Download Biochemistry of Sulfur

PD Dr. Arnulf Kletzin
Institut für Mikrobiologie und Genetik
TU Darmstadt
Schnittspahnstraße 10
64287 Darmstadt
Germany
Sulfur Oxidation and Reduction in Acidianus ambivalens
Numerous (micro-) organisms
oxidize sulfur (S˚) and
inorganic sulfur compounds
(ISCs) for energy conservation.
The oxidation of S˚ to sulfuric
acid proceeds in at least two
steps, often more involving
intermediates like sulfite,
thiosulfate, tetrathionate, etc.
(Fig. 1). Thermoacidophilic
Archaea oxidize S˚ with a cytoplasmic sulfur-disproportionating enzyme. The products
thiosulfate and sulfite are
oxidized by membrane-bound
oxidoreductases.
Figure 1:
1: The
The biological
biological sulfur
sulfur cycle
cycle
Figure
shown by
by reactions
reactions involving
involving inorganic
inorganic
shown
sulfur compounds.
compounds. Enzymes:
Enzymes: 1,
1, polypolysulfur
sulfide reductase;
reductase; 2,
2, sulfur
sulfur reductase;
reductase; 3,
3,
sulfide
sulfide: quinone
quinone oxidoreductase
oxidoreductase or
or
sulfide:
sulfide:cytochrome cc oxidoreductase;
oxidoreductase; 4,
4,
sulfide:cytochrome
sulfur oxygenase;
oxygenase; 5,
5, sulfite:
sulfite: acceptor
acceptor
sulfur
oxidoreductase; 6,
6, ATP
ATP sulfurylase;
sulfurylase; 7,
7, ATP
ATP
oxidoreductase;
sulfurylase or
or adenylylsulfate:
adenylylsulfate: phosphate
phosphate
sulfurylase
adenylyltransferase; 8,
8, adenylylsulfate
adenylylsulfate
adenylyltransferase;
(APS) reductase;
reductase; 9,
9, sulfite
sulfite reductase;
reductase; 10,
10,
(APS)
tetrathionate reductase;
reductase; 11,
11, thiosulfate:
thiosulfate:
tetrathionate
acceptor oxidoreductase;
oxidoreductase; 12,
12, Sox
Sox
acceptor
complex; 13,
13, sulfur
sulfur oxygenase
oxygenase reductase;
reductase;
complex;
14, thiosulfate
thiosulfate reductastase;
reductastase; 15,
15,
14,
tetrathionate hydrolase
hydrolase (there
(there is
is also
also aa
tetrathionate
trithionate hydrolase
hydrolase in
in addition);
addition); 16,
16, OOtrithionate
acetylserin or
or O-phosphoserine
O-phosphoserine sulfhydrosulfhydroacetylserin
lases; 17,
17, cysteine
cysteine desulfurase.
desulfurase. Gray
Gray
lases;
circles denote
denote disproportionation
disproportionation reactreactcircles
ions. Reactions
Reactions of
of cysteine
cysteine breakdown
breakdown in
in
ions.
eukaryotes and
and of
of phosphoadenosinphosphoadenosineukaryotes
phosphosulfate formation
formation and
and reduction
reduction
phosphosulfate
are omitted.
omitted.
are
Sulfur Oxidation, Sulfur Oxygenase Reductase: The initial enzyme in the S˚ oxidation pathway is
unique in several aspects. The soluble protein was termed sulfur oxygenase reductase (SOR) because
it is the only S˚-disproportionating enzyme. Sulfite, thiosulfate and hydrogen sulfide are products.
Oxygen is required for activity but no organic cofactors:
(Eq. 1)
4 S˚ + O2 + 4 H2O ---- > 2 HSO3- + 2 H2S + 2 H+
Optimal activity was observed at 85 ˚C and pH 7, maximal
activity at 108 ˚C. Three conserved cysteine residues are present
in the known SORs. Site-directed mutagenesis showed that one
of these was indispensable, while mutagenesis of the other two
resulted in reduced activities. EPR spectroscopy and redox
titration showed that the SOR contains a mononuclear non-heme
iron center in the high-spin Fe3+ state and with a low reduction
potential (Eo' = -268 mV). It was intriguing to find that the
reduction potential was more than 300 mV lower than usually
found for this type of iron centers and low enough to explain the
S˚ reducing activity of the enzyme (Eo' [H2S/S˚] = -270 mV).
Figure 2: Electron Micrograph of the
SOR (Reinhard Rachel, Regensburg)
Figure 3: Molecular model of the SOR. A, Cartoon representation with α-helical regions in blue and βsheet in violett, Fe atom red balls. B, Surface representation with the subunits coloured differently. C,
Section with the surface coloured according to charge (blue=positive, red = negative).
SOR structure and mechanism: Hollow globular particles of 15.5 nm in diameter appeared in
electron microscopic pictures of the purified Ac. ambivalens SOR (Fig. 2). X-ray crystallographic
analysis to 1.7 Å resolution showed that the SOR is a spherical homo-icosatetramer (i.e. 24 subunits).
It surrounds an empty cavity with a diameter of 71-107 Å Fig. (Fig. 3) and a molecular mass of
844.000 for the native SOR (871.000 for the recombinant). Each subunit consisted of a β-barrel core
surrounded by α-helices. The iron center was coordinated by two histidine ligands, a bidentate
glutamate and two water molecules in a structural motif, which is known as "2-His 1-Carboxylate
facial triad" (Fig. 4). Mutation of any of the three iron ligands to alanine resulted in the complete loss
of activity and iron-binding capabilities. The residue C31 was modified to a persulfide. The iron center
and the three cysteines are buried in a pocket in the interior of each monomer.
Some conclusions for the reaction mechanism could be
derived from the structure. The core active site is composed
of the iron site and the modified C31. It is accessible only
from the interior cavity. Substrate entry has to proceed
through the hydrophobic channels along the 4-fold axes of
the sphere (Fig. 2). S˚ is most probably bound covalently to
C31. The linear sulfur chain is aligned to the iron site and
replaces the water ligands, poising the iron site for oxygen
binding and activation.
Figure 4: The catalytic pocket of the SOR containing the conserved
cysteines and the iron. (A) Stereo view of the mononuclear non-heme
iron center with Fe ligands, Fe ion; red, waters; ball-and-stick, protein
ligands. (B) Cavity surface representation of the catalytic pocket with
Cys and Fe highlighted; gray arrow, cavity entrance. (C) Identification of
an additional sulfur atom at Cys31.
Seite: 2/6
Thiosulfate Oxidation: A tetrathionate-forming, membrane-bound thiosulfate:quinone
oxidoreductase (TQO) was isolated from aerobically grown Ac. ambivalens cells. Optimal activity was
observed at 85 ˚C and pH 5. The 102 kDa glycosylated holoenzyme had a α2β2 stoichiometry. Oxygen
electrode measurements showed an electron transport from thiosulfate to molecular oxygen via the
terminal heme copper quinol:oxygen oxidoreductase (Fig. 5).
The fate of the tetrathionate formed by the Ac. ambivalens TQO has not been investigated yet when
the organism grows on S˚. However, there is a possibility that a thiosulfate/tetrathionate cycle exists.
Tetrathionate is unstable in the presence of strong reductants and is reduced to thiosulfate in vitro at
high temperatures. Therefore, H2S and sulfite might re-reduce tetrathionate formed by the TQO and
thus feed electrons indirectly from the S˚ disproportionation reaction catalyzed by the SOR into the
quinone pool (Fig. 5).
Figure 5: Hypothetical model of S˚ oxidation in A. ambivalens derived from known enzyme activities (in italics) and possible
non-enzymic reactions: SAOR, sulfite:acceptor oxidoreductase; TQO, thiosulfate:quinone oxidoreductase; SOR, sulfur
oxygenase reductase; APS, adenylylsulfate; APAT, adenylylsulfate:phosphate adenylyltransferase, CQ, caldariella quinone.
Seite: 3/6
Anaerobic Sulfur Reduction with Hydrogen as Electron Donor: Many microorganisms reduce sulfur
when growing anaerobically. Several metabolic types with S˚ as electron acceptor can be
distinguished. Many heterotrophs require the addition of S˚ to the media as terminal electron
acceptors. The products are either CO2 or small organic compounds like acetate. H2S is produced in
the presence and H2 in the absence of S˚. Alternatively, numerous S˚ reducers like Acidianus
ambivalens gain energy from H2 oxidation; ATP and reduction equivalents are used for CO2 fixation:
H2 + S˚ ---- > H2S
(or HS-, depending on the pH)
Acidianus ambivalens sulfur reductase and hydrogenase: A sulfur reductase (SR; H2:S˚
oxidoreductase) purified from membrane fractions of anaerobically grown Ac. ambivalens cells
showed activity in the presence of a co-purified hydrogenase. The A. ambivalens hydrogenase encoded
by a polycistronic cluster including genes for a NiFe and an FeS subunit was rather dissimilar to other
hydrogenases (Fig. 6). The Isp1 protein is probably the membrane anchor. The FeS subunit contained
a leader peptide with a twin-arginine translocation motif (TAT) not present in the mature protein
suggesting a transport across the membrane (Fig. 7). Fe and Ni were present in membrane and in
enriched hydrogenase fractions in accordance with the observed sequence similarity.
The Ac. ambivalens SR gene cluster consisted of five ORFs, sreABCDE (Fig. 6). The deduced amino
acid sequences of sreA encoding a 110 kDa subunit visible on SDS gels and sreB showed similarity to
the molybdopterin and the FeS subunits of the DMSO/Nitrate reductase family, respectively. SreA also
contained a twin arginine motif suggesting an export by the TAT pathway. The sreC gene encoded a
protein with 10 predicted transmembrane helices, sreD an unknown polyferredoxin with 26 cysteine
residues and sreE a small system-specific chaperone. Molybdenum, but not tungsten was found in
solubilized membrane fractions suggesting that the SR is a molybdoprotein in accordance with the
observed sequence similarity.
Figure 6: Gene clusters encoding the NiFe hydrogenase and the SR in Ac. ambivalens. Small black circles indicate cysteinecontaining sequence motifs potentially coordinating FeS clusters; Ni and Mo indicate potential metal binding sites; m,
predicted transmembrane protein. RR twin arginine protein translocation motif, the tat cluster encodes proteins required for
twin arginine translocation pathway; HynS, HynL, small and large subunit of the hydrogenase; isp1, membrane anchor; hyp,
hydrogenase maturation genes/proteins; hoxM, protease of HynL maturation.
Seite: 4/6
The predicted orientation and the molecular composition of the Ac. ambivalens SR deduced from the
biochemical results and the sequence analysis are similar to the W. succinogenes PSR (Fig. 7). Both
enzymes consist of homologous catalytic and electron transfer subunits (SreA/PsrA and SreB/PsrB,
respectively) and non-homologous membrane anchors (SreC/PsrC). The hydrogenases also have
comparable quaternary structures; they are composed of at least three subunits each, the homologous
Ni-containing catalytic (HynL/HynB; Fig. 7), the FeS-containing electron transfer subunits
(HynS/HynA) and the non-homologous membrane anchors (Isp1/HynC).
The only other probably orthologous sreABCDE gene clusters have been identified in the genomes of
Su. solfataricus and Su. islandicus (but not in Su. acidocaldarius and Su. tokodaii) suggesting that they
should grow by heterotrophic anaerobic S˚ respiration; however, this has not been demonstrated yet.
Phylogenetic analysis showed that other homologs of SreA are present in the genomes of other
facultative sulfur reducers and that they form a branch in the DMSO reductase family tree together
with other enzymes with unrelated substrates. It can be concluded that these organisms have a
mutually similar set of membrane-bound SRs with related but diverse molybdopterin and electron
transfer subunits while additional subunits might vary. The membrane-bound hydrogenases diversify
in a similar way, although the analysis is more complicated because NiFe hydrogenases fall into four
subfamilies, which are all realized in Archaea. It was interesting to see that the Sulfolobus species
sequenced so far do not have hydrogenase genes, showing that they should not be able to grow
lithotrophically with H2.
Figure 7: Schematic representation of the sulfur and polysulfide respiration in Ac. ambivalens and Wolinella succinogenes.
The Ac. ambivalens model was developed in analogy to Wo. succinogenes from the results of the sequence comparison and the
biochemical data. Homologous subunits are shown in identical shading. CM cytoplasmic membrane, OM, outer membrane,
MQ, menaquinone, SQ, sulfolobus quinone.
Seite: 5/6