the bacterial soxax cytochromes - springer · the soxax heme thiolate proteins also belong to this...

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: [email protected]

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

http://dx.doi.org/10.1007/s00018-012-1098-y

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


123


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


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)


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


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


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


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]


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].


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


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.


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.

References

1. Bamford VA, Bruno S, Rasmussen T, Appia-Ayme C, Cheesman

MR, Berks BC, Hemmings AM (2002) Structural basis for the

oxidation of thiosulfate by a sulfur cycle enzyme. EMBO J

21(21):5599–5610

2. Lu WP, Kelly DP (1988) Respiration-driven proton translocation

in Thiobacillus versutus and the role of the periplasmic thiosul-

phate-oxidizing enzyme system. Arch Microbiol 149:297–302

3. Omura T (2005) Heme-thiolate proteins. Biochem Biophys Res

Comm 338(1):404–409

4. Green MT (2009) CH bond activation in heme proteins: the role

of thiolate ligation in cytochrome P450. Curr Opin Chem Biol

13(1):84–88

5. Pazicni S, Cherney MM, Lukat-Rodgers GS, Oliveriusova J,

Rodgers KR, Kraus JP, Burstyn JN (2005) The heme of cysta-

thionine beta-synthase likely undergoes a thermally induced

redox-mediated ligand switch. Biochemistry 44(51):16785–

16795

6. Youn H, Conrad M, Chung SY, Roberts GP (2006) Roles of the

heme and heme ligands in the activation of CooA, the CO-

sensing transcriptional activator. Biochem Biophys Res Comm

348(2):345–350

7. Dhawan IK, Shelver D, Thorsteinsson MV, Roberts GP, Johnson

MK (1999) Probing the heme axial ligation in the CO-sensing

CooA protein with magnetic circular dichroism spectroscopy.

Biochemistry 38(39):12805–12813

8. Alric J, Tsukatani Y, Yoshida M, Matsuura K, Shimada K,

Hienerwadel R, Schoepp-Cothenet B, Nitschke W, Nagashima

KVP, Vermeglio A (2004) Structural and functional character-

ization of the unusual triheme cytochrome bound to the reaction

center of Rhodovulum sulfidophilum. J Biol Chem 279(25):26090–

26097

9. Grein F, Venceslau SS, Schneider L, Hildebrandt P, Todorovic S,

Pereira IAC, Dahl C (2010) DsrJ, an essential part of the

DsrMKJOP transmembrane complex in the purple sulfur bacte-

rium Allochromatium vinosum, is an unusual triheme cytochrome

c. Biochemistry 49(38):8290–8299

10. Grein F, Pereira IAC, Dahl C (2010) Biochemical characteriza-

tion of individual components of the Allochromatium vinosumDsrMKJOP transmembrane complex aids understanding of

complex function in vivo. J Bacteriol 192(24):6369–6377

11. Lu WP, Swoboda BEP, Kelly DP (1985) Properties of the thio-

sulphate oxidising multi-enzyme system from Thiobacillusversutus. Biochim Biophys Acta 828:116–122

12. Lu WP, Kelly DP (1984) Properties and role of sulphite:cyto-

chrome c oxidoreductase purified from Thiobacillus versutus.

J Gen Microbiol 130:1683–1692

13. Lu W-P, Kelly DP (1984) Purification and characterization of two

essential cytochromes of the thiosulphate-oxidizing multi-

enzyme system from Thiobacillus A2 (Thiobacillus versutus).

Biochim Biophys Acta 765(2):106–117

14. Katayama Y, Hiraishi A, Kuraishi H (1995) Paracoccus thioc-yanatus sp. nov., a new species of thiocyanate- utilizing

facultative chemolithotroph, and transfer of Thiobacillus versutusto the genus Paracoccus as Paracoccus versutus comb. nov. with

emendation of the genus. Microbiology 141:1469–1477

15. Kelly DP, Shergill JK, Lu WP, Wood AP (1997) Oxidative

metabolism of inorganic sulfur compounds by bacteria. Antonie

Van Leeuwenhoek 71(1–2):95–107

16. Mittenhuber G, Sonomoto K, Egert M, Friedrich CG (1991)

Identification of the DNA region responsible for sulfur-oxidizing

ability of Thiosphaera pantotropha. J Bacteriol 173(2):7340–

7344

17. Wodara C, Kostka S, Egert M, Kelly DP, Friedrich CG (1994)

Identification and sequence analysis of the soxB gene essential

for sulfur oxidation of Paracoccus denitrificans GB17. J Bacte-

riol 176:6188–6191

18. Ludwig W, Mittenhuber G, Friedrich CG (1993) Transfer of

Thiosphaera pantotropha to Paracoccus denitrificans. Int J Sys

Bacteriol 43(2):363–367

19. Quentmeier A, Li L, Friedrich CG (2008) Identification of two

inactive forms of the central sulfur cycle protein SoxYZ of

Paracoccus pantotrophus. FEBS Lett 582(25–26):3701–3704

20. Reijerse EJ, Sommerhalter M, Hellwig P, Quentmeier A, Rother

D, Laurich C, Bothe E, Lubitz W, Friedrich CG (2007) The

unusual redox properties of SoxXA, a novel c-type heme-enzyme

essential for chemotrophic sulfur-oxidation of Paracoccus pan-totrophus. Biochemistry 46(26):7804–7810

21. Bardischewsky F, Quentmeier A, Friedrich CG (2006) The fla-

voprotein SoxF functions in chemotrophic thiosulfate oxidation

of Paracoccus pantotrophus in vivo and in vitro. FEMS Micro-

biol Lett 258(1):121–126

22. Bardischewsky F, Quentmeier A, Rother D, Hellwig P, Kostka S,

Friedrich CG (2005) Sulfur dehydrogenase of Paracoccus pan-totrophus : the heme-2 domain of the molybdoprotein

cytochrome c complex is dispensable for catalytic activity. Bio-

chemistry 44(18):7024–7034

23. Friedrich CG, Rother D, Bardischewsky F, Quentmeier A,

Fischer J (2001) Oxidation of reduced inorganic sulfur com-

pounds by bacteria: emergence of a common mechanisms? Appl

Environ Microbiol 67(7):2873–2882

24. Quentmeier A, Kraft R, Kostka S, Klockenkamper R, Friedrich

CG (2000) Characterization of a new type of sulfite dehydroge-

nase from Paracoccus pantotrophus GB17. Arch Microbiol

173(2):117–125

25. Rother D, Ringk J, Friedrich CG (2008) Sulfur oxidation of

Paracoccus pantotrophus: the sulfur-binding protein SoxYZ is

the target of the periplasmic thiol-disulfide oxidoreductase SoxS.

Microbiology 154:1980–1988

26. Bardischewsky F, Fischer J, Holler B, Friedrich CG (2006) SoxV

transfers electrons to the periplasm of Paracoccus pantotro-phus—an essential reaction for chemotrophic sulfur oxidation.

Microbiology 152:465–472

27. Rother D, Orawski G, Bardischewsky F, Friedrich CG (2005)

SoxRS-mediated regulation of chemotrophic sulfur oxidation in

Paracoccus pantotrophus. Microbiology 151:1707–1716

28. Bardischewsky F, Friedrich CG (2001) Identification of ccdA in

Paracoccus pantotrophus GB17: disruption of ccdA causes

complete deficiency in c-type cytochromes. J Bacteriol 183(1):

257–263

29. Bardischewsky F, Friedrich CG (2001) The shxVW locus is

essential for oxidation of inorganic sulfur and molecular hydro-

gen by Paracoccus pantotrophus GB17: a novel function for

lithotrophy. FEMS Microbiol Lett 202:215–220

30. Friedrich CG, Bardischewsky F, Rother D, Quentmeier A,

Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Micro-

biol 8(3):253–259

31. Verte F, Kostanjevecki V, De Smet L, Meyer TE, Cusanovich

MA, Van Beeumen JJ (2002) Identification of a thiosulfate uti-

lization gene cluster from the green phototrophic bacterium

Chlorobium limicola. Biochemistry 41(9):2932–2945


123

32. Klarskov K, Verte F, VanDriessche G, Meyer TE, Cusanovich

MA, Van Beeumen J (1998) The primary structure of soluble

cytochrome c551 from the photographic green sulfur bacterium

Chlorobium limicola, strain Tassajara, reveals a novel c-type

cytochrome. Biochemistry 37(30):10555–10562

33. Frigaard NU, Dahl C, Robert KP (2008) Sulfur metabolism in

phototrophic sulfur bacteria. In: Advances in Microbial Physiol-

ogy, vol 54. Academic Press, pp 103–200

34. Hensen D, Sperling D, Truper HG, Brune DC, Dahl C (2006)

Thiosulphate oxidation in the phototrophic sulphur bacterium

Allochromatium vinosum. Mol Microbiol 62(3):794–810

35. Sauve V, Bruno S, Berks BC, Hemmings AM (2007) The SoxYZ

complex carries sulfur cycle intermediates on a peptide swinging

arm. J Biol Chem 282(32):23194–23204

36. Ghosh W, Roy P (2007) Chemolithoautotrophic oxidation of

thiosulfate, tetrathionate and thiocyanate by a novel rhizobacte-

rium belonging to the genus Paracoccus. FEMS Microbiol Lett

270(1):124–131

37. Sauve V, Roversi P, Leath KJ, Garman EF, Antrobus R, Lea SM,

Berks BC (2009) Mechanism for the hydrolysis of a sulfur–sulfur

bond based on the crystal structure of the thiosulfohydrolase

SoxB. J Biol Chem 284(32):21707–21718

38. Zander U, Faust A, Klink BU, de Sanctis D, Panjikar S, Quent-

meier A, Bardischewsky F, Friedrich CG, Scheidig AJ (2011)

Structural basis for the oxidation of protein-bound sulfur by the

sulfur cycle molybdohemo-enzyme sulfane dehydrogenase So-

xCD. J Biol Chem 286(10):8349–8360. doi:10.1074/jbc.M110.

193631

39. Frigaard NU, Dahl C (2009) Sulfur metabolism in phototrophic

sulfur bacteria. Adv Microb Phys 54:103–200

40. Kappler U, Aguey-Zinsou KF, Hanson GR, Bernhardt PV,

McEwan AG (2004) Cytochrome c551 from Starkeya novella:

characterization, spectroscopic properties, and phylogeny of a

diheme protein of the SoxAX family. J Biol Chem 279(8):6252–

6260

41. Ogawa T, Furusawa T, Nomura R, Seo D, Hosoya-Matsuda N,

Sakurai H, Inoue K (2008) SoxAX binding protein, a novel

component of the thiosulfate-oxidizing multienzyme system in

the green sulfur bacterium Chlorobium tepidum. J Bacteriol

190(18):6097–6110

42. Orawski G, Bardischewsky F, Quentmeier A, Rother D, Friedrich

CG (2007) The periplasmic thioredoxin SoxS plays a key role in

activation in vivo of chemotrophic sulfur oxidation of Paracoc-cus pantotrophus. Microbiology 153:1081–1086

43. Gregersen LH, Bryant DA, Frigaard N-U (2011) Mechanisms and

evolution of oxidative sulfur metabolism in green sulfur bacteria.

Frontiers Microbiol 2:116. doi:10.3389/fmicb.2011.00116

44. Sakurai H, Ogawa T, Shiga M, Inoue K (2010) Inorganic sulfur

oxidizing system in green sulfur bacteria. Photosynth Res

104(2–3):163–176. doi:10.1007/s11120-010-9531-2

45. Kilmartin JR, Maher MJ, Krusong K, Noble CJ, Hanson GR,

Bernhardt PV, Riley MJ, Kappler U (2011) Insights into structure

and function of the active site of SoxAX cytochromes. J Biol

Chem 286(28):24872–24881

46. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S

(2011) MEGA5: molecular evolutionary genetics analysis using

maximum likelihood, evolutionary distance, and maximum par-

simony methods. Mol Biol Evol 28:2731–2739

47. Rother D, Henrich HJ, Quentmeier A, Bardischewsky F, Fried-

rich CG (2001) Novel genes of the sox gene cluster, mutagenesis

of the flavoprotein SoxF, and evidence for a general sulfur-oxi-

dizing system in Paracoccus pantotrophus GB17. J Bacteriol

183(15):4499–4508

48. Dambe T, Quentmeier A, Rother D, Friedrich C, Scheidig AJ

(2005) Structure of the cytochrome complex SoxXA of Para-coccus pantotrophus, a heme enzyme initiating chemotrophic

sulfur oxidation. J Struct Biol 152(3):229–234

49. Friedrich CG, Quentmeier A, Bardischewsky F, Rother D, Kraft

R, Kostka S, Prinz H (2000) Novel genes coding for lithotrophic

sulfur oxidation of Paracoccus pantotrophus GB17. J Bacteriol

182(17):4677–4687

50. Kappler U, Bernhardt PV, Kilmartin J, Riley MJ, Teschner J,

McKenzie KJ, Hanson GR (2008) SoxAX cytochromes, a new

type of heme copper protein involved in bacterial energy gener-

ation from sulfur compounds. J Biol Chem 283(32):22206–22214

51. Frausto da Silva JJR, Williams RJP (2001) The biological

chemistry of the elements—the inorganic chemistry of life.

Oxford University Press, Oxford

52. Cheesman MR, Little PJ, Berks BC (2001) Novel heme ligation

in a c-type cytochrome involved in thiosulfate oxidation: EPR

and MCD of SoxAX from Rhodovulum sulfidophilum. Bio-

chemistry 40:10562–10569

53. Kappler U, Hanson GR, Jones A, McEwan AG (2005) A

recombinant diheme SoxAX cytochrome-implications for the

relationship between EPR signals and modified heme-ligands.

FEBS Lett 579:2491–2498

54. Welte C, Hafner S, Kratzer C, Quentmeier A, Friedrich CG, Dahl

C (2009) Interaction between Sox proteins of two physiologically

distinct bacteria and a new protein involved in thiosulfate oxi-

dation. FEBS Lett 583(8):1281–1286

55. Multhaup G, Schlicksupp A, Hesse L, Beher D, Ruppert T,

Masters CL, Beyreuther K (1996) The amyloid precursor protein

of Alzheimer’s disease in the reduction of copper(II) to copper(I).

Science 271(5254):1406–1409


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http://dx.doi.org/10.1074/jbc.M110.193631

http://dx.doi.org/10.1074/jbc.M110.193631

http://dx.doi.org/10.3389/fmicb.2011.00116

http://dx.doi.org/10.1007/s11120-010-9531-2

the bacterial soxax cytochromes - springer · the soxax heme thiolate proteins also belong to this...

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