Biochemical and Biophysical Research Communications 369 (2008) 919–923
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure studies on sulfur oxygenase reductase
from Acidianus tengchongensis
Mei Li a, Zhiwei Chen b, Pingfeng Zhang a,c, Xiaowei Pan a,c, Chengying Jiang b, Xiaomin An a,
Shuangjiang Liu b, Wenrui Chang a,*
a
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, PR China
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
c
Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
b
a r t i c l e
i n f o
Article history:
Received 20 February 2008
Available online 7 March 2008
Keywords:
Sulfur oxygenase reductase
Crystal structure
Non-heme iron center
Channels
Monomer tunnel
a b s t r a c t
Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur
to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal
structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere.
Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the
substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from
Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly,
Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than
six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost.
Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.
Ó 2008 Elsevier Inc. All rights reserved.
Biological oxidation and reduction of elemental sulfur are
important reactions involved in the biogeochemical cycles of sulfur
element. Members of the thermoacidophilic archaea, such as Sulfolobus and Acidianus, have been frequently isolated and detected
from sulfur-containing thermal vents [1–3], suggesting that they
are popular inhabitants and may play important ecological roles
in those environments. Some members of the genera Sulfolobus
and Acidianus oxidize sulfur for energy and their first step of sulfur
oxidation was catalyzed by sulfur oxygenase reductases (SORs).
SOR was first described by Emmel et al. [4]. Currently, SOR and
its homologs have been identified in thermophilic and acidophilic
archaea [5–8] and bacteria [9,10].
SOR simultaneously catalyzes the oxidation and reduction of
elemental sulfur to produce sulfite, thiosulfate, and sulfide in the
presence of molecular oxygen [4,7]. No requirements for cofactors
or external electron donors/acceptors for the reaction have been
reported [11,12]. The optimal catalytic temperatures for SORs from
Acidianus tengchongensis and Acidianus ambilvalens were reported
to be 65–85 °C.
The active site of SOR is composed of a mononuclear non-heme
iron site and three conserved cysteine residues. The crystal structure of the SOR-AA showed that the enzyme is a multimer containing 24 identical subunits, forming a large hollow sphere. Six
* Corresponding author. Fax: +86 10 64889867.
E-mail address: [email protected] (W. Chang).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.02.131
chimney-like protrusions, each composed of four helices that belonged to four individual monomers, were observed in the spherical structure. It was proposed that the chimney-like protrusions
were the entry routes of the substrate [13].
However, there are some questions that remain unanswered,
such as whether any channel that functions as the exit route for
products exists within the sphere? Moreover, the detailed process
of the SOR catalytic reaction remains to be accounted. In the present work, we studied enzymatic activities on wild type and mutants of SOR-AT and solved the crystal structures of recombinant
SOR in two different crystal forms (I432 and R3). Although the
overall folding of the SOR-AT structure is similar to that of the
SOR-AA structure, the catalytic sites in the two are quite different.
The detailed catalytic process and the exit routes for products were
analyzed based on our structures. These results may provide new
insights for understanding the catalytic process and the mechanism of SORs.
Materials and methods
Protein expression, purification, and enzymatic activity assays. The expression of
wild type and mutants of SOR were performed as described previously [14]. Protein
concentration was determined using a BCA protein assay kit (Sigma), by following
the instructions of the manufacturer (BCA1, Sigma, USA).
The oxygenase activity of SOR was determined as described before [15]. One
unit (U) is defined as the amount of enzyme required for the formation of 1 lmol
of sulfite plus thiosulfate per minute. The concentrations of SO32 and S2O32 were
determined by basic fuschnin and methylene blue methods, respectively [8].
920
M. Li et al. / Biochemical and Biophysical Research Communications 369 (2008) 919–923
Crystallization and data collection. Crystallization trials were set up using the
Hampton Research Screen Kit I & II (98 conditions) by applying the hanging drop
vapor diffusion technique with drops of 1 ll protein solution and 1 ll crystallization
reagent, which were allowed to equilibrate against 500 ll of crystallization reagent
in a well reservoir. Finally, two crystal forms of SOR suitable for diffraction studies
were obtained using optimized conditions (crystal form I: 1.08 M NaCitr3, 0.1 M
Hepes buffer, pH 7.5, 303 K; crystal form II: 25% PEG400, 0.1 M NaI, 0.1 M Hepes
buffer, pH 7.1, 289 K).
For crystal form I, a 2.7 Å resolution dataset of wild type SOR was collected at
BL6B in the Photon Factory, Tsukuba, Japan. A separate dataset of a mutant SOR
(C101S) was collected to 3.2 Å resolution from crystal form II, by utilizing a Rigaku
R-AXIS IV image plate detector at Institute of Biophysics, CAS. Both diffraction data
were collected at 100 K and were integrated and scaled with programs DENZO and
SCALEPACK [16]. The results showed that crystal form I belongs to the space group
I432, crystal form II belongs to the space group R3.
Structure determination and refinement. The structure was solved by molecular
replacement with MolRep [17] in the CCP4 suite [18]. The search model was SORAA structure (PDB code: 2CB2), which displays 88% sequence identity to SOR-AT.
The initial model was manually rebuilt with the program O [19] and refined by
ARP/warp [20] and refmac5 [21]/CNS [22]. The stereochemical quality of the refined
structures was verified with the program PROCHECK [23]. A summary of the data
collection and structure refinement statistics is provided in Table 1.
The atomic coordinate of the SOR crystal structure had been deposited in the
Protein Data Bank with Accession No. 3BXV. Figs. 1–4a were created using the program PyMol (DeLano Scientific, LLC).
Results and discussion
In the present study, crystal structures of wild type and mutant
(C101S) of SOR-AT were solved in two different crystal forms: I432
and R3. It should be noted that the sequence of the SOR protein of
the I432 form differs from the NCBI protein sequence AAK58572 at
two positions (K174E and V238A). These changes might be due to
errors introduced during PCR amplification or because of the passing of the generation in Escherichia coli. However, both sites are not
conserved among SOR proteins, as shown in Supplementary Fig. 1.
Further, these mutations did not affect the SOR enzymatic activity
at all (Supplementary Table 1).
Table 1
Data collection and refinement statistics of SOR
Crystal form
I
II
Space group
Cell parameters
Resolution (Å)
No. of total reflections
No. of unique reflections
Completeness (%)
I/Sigma
Rmerge (on I) (%)
Molecules (A. U.)
No. of residues
No. of Fe atoms
No. of water molecules
Rcryst (%)
Rfree (%)
I432
a = b = c = 159.1 Å,
a = b = c = 90°
30–2.7 (2.80–2.70)
90,202
9396
99.6 (98.7)
22.7 (3.8)
8.2 (37.7)
1
308
1
53
19.2
21.3
R3
a = b = 225.2 Å, c = 137.1 Å,
c = 120°
50–3.2 (3.31–3.20)
123,519
41,173
96.2 (93.9)
6.6 (2.8)
13.9 (29.3)
8
2456
8
253
21.6
25.3
RMS deviations
Bond (Å)
Angle (°)
0.010
1.73
0.011
1.83
Average B-value (Å2)
Main chain
Side chain
Fe atoms
Water molecules
30.4
31.2
34.7
32.1
14.0
14.9
13.0
13.1
90.7
9.3
81.6
18.3
Ramachandran
Most-favored regions (%)
Additional allowed
regions (%)
Generously allowed
regions (%)
0.1
Overall structure and the active site of SOR monomer
The architectures of two independently refined structures of
SOR-AT are nearly identical. The monomer consists of 308 amino
acid residues and belongs to the a/b folding type. The core structure is a b-barrel composed of eight b-strands (E1–E8) surrounded
by nine a-helices (H1–H9) (Fig. 1A).
The active sites of the two structures in different crystal forms
are also very similar. They are composed of three cysteine residues
and an iron atom, which is coordinated by His86, His90, Glu114,
and a water molecule (Fig. 1B). Replacements of His86 or His90
by a phenylalanine residue resulted in a dramatic reduction in
SOR activity. Mutation of any cysteine residues (C31S, C101S, and
C104S) at the active site also leads to complete loss of SOR catalytic
ability (Supplementary Table 1). The importance of these residues
is also emphasized by their 100% conservation in all SOR proteins
(Supplementary Fig. 1).
A comparison of the two structures showed that the conformation of Ser101 of the inactive mutant C101S, which was solved as
the R3 form, is the same as that of Cys101 in the I432 form, the
wild type structure (Fig. 1B), with only a c-O atom substituted
for a c-S atom. This indicated that only the c-S atom contributed
to the SOR function.
The cysteine–cysteine and cysteine–iron distances are in the
range of 5.5–12.4 Å, which is quite reasonable considering the size
of elemental sulfur, one of the substrates of SOR. The two mutated
residues (K174E and V238A) are located far away from the active
site in the I432 form (Fig. 1A), thus having little influence on its
enzymatic activity.
Tetramer and trimer channels in SOR icosatetramer sphere might be
routes for substrate entry and product exit, respectively
In both crystal forms, 24 identical monomers are assembled
into a hollow sphere according to point group symmetry 432 (Supplementary Fig. 2), which is consistent with the SOR-AA crystal
structure. The distance among different active sites of neighboring
monomers is greater than 37 Å, which makes it almost impossible
for cooperative catalysis by individual monomers. Since the active
site of each monomer is accessible to the solvent only from the inside of the sphere, it was postulated that the possible function of
this packing is to form an isolated environment. Therefore, there
must be passages for the entry of substrates and the exit of
products.
There are six protruding channels, referred to as tetramer channels, which were formed by identical a-helices (H4) of four individual monomers along the fourfold symmetry axes. Their inner
surface is highly hydrophobic, formed by residues Phe133,
Val137, and Phe141. The phenyl groups of Phe133 and Phe141 residues of each monomer stick out at the inner surface of the channel
(Fig. 2A). Given the size and strong hydrophobic property fulfill the
requirement of substrate [S0], these channels are likely to be the
paths for substrate entry into the sphere; this was previously noticed by Urich et al. [13].
However, the question of how and where the products leave remains. Are the tetramer channels also the paths for products exit,
or are there other channels for products? Careful examination of
our structures suggested that eight channels, each composed of
H3 and a loop region (H6–E7) of three individual monomers, might
be the product channels (Fig. 2B), referred to as trimer channels.
Unlike the tetramer channels, trimer channels are more polar, with
Ser95, Arg99, Ser103, and Ser226 contributing to their inner surface. The side chains of Arg99 and Ser226 stick out, forming a narrow gorge of the trimer channel (Fig. 2B). Considering the smaller
size and the positively charged nature, it was assumed that trimer
channels might be used for products exit.
M. Li et al. / Biochemical and Biophysical Research Communications 369 (2008) 919–923
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Fig. 1. (A) Superposition of the overall structures of SOR-AT between the two crystal forms. The core structure is composed of nine a-helices and eight b-strands. The
secondary structure is labeled as H1–H9 for helices and E1–E8 for strands. The structure of the I432 form is shown in blue and that of the R3 form, in green. The iron atom is
represented as a large sphere and the ligated water molecule as a small sphere. Two mutated residues in the I432 form are represented as sticks and labeled in red italic fonts.
(B) Superposition of the active site of SOR-AT structures between the two crystal forms. The color designation is the same as that in (A). The iron atom and water molecule are
represented as spheres. The residues constituting the active site are shown as sticks. The conformation of C101 in the I432 form and that of S101 in the R3 form are similar.
The residue is labeled by a letter within parentheses; the letters refer to the amino acid type in the R3 form. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
Fig. 2. Ribbon representation of the tetramer channel and trimer channel in SOR-AT structure. (A) Tetramer channel. Four individual monomers are shown in red, orange,
green, and purple. The side chains of residues Phe133, Val137, and Phe 141 at each monomer, which contributed to the inner surface of the tetramer channel, are shown as
sticks. View along the crystallographic fourfold axis. (B) Trimer channel. Three individual monomers are shown in red, blue, and pink. The side chains of residues Arg99 and
Ser 226 at each monomer, which contributed to the inner surface of the trimer channel, are shown as sticks. View along the crystallographic threefold axis. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
Monomer tunnel in SOR structure
Besides tetramer and trimer channels, monomer tunnels were
also observed in our structures. The iron atom is buried inside
the SOR molecule and is accessible to the solvent only through
the monomer tunnel, which can also be described as the joining
of two differently orientated pockets. One pocket is wider; its
opening at the SOR surface is formed by Phe20, Phe23, Ser24,
Gly27, Val47, Val296, and Met297 (Fig. 3A). The other is relatively
narrow, the opening being made up of Ala105, Met108, Pro112,
and Phe210 (Fig. 3B).
The observation of the monomer tunnel in the whole icosatetramer sphere structure revealed that the wider opening lies near
the tetramer channel, while the narrower one lies close to the tri-
mer channel. However, the narrow opening is covered by a loop
from another monomer, with Glu560 , Arg570 , Phe580 , and Gly590
in its vicinity. Interestingly, the side chains of all these residues
swing away, maintaining this pocket in the open state (Supplementary Fig. 3). Moreover, the side chain of Arg570 swings back
and is just located near the narrow opening of the monomer tunnel, which may facilitate the movement of negatively charged
products toward it. Thus, it was proposed that the wider opening
of the tunnel is the entrance, which is used by the substrate to access the active site, while the narrower one is the exit.
At the active site, the iron atom and its binding residues are
located close by, while the three cysteine residues are located far
from them. Relative to the iron atom, Cys31 is located on the
entrance side of the monomer tunnel, while Cys101 and 104 are
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M. Li et al. / Biochemical and Biophysical Research Communications 369 (2008) 919–923
Fig. 3. Surface representations of SOR monomer. Red represents a negative charge, and blue represents a positive charge. The monomer surface is shown as a transparent
layer. There are two openings of the monomer tunnel at the SOR surface. The residues constituting the openings are labeled. The iron atom is represented as a sphere. (A) The
entrance of the monomer tunnel. (B) The exit of the monomer tunnel. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
Fig. 4. (A) Superposition of the active site between the SOR-AT (I432 form) and SOR-AA structures. The iron atom is represented as a large sphere and water molecules as
small spheres. The residues constituting the active site are shown as sticks. The SOR-AA structure is shown in green, while the color of the SOR-AT structure is represented by
the atom type: yellow, carbon atom; red, oxygen atom (water molecule is included); blue, nitrogen atom; orange, sulfur atom; magenta, iron atom. (B) The stereo view of
electron density maps at the active site of SOR. The color designation is the same as that in (A). The structures are shown by a ball and stick model. The 2 Fo–Fc electron density
map countered at 1r is shown in blue, the Fo–Fc electron density map countered at 2.5r is shown in red. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
M. Li et al. / Biochemical and Biophysical Research Communications 369 (2008) 919–923
923
located on the exit side, implying that the function of these cysteine residues might be different during the catalytic reaction process. Since the whole catalytic reaction of SOR could be split into
sulfur oxidation and sulfur disproportionation reactions, it was
postulated that Cys31 is involved in the oxidation reaction, while
Cys101 and 104 are involved in the latter reaction.
Appendix A. Supplementary data
Comparison of SOR-AT and SOR-AA structures
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The superposition of SOR-AT (I432 form) and SOR-AA structures
showed that their overall architectures are quite similar; however,
significant differences were found with regard to the active site. A
very special residue, Css31 (cysteine persulfide), was refined in the
SOR-AA structure. Previous reports showed that the sulfane sulfur
atoms are labile species, usually generated in the catalytic cycles of
the synthesis of sulfur-containing biomolecules [24,25]. In contrast, the residue in SOR-AT structures is a cysteine, and no extra
Fo–Fc electron density around the c-S atom of Cys31 was found
(Fig. 4). Another difference is that the iron atom is five-coordinated, and only one of the two water molecules (Wat1 and
Wat2) that coordinate the iron atom in the SOR-AA structure was
found in the SOR-AT structures (Fig. 4A). As Fig. 4B shows, the
Fo–Fc density countered at 2.5r was clearly present at the positions
of Wat1 and the adjacent solvent molecule, while no Fo–Fc density
exists at the positions of Wat2 and the d-S atom of Css31.
Postulated catalytic reaction process of SOR
Since Cys31 is located at the entrance of the monomer tunnel,
we proposed that it might be the real residue that binds the substrate [S0] at the initial step of the catalytic reaction; the binding
site should be the position of the d-S atom of Css31 in the SORAA structure. Accordingly, the position of Wat2 in the SOR-AA
structure was suggested to be the binding site of molecular oxygen.
The two crystal structures might represent two different statuses
in the catalytic reaction process of SOR, which is probably due to
the different characteristics, different resources and/or the different crystallization conditions of the two proteins.
Based on our structures, on combining the SOR-AA structure, a
preliminary and hypothetical process of catalytic reaction of SOR
can be proposed. The substrate sulfur initially entered the inner
part of the icosatetramer sphere through the hydrophobic tetramer
channel. It then moved toward the entrance of the monomer tunnel
to access the active site by binding to the c-S atom of Cys31. Simultaneously, the oxygen molecule settled near the iron atom at the
position of Wat2, thereby trigger the oxidation reaction. Cys101
and 104 might assist in sulfur binding, or they might probably be
involved later in the disproportionation reaction. After the reaction
was completed, the products moved via the exit of the monomer
tunnel and then out of the sphere through the trimer channel.
In this study, we observed trimer channels and monomer tunnels in SOR-AT structures for the first time. Besides, the structural
comparison of SOR-AT and SOR-AA revealed the remarkable differences in their active sites, leading to the assumption that they
might represent different statuses of SOR. Thus, a detailed process
of the SOR catalytic reaction was suggested, which may provide
new insights for better understanding SOR proteins.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant Nos. 30500094, 10490193, and 30621005),
the 973 Project (Grant No. 2006CB911001). We are grateful to Drs.
N. and K. Sakabe at Photon Factory, Japan, Dr. Yu-hui Dong and Dr.
Peng Liu at the Institute of High Energy Physics, CAS, Mr. Yi Han at
the Institute of Biophysics, CAS for data collection.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2008.02.131.
References