Bacteriol. 179, 889^898.
Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee,
H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D.,
Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P.,
Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R.,
Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso,
A., Bush, D., Safer, H., Patwell, D., Prabhakar, S., McDougall, S., Tulig, C., Shimer, G., Goyal, A., Church, G., Daniels, C.J., Mao, J., Rice, P., Pietrokovski, S., Noëlling, J. and
Reeve, J.N. (1997) The complete genome sequence of Methanobacterium thermoautotrophicum strain vH: functional
analysis and comparative genomics. J. Bacteriol. 179, 7135^
7155.
Friedrich, T. and Weiss, H. (1997) Modular evolution of the
respiratory NADH :ubiquinone oxidoreductase and the origin of its modules. J. Theor. Biol. 187, 529^540.
Bult, C.J., White, O., Olsen, G.J., Zhou, L., Fleischmann,
R.D., Sutton, G.G., Blake, J.A., FitzGerald, L.M., Clayton,
R.A., Gocaine, J.D., Kerlavage, A.R., Dougherty, B.A.,
Tomb, J.F., Adams, M.D., Reich, C.I., Overbeek, R., Kirkness, E.F., Weinstock, K.G., Merrik, J.M., Glodek, A.,
Scott, J.L., Geoghagen, N.S.M., Weidman, J.F., Fuhrmann,
J.L., Nguyen, D., Utterback, T.R., Kelley, J.M., Peterson,
J.D., Sadow, P.W., Hanna, M.C., Cotton, M.D., Roberts,
K.M., Hurst, M.A., Kaine, P.P., Borodovsky, M., Klenk,
H.P., Fraser, C.M., Smith, H.O., Woese, C.R. and Venter,
J.C. (1996) Complete genome sequence of the methanogenic
archaeon Methanococcus jannaschii. Science 273, 1058^1073.
Neijssel, O.E. and Teixera de Mattos, M.J. (1994) The energetics of bacterial growth : a reassessment. Mol. Microbiol.
13, 179^182.
Schwoërer, B. and Thauer, R.K. (1991) Activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin
reductase, and heterodisul¢de reductase in methanogenic
archaea. Arch. Microbiol. 155, 459^465.
Sorgenfrei, O., Muëller, S., Pfei¡er, M., Sniezko, I. and Klein,
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart
R. Hedderich et al. / FEMS Microbiology Reviews 22 (1999) 353^381
[157]
[158]
[159]
[160]
[161]
[162]
[163]
[164]
A. (1997) The [NiFe] hydrogenases of Methanococcus voltae :
genes, enzymes and regulation. Arch. Microbiol. 167, 189^
195.
Bobik, T.A. and Wolfe, R.S. (1989) An unusual thiol-driven
fumarate reductase in Methanobacterium with the production
of the heterodisul¢de of coenzyme M and N-(7-mercaptoheptanoyl)threonine-O3 -phosphate. J. Biol. Chem. 264, 18714^
18718.
Heim, S., Kuënkel, A., Thauer, R.K. and Hedderich, R.
(1998) Thiol :fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum: identi¢cation of the catalytic sites
for fumarate reduction and thiol oxidation. Eur. J. Biochem.
253, 292^299.
Staples, C.R., Ameyibor, E., Fu, W., Gardet-Salvi, L., StrittEtter, A.-L., Schuërmann, P., Kna¡, D.B. and Johnson, M.K.
(1996) The function and properties of the iron-sulfur center
in spinach ferredoxin:thioredoxin reductase : a new biological
role for iron-sulfur clusters. Biochemistry 35, 11425^
11434.
Staples, C.R., Gaymard, E., Stritt-Etter, A.-L., Telser, J.,
Ho¡man, B.M., Schuërmann, P., Kna¡, D.B. and Johnson,
M.K. (1998) Role of the [Fe4 S4 ] cluster in mediating disul¢de
reduction in spinach ferredoxin:thioredoxin reductase. Biochemistry 37, 4612^4620.
Williams, C.H. Jr. (1995) Flavoprotein structure and mechanism. 6. Mechanism and structure of thioredoxin reductase
from Escherichia coli. FASEB J. 9, 1267^1276.
Kunst et al. (1997) The complete genome sequence of the
Gram-positive bacterium Bacillus subtilis. Nature 390, 249^
256.
Oliver, K. and Harris, D., EMBL accession number z97991.
Deckert, G., Warren, P.V., Gaasterland, T., Young, W.G.,
Lenox, A.L., Graham, D.E., Overbeek, R., Snead, M.A.,
Keller, M., Aujay, M., Huber, R., Feldman, R.A., Short,
J.M., Olsen, G.J. and Swanson, R.V. (1998) The complete
[165]
[166]
[167]
[168]
[169]
[170]
[171]
381
genome of the hyperthermophilic bacterium Aquifex aeolicus.
Nature 392, 353^358.
Rakhely, G., Colbeau, A., Garin, J., Vignais, P.M. and Kovacs, K.L. (1998) Unusual organization of the genes coding
for HydSL, the stable [NiFe] hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS. J. Bacteriol.
180, 1460^1465.
Janssen, S., Schaëfer, G., Anemuëller, S. and Moll, R. (1997) A
succinate dehydrogenase with novel structure and properties
from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization. J. Bacteriol.
179, 5560^5569.
Nakamura, Y., Kaneko, T., Hirosawa, M., Miyajima, N.
and Tabata, S. (1998) CyanoBase, a WWW database containing the complete nucleotide sequence of the genome of
Synechocystis sp. strain PCC6803. Nucleic Acids Res. 26, 63^
67.
Rossi, M., Pollock, W.B.R., Reij, M.W., Keon, R.G., Fu, R.
and Voordouw, G. (1993) The hmc operon of Desulfovibrio
vulgaris subspec. vulgaris Hildenborough encodes a potential
transmembrane redox protein complex. J. Bacteriol. 175,
4699^4711.
Pott, A.S. and Dahl, C. (1998) Sirohaem sul¢te reductase
and other proteins encoded by genes at the dsr locus of
Chromatium vinosum are involved in the oxidation of intracellular sulfur. Microbiology 144, 1881^1894.
Cole, S.T., Eiglmeier, K., Ahemd, S., Honore, N., Elmers,
L., Anderson, W.F. and Weiner, J.H. (1988) Nucleotide sequence and gene-polypeptide relationships of the glpABC
operon encoding anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12. J. Bacteriol. 170, 2448^2456.
Pellicer, M., Badia, J., Aguilar, J. and Baldoma, L. (1996) glc
Locus of Escherichia coli: characterization of genes encoding
the subunits of glycolate oxidase and glc regulator protein.
J. Bacteriol. 178, 2051^2059.
FEMSRE 636 25-1-99 Cyaan Magenta Geel Zwart