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REVIEW
The bacterial SoxAX cytochromes
Ulrike Kappler • Megan J. Maher
Received: 25 March 2012 / Revised: 9 July 2012 / Accepted: 17 July 2012 / Published online: 21 August 2012
� Springer Basel AG 2012
Abstract SoxAX cytochromes are heme-thiolate proteins
that play a key role in bacterial thiosulfate oxidation, where
they initiate the reaction cycle of a multi-enzyme complex
by catalyzing the attachment of sulfur substrates such as
thiosulfate to a conserved cysteine present in a carrier
protein. SoxAX proteins have a wide phylogenetic distri-
bution and form a family with at least three distinct types
of SoxAX protein. The types of SoxAX cytochromes differ
in terms of the number of heme groups present in the
proteins (there are diheme and triheme versions) as well as
in their subunit structure. While two of the SoxAX protein
types are heterodimers, the third group contains an addi-
tional subunit, SoxK, that stabilizes the complex of the
SoxA and SoxX proteins. Crystal structures are available
for representatives of the two heterodimeric SoxAX protein
types and both of these have shown that the cysteine ligand
to the SoxA active site heme carries a modification to a
cysteine persulfide that implicates this ligand in catalysis.
EPR studies of SoxAX proteins have also revealed a high
complexity of heme dependent signals associated with this
active site heme; however, the exact mechanism of catal-
ysis is still unclear at present, as is the exact number and
types of redox centres involved in the reaction.
Keywords SoxAX cytochromes � Cytochromes �Heme thiolate proteins � Sulfur oxidation �Crystal structure � Redox centres
Introduction
The so-called SoxAX cytochromes are a group of c-type
cytochromes that catalyze the formation of heterodisul-
fide bonds between inorganic sulfur compounds and a
conserved cysteine on a sulfur carrier protein [1, 2]. The
reaction involves a heme group located in the SoxAX
active site, which has a His/Cys axial ligation. Unlike the
well-known His/Met ligated hemes present in many pro-
and eukaryotic cytochromes, proteins containing His/Cys
ligated heme groups are relatively rare in nature, and
fulfill a range of special and diverse functions [3].
A well-known example of heme thiolate proteins are the
cytochrome P450s that play a role in xenobiotic metab-
olism; however, in these proteins, the heme group is
essentially five coordinate, which increases its catalytic
reactivity [3, 4]. There are several examples of proteins
that contain six coordinate heme thiolate groups,
including human cystathionine beta synthase, an enzyme
involved in the formation of cystathionine from homo-
cysteine where the His/Cys ligated heme has been
proposed to play a role in redox sensing and also influ-
ences catalytic activity [5]. Other examples are the
bacterial CooA carbon monoxide sensor protein in which
the heme group acts as a redox sensor [6, 7], a reaction
centre cytochrome (PufC) from the phototrophic bacte-
rium Rhodovulum sulfidophilum [8], and the DsrJ triheme
cytochrome that is part of a membrane protein complex
found in both dissimilatory sulfate reducing and sulfur
oxidizing bacteria [9, 10].
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00018-012-1098-y) contains supplementarymaterial, which is available to authorized users.
U. Kappler (&)
School of Chemistry and Molecular Biosciences,
The University of Queensland, St. Lucia, QLD 4072, Australia
e-mail: u.kappler@uq.edu.au
M. J. Maher
School of Molecular Sciences, La Trobe University,
Melbourne, VIC 3086, Australia
Cell. Mol. Life Sci. (2013) 70:977–992
DOI 10.1007/s00018-012-1098-y Cellular and Molecular Life Sciences
123
The SoxAX heme thiolate proteins also belong to this
diverse group of proteins and play a key role in bacterial
sulfur oxidizing photo- and chemolithotrophs, i.e. bacteria
that are capable of using inorganic sulfur compounds as
electron donors for photosynthesis or for energy genera-
tion. Several related but structurally distinct types of
SoxAX cytochromes are known, but the properties and
types of redox centres present in these enzymes as well as
their respective roles in catalysis are still only partly
understood.
Discovery of SoxAX cytochromes
The SoxAX cytochromes were first discovered by Lu and
Kelly [11–13] during studies of sulfur oxidation in the
facultatively chemolithotrophic bacterium Paracoccus
versutus (known at the time as Thiobacillus versutus or
Thiobacillus A2 [14]). Kelly and coworkers were the first
to isolate and partially characterize the constituents of a
bacterial thiosulfate oxidizing enzyme complex to which
the SoxAX cytochromes belong. This enzyme complex
was called either the Paracoccus sulfur oxidation system
(PSO), or the thiosulfate oxidizing multi-enzyme system
(TOMES) (today it is often simply referred to as the Sox
system). Kelly referred to the components of the complex
as enzymes A and B (equivalent to SoxYZ and SoxB), a
cytochrome c552.5 (eq. to SoxAX) and a sulfite: cytochrome
c oxidoreductase/cytochrome c551 complex (eq. to SoxCD).
In these early studies, many of the properties of the indi-
vidual components of the enzyme system, such as the
ability of enzyme A to bind thiosulfate or the fact that the
cytochrome c552.5 is an essential component of the enzyme
complex, were correctly inferred although the molecular
details underlying these observations could not be analyzed
in depth. Characterization of the TOMES also included the
first EPR spectroscopic studies of the metal centres present
in the different enzymes (summarized in [15]).
In the early 1990s, the group of Friedrich used trans-
poson mutagenesis to identify and then sequence a gene
region involved in thiosulfate oxidation in another bacte-
rium that is today known as Paracoccus pantotrophus
GB17 [16, 17] (at the time: Thiosphaera pantotropha
GB17, then Paracoccus denitrificans GB17 [18]). This
gene region turned out to encode proteins of an enzyme
complex similar to the one that had been studied by Kelly
and coworkers. The identification of the gene region
encoding the TOMES opened up many possibilities for
comparative and in depth molecular and biological studies,
and the group of Friedrich subsequently reported the
purification and characterization of several TOMES com-
ponents [19–24]. More than 15 genes have today been
identified as belonging to the sox gene region in
P. pantotrophus, but the core enzyme complex only requires
the SoxAX, SoxYZ, SoxB and SoxCD proteins for function
while the remaining genes encode proteins that are
involved in regulation and (re-)activation of core Sox
proteins [21, 25–29]. The fact that the genes found in the
P. pantotrophus sulfur oxidation gene region were referred
to as ‘sox’ genes laid the foundation for the current
nomenclature of the encoded proteins, all of which are
today referred to as ‘Sox’ proteins.
While initial studies of the Sox system all focussed on
chemolithotrophic sulfur oxidizing bacteria, it was soon
recognized that not only these bacteria, but also the
phototrophic purple and green sulfur bacteria contained
homologues of Sox proteins [30]. In the phototrophic
bacteria, the Sox proteins occur in addition to the proteins
of the dissimilatory sulfite reductase (Dsr) complex that
have been known to be essential for sulfur oxidation pro-
cesses in these bacteria for over 40 years [31–34]. The
combination of Dsr and Sox proteins can occur in both
photo- and chemotrophic bacteria that form sulfur deposits
as an intermediate of the sulfur oxidation process. In these
dsr-gene cluster containing photo- and chemotrophic bac-
teria the Sox system is thought to be specifically involved
in the utilization of thiosulfate but not sulfide as an electron
donor [33]. In contrast, chemotrophic bacteria that contain
only a Sox system appear to be capable of using thiosulfate
as well as sulfide and sulfite as substrates for Sox system
mediated sulfur oxidation [35]. In one case even sulfur and
tetrathionate have been reported to be substrates of a Sox
multi-enzyme complex [36].
SoxAX cytochromes and their roles in different
pathways for dissimilatory sulfur oxidation
The functions of the homologous Sox proteins from bac-
teria relying solely on the Sox pathway and those using the
Dsr/Sox pathway are conserved, and although more than 15
individual Sox proteins are known at present, only three or
four protein complexes, respectively, are essential for the
functioning of the enzyme system, depending on whether a
bacterium uses the Dsr/Sox or the Sox pathway. The three
essential protein complexes common to all Sox systems are
the SoxAX cytochromes, the cofactor-less SoxYZ proteins
that carry a conserved ‘GGCGG’ motif [35] and the SoxB
proteins that contain a dimanganese centre [37], while the
molybdenum- and heme-containing SoxCD sulfur dehy-
drogenase is only found in bacteria using the Sox pathway
but is essential for sulfur oxidation in these microorgan-
isms [38].
Based on the properties of the essential complex pro-
teins and the observation that no free sulfur intermediates
appear to occur during thiosulfate oxidation, the following
978 U. Kappler, M. J. Maher
123
model for the Sox pathway reactions has been proposed
(Fig. 1) [30]. Throughout the Sox system reaction cycle,
the heterodimeric SoxYZ protein acts as a ‘carrier protein’
to which the reduced sulfur compounds remain attached at
all stages of the oxidation process (Fig. 1) [30, 35]. The
attachment of the sulfur compounds to a conserved
‘GGCGG’ motif at the C-terminus of SoxY is mediated by
the SoxAX cytochrome, which is thus essential for initi-
ating the sulfur compound oxidation process [1, 35]. If the
sulfur substrate is thiosulfate, the next reaction is a SoxB
mediated hydrolysis of the sulfone group of the bound
thiosulfate molecule, leading to the formation of one
molecule of sulfate, the ultimate product of thiosulfate
oxidation. Following the hydrolytic step, the SoxCD sulfur
deydrogenase catalyzes the six electron oxidation of the
remaining thiosulfate ‘sulfane’ sulfur atom to a sulfone
group, which is then liberated from the SoxYZ carrier
protein as a second molecule of sulfate through a second
reaction with SoxB [23, 35, 37] (Fig. 1).
In cases where other sulfur substrates are being oxi-
dized, the reaction cycle has been proposed to be shorter
[35], e.g. if the reaction substrate were sulfide, then the
reaction cycle would start with SoxAX mediated attach-
ment of the substrate to SoxYZ, followed by the SoxCD
mediated six electron oxidation of the SoxYZ bound sul-
fane sulfur molecules and a reaction with SoxB (Fig. 1)
[35], while in the case of sulfite, only one SoxB mediated
hydrolysis reaction would be predicted to take place
(Fig. 1).
In bacteria using the Dsr/Sox pathway the absence of
SoxCD requires a further modification of the Sox reaction
cycle (Fig. 2). The current view is that following the initial
SoxAX and SoxB mediated reactions, the sulfane sulfur
atom of the thiosulfate molecule will remain bound to
SoxYZ [33, 34, 39]. The cycle will then repeat and
incoming, additional thiosulfate molecules will be attached
to these SoxY bound sulfur atoms rather than directly to the
conserved SoxY cysteine. This process is thought to lead to
the formation of chains of sulfur atoms, that can eventually
be transferred to the sulfur globules formed by many of
these bacteria (Fig. 2), although the mechanism by which
the ‘sulfur chain’ is transferred from SoxYZ to the sulfur
globules as well as the length of the sulfur atom chain
required for the transfer reaction is currently unknown.
In summary, in both types of Sox systems, the role of the
SoxAX cytochromes in sulfur oxidation is to catalyze the
formation of a heterodisulfide bond between the conserved
SoxY cysteine (‘GGCGG’ motif) and thiosulfate (eq. 1) or
other sulfur substrates.
SoxZY-SHþ S-SO2�3 þ 2 ferricytochrome c
! SoxZY-S-S-SO�3 þ 2 ferrocytochrome c ð1Þ
In each case, SoxAX proteins have been postulated to be
able to interact with a variety of sulfur substrates or
Fig. 1 Schematic representation of the Sox pathway reaction cycle
with different sulfur substrates, a thiosulfate as substrate, b sulfide as
substrate, c sulfite as substrate
Fig. 2 Schematic representation of the reaction cycle of Sox proteins
involved in thiosulfate oxidation via the Dsr/Sox pathway
The bacterial SoxAX cytochromes 979
123
modified version of the SoxYZ protein; however, it is
unknown at present whether differences in substrate
specificities exist between SoxAX proteins from different
bacteria or those employing different pathways for sulfur
oxidation.
The heterodisulfide bond formation leads to the libera-
tion of two electrons, which are thought to be transferred
from the SoxAX cytochrome to a cytochrome c that
channels them into the electron transport chain, thus con-
tributing to energy conservation [1].
Types of SoxAX proteins and their encoding operons
Although the SoxAX proteins from bacteria employing the
Sox and the Dsr/Sox pathway are thought to catalyze the
same or similar reactions, differences exist in the basic
structure of these proteins. As their name suggests, most
SoxAX proteins are heterodimeric complexes of two c-type
heme bearing subunits, where the SoxA subunits generally
have a molecular mass of around 29 kDa and contain either
one or two heme groups, while the SoxX subunits can vary
in their molecular mass, from *11–20 kDa for the mature
proteins [40]. A further difference between the subunits is
that the SoxA heme groups always have a His/Cys axial
ligation while the single SoxX heme has a His/Met axial
ligand pair.
In addition to heterodimeric forms of SoxAX there also
exist heterotrimeric forms which possess a third, low
molecular weight subunit (‘SoxK’, 9.4 kDa, also known as
the ‘SoxAX binding protein’) that stabilizes the complex of
the associated SoxA and SoxX subunits [41]. At present
there is no data suggesting that the SoxK subunit has
additional functions beyond stabilization of the SoxAX
complex.
The structure of the gene clusters (or regions) encoding
the Sox proteins also appears to differ depending on the
type of bacteria and/or the sulfur oxidation pathways
present. In sox gene clusters from most chemolithotrophic
sulfur oxidizers such as P. pantotrophus, the genes
encoding the core components of the enzyme complex
occur together with genes encoding specialized proteins
involved in maintaining the complex components in their
active state as well as other proteins that regulate expres-
sion of the gene cluster (SoxR) [26, 27, 42]. In bacteria
with Dsr/Sox pathways, however, the genes encoding these
sox–specific accessory proteins appear to be absent or are
located elsewhere on the chromosome, and the sox genes
themselves either form shortened sox gene clusters
(soxJXYZAKBW, found especially in members of the Chlorobi
[43]) that lack soxCD genes or can be located in several
independent loci [30, 41, 44]. As already indicated above,
the absence of soxCD genes is thought to be typical of
bacteria that oxidize reduced sulfur compounds with ele-
mental sulfur as an intermediate [33].
Phylogeny of SoxAX proteins
The phylogenetic relationships between the different forms
of SoxAX proteins have been explored [40, 41] and while
initially only a significant divergence of sequences origi-
nating from Starkeya and Ralstonia species relative to
those from Paracoccus and Chlorobium sp. was apparent.
Due to the limited number of available sequences [40],
recent analyses by Ogawa and coworkers [41] clearly
demonstrated the existence of three distinct types of
SoxAX proteins. Type I and II SoxAX proteins are het-
erodimers, while the heterotrimeric SoxAXK proteins form
the third group. Type I SoxAX proteins have SoxA sub-
units with two heme groups and SoxX subunits with a
molecular mass of *14 kDa, while the Type II SoxA
proteins only contain one heme group, and are associated
with SoxX proteins that have a molecular mass of
approximately 20 kDa. The increased molecular mass in
these SoxX proteins is largely due to the presence of an
N-terminal extension [45]. The Type III SoxAXK proteins
also have single heme group SoxA subunits and distinct
SoxX subunits with a molecular mass of *10 kDa.
According to Ogawa and coworkers [41], the loss of
20–30 N-terminal amino acids distinguishes the single
heme SoxA subunits of Type II from Type III SoxA pro-
teins. Despite these differences, the overall levels of amino
acid sequence identity and similarity are similar for the
three groups.
The phylogenetic analysis carried out by Ogawa [41]
focusses on the SoxAXK proteins and also includes a
number of sequence alignments showing differences and
conserved regions in the primary sequence of the SoxAX(K)
proteins; however, the analysis was limited to sequences from
*40 bacterial species in total.
A recent database search revealed that, at present, there
are over 200 SoxA related protein sequences available, and
we have conducted an analysis of 216 SoxA amino acid
sequences originating from a wide variety of bacterial
species including not only all groups of Proteobacteria,
green sulfur bacteria and Aquificales, but also members of
the Cytophaga group, the Firmicutes and many general
Fig. 3 Phylogenetic relationship of SoxA proteins. Two hundred
sixteen SoxA amino acid sequences were analyzed using Mega 5.0.
The tree shown was generated using the Neighbor-joining algorithm
(Poisson model; uniform rate of evolution for all sites; gap treatment:
pairwise-deletion; robustness testing: bootstrap method with 500
resampling cycles). The different types of SoxAX proteins are
indicated by black bars and labels TI-TIV. A group of several SoxA
related cytochromes was used as the outgroup
c
980 U. Kappler, M. J. Maher
123
The bacterial SoxAX cytochromes 981
123
members of the domain bacteria. Five SoxA related protein
sequences were used as an outgroup (Figs. 3, S1). Phylo-
genetic trees were generated using neighbor-joining,
minimum evolution and maximum likelihood algorithms as
integrated into Mega 5.0 [46] and indicated that in addition
to the three types of SoxA proteins described above, a fourth
group of SoxA proteins may be emerging as more sequences
are available in the database (Fig. 3). At present the putative
fourth SoxA group comprises 13 sequences from several
e-Proteobacteria such as Sulfurimonas and Sulfurihydro-
genibium sp. as well as SoxA from the haloalkaliphilic
c-Proteobacterium Thioalkalivibrio sulfidophilus. The soxA
genes encoding these proteins are mostly located in
soxXYZAB gene clusters, with the exception of the sequences
from Beggiatoa and Halomonas halophila, where the soxA
encoding genes are not found in the vicinity of a soxX gene,
and in fact, genes encoding the second subunit of the SoxAX
protein appear to be absent from the genomes of these bac-
teria. It is possible that these two ‘SoxA’ proteins have a
different function from the remaining proteins present in the
phylogenetic tree; however, as they are clearly related to the
new group of SoxAX proteins it was decided not to remove
them from the alignment (Fig. 3). The soxX genes associated
with the genes encoding the tentatively named ‘Type IV’
SoxA proteins encode proteins with *150 amino acids
(mature protein, mol. mass *16.7 kDa) and contain an
N-terminal extension region. However, database searches
using the ‘Type IV’ SoxX proteins did not reveal any close
relationship of these proteins to the SoxX proteins found in
Type II SoxAX proteins. The properties of the putative
‘Type IV’ SoxAX proteins will need to be evaluated in more
depth as more and especially biochemical data become
available.
Another feature that becomes apparent when examining
the SoxA phylogenetic tree (Fig. 3) is that several bacteria
contain multiple copies of SoxAX which can belong to
several SoxAX types. An example are the Bradyrhizobium
species, which contain at least two Type I and one Type III
SoxAX protein (Figs. 3, S1), but multiple copies of SoxAX
proteins are also found in Starkeya novella (Types I and II),
Thiobacillus denitrificans (2 9 Type III), Thioalkalivibrio
sulfidophilus (2 9 Type II, 1 9 Type ‘IV’) and other
bacteria. The genes encoding these proteins can either be
located in different gene loci or form concatenations, as is
the case in Thioalkalivibrio sulfidophilus where genes
Tgr7_853-858 correspond to three consecutive copies of
SoxAX genes encoding the two different types of SoxAX
proteins found in this organism. The biological significance
of these multiple soxAX gene copies is unknown at present,
but could be indicative of either specialized functions
associated with the different copies of soxAX genes or
could indicate biological redundancy within the Sox sys-
tems of different organisms.
Ogawa et al. [41] also analyzed the structure of gene loci
encoding SoxAX proteins with a special emphasis on genes
encoding Type III SoxAX proteins for which they uncov-
ered a remarkable diversity in the organization of gene
clusters while only a limited analysis of gene clusters
encoding Type I and Type II SoxAX proteins was carried
out [41]. For Type I SoxAX proteins, the P. pantotrophus
gene cluster is shown, while for the Type II proteins two
sox gene clusters with an organization of the core sox genes
(in bold) in two transciptional units-soxWVXA-sox-
YZBCDxF are presented. It should be noted, however, that
in particular the genes encoding Type II SoxAX proteins
are not always found in gene clusters consisting of two
transcriptional units (this is the case for S. novella and
several Rhodopseudomonas sp. as shown by [41]). For
example, in Comamonas species the genes encoding Type
II SoxAX proteins are part of a soxCDYZAXB gene cluster
which forms only one transcriptional unit and also differs
from the ‘canonical’ organization of the core sox genes
(soxXYZABCD) seen, e.g., in P. pantotrophus [47].
An interesting detail of Ogawa’s extensive analysis of
the Type III soxAX gene clusters is that the SoxK subunit
is not always encoded in close proximity to the soxA
genes (e.g. in Thiobacillus denitrificans) and in one case
(Thiomicrospira crunogena) it appears to be absent from
the genome [41]. It would be interesting to investigate
whether a SoxK subunit is an essential component of all
Type III SoxAX proteins or not, and whether other small
proteins can take over the function of SoxK in bacteria
that contain Type III SoxAX proteins but lack a SoxK
homologue.
Crystal structures of SoxAX proteins
At present, crystal structures have been solved for two
Type I SoxAX proteins from R. sulfidophilum (RsSoxAX,
PDB code 1H33, 1.75 A resolution; [1]) and P. pantotrophus
(PpSoxAX, PDB code 2C1D, 1.92 A resolution; [48]),
respectively and one Type II protein from S. novella
(SnSoxAX, PDB code 3OA8, 1.77 A resolution; [45]).
The SoxA structures
In all three structures, the SoxA subunits have a two-
domain structure, with pseudo twofold symmetry between
the domains. Each domain has a mitochondrial c-type
cytochrome fold. In the case of the Type I proteins, each
domain binds a heme cofactor (hemes 1 and 2, in the
nomenclature used for the RsSoxAX structure) with His/
Cys axial coordination. The structure of the SnSoxA
subunit has a similar domain configuration, but binds only
982 U. Kappler, M. J. Maher
123
a single heme cofactor (the equivalent of Type I, heme 2).
In the place of heme 1, there is a disulfide bond (Cys A74-Cys
A110) (Fig. 4). The presence of the disulfide bond in the
SnSoxA structure accompanies a difference in the confor-
mation of a loop comprised of residues 73–83, when
compared with the RsSoxA protein. This loop reaches in
toward the core of the molecule in the SnSoxA structure,
but faces away and forms part of the surface of the protein
in the RsSoxA model (Fig. 4).
The Type I SoxA structures are distinguished by the
presence of a significant N-terminal extension of approxi-
mately 50 residues, which ‘caps’ the heme 2 binding site
(Fig. 4) and restricts the solvent accessibility to the heme
cofactor. Significantly, the propionate groups of the heme 2
cofactor are solvent-accessible in the SnSoxA protein, but
buried in the RsSoxA and PpSoxA structures (Fig. 5). This
potentially restricts access to the active site, and may
influence the (as yet undefined) substrate specificities of the
respective proteins.
Despite the differences in the polypeptide structures of
the SoxA subunits between proteins from different fami-
lies, the coordination structures of the heme 2 binding sites
are remarkably conserved. However, while in the two
crystal structures of Type I SoxAX proteins the cysteine
ligands to the active site hemes were found to be quanti-
tatively modified to a cysteine persulfide (CSS) [1, 48], in
the SnSoxAX structure, the coordinating residue A236 was
modelled as an equal mixture of Cys and post-transla-
tionally modified cysteine persulfide (CSS) [45]. The CSS
modification has been suggested to result from incomplete
catalysis, where only the thiosulfate sulfone sulfur (rather
than the entire thiosulfate moiety) is transferred to SoxYZ
[1]. Given that the SnSoxAX protein was produced
recombinantly and not exposed to the SoxYZ protein
Fig. 4 Comparison of the SoxA and SoxX structures from Starkeyanovella and Rhodovulum sulfidophilum. a The structure of SnSoxA.
The heme cofactor (heme 2, using RsSoxA nomenclature) is
highlighted in orange and the Cys A74 and Cys A110 Sc atoms,
which participate in the disulfide linkage, which replaces the heme 1
site, are shown as yellow spheres. b The structure of RsSoxA. Heme
cofactors 1 and 2 are highlighted in orange. The N-terminal extension
(residues 1–51), which ‘caps’ the heme 2 binding site is represented in
dark blue. For both structures, the loop (residues 73–83, SnSoxA;
residues 79–87, RsSoxA) which shows a different conformation in the
SnSoxA and RsSoxA structures is represented in pink. c The structure
of SnSoxX. The N-terminal extension present in the SnSoxX structure
is represented in blue and the disulfide linkage, between residues Cys
B64 and Cys B175 is shown as yellow spheres (the positions of the
Cys Sc atoms are represented). d The structure of RsSoxX. For both
c and d, the heme cofactors and their axial Met and His ligands are
shown in orange. Features, which show different conformations in the
two structures are highlighted in pink (residues B109–B121 for
SnSoxX and residues B97–B119 for RsSoxX)
The bacterial SoxAX cytochromes 983
123
during its production for crystallisation [45], the reason for
the presence of a partial modification of the active site
cysteine residue in the SnSoxAX structure is presently
unclear.
The SoxX structures
At the core of all three SoxX structures is a tightly folded
heme-binding domain, with the heme ligated by His and
Met axial ligands (Fig. 4). In all three structures the heme
cofactor and propionate groups form part of the solvent-
exposed surface of the protein (Fig. 6). This correlates with
the proposal that the SoxX heme is the site of electron
storage and transfer to the electron-transfer partner cyto-
chrome c during the turnover of the enzyme. The exposed
part of the SoxX heme cofactor is surrounded by a ring of
hydrophobic surface residues, which is bordered by regions
of negative potential (Fig. 6). The sequence of the putative
electron acceptor for SnSoxAX, cytochrome c550 from
S. novella, predicts a basic pI (*8.5) for the protein, in
addition, both the assay for the entire Sox system and the
SoxAX assay can be carried out using horse heart cyto-
chrome c (pI *10) [49, 50]. It is likely that the region
described represents a docking site between SoxAX and the
external, electron accepting cytochrome c during electron
transfer.
The SoxAX dimer
The structure of the SnSoxX protein (Type II) shows an
N-terminal extension of the SnSoxX subunit by *65 res-
idues (residues B29-B95 in SnSoxX) that is not present in
the RsSoxAX and PpSoxAX structures and is tethered to
the heme-binding domain through a disulfide bridge
(SnSoxX residues Cys B64–Cys B175) and three hydro-
gen-bonding interactions. The presence of this N-terminal
extension correlates with the buried surface area in the
dimer being significantly greater for the Type II protein
than for the Type I proteins (19 and 16 % buried
surface areas on complex formation for the Type II and
Fig. 5 Active sites of the SnSoxAX and RsSoxAX structures. Panels
a and b SnSoxAX with and without surface representations,
respectively; Panels c and d RsSoxAX. In all panels the heme 2
cofactors is represented in orange and the Cys active site residue in
hot pink. In the SnSoxAX and RsSoxAX structures, residues Gln 197
and Asp 192, respectively (indicated in stick representations) ‘gate’
access to the active site
984 U. Kappler, M. J. Maher
123
Type I proteins, respectively). Presumably, this results in
increased dimer stability for the Type II proteins (Fig. 6).
In all structures the SoxAX dimer features a deep trough
along the SoxA protein, near the interface between sub-
units. The heme 2 cofactor and the active site Cys/CSS
residue lie at the bottom of this trough. An electrostatic
surface calculation indicates that this area is positively
charged (Fig. 6). This region of the structure has been
proposed to represent the docking site for the ‘swinging
arm’ of the SoxYZ protein [35] and other substrates [45],
such as thiosulfate, sulfide (HS-) and sulfite (HSO3-)
[35, 47]. Many of these substrates are negatively charged at
neutral pH, so that the positively charged binding pocket
would help to attract the substrate molecule.
Interestingly, a single residue (Gln 197 in SnSoxAX and
Asp 192 in RsSoxAX) seems to ‘gate’ access to the active
site (Fig. 5). In fact, the sequence alignments presented by
Ogawa et al. [41], indicate that the identity of this residue
is different, depending on the classification of the SoxAX
protein: the sequences of the Type I proteins show an
aspartate residue in this position, with glutamine and glu-
tamic acid residues for the Type II and Type III proteins,
respectively. The consequences of this observation for the
activities and substrate specificities of these enzymes have
not been investigated.
Properties of the SoxAX redox centres
An important parameter for the biological function of heme
groups and their ability to participate in electron transfer or
other reactions is their redox potential, which is influenced
to a large extent by the axial ligands present [51]. The His/
Cys axial ligation of the SoxA hemes confers an extremely
low redox potential on these heme groups, with values of
-432 ± 15 and -479 ± 10 mV versus NHE at pH 7.0
having been reported for the P. pantotrophus Type I and
the S. novella Type II SoxAX proteins, respectively [20,
50]. The SoxX heme groups of these two proteins had
potentials of 189 ± 15 and 183 ± 10 mV versus NHE at
the same pH, which is typical for His/Met ligated heme
groups [20, 50]. While for both proteins the SoxX heme
potential was essentially invariant, the potential of the
SoxA heme was reported to change by approximately
45 mV/pH unit for the P. pantotrophus SoxA heme, and a
similar trend was observed for the S. novella protein
[20, 50].
The low redox potential of the SoxA heme means that at
physiological pH values, this heme group is very unlikely
to participate in electron transfer reactions, i.e. the heme
will not be reduced during the reaction catalyzed by the
SoxAX proteins [20, 50]. This poses a potential problem,
Fig. 6 Dimeric structures of the SnSoxAX and RsSoxAX structures.
a SnSoxAX; b RsSoxAX. For both structures, the SoxA subunit is
shown as a surface representation in light blue. The SoxX subunit is
shown as a cartoon in light green and the SoxX heme in orange. c and
d Electrostatic surfaces of the SnSoxAX dimer. Regions of positive
charge are coloured blue and regions of negative charge, red.
Hydrophobic surfaces are represented in white. The heme cofactors
are represented in orange. Panel d is related to c by a 90� rotation
about the x axis
The bacterial SoxAX cytochromes 985
123
as the SoxAX reaction (Eq. 1) requires the transient storage
of two electrons in the SoxAX redox centres, but only one
of the two heme groups will be capable of storing elec-
trons. As so far no evidence for the presence of a radical
species in any of the SoxAX proteins has been found, this
suggests that additional redox centres may be present/
required for the SoxAX reaction. The S. novella SoxAX
protein (Type II) has recently been shown to specifically
bind 1 equivalent of Cu/protein molecule, and this Cu
centre was reported to have a redox potential of
?196 ± 18 mV versus NHE at pH 8.0 which is similar to
the redox potential of the SoxX heme [50]. The exact
location of the Cu centre is unknown at present and could
not be determined from the crystal structure of the corre-
sponding protein as only the ‘as prepared’ form of the
protein gave rise to crystals with suitable diffraction
properties [45]. However, the Cu centre appears to be
located in the vicinity of the SoxA heme, as Cu-loading of
the S. novella SoxAX caused changes in the EPR properties
of the SoxA heme (see below) as well as to its redox
potential: for Cu-loaded S. novella SoxAX the redox
potential at pH 7 was -455 ± 10 mV versus NHE as
opposed to -479 ± 10 mV versus NHE for the as pre-
pared protein [50]. At present, however, the S. novella
SoxAX protein is the only SoxAX protein that has been
shown to bind Cu and thus to contain a non-heme redox
centre and investigation of other SoxAX proteins for their
ability to bind Cu or other metal centres will be required to
confirm the general presence/role of a Cu centre in SoxAX
cytochromes.
Spectroscopy of the SoxAX redox centres
From the beginning the SoxAX cytochromes have been
noted for the complexity of their EPR spectra. The first
EPR spectra of a SoxAX cytochrome were published by
Kelly [15], who identified several regions containing heme-
dependent EPR signals for the SoxAX protein from
Paracoccus versutus. These included a high spin signal
(g * 6), some signals in the g * 3 region, that would
most likely correspond to a His/Met ligated heme such as
the one found in SoxX, and three additional, overlapping
spectra in the g * 2 region. Kelly showed that following
reduction of the protein with dithionite, only the signals in
the g * 2 region remained, which is in keeping with the
low redox potentials of the SoxA hemes that have been
determined since then [20, 50]. Of the three heme-related
features present in the g * 2 region, one did not undergo
changes, while the relative proportions of the other two
varied, e.g. in response to changes in the buffering system
used [15] (Table 1). This led to the suggestion that the
variable components were linked to the same heme group
and would most likely arise from changes in the coordi-
nation environment of the central heme Fe atom. Kelly also
concluded that at least one of the heme groups present in
the P. versutus SoxAX protein might undergo a ligand
switch from a six to a five coordinate state, giving rise to
the observed high spin feature [15].
Subsequent studies of the EPR and MCD properties of
the three heme, Type I SoxAX protein from R. sulfido-
philum largely confirmed these findings and extended the
analyses to an identification of the heme axial ligands
through a combination of EPR and MCD spectroscopy
[52]. In addition to a high spin signal at g * 5.8, the three
heme groups of the R. sulfidophilum protein gave rise to
four heme dependent EPR spectra, a readily reducible
HALS (high anisotropy low spin) heme signal at g * 3.5
(LS3) and three additional heme species in the g * 2
region, that were shown to correspond to hemes with a
Table 1 g-values associated with heme derived EPR species in
different SoxAX cytochromes
Heme Type gz gx gy Ref
S. nov. rSoxAX LS1a II 1.859 2.531 2.349 [53]
S. nov. rSoxAX LS1b II 1.835 2.574 2.348 [53]
S. nov. rSoxAX LS2 II 1.913 2.433 2.271 [53]
S. nov. rSoxAX LS3 I 3.502 – – [53]
S. nov. SoxAX LS1a II 1.853 2.531 2.348 [40]
S. nov. SoxAX LS1b II 1.835 2.556 2.348 [40]
S. nov. SoxAX LS2 II 1.912 2.417 2.268 [40]
S. nov. SoxAX LS3 I 3.502 – – [40]
R. sulf. SoxAX LS1a II 1.870 2.580 2.300 [52]
R. sulf. SoxAX LS1b II 1.840 2.520 2.230 [52]
R. sulf. SoxAX LS2 II 1.910 2.420 2.260 [52]
R. sulf. SoxAX LS3 I 3.50 – – [52]
P. vers. SoxAX LS1 II 1.86 2.55 2.31 [15]
P. vers. SoxAX LS2 II 1.89 2.43 2.27 [15]
P. vers. SoxAX LS3 I 3.5 – – [15]
P. vers.B SoxAX LS1a II 1.834 2.583 2.395 [15]
P. vers.B SoxAX LS1b II 1.875 2.516 2.302 [15]
P. vers.B SoxAX LS2 II 1.915 2.40 2.245 [15]
P. vers.B SoxAX LS3 I 3.5 – – [15]
P. panto. SoxAX LS1a II 1.87 2.54 2.3 [20]
P. panto. SoxAX LS2 II 1.9 2.43 2.26 [20]
P. panto. SoxAX LS3 I 3.45 – – [20]
S. nov. C236 M 1 II 1.634 2.879 2.25 [45]
S. nov. C236 M 2 II 1.379 2.979 2.3 [45]
S. nov. C236 M 3 I 3.174 – – [45]
S. nov. C236 M LS3 I 3.495 – – [45]
S. nov., Starkeya novella; R. sulf., Rhodovulum sulfidophilum;P. vers., Paracoccus versutus samples (no mark, B) for this organism
differ in the buffering systems used; P. panto., Paracoccuspantotrophus
986 U. Kappler, M. J. Maher
123
histidine/thiolate axial ligation [52] (Table 1). In keeping
with the suggestions of Kelly, it was postulated that the
single heme group giving rise to two preparation-depen-
dent EPR signals (LS1A and B), was likely to undergo a
ligand switch [52]. The alternative ligand was suggested to
be a cysteine-persulfide [52], which was later confirmed to
exist in the crystal structure of the same protein [1]. The
invariant signal in the g * 2 region (LS2) was thought to
be linked to the second His/Cys ligated heme group present
in the R. sulfidophilum diheme SoxA subunit [52].
The strong influence of the exact experimental condi-
tions as well as the preparation method on the relative
abundance of different EPR active forms of the SoxA
thiolate ligated hemes was highlighted by studies of the
triheme Type I SoxAX protein from P. pantotrophus, in
which a high spin heme component was nearly absent, and
the g * 2 region only contained two heme dependent
spectra, LS1 and LS2 [20].
The assignment of the LS1 and LS2 components of the
SoxA EPR spectra to the two heme groups present in the
SoxA subunits of the triheme Type I SoxAX proteins,
however, appears to be an oversimplification. EPR spectra
of the diheme, Type II SoxAX protein from S. novella also
contained EPR active components corresponding to LS1A
and B, LS2 and LS3 [53] although this protein contains
only a single active site SoxA heme group [40] (Fig. 7). If
the LS2 spectrum was exclusively caused by the second
SoxA heme it should have been absent from EPR spectra of
a Type II SoxAX protein. This result clearly demonstrated
that the active site SoxA heme group contributes not only
to the LS1 but also the LS2 component of the EPR spectra,
and indicated that this heme group can adopt a larger
number of conformations than previously thought [40].
This conformational flexibility of the SoxA active site
heme was also evident in EPR studies of a variant of the S.
novella SoxAX protein (SnSoxAXC236M), in which the
SoxA heme ligating cysteine was replaced by a methionine
in an attempt to create a stable ligand environment around
the SoxA heme [45]. Instead of the intended simplification
of the SnSoxAXC236M EPR properties, the EPR spectra
were found to be more complex, and contained four new
signals (three in the g * 2 region, one in the g * 3
region) in addition to the LS3 SoxX heme signal and an
intense high spin signal in the g * 6 region [45] (Table 1).
The crystal structure of this protein showed that the heme
ligating Met236 was in an unusual rotamer conformation,
which would influence the EPR properties of the system
due to changes in the orbital interactions between the Fe
atom and the axial ligand [45]. The distances of the heme
ligands to the central Fe atom also varied considerably
(e.g. 2.56–2.9 A for the Fe-Met bondlength) between the
two SnSoxAXC236M structures present in one asymmetric
unit [45], with a bondlength of 2.9 A for the Fe-Met bond
in one of these two structures essentially suggesting a heme
group in transition to a high spin state. These variations
were observed despite the fact that the amino acid substi-
tution did not affect the structure of the backbone of the
SnSoxAXC236M protein [45].
Together, all these observations indicate that the SoxA
heme site has a significant inherent structural flexibility,
which in turn gives rise to the observed microheterogene-
ity in the EPR spectra associated with this heme group
[15, 20, 40, 45, 50, 52, 53]. The varying extents of flexi-
bility observed for SoxAX proteins isolated from different
species could either reflect differences in the preparation
methods and buffering systems used, or might be caused by
subtle changes in the protein environment of the SoxA
active site heme although this is not apparent from the
currently available crystal structures. Whether the struc-
tural flexibility of the SoxA site is important for catalysis
and the proposed ligand exchange reaction that might
underlie the formation of the heterodisulfide bond remains
to be elucidated, although the structural flexibility may be
necessary as SoxAX proteins are required to interact with a
variety of substrates.
Fig. 7 EPR spectra of the Type II SoxAX protein from Starkeyanovella Panel a CW-EPR spectrum of S. novella SoxAX (50 mM, in
20 mM Tris–HCl, pH 8.0, T = 2 K), Panel b S. novella SoxAX
CW-EPR signals in the g * 2 region (50 mM, in 20 mM Tris–HCl,
pH 8.0, T = 2 K), a experimental spectrum, b,c,d simulations of
LS1A, LS1B and LS2, respectively (adapted from [53]) Panel
c Spectrum i, experimental CW-EPR spectrum of Cu(II)-loaded
S. novella SoxAX, (T = 60.0 K), ii spectrum i corrected for heme
dependent components (adapted from [50]
The bacterial SoxAX cytochromes 987
123
EPR studies of the Type II SoxAX Cu centre
Cu related resonances have been observed in the EPR
spectra of as prepared SoxAX proteins from R. sulfidophi-
lum, P. pantotrophus and S. novella, and in all cases were
initially attributed to adventitiously bound Cu(II) [20, 40,
52]. However, following the observation that the S. novella
SoxAX binds one equivalent of Cu per molecule EPR studies
of the Cu centre in this protein were carried out [45, 50] and
suggest that it is a tetragonally distorted, square planar
Cu(II) centre [50]. Partially resolved nitrogen hyperfine
resonances were also present, and the authors concluded
that the Cu was most likely coordinated by three nitrogen
and one oxygen ligand, or four nitrogen ligands [50]
(Fig. 7). EPR spectra of Cu-loaded S. novella SoxAX
showed line broadening for the LS2 species associated with
the SoxA heme, indicating the presence of dipole–dipole
interactions between the Cu centre and the SoxA heme,
which would suggest a distance of 10–15 A between the
two redox centres [50]. Changing the axial ligand field of
the SoxA heme, e.g. in the SoxAXC236M protein, resulted in
changes of the EPR properties of the Cu centre, which was
also taken as evidence that the two centres would most
likely be located in close proximity [45]. The differences in
the SnSoxAXWT and SnSoxAXC236M Cu EPR were attrib-
uted either to a change in the number of coordinated
nitrogen nuclei or a change in the charge state of the Cu
centre [50].
The most important issue regarding the SoxAX Cu
centre at this stage is the identification of its exact location
and the ligands involved in binding the Cu. Histidine res-
idues are typical ligands for Cu centres, but the S. novella
SoxA subunit contains only two His residues that are not
axial ligands for heme groups, and only one of these His
residues is located in proximity to the SoxA heme. While
this histidine residue that could potentially be involved in
binding the Cu centre is not conserved in the Type I
SoxAX proteins, it is found in the majority of Type II
SoxAX proteins that are currently in the database.
SoxAX activity measurements
The proposed reaction for SoxAX in all pathways in which
it has been shown to play a role is the formation of a
heterodisulfide bond between an incoming sulfur substrate
molecule (e.g. thiosulfate) and the conserved cysteine
residue present at the C-terminus of the SoxY subunit of
the SoxYZ carrier protein (Eq. 1). Details of the reaction as
well as the SoxAX reaction mechanism, however, remain
to be elucidated. Efforts to study details of the SoxAX
reaction have been hampered by the absence of a suitable
assay for the SoxAX catalyzed reaction. Most assays of
SoxAX activity have been carried out using a reconstituted
Sox system, in which all essential Sox proteins are present
in small amounts, and such assays have clearly shown that
in photo- and chemotrophic sulfur oxidizers, the reaction
catalyzed by SoxAX is essential for the function of the Sox
pathway [41, 49] as in its absence only a residual amount
of activity (*4.5 %) remains [49].
In some cases a reconstituted Sox system containing a
mixture of components from a photo- and a chemotrophic
bacterium representing the Dsr/Sox and the Sox pathway,
respectively, may also be functional [54]. However, this
depended strongly on which components of the system
originated from the photo- or chemotroph: While the
phototroph SoxYZ interacted well with the proteins
derived from a chemotrophic bacterium, the SoxB protein
from the phototrophic bacterium did not [54]. This work
highlights the difficulties inherent in developing assays for
the activities of individual Sox proteins–either purified
samples of all the core Sox proteins from the same
organism are required, or, in order to create a system that
isolates a single reaction, large amounts of purified SoxYZ,
which participates in all reactions of the Sox system as a
substrate will be needed (Figs. 1, 2).
Some efforts have been made to create an in vitro assay
system for SoxAX. The first assay system reported contained
20 mM of a suitable buffer (MES pH 6.0 or Tris–acetate pH
7.0), 0.04 mM cytochrome c from horse heart, a catalytic
amount of purified SoxAX and 1 mM reduced glutathione
[50]. In this assay, the reduced glutathione was proposed
to take the place of both thiosulfate and SoxYZ as sulfur
substrates (Eq. 2), and the reaction is thought to lead to the
formation of oxidized glutathione and reduced cytochrome c,
with the latter being monitored spectrophotometrically
(Eq. 3). Combinations of GSH with, e.g. thiosulfate or other
second sulfur substrates could not be used in this assay system
due to high background activity being observed.
SoxZY-SH þ S-SO2�3 þ 2 ferricytochrome c
! SoxZY-S-S-SO�3 þ 2 ferrocytochrome c ð2Þ
2GSH þ 2 ferricytochrome c! GSSGþ 2 ferrocytochrome c
ð3Þ
Using this system the activity of SoxAX from S. novella
was assayed, and revealed KM_GSH values of 0.49 ±
0.12 mM and 0.195 ± 0.012 mM at pH 6 and 7, respec-
tively (Table 2). The corresponding turnover numbers
(kcat) were 8.72 ± 0.84 s-1 and 3.7 ± 0.25 s-1 [45, 50].
The assays were conducted using Cu-loaded SoxAX
protein after it was observed that relative to the ‘as
prepared’ protein, Cu-loaded protein had a 14 times
increased activity in the assay system (1.54 U/mg vs.
0.124 U/mg for the as prepared protein) [50].
988 U. Kappler, M. J. Maher
123
A second assay system was also developed where
thiosulfate and purified, recombinant SoxYZ were used as
the sulfur substrates to mimic the in vivo reaction of
SoxAX more closely. However, as SoxYZ proteins easily
undergo oxidation and thus inactivation following purifi-
cation [19], a major issue with such a system is how SoxYZ
can be kept in a stable and active redox state, and how this
redox state can be consistently reproduced. In vivo this is
accomplished by the action of various accessory Sox pro-
teins [25, 54]; for the in vitro assay the amount of free SH
groups present on SoxYZ was used as a guide to the
amount of ‘active’ SoxYZ in the preparation [45]. The
limited availability of recombinant SoxYZ, however, pre-
cluded the determination of catalytic parameters using this
assay system [45]. Using a tenfold excess of SoxYZ over
SoxAX (4.86 lM and 0.048 lM concentrations were used,
respectively), the activity of Cu-loaded S. novella wild type
SoxAX was 0.165 ± 0.021 U/mg (Table 2).
The SoxAX reaction mechanism
Several proposals for the catalytic mechanism of SoxAX
cytochromes have been made, and the suggestions vary
largely depending on whether additional redox centres
(such as a Cu centre) are thought to be present in the
SoxAX protein or not.
The first proposal for a SoxAX reaction mechanism was
made based on the crystal structure of the Type I SoxAX
protein from R. sulfidophilum [1] and used elements of the
reaction mechanisms of sulfur transferase/rhodanese
enzymes as a model. Sulfurtransferases [EC 2.8.1.-] cata-
lyze the transfer of a sulfane sulfur atom between substrate
molecules.
The R. sulfidophilum SoxAX crystal structure revealed
important similarities between the active sites of sulfur
transferases and the one found in SoxAX, namely the
presence of a strongly positively charged environment and
a catalytically active cysteine residue, i.e. the cysteine
ligand to the SoxA active site heme [1]. The fact that this
heme group carries a cysteine-persulfide modification
(which has also been identified in all subsequently solved
structures of SoxAX proteins [45, 48]) was taken as evi-
dence for its catalytic function; however, it was noted that a
main difference between the sulfur transferase mechanism
and that of SoxAX is that SoxAX is a redox active enzyme,
while rhodaneses are non redox active enzymes that only
perform a group transfer reaction [1]. An arginine (Arg218,
R. sulfidophilum numbering) close to the SoxA active site
heme and the cysteine ligand were proposed to be involved
in orienting the incoming thiosulfate molecule so that the
thiosulfate sulfane sulfur would be in close proximity to the
catalytic cysteine residue [1]. Initially, a covalent bond
would be formed between the cysteine and the thiosulfate
molecule, leading to a two electron reduction of the SoxAX
protein, and it was proposed that the two electrons liberated
by the reaction would be stored in the SoxX and the SoxA
active site heme [1], (hemes 2 and 3 in the nomenclature of
[1]).
In a second reaction an incoming SoxYZ molecule
would react with the SoxA-thiocysteine-S-sulfate complex,
which could result either in a transfer of the entire thio-
sulfate moiety to SoxYZ, or could lead to an incomplete
reaction where only the thiosulfate sulfone sulfur would be
transferred, leaving the crystallographically observed per-
sulfide modified cysteine which could be regenerated in a
second reaction with another SoxYZ molecule [1].
The fact that the SoxA heme cysteine ligand is known to
undergo modification on incubation with different sulfur
substrates supports the suggestion that the heme-ligating
cysteine is active in catalysis [53].
This is a very elegant suggestion for a potential reaction
mechanism, however, it requires that the SoxA heme par-
ticipates in the transient storage of one of the two electrons
that are liberated during the formation of the disulfide
bond, and as has subsequently been shown [20, 50], the
extremely low redox potential of this heme group prevents
it from storing electrons under physiological conditions.
This then leads to the question of what could be hap-
pening to the second electron liberated during heterodisulfide
bond formation and how it could be stored in the SoxAX
protein. The additional heme group that is present in
Type I SoxAX proteins has the same axial ligation as the
active site heme and thus will also have an extremely low
redox potential that would stop it from acting as an
electron sink even if it was located close enough to the
active site to easily accept electrons which is not the case
[1, 48].
Based on the observation that the S. novella Type II
SoxAX protein is capable of binding exactly one equiva-
lent of Cu and that Cu-loading of this protein resulted in
Table 2 Activity of the Type II SoxAX protein from Starkeyanovella in in vitro SoxAX activity assays
S. novella SoxAXWT S. novella SoxAXC236M
GSH-based assay, pH 7.0
KM_GSH (mM) 0.49 ± 0.12 n.r. [50]
kcat (s-1) 8.72 ± 0.84 n.r. [50]
GSH-based assay, pH 6.0
KM_GSH (mM) 0.195 ± 0.012 0.228 ± 0.027 [45]
kcat (s-1) 3.7 ± 0.3 2.0 ± 0.5 [45]
SoxYZ assay, pH 6
U/mg 0.165 ± 0.021 0.114 ± 0.022 [45]
n.r. not reported
The bacterial SoxAX cytochromes 989
123
changes to the EPR properties of the SoxA active site
heme, it has been suggested that the Cu centre could be
important in SoxAX catalysis [50]. The Cu atom would
provide a redox centre capable of storing one electron and
Cu atoms have also been noted for their reactivity towards
sulfur compounds [55], which could enhance SoxAX
catalysis. In the presence of a catalytically active Cu cen-
tre, the proposed SoxA thiocysteine–S-sulfate form of the
protein might be an intermediate that would ‘trap’ thio-
sulfate inside the SoxA protein, ready for reaction with an
incoming SoxYZ protein. Given that SoxYZ is a protein
complex (*28 kDa) and thus interaction not only requires
proximity to SoxAX but also the correct orientation of both
proteins as well as a suitable position of the mobile
GGCGG motif [35], it is to be expected that interactions
with SoxYZ would form on a different timescale and be
less frequent than interactions with a small molecule such
as thiosulfate that can easily diffuse into the SoxAX active
site. Thus, the interaction with SoxYZ might be a rate-
limiting step in SoxAX catalysis. If the Cu centre were
located within 10–15 A of the SoxA active site heme it
could be involved in promoting the formation of either
modifications to the SoxA heme cysteine ligand and/or the
subsequent reaction with the incoming SoxYZ protein.
This mechanism would also explain why in the
GSH-based assay system the Cu-loaded S. novella SoxAX
protein had a higher activity than the ‘as prepared’ SoxAX
that only contained about 10–15 % Cu. Further evidence
in favor of the involvement of the Cu centre comes
from SoxAX activity assays with the already mentioned
S. novella SoxAXC236M protein in which the cysteine heme
ligand has been replaced. It was expected that if the cys-
teine were crucial to the reaction mechanism as it would be
if no additional redox centres were involved in the reaction,
this protein should have no catalytic activity. How-
ever, Cu–loaded SnSoxAXC236M was catalytically active
although turnover was reduced by nearly 50 % (Table 2).
Binding of the artificial sulfur substrate GSH was not
significantly affected (KM_GSH values were similar for both
SnSoxAXC236M and SoxAXWT protein). In the SoxYZ
based assay, SnSoxAXC236M had *70 % of the activity of
the wild type enzyme (0.114 U/mg) when assayed under
the same conditions as the wild type protein [45]. Together
these observations suggest that the SoxA heme cysteine is
important but not crucial for SoxAX activity and that the
proposed Cu centre could be involved in speeding up the
heterodisulfide bond formation [45].
Further work is clearly needed, however, to unravel the
molecular details of the SoxAX reaction, including the
exact location and properties of the Cu centre, its presence
in other types of SoxAX proteins as well as experiments
detailing the role of the active site arginine in catalysis and
the actual products produced in a SoxYZ based assay
system.
Concluding remarks
SoxAX cytochromes are a unique type of heme-con-
taining enzymes that are essential for the bacterial
oxidation of thiosulfate because they initiate the reaction
of the Sox system in both photo- and chemotrophic sulfur
oxidizing bacteria. They are found in nearly all known
groups of bacteria, and additional forms of these proteins
may be discovered as more genome sequences become
available. The structural features underlying the forma-
tion of the SoxAX complex are worth investigating as in
some cases stable complexes are formed between the
SoxA and SoxX subunits while in other cases a third
protein, SoxK (or SAXB) is required to achieve complex
formation.
Features of central interest are the redox centres of
SoxAX and how they shape the catalytic mechanism of
these proteins. There is consensus regarding the nature
of the reaction catalyzed by SoxAX, namely the formation
of a heterodisulfide bond between the SoxYZ carrier pro-
tein and sulfur substrates such as thiosulfate. Details of the
mechanism are unclear, however, partly due to the absence
of an assay system that would be readily available and
closely mimic the interactions of SoxAX with both its
protein and its inorganic sulfur substrate. Another feature
that requires further investigation to confirm or disprove its
general role in sulfur oxidation is the Cu centre that appears
to be present in the Type II SoxAX protein from S. novella.
Further work should focus on establishing the presence of
this redox centre in other SoxAX proteins and also aim to
more clearly define the binding site required. If the SoxAX
reaction mechanism would involve this Cu centre, the
binding site would have to be capable of accommodating
both Cu(I) and Cu(II), and it could also be expected to be
conserved in other SoxAX proteins.
Another open issue is the exact role of the SoxAX active
site heme and its cysteine ligand. The emerging picture is
that the SoxA heme site, which is clearly implicated in
catalysis is also a site of inherent structural flexibility that
underlies the complexity of the EPR signals observed for
all SoxAX proteins studied to date. Whether this flexibility
is a prerequisite for catalysis is unknown, but it is possible
that this is the feature enabling the formation of the high-
spin heme signals that have been observed in EPR studies
of many SoxAX proteins. Based on current knowledge it
seems reasonable to suggest that if the heme-ligating cys-
teine is catalytically active it could at least temporarily
cease to be a direct axial ligand to the SoxA heme.
990 U. Kappler, M. J. Maher
123
Acknowledgments This work was supported by a fellowship
(Australian Research Fellowship, DP0878525) from the Australian
Research Council to UK. MJM is supported by a La Trobe Institute
for Molecular Science Senior Research Fellowship.
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