Reductive elimination of superoxide: Structure and mechanism of superoxide reductases

Biochim Biophys Acta. 2010 Feb;1804(2):285-97. doi: 10.1016/j.bbapap.2009.10.011. Epub 2009 Oct 24.

Reductive elimination of superoxide: Structure and mechanism ofsuperoxide reductases.

Pinto AF, Rodrigues JV, Teixeira M.

This paper was published in:

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Reductive elimination of superoxide: structure and mechanism of

superoxide reductases

Ana Filipa Pinto, João V. Rodrigues, Miguel Teixeira

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da

República (EAN), 2780-157 Oeiras, Portugal

Corresponding Author

Miguel Teixeira

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da

República (EAN), 2780-157 Oeiras, Portugal

E-mail: [email protected]

* Manuscript

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ABSTRACT

Superoxide anion is among the deleterious reactive oxygen species, towards

which all organisms have specialized detoxifying enzymes. For quite a long time

superoxide elimination was thought to occur through its dismutation, catalyzed by Fe, Cu,

Mn or Ni containing enzymes. However, during the last decade, a novel type of enzyme

was established, that eliminates superoxide through its reduction: the superoxide

reductases, which are spread among anaerobic and facultative microorganisms, from the

three life kingdoms. These enzymes share the same unique catalytic site, an iron-ion

bound to four histidines and a cysteine, which in its reduced form reacts with superoxide

anion with a diffusion-limited second order rate constant of ~109 M-1s-1. In this review, the

properties of these enzymes will be thoroughly discussed.

1. Introduction

Although dioxygen, in its fundamental state, is a relatively inert molecule, due to its

spin triplet ground state, it can be rapidly converted into reactive species, starting with its

one electron reduction product, the superoxide anion, which may be formed by reaction of

the O2 molecule with transition metals or radical species, such as, for example,

semiquinones. This reduction initiates a cascade of reactions, which, before reaching the

formation of the water molecule, have as products toxic species, such as hydrogen

peroxide or the hydroxyl radical:

Therefore, all living species have developed systems to detoxify reactive oxygen species

(ROS), namely the superoxide anion and hydrogen peroxide. It should be emphasized

O2 e O2.- e H2O2 e OH. e H2O O2 e O2.- e H2O2 e OH. e H2O

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that detoxification of ROS is equally important in aerobic or anaerobic organisms, since

an anaerobic environment may be transiently exposed to oxygen. The best known

enzymatic systems are the superoxide dismutases (SODs, reactions 1 and 2):

Mn+ + O2-. → M(n-1)+ + O2 1)

M(n-1)+ + O2-. + 2 H+ → Mn+ + H2O2 2)

__________________________________

2H++ 2 O2-. → O2 + H2O2 (1+2),

and the peroxidases (of which catalases are a particular case, if X=O2)

XH2 + H2O2 → X + 2 H2O 3)

The combination of SODs and peroxidases/catalases leads ultimately to the formation of

water.

Another way of dealing with dioxygen is to fully reduce it to water, without the

release of those intermediates, which is accomplished by the membrane-bound oxygen

reductases of the haem-copper, bd or diiron types, in a process that is in general coupled

with energy conservation by oxidative phosphorylation. More recently, a possible

alternative enzymatic system has been described for oxygen reduction to water, not

coupled to respiration, constituted by the flavodiiron oxygen reductases (for a recent

review see [1]).

Apart from the above mentioned enzymes, mainly during the last decade, another

type of enzymes has been elucidated, which detoxifies the superoxide anion not through

its dismutation (equations 1 and 2), but only through its reduction (reaction 2). These

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enzymes, which are named superoxide reductases (SOR), are the subject of this review.

After a short historical background, we will analyze the primary and tertiary structures of

these enzymes; it will follow a brief discussion of the main physico-chemical properties of

SORs, and a larger section with a detailed analysis of the catalytic mechanism. At the

end, questions still remaining to be answered will be addressed. We call the attention of

the reader to other earlier reviews on this subject [2-8].

2. Historical background

The two first examples of superoxide reductases were isolated from sulfate

reducing bacteria of the Desulfovibrio genus [9, 10]. In 1990, Moura and coworkers,

reported the isolation and characterization of a novel protein from Desulfovibrio (D.)

desulfuricans (ATCC 27774) and from D. vulgaris Hildenborough, containing two iron

sites: centre I, similar to that of desulforedoxin, a small iron protein having a rubredoxin-

like FeCys4 centre, so far only isolated from D. gigas [11], and centre II, a new type of iron

site, which in that preparation was in the ferrous form; for these reasons the protein was

named desulfoferrodoxin, Dfx (a contraction of desulforedoxin, ferrous and redoxin).

Subsequently, the cloning of a fragment of D. vulgaris chromosomal DNA revealed that

desulfoferrodoxin is adjacent to a gene encoding a type I rubredoxin forming a single

transcriptional unit, which led to the hypothesis that rubredoxin could be its electron

donor, and hence to an alternative name to Dfx: rubredoxin: oxidoreductase, or rbo ([12].

Later on, another interesting protein was isolated from D. gigas [9]. It contained an iron

centre similar to that of Dfx centre II, and due to its blue color was named neelaredoxin

(from the Sanskrit word for blue, neela). In 1996, a role for desulfoferrodoxin was

proposed, since an Escherichia coli sodAB deletion strain was successfully

https://www.researchgate.net/publication/11301808_The_mechanisms_of_superoxide_reduction_by_superoxide_reductases_in_vitro_and_in_vivo?el=1_x_8&enrichId=rgreq-2169656b3aa3813643d23642029920b4-XXX&enrichSource=Y292ZXJQYWdlOzM4MDM2OTQ3O0FTOjEwMTIxODY3NjM3OTY1NUAxNDAxMTQzODIxNDI5

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complemented with a plasmid carrying a dfx gene from Desulfoarculus baarsii [13]; short

after, Liochev and Fridovich [14] proposed that these enzymes could eliminate

superoxide through its reduction only, which was afterwards proven by Adams and co-

workers [15] and Nivière and colleagues [16], and is now well established.

According to the number of metal centres, there are two types of superoxide

reductases: neelaredoxins, or 1Fe-SORs, and desulfoferrodoxins, or 2Fe-SORs. As will

be described in the next sections, considering the three dimensional structures and the

amino acid sequences, the 1Fe-SORs have been classified as Class II and Class III

SORs, and the 2Fe-SORs as the Class I enzymes [3, 8]. When considered necessary, a

combination of both classifications will be used; otherwise, we will use the simplest

classification based on the number of metal sites.

3. Three dimensional structures

The three dimensional crystallographic structures of a few superoxide reductases

have been determined (Table I), for the oxidized and reduced states, as well as for a

catalytic intermediate. The enzyme active centre of SORs is in a domain common to 1Fe-

and 2Fe-SORs, which is organized in a seven-stranded (3+4 sheet stranded) β-barrel that

adopts an immunoglobulin-like fold, preceded by a one turn 310 helix (at the N-terminus

for 1Fe/Class II SORs) that connects to the barrel by a ~15-residue loop (Figure 1-A). In

1Fe/Class III SORs or in 2Fe-SORs, the loop connects to another domain, with a

spherical shape, almost identical to that of D. gigas desulforedoxin [17]; as mentioned

above, in 2Fe-SORs this extra domain contains iron centre I. The oligomerisation states

and the inter-monomers interactions are quite distinct among SORs (Figure 1-B):

1Fe/Class II SORs are homotetramers, while 2Fe-SORs or 1Fe/Class III SORs are


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homodimers; so far, there is no evidence for any correlation between the quaternary

structure and the enzymatic function. Another difference between Class II SORs and

both Class I and Class III SORs, is that the later have shorter loops connecting the beta

strands (Figures 1 and 2).

Metal Sites

In SORs, both iron sites are close to the molecular surfaces, and exposed to the

solvent, and its ligands are located in loops that connect the β strands (Figure 1). This

situation is different from that of superoxide dismutases, in which the metal centres are

embedded inside the protein, at the end of a substrate channel [18]. In the 2-Fe SORs,

the centre I has an iron coordinated to four cysteines, in a distorted tetrahedral geometry,

as in desulforedoxin (Figure 1-C) [19] [17].The common site for the two types of SOR,

called centre II in 2-Fe SORs, has a so far unique geometry (Figure 1-C). In the ferrous

state, the iron is coordinated in a square-pyramidal geometry to four nitrogens (three εN

and one δN) from histidine-imidazoles, in the equatorial plane, and the fifth axial position

(at the inner side of the protein) is occupied by a cysteine-sulfur (Table 2). It has been

generally assumed that no solvent molecule occupies the sixth axial position. The

structure of the ferric form has been unambiguously determined only for the 1Fe enzymes

from Pyrococcus (P.) furiosus and Pyrococcus horikoshi, which show that the ferric ion

becomes bound to a monodentate carboxylate from a glutamate residue, establishing an

octahedral geometry around the iron ion (Figure 1-C). This change in iron-coordination

upon oxidation/reduction is accompanied by a significant movement of two loop regions

(Gly9-Lys15 and Gly36-Pro40, in P. furiosus 1Fe-SOR), which widens the accessibility to

the ferrous iron site. These structural modifications have not been clearly established in

the 2Fe-SORs or in the Treponema (T). pallidum 1Fe/Class III enzyme, using x-ray

crystallography, namely due to the difficulty of maintaining the enzymes in the ferric state,

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in the absence of an oxidant; in fact, these enzymes have been shown to be readily

photoreducible and, if ferricyanide is added, it binds to the iron site [19] [20]. However,

several spectroscopic data strongly suggest that in the 2Fe-SORs from D. baarsii and D.

vulgaris the binding of the glutamate coupled to the redox process also occurs [21]. For

the other available structures, there is no indication of the iron oxidation state, but since

the glutamate residue is not bound to the iron ion, they presumably correspond to the

reduced form of the enzymes (Table 1). It may be anticipated that in some cases the sixth

axial position will be vacant in the oxidized form, or occupied by a solvent molecule, as

the glutamate residue is not strictly conserved (see next section); for example,

spectroscopic and kinetic data for the 1Fe-SOR from Nanoarchaeum (N) equitans, which

lacks that residue, strongly suggests that the iron is bound only to five aminoacid ligands

in the oxidized state [22].

The geometry of the catalytic centre and the type of the iron amino acid ligands of

SORs is quite distinct from that of the Fe superoxide dismutases, where the metal is in a

trigonal bipyramidal geometry, bound to two histidines and one monodentate aspartate in

the equatorial plane, and another histidine and one solvent molecule in the axial

positions. The electrostatic surface close to centre II has a positive character, mainly due

to the metal ion and to a lysine residue (Lys 15, in P. furiosus 1Fe-SOR); this fact has

been rationalized as a strategy to attract the anionic substrate, contributing to the very

high rate constant for the first bimolecular reaction between the enzyme and superoxide.

In this respect, SORs resemble superoxide dismutases, which have also a positive

surface surrounding the substrate channel [18].

In 2Fe-SORs, the two metal sites are, within each monomer, ca 22 Å apart (32 Å if

from different monomers), which hinders an efficient electron transfer between them.

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4. Amino acid sequence analysis

A comprehensive search for superoxide reductases homologues was performed,

using more then one type of SOR as query. As for June 2009, 182 homologous were

retrieved. The amino acid sequences were aligned (see Figure 2 for a selected subset)

and an unrooted dendogram (Figure 3) was calculated using the neighbor-joining

methodology of ClustalX [23].The amino acid sequences cluster in two major groups,

corresponding to 1Fe- and 2Fe-SORs, and a smaller one, which comprises several Class

III 1Fe-SORs and is closer to the cluster of 2Fe-SORs; however, Class III enzymes are

also scattered among the 2Fe-SORs, i.e., they do not appear to form an homogeneous

clade. The distribution of Class III enzymes suggests that they may have evolved more

than once from 2Fe-SORs, by loss of the cysteine ligands to centre I. Another observation

from the dendogram is that the enzymes do not cluster according to the organismal

phylogeny, namely according to the bacterial or archaeal kingdoms, indicating a high level

of lateral gene transfer events throughout evolution.

It was initially thought that SORs were restricted to anaerobic prokaryotes, but this

is not the case: they are also present in facultative microorganisms, and at least one

example of an eukaryotic enzyme is already known, from the microaerophilic protozoan

Giardia intestinalis (www.giardiadb.org). Superoxide reductases coexist in many

organisms with other superoxide detoxifying enzymes, i.e., superoxide dismutases of the

iron/manganese and/or CuZn types, while other organisms appear to rely solely on those

enzymes for superoxide detoxification. It seems that the reductases are another example

of the nature evolutionary diversity, rather than being a specific type of enzymes designed

for protection of particular anaerobes. Nevertheless, it should be pointed out that virtually

nothing is yet known regarding the regulation of the expression of these enzymes. The

analysis of the complete genomes reveals also that the genes coding for SORs are found

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in quite diverse genetic loci; only in a few cases they are found together with genes

encoding either their electron donors (rubredoxins, as in D. vulgaris, for example), or

other oxidative stress responsive genes. Furthermore, in many of the organisms

containing sor genes, genes encoding rubredoxins or desulforedoxins are not present,

which indicates that electron donors other than those have to be operative.

The extensive number of sequences available reveals some more interesting

aspects (Figure 2). Apart from the N-terminal domain of the 2Fe and 1Fe Class III SORs,

one (so far) unique example was found, from an uncultured and still unclassified

bacterium (YP_001956215.1): a domain which instead of the desulforedoxin signature -

CXXC-Xn-CC, has that of type I rubredoxins -CXXC-Xn-CXXC-.The spacing between the

two cysteines pairs (ca 12 residues) is similar to those of the rubredoxin-like domains of

rubrerythrins (another type of oxidative stress enzymes [6]), rather than to those of

canonical, isolated, rubredoxins (ca 30 residues); furthermore, this domain has a higher

amino acid similarity with the equivalent domains of rubrerythrins than with those from

desulforedoxins or superoxide reductases. A second interesting case are the enzymes

from a few organisms (e.g., Desulfuromonas acetoxidans DSM 684 and Geobacter

sulfurreducens) whose sequences are preceded by twin arginine signal peptides,

suggesting their periplasmic location in those organisms, in contrast to what is generally

believed for the remaining enzymes, on the basis of the lack of recognizable translocation

signals.

In terms of conservation of amino acids, it is remarkable that very few residues are

strictly conserved (Figure 2): i) the cysteine ligands to the FeCys4 centre (in the 2Fe-

SORs), and the four histidines and the cysteine bound to the catalytic centre; and ii) the

proline at the characteristic motif -(E)(K)H(V)P- and an isoleucine after the third histidine

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ligand (Figure 2). Other residues that were considered strictly conserved and catalytically

important, are not at all conserved, such as the above mentioned glutamate residue

bound to centre II in oxidized SORs, or the lysine residue, also at the -(E)KHVP- motif,

and located close to the catalytic site. In Figure 2, examples of this diversity are depicted.

This lack of conservation, translates in quite low values of amino acid sequence

identities/similarities among these enzymes, and indicates a robustness of the protein

scaffold towards mutational changes. This very low amino acid conservation also

establishes the minimal requisites for the catalytic mechanism, as will be discussed in the

last section.

5. Physiological studies

The first evidence for a possible function of these enzymes came from the

observation by D. Touati and coworkers that a DNA fragment of the sulphate reducing

bacterium D. baarsii was able to complement an E. coli sodAB deletion mutant [13]. It

came as a surprise that this fragment encoded not a superoxide dismutase, but a

desulfoferrodoxin. These authors further established that site II was the catalytic one, as

expression of the Dfx first domain was not able to complement the E. coli mutant strain

[16]. Later, other SORs (1Fe- or 2Fe-) were also shown to complement the same E. coli

deletion strain (e.g., [24, 25]). These studies prompted the search for a superoxide

dismutase activity by these enzymes and indeed, a low activity was obtained for the D.

desulfuricans ATCC 27774 Dfx [26] and for the D. gigas and Archaeoglobus (A.) fulgidus

neelaredoxins [27] [28], as well as for the enzymes of D. baarsii [16] and D. vulgaris [29].

It was also reported in those studies that superoxide, generated by the xanthine/xanthine

oxidase system, was able to oxidize centre II. However, those activities are approximately

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two orders of magnitude lower than those of superoxide dismutases (ca 40 s-1 versus

2000-4000 s-1), and too low to explain the ability of the enzymes to complement the E. coli

SOD deficient strain. Since the activities of the enzymes in which the “conserved”

glutamate was replaced by aliphatic residues were also very low [30] [31], the lack of a

significant SOD activity could not be attributed to a blocking of the access to the ferric

site, which could lead to the abolishment of the oxidative part of the dismutation process

(reaction 1). It was also reported that the activity of the enzyme form having centre II

reduced appeared to be higher than that of the oxidized ones, which could not be

correctly explained by a superoxide dismutase activity [26]. Fridovich and coworkers [14],

proposed that the activity observed was not of dismutation (equations 1 and 2), but of

superoxide reduction (equation 2), leading to elimination of superoxide and formation of

hydrogen peroxide, which then led to the now accepted superoxide reductase activity.

Apart from the heterologous complementation assays, very few in vivo studies

have been performed for these enzymes. A D. vulgaris mutant strain with increased

resistance to oxygen was found to have dfx transcriptional levels higher than those of the

wild type strain [32]; in concordance with those results, a D. vulgaris dfx deletion mutant

had a higher sensitivity to oxygen [33]. An up-regulation of the transcriptional level of

SORs encoding genes was also observed in C. acetobutylicum [34] and Thermotoga

maritima [35] upon oxygen stress and. In contrast, these levels apparently do not change

in D. vulgaris and P. furiosus, under oxidative stress, what may indicate a constitutive

expression of the SOR genes [36] [37]. Clearly, this is a field essentially unexplored.

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6. Properties of the iron sites

Superoxide reductases have been extensively studied using a wide range of

spectroscopic tools, namely UV-Visible, Resonance Raman, EPR, Mössbauer and FT-

Infrared. Both iron sites are in a high spin state, in the oxidized (S = 5/2) and reduced (S =

2) forms. Therefore, both exhibit characteristic EPR resonances in the ferric state with

variable rhombicities (E/D ~0.1 or ~0.3) [9] [38] [39]. The signatures from Centres I and II

overlap considerably, which together with the low value for the zero-field splitting, which

leads to the population of more than one Kramers doublet at low temperature, thus further

complicating the EPR spectra, make EPR not a good technique to distinguish

unambiguously the two sites, in 2Fe-SORs, or to monitor changes at the catalytic site and

differentiate active and inactive forms of the enzymes. On the contrary, the two sites are

easily discernible by electronic absorption (Figure 4): centre I has the characteristic

features of a desulforedoxin, FeCys4 site, with maxima at 375 and 495 nm and a broad

shoulder at 560 nm; centre II has a broad absorption band at ~ 660 nm, which is

responsible for the blue color of 1Fe-SORs, or the grey color (the mixture of pink and

blue) for the oxidized 2Fe-SORs, and a shoulder at ca 330 nm. The 660 nm band has

been attributed to a sulfur to iron charge transfer transition [39]. In the fully reduced state

both types of SORs are colorless, while the half-reduced (centre I oxidized, centre II

reduced) 2Fe-SORs are pink. These features have been essential to elucidate the

catalytic mechanism of SORs, coupling absorption spectroscopy to fast kinetics methods

(pulse radiolysis and stopped-flow).

The two centres have quite distinct reduction potentials: centre I close to 0 mV and

centre II between 190 and 365 mV, at neutral pH (Table 3). This large difference has

been also important to study in detail the events on the catalytic centre II, without

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interference of centre I. The potentials for the catalytic centre are similar to those reported

for superoxide dismutases, and perfectly adequate for superoxide reduction (E(O2-/H2O2)

= 890 mV, at pH 7).

The catalytic centre of both types of SORs is able to bind small anionic ligands,

such as cyanide or fluoride [27], [26], [40, 41]. The binding of cyanide leads to the

formation of a quasi-axial EPR spectrum of an S=1/2 species, while with fluoride or

hydroxide the spin state does not change.

Proton equilibria

Since the isolation of the first 1Fe-SOR, the D.gigas neelaredoxin [9], there has

been increasing evidence for pH dependent equilibria at or near the catalytic site, some of

which are of mechanistic relevance: in fact, the catalytic reaction (reaction 2) involves

also the consumption of two protons, since at physiological pH values the superoxide

anion is in the basic, deprotonated form (pKa ~4.8).

The enzymes, in the oxidized state, at pH above 9 exhibit a drastic change of the

electronic absorption spectra, with the absorption band shifting to ~590 nm (Table 3,

Figure 4-C). This transition has an apparent pKa of ~9.5. The chemical identity of the

basic form was established by Resonance Raman spectroscopy: the detection of a

vibrational band at 466-471 cm-1, characteristic of a high-spin Fe3+-OH stretching mode

vibration, in the wild type SOR from D. baarsii, and its E47A and K48I mutants, which

disappears at acidic pH values and exhibits a clear shift when the samples were prepared

in H218O or D2O, established it as an iron-hydroxide form [42]. The same observation was

recently reported for the enzymes from Archaeoglobus (A.) fulgidus and Nanoarchaeum

equitans [43]. Therefore, this transition corresponds in fact to a ligand-exchange, being

the glutamate ligand substituted by an hydroxide anion (Scheme I-A), and the value of the

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apparent pKa gives an indication of the relative affinities of the iron ion to each ligand, i.e.,

the pKa is not a true proton ionization constant, for the glutamate-bound enzymes.

This ligand exchange has been also observed by EPR spectroscopy (a rhombic EPR

spectrum, with E/D~0.25 appears at high pH [5, 9, 39], and by Fourier–transform infrared

spectroscopy [21]. In site-directed mutants of the glutamate ligand, as well as for the N.

equitans enzyme, a pH dependent equilibrium is also observed, but with a much lower

apparent pKa, ca. 2 units lower [5, 41]. Again, Resonance Raman data has shown that

the basic form corresponds to an hydroxide-bound iron. However, in these cases, the pH

dependence can be attributed to a true protonic equilibrium (Scheme 1-B), the

protonation of the hydroxide ligand at acidic pH values. As we will see in the next section,

these processes are essential to understand the reactivity of these enzymes with

superoxide.

Fe3+

His

His

His

Cys

His

H2O

Fe3+

His

His

His

Cys

His

HO

pKa= 6.1

Glu

O

O C

Fe3+

His

His

His

Cys

His Fe3+

His

His

His

Cys

His

HO

pKaapp = 9.6

Scheme I

I A

I B

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The same pH dependent processes were shown to affect (decrease) the reduction

potentials of centre II from the D. baarsii 2Fe-SOR, but not that of centre I [5].

7. Mechanism and catalytic intermediates of superoxide reduction

Overview of the oxidative cycle

The mechanism of superoxide reduction has been scrutinized mainly using pulse

radiolysis. This approach allows the production of defined amounts of the superoxide

anion in a very fast time scale (superoxide is formed in the first microseconds after

pulsing an air-saturated solution with electrons [44]), and in a rather “clean” way, and was

determinant to establish the oxidative part of the catalytic mechanism of these enzymes

(reduction of superoxide to hydrogen peroxide and concomitant oxidation of the ferrous

enzyme to the ferric, resting state). The main results are here discussed, using as an

example the 1Fe-SOR of Archaeoglobus fulgidus, and referring to other enzymes when

complementary information is relevant. The reaction is initiated having the enzyme in the

reduced state (colorless, as there is no electronic absorption in the visible region), taking

profit from the fact that in this form it is essentially unreactive towards oxygen; the

enzyme solution is then pulsed with superoxide and the reaction is monitored by single

wavelength optical spectroscopy. The electronic features of each intermediate are

obtained by measurements at a sufficient number of wavelengths (which enables to

reconstruct the electronic spectrum) and for a sufficiently long time range (up to seconds).

Using this method, the same sample can be pulsed several times, and the amount of

protein that reacted may be quantified, which allows to determine the extinction

coefficients of each species. The first step of the reaction appears to be common to all

enzymes so far studied (Figure 5, Table 3): upon the superoxide pulse, a first

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intermediate, T1, is formed, with an absorption maximum at ca 620 nm. This process

occurs with a second order rate constant of ~109 M-1s-1, i.e., at a diffusion limited rate

(Table 3). The observation of the visible absorption of T1, tells immediately that at this

stage the iron is already in a ferric, Fe (III), form, which implies that the superoxide anion

was concomitantly reduced to the peroxide level, and there is a general agreement that

T1 corresponds to a ferric-(hydro)peroxo species (see below). This intermediate, in the A.

fulgidus enzyme, decays subsequently, in a pseudo first order, unimolecular process, to

another species T2, with optical properties identical to those observed for the basic form

of ferric SOR, i.e., to a species to which an hydroxide is bound [41]. This observation

establishes that at this stage the product was already released from the enzyme. For the

wild type enzyme, the transient state T2 decays further to the resting, oxidized, state,

again in a unimolecular process, presumably re-binding the glutamate ligand. For the

glutamate mutants, as for the N. equitans enzyme, T2 is the final state of the oxidative

part of the catalytic cycle. For the 2Fe-SOR from Desulfoarculus baarsii and the 1Fe-SOR

from Treponema pallidum, two intermediates are also detected, while for the D. vulgaris

enzyme, T1 decays directly to the resting form, also in a unimolecular process i.e., no

intermediates could be detected (Figure 6).

Role of centre I

The large difference in midpoint reduction potentials between centre I and II, and

their distinct absorption features, allowed to perform the fast kinetic studies monitoring

each centre individually. It has been unequivocally shown that centre I does not

participate in the oxidative part of the catalytic cycle, i.e., in the reduction of superoxide to

hydrogen peroxide. The definite proof was obtained by the study of mutants lacking

centre I, whose function remains therefore elusive [45].

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Nature of intermediate T1

This first intermediate is observed for all enzymes so far studied, and displays

maxima at 580-620 nm (extinction coefficient of 3000-4000 M-1 cm-1). It is unequivocally a

ferric species, which means that at this stage of the catalytic cycle, superoxide was

already reduced; its rate of formation is, within the experimental error, independent of pH,

which establishes that the rate is not limited by a proton transfer event. The identity of this

transient has been difficult to prove, since due to its fast decay rate (>50 s-1) it has not

been possible to trap the enzyme in this state, starting from the reaction of the reduced

enzyme with superoxide. Therefore, further evidences for its nature came from indirect

experiments, using hydrogen peroxide, and from theoretical studies. The use of hydrogen

peroxide is not without risk, since either long incubation or high amounts of it lead to

destruction of the enzyme. A peroxide-intermediate was tentatively reconstituted by

incubation of the ferrous enzyme with slight excess of H2O2 [46]. This trapped-species

was assigned to a side-on η2-Fe3+-peroxo species by Mössbauer and Raman

spectroscopies [46, 47]; however, its visible spectrum (absorption maximum at 560 nm

and extinction coefficient of 1000 M-1 cm-1) is clearly different from the transient species

observed in the pulse radiolysis experiments. Silaghi-Dumitrescu and co-workers

addressed this problem by computational methods, studying models of the SOR active

site and different iron peroxo-adducts [48]. These calculations suggested that a ferric end-

on (hydro)peroxide was favored over a side-on peroxide. Indirect evidence comes also

from the pH dependence behavior for the transition from T1 to T2. This is the only

observable pH-dependent process, which, knowing that two protons are needed for the

formation of H2O2, strongly suggests that in T1 the first proton is already bound to O22-, as

proposed by the theoretical studies. Furthermore, the reduction of O2- by synthetic

analogues of SOR can only occur if a source of protons is present (even if it is a weak

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acid such as ethanol) [49], which is indicative of a proton-coupled electron transfer

process for the first step. This observation might be an important clue on how protons are

transferred in SOR proteins.

A quite interesting study was performed combining X-ray diffraction and Raman

spectroscopy of SOR crystals [50]. Diffraction data obtained after incubation of the

oxidized enzyme with H2O2 was interpreted as showing the formation of the

(hydro)peroxo bound form; it was also proposed the involvement of several water

molecules in the catalytic process, as anticipated by Cabelli and co-workers [51].

In summary, there is a general consensus that T1 is a ferric peroxo species, for

which theoretical calculations favor an end-on hydroperoxo form, but definitive

experimental evidence is still lacking.

Nature of Intermediate T2 - How many reaction intermediates?

For the A. fulgidus enzymes (1Fe- and 2Fe-SORs), the iron-(hydroperoxo) T1,

decays in an apparent first order process to a second intermediate, T2. The chemical

identity of this species, which is the resting, final state, for the glutamate mutants of A.

fulgidus 1Fe-SOR (E12V, E12Q), or for the glutamate lacking 1Fe-SOR from N. equitans,

was established by Resonance Raman spectroscopy of the basic form of the enzymes,

as reported above. Accordingly, T2 corresponds to the acidic form of those species, i.e.,

to a ferric-H2O form, if the reaction is performed at pH values lower than those of the

respective equilibrium. The mechanistic implication of these results is that the only

species that can be assigned to a peroxo-bound intermediate is T1, and that the reduction

of superoxide proceeds via a single observable reaction intermediate. For the D. vulgaris

2Fe-SOR also a single putative peroxo intermediate is detected, even at high

temperatures [52]; in preliminary experiments with the 2Fe-SOR from D. desulfuricans, an

19

enzyme which is highly homologous to that of D. vulgaris, we could observe exactly the

same kinetic behavior (our unpublished results).

The rate of decay (k2) of T1 to T2 is pH dependent, i.e., it involves a rate limiting

protonation step, within a certain pH range (up to pH ~8.5); above pH ~8.5, the rate is pH

independent (~50s-1) or increases with pH. This increase with pH may be related to the

substitution of the product by the hydroxide anion, which is expected to be faster at basic

pH values, while the pH independent process may be explained by a direct attack of T1

by a water molecule, a pseudo-first order process. The behavior at low pH can be

described by a second order process, with k2=k2´[H+], with a value for k2´ of ~109 M-1 s-1,

i.e., it is a diffusion limited protonation step. In accordance with this interpretation, k2

shows a clear deuterium isotopic effect [3].

A major question is why in the 1Fe-SORs from A. fulgidus and N. equitans and in

the 2Fe-SOR from A. fulgidus the Fe3+-OH species is formed, and in other enzymes the

transient intermediate (T1) apparently decays directly to the Fe3+-Glu resting form. To

clarify whether that intermediate was associated to the catalytic reaction, we followed by

stopped flow coupled to spectrophotometry the chemical oxidation of reduced SORs: if an

intermediate would be detected, it could not be associated to the enzymatic reaction,

such as a product bound enzyme form, for example. In fact, a transient with optical

properties identical to T2, and which decayed with a similar unimolecular rate constant

was observed in those experiments, further proving that T2 does not correspond to a form

in which the product is still bound (Scheme II).

20

We attribute these differences in the number of detected intermediates to distinct rates for

the several molecular processes: protonation of the hydroperoxo, release of hydrogen

peroxide, OH- binding and glutamate binding. In fact, those individual rates could not be

determined and their relative values dictate whether the Fe3+-OH species is

observed/formed or not. It is possible that the distinct kinetic behavior observed for these

enzymes is also related with the different thermophilic nature of their source organisms. It

is known that thermophilic enzymes at room temperature exhibit a slower dynamics and

lower catalytic rates, as compared to their optimal rates, generally close to the optimum

growth temperatures of the parental organism. The effect of the temperature on the

reaction rates, ligand binding and pH-equilibrium constants is yet to be established for

those enzymes but, nevertheless, the rates determined for the A. fulgidus and N. equitans

enzymes, organisms with optima growth temperatures of ~80ºC, are within the range of

those determined for the enzymes from the mesophilic organisms.

In apparent contradiction with these results, that support a kinetic mechanism

involving one single observable reaction intermediate having a substrate-derived form

bound (the (hydro)peroxo), Nivière and co-workers reported that in D. baarsii 2Fe-SOR or

T. pallidum 1Fe-SOR the decay of T1 produces a second iron-peroxide species [47]. This

Glu

O

O C

Fe3+

His

His

His

Cys

His Fe3+

His

His

His

Cys

His

HO

Fe2+

His

His

His

Cys

His

e

Scheme II

21

proposal was based on the fact that this intermediate is spectroscopically different from

the final resting state. We have compared the spectrum of this species with that of the

final product and found that they are in fact superimposable, with the notable exception of

two data points. These points define together an intense sharp peak (λmax~620 nm,

ε~4000 M-1 cm-1) protruding from a broad band characteristic of the “resting”-oxidized

enzyme. To our knowledge no similar spectra has been ever observed in non-haem iron

compounds (either synthetic or in proteins). Our interpretation of these data is that the

second species observed in these experiments is, in fact, the final Fe3+-Glu species,

supporting that superoxide reduction involves only one observable peroxo intermediate

(T1).

Effect of phosphate binding

In our previous pulse radiolysis studies with A. fulgidus 1Fe-SOR we came

across with the unexpected finding that phosphate, a widely used buffer, interferes

with the reaction kinetics in two different ways. First, phosphate acts as a proton

donor for the hydroperoxide intermediate (T1), increasing the rate of product

formation in a concentration-dependent manner [41]. Similar effects were observed

for D. vulgaris 2Fe-SOR in a study involving phosphate, formate, fluoride and azide,

showing that all these compounds acted as general acids, accelerating the release of

the reaction product, thus mimicking the solvent-mediated protonation of the

hydroperoxide intermediate [53]. The second interference seen in the presence of

phosphate is the formation of an additional intermediate species, with distinct

absorption properties, which was assigned to the binding of phosphate to the active

site. In the wild type SOR phosphate is displaced from the active site by the Glu12

(A. fulgidus) ligand, whereas in the E12 mutants it remains bound to the iron as a

22

stable end-product [41]. Similarly, azide has also been observed to bind the active

site in D. vulgaris 2Fe-SOR after the decay of T1, again being displaced by the

glutamate ligand [53]. In addition to azide and phosphate, other anions have been

reported to bind the active site of SOR, such as CN-, ferricyanide, fluoride and

formate [27], [26], [40, 41], clearly revealing the high propensity of the active site to

accept external ligands, although in some cases the anions only form stable adducts

in the glutamate mutant. Therefore, the presence of these ligands may interfere in the

kinetics in a way that is dependent on the dissociation constant of the anion and its

binding rate. Nevertheless, only the effect of phosphate should be relevant in the

biological context, as it is an abundant compound. Although it is likely that formation

of the ferric-phosphate adduct occurs inside the cells, inclusion of phosphate in

steady state kinetic measurements did not affect the turnover rate [54], supporting

that binding of phosphate is not an inhibition factor.

Role of specific aminoacid residues

As already mentioned, two particular amino acid residues have been considered

important for the mechanism of superoxide reduction, apart from the metal ligands: the

glutamate ligand (E12 in A. fulgidus 1Fe-SOR, E47 in D. vulgaris 2Fe-SOR– see Table

2), and the lysine of the motif –EKHVP- (K13 in A. fulgidus 1Fe-SOR, Table 2). The

glutamate was proposed to assist the release of the product from the catalytic site, or to

be involved in some proton transfer event. However, the study of several site-directed

mutants in which the glutamate residue was substituted by other amino acids (alanine,

valine or glutamine), in the A. fulgidus, D. vulgaris and D. baarsii enzymes, as well as the

study of the glutamate-lacking N. equitans 1Fe-SOR, revealed that the same rate

constants and pH dependence behavior was observed, i.e., there is no evidence that the

23

glutamate binding is important for product release (in agreement with the proposal that

the product is released already at the T2, Fe3+-OH intermediate), nor for proton delivery to

the catalytic site. In particular, it has been shown by stopped-flow spectrophotometry that

the protonation of the hydroperoxide intermediate occurs mainly via solvent-mediated

pathways [53].

The lysine residue was considered either to provide a positive surface charge near

the active site, thereby contributing to increase the rate constant for binding of the anionic

substrate, or to provide a proton and/or to stabilize through an hydrogen-bond the

hydroperoxide ligand. Indeed, a site-directed mutant of the D. baarsii enzyme, showed a

decreased rate constant for the formation of T1 [30] (Table 3). However, as for the

glutamate, there are examples of enzymes lacking this residue (Figure 2) but it remains to

be determined if these enzymes are as effective as the lysine-containing ones.

In fact, the low conservation of aminoacid residues, strongly suggest that only the

metal ligands are important for catalysis. On one hand, the fact that the center is exposed

to the solvent somehow abolishes the need for proton conducting aminoacids as a

constant source for protons is always available. There is accumulating evidence that

protons are indeed supplied by the solvent [47] [41]. In particular, the direct and fast

protonation of T2 at the oxygen bound to the iron is a way of avoiding the split of the O2

bond and the formation of a highly oxidizing ferryl species. On the other hand the

histidines also establish a stereochemical restriction, favoring the formation of an end-on

peroxo intermediate [47]. Finally, the strength of the Fe-sulfur bond has been correlated

with the catalytical activity: for example, the resonance raman data show a possible

correlation between the Fe-sulfur cysteine bond and the rate of release of the product

molecule, H2O2 [43] [55]: the bond is weaker in 2Fe-SORs and in N. equitans 1Fe-SOR,

and these enzymes have a higher rate constant fro the decay of T2.

24

Physiological electron donors – Reductive path

It has been generally assumed that rubredoxins are the direct electron donors to

superoxide reductases, which was initially suggested due to the genomic organization of

the respective genes in D. vulgaris [12]. Indeed, for 1Fe- and 2Fe-SORs, it has been

demonstrated that reduced rubredoxins are quite efficient electron donors, with second

order rate constants in the order of ~106-107 M-1s-1 [54, 56, 57]. These rubredoxins, on its

turn, are reduced by NAD(P)H dependent oxidoreductases. In addition to rubredoxin, it

was also demonstrated, by steady-state kinetics, that desulforedoxin is able to function as

an electron donor to D. gigas SOR [20]. However, it is now quite clear that other

physiological electron donors have to exist, as a large number of organisms having genes

encoding SORs do not contain genes coding for rubredoxins.

Regarding the “reductive path”, a question remains also to be answered: since, at

least for some enzymes, the ferric, product-free enzyme is formed prior to glutamate

binding, its reduction may occur before that process, i.e., reduction of SORs may occur at

the level of the T2 intermediate.

Final remarks

In summary, a novel form of superoxide elimination, through its reduction, was

unambiguously identified in the last years, performed by the 1Fe or 2Fe superoxide

reductases, using as the catalytic site an iron ion bound to four histidines and a cysteine,

in a square pyramidal geometry. In many organisms this appears to be the only route for

superoxide detoxification, as their genomes to not encode for, at least, the other already

known superoxide detoxifying enzymes, while in others these enzymes co-exist with

superoxide dismutases.

In spite of the studies so far reported, several issues remain to be established,

namely the number of catalytic intermediates, as already discussed. A puzzling question

25

is also why SORs show such a low superoxide dismutase activity although in pure

thermodynamic grounds SOR’s should be capable of equally oxidizing or reducing

superoxide. In fact, it is now generally accepted that the relevant activity is the reduction

of superoxide. Nevertheless, one should consider that the low SOD activity of these

proteins (5 × 106 M-1 s-1 measured for A. fulgidus 1Fe-SOR) can be high enough to

compete with the spontaneous rate of disproportionation of superoxide (~105 M-1 s-1, pH

7), and thus might be important. Its significance in biological systems depends on both

the concentrations of the enzyme and of O2-, and on the rate at which O2

- self-dismutates,

which is a function of pH. For instance, some preliminary results suggest that the SOD

activity of 1Fe-SORs is still in the order of 106 M-1 s-1 at high pH where the spontaneous

disproportionation rate is dramatically lowered (104-103 M-1 s-1, pH 8-9). It is,

nevertheless, pertinent to consider the reasons why SORs do not show a significant SOD

activity. Since both SOR and SOD have reduction potentials in the range of +200-350

mV, and thus are able to reduce and oxidize O2-, in the case of SORs there must be a

kinetic barrier that prevents the ferric ion to be reduced by O2-. It has been initially

proposed by D. Kurtz that this can be due to the reorganizational energy required for the

change between hexa- and pentacoordination in the active site, or due to the competition

for the sixth coordination site between superoxide and either OH- or the glutamate residue

[58]. In agreement with this proposal, the mechanism of inhibition of Fe-SOD reduction by

superoxide at high pH is believed to involve binding of an extra OH- anion to the five-

coordinated active site, resulting in a six-coordinated ferric centre [59]. This is supported

by the fact that the SOD activity is decreased at high pH showing an apparent pKa similar

to that of binding of OH- (pKaapp ≈ 8.5), and by the apparent increase in KM for superoxide

at high pH, both compatible with competitive inhibition by OH- [60]. It is also interesting to

note that in Fe-substituted Mn-SOD from E. coli, the pKaapp for OH- binding is lowered to

26

6.4, and the activity, which is much lower than that of the Mn-SOD, increases at low pH

[61]. The pKaapp

for OH- binding in Fe-substituted Mn-SOD has been modulated by site-

directed mutagenesis which resulted in an increase to 7.8, and the overall SOD activity

also increased [62], again supporting that binding of superoxide is favored by decreasing

the bond strength of the sixth ligand (i.e. by protonation of bound OH-) in the hexa-

coordinated ferric ion (Figure 7). It is noteworthy that SORs show a pKa for the OH-/OH2

equilibrium (pKa~6) that is similar to that obtained in Fe-substituted Mn-SOD. It remains to

be verified if SORs also show an increased SOD activity at low pH (<6). Another aspect

that may facilitate reduction of the metal by O2- is the coupling of the electron transfer to

the uptake of a proton by the protein, which is proposed to occur in SOD but not SOR

[18].

This exquisite preference for the superoxide reduction by SORs, as compared to

SODs, remains certainly an unsolved issue in this field. Also, the nature of the proton

donor is not yet established, but in this context it seems quite plausible that, due to the

solvent exposure of the catalytic centre, water may play a determinant role, as particularly

well illustrated by the x-ray crystallographic structures of the iron-peroxide intermediate

[50], which reveals a complex network of hydrogen bonded water molecules, close to the

iron sites.

Another relevant issue also remains to be explored: the relative roles of superoxide

dismutases or reductases in the organisms that contain both types of enzymes. Does this

fact just confer robustness to the organisms? In this respect, it should be recalled that

superoxide dismutases have also been proposed to act only as reductases under certain

conditions [63], i.e., the balance between oxidation and reduction will depend on the

superoxide flux as well as on the cellular redox states.

27

Acknowledgements

The work presented in this article has been financed by Projects from Fundação para a

Ciência e Tecnologia, Portugal. We would like to thank our long-term collaborators in this

field, namely L.M. Saraiva and C. M. Soares (ITQB), and D. Cabelli (Brookhaven National

Laboratory).

28

Figure Legends

Figure 1 - Three dimensional structures of superoxide reductases. A, B: left, D.

desulfuricans Dfx (pdb 1Dfx); right, P. furiosus neelaredoxin (pdb 1DO6). A-

structures of each monomer, colored according to the secondary structure, B-

quaternary structures (each monomer is represented by a different color); C-

Structures of Dfx centre I (left), neelaredoxin centre II, oxidized (middle) and reduced

(right). Figure prepared using Pymol [64]. Iron ion in silver (center I) or blue (center II)

spheres.

Figure 2 – Amino acid sequence alignment of SORs, using ClustalX [23]. Residues

that bind the catalytic centre are indicated by an *; the cysteines binding centre I of

2Fe-SORs are encircled by boxes. Strictly conserved residues are shaded in black;

the lysine of the (E)(K)H(V)P(V) motif is marked with an arrow. Neelaredoxins:

Pyrococcus furiosus DSM 3638 (gi|18977653|); Desulfovibrio gigas (gi|4235394|); A.

fulgidus (gi|11497956|); Nanoarchaeum equitans (gi|41614807|); Thermofilum

pendens Hrk 5 (gi|119720733|); Methanosarcina acetivorans C2A (gi|20092535|).

Desulfoferrodoxins: Clostridium phytofermentans ISDg (gi|160880064|); Desulfovibrio

vulgaris subsp. vulgaris str. Hildenborough (gi|46581585|); Desulfovibrio

desulfuricans ATCC 27774 (gi|157830815|); Desulfoarculus baarsii (gi|3913458|);

Geobacter uraniireducens Rf4 (gi|148264558|); Archaeoglobus fulgidus

(gi|11498439|). Class III neelaredoxin from Treponema pallidum subsp. pallidum str.

Nichols (gi|15639809|). Neelaredoxin with a Rd-like domain from an uncultured

termite group 1 bacterium phylotype Rs-D17 (gi|189485274|).

29

Figure 3 – Dendogram of SORs, displayed with TreeView

(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). 1Fe-SORs, from Class II, are

represented in blue, 2Fe-SORs in red, and Class III, 1Fe-SORs in light brown. The

sequences presented in Figure 2 are highlighted. Archaeoglobus fulgidus (NlrAf and

DfxAf), Clostridium phytofermentans (DfxCp), Desulfovibrio desulfuricans (DfxDd),

Desulfovibrio gigas (NlrDg), Desulfovibrio vulgaris (DfxDv), Desulfoarculus baarsii

(DfxDb), Geobacter uraniireducens (DfxGu), Methanosarcina acetivorans (NlrMa),

Nanoarchaeum equitans (NlrNeq), Pyrococcus furiosus (NlrPfur), Thermofilum

pendens (NlrTp), Treponema pallidum (Class III Tp) and an uncultured bacterium

(RdNlr); ClassIII SORs from Fusobacterium mortiferum ATCC 9817 (gi|237736311|),

ClassIII Fm, and Eubacterium biforme DSM 3989 (gi|218283678|), ClassIII Eb.

Figure 4 - Characteristic spectra of superoxide reductases. a) Spectra of A. fulgidus

2Fe-SOR in the half-reduced state (centre I oxidized, centre II reduced, solid line)

and fully oxidized (both centres oxidized, dashed line); inset shows the difference

spectrum, i.e., the features of centre II. b) Spectra of A. fulgidus 1Fe-SOR in the

oxidized (solid line) and reduced states (dotted line). c) pH-induced spectral changes

of the characteristic absorbance band at 660 nm, of A. fulgidus 1Fe-SOR; inset

shows the pH-titration followed by Visible spectroscopy.

Figure 5 – Reconstituted spectra of reaction intermediates observed upon reaction of

superoxide with A. fulgidus 1Fe-SOR, and respective putative assignments.

30

Figure 6 – Catalytic cycle of superoxide reductase, showing only the observable

intermediates. Larger arrow: reductive cycle; narrow arrows: oxidative cycle.

Figure 7 – Possible inhibition mechanism of the reduction of SOD and SOR by O2-

through competition with OH-.

31

Table 1 – SOR’s crystallographic structures.

* These data contains both the oxidized, glutamate bound and the reduced forms.

SOR Type Kingdom Organism PDB Resolution (Å)

Oxidation state Wild Type/Mutants

References

1Fe-SOR Archaea Pyrococcus

horikoshii Ot3 2HVB 2.5 Oxidized/ Glu bound WT Structural Genomics

Archaea Pyrococcus furiosus

1DQI 1.7 Oxidized/ Glu bound WT [65]

1DO6 2 Oxidized* WT [65] 1DQK 2 Reduced WT [65] Bacteria Thermotoga

maritima 2AMU 2 Reduced WT Structural Genomics

2Fe-SOR

Bacteria Desulfovibrio desulfuricans ATCC 27774

1DFX 1.8 Native/ ferricyanide bound WT [19]

Bacteria Desulfoarculus baarsii

2JI3 1.9 Fe-Peroxide intermediate E114A [50]

2JI2 1.9 Reduced E114A [50] 2JI1 1.9 Reduced WT [50] 1VZI 1.7 X-Ray

photoreduction/Ferrocyanide bound

E47A [66]

1Fe-SOR/ClassIII

Bacteria Treponema pallidum

1Y07 1.55 Native/Glu unbound WT [67]

32

Table 2 – Numbering of center II aminoacid ligands of the SORs represented in the

sequence alignment and in the dendogram (“ –“ : residues not conserved)

Fe ligands 1Fe-SOR Glu His His His His Cys Lys P.furiosus E14 H16 H41 H47 H114 C111 K15 D.gigas E15 H17 H45 H51 H118 C115 K16 A.fulgidus E12 H14 H40 H46 H113 C110 K13 N.equitans - H10 H35 H41 H100 C97 K9 T.pendens E24 H26 H51 H57 H113 C110 - M.acetivorans - H27 H54 H60 H114 C111 K26 2Fe-SOR C.phytofermentans E47 H49 H69 H75 H121 C118 K48 D.vulgaris E47 H49 H69 H75 H119 C116 K48 D.desulfuricans E47 H49 H69 H75 H119 C116 K48 D.baarsii E47 H49 H69 H75 H119 C116 K48 G.uraniireducens E47 H49 H69 H75 H119 C116 K48 A.fulgidus E47 H49 H69 H75 H119 C116 K48 1Fe-SOR/Class III

T.pallidum E48 H50 H70 H76 H122 C119 K49 Rd 1Fe-SOR uncultured bacterium - H53 H83 H89 H137 C134 K52

33

Table 3 – Spectroscopic and redox properties of SOR’s catalytic center

Oxidized λmax (nm) Eº (mV) center II pKa T1- Fe 3+ "hydroperoxo" λmax (nm)

K1(T1) K2(T2) (neutral

pH)

References

Low pH High pH (x 109 M-1s-1) (s-1) 2Fe-SOR

A.fulgidus 630 540 365 8.5 580 0.6 57 [57] D.vulgaris 647 560 250 ─ 590 1.5 40 [52]

D.vulgaris E47A ─ ─ ─ ─ 600 1.5 65 [52] D.vulgaris K48A ─ ─ ─ ─ 600 0.21 25 [52]

D.baarsii 644(pH 7.6) ─ 350 (pH 6-9) 9 610 1.1 500 [5, 30] D.baarsii E47A 580(pH 7.6) ─ 520 (pH5.5-6.5) 6.6 630 1.2 440 [5, 30] D.baarsii K48A 635(pH 7.6) ─ 520 (pH5.5-6.5) 7.6 600 0.38 300 [5, 30]

1Fe-SOR

A.fulgidus 666 590 250 9.6 620 1.2 ~400 [41] A.fulgidus E12V 670 590 302 6.3 620 0.22 ~400 [41]

N.equitans 655 550 350 6.5 590 1 <10 [22] T.pallidum 650 560 ~200 6 610 0.6 4800 [68]

T.pallidum E48A 650 560 ─ 6 ~600 0.6 2080 [47] D.gigas 666 590 190 >9 ─ ─ ─ [9]

34

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Table 1 – SOR’s crystallographic structures.

* These data contains both the oxidized, glutamate bound and the reduced

forms.

SOR Type Kingdom Organism PDB Resolution (Å)

Oxidation state Wild Type/Mutants

References

1Fe-SOR Archaea Pyrococcus

horikoshii Ot3 2HVB 2.5 Oxidized/ Glu bound WT Structural Genomics

Archaea Pyrococcus furiosus

1DQI 1.7 Oxidized/ Glu bound WT [65]

1DO6 2 Oxidized* WT [65] 1DQK 2 Reduced WT [65] Bacteria Thermotoga

maritima 2AMU 2 Reduced WT Structural Genomics

2Fe-SOR

Bacteria Desulfovibrio desulfuricans ATCC 27774

1DFX 1.8 Native/ ferricyanide bound WT [19]

Bacteria Desulfoarculus baarsii

2JI3 1.9 Fe-Peroxide intermediate E114A [50]

2JI2 1.9 Reduced E114A [50] 2JI1 1.9 Reduced WT [50] 1VZI 1.7 X-Ray

photoreduction/Ferrocyanide bound

E47A [66]

1Fe-SOR/ClassIII

Bacteria Treponema pallidum

1Y07 1.55 Native/Glu unbound WT [67]

Table 1

Table 2 – Numbering of center II aminoacid ligands of the SORs represented in

the sequence alignment and in the dendogram (“ –“ : residues not conserved)

Fe ligands 1Fe-SOR Glu His His His His Cys Lys P.furiosus E14 H16 H41 H47 H114 C111 K15 D.gigas E15 H17 H45 H51 H118 C115 K16 A.fulgidus E12 H14 H40 H46 H113 C110 K13 N.equitans - H10 H35 H41 H100 C97 K9 T.pendens E24 H26 H51 H57 H113 C110 - M.acetivorans - H27 H54 H60 H114 C111 K26 2Fe-SOR C.phytofermentans E47 H49 H69 H75 H121 C118 K48 D.vulgaris E47 H49 H69 H75 H119 C116 K48 D.desulfuricans E47 H49 H69 H75 H119 C116 K48 D.baarsii E47 H49 H69 H75 H119 C116 K48 G.uraniireducens E47 H49 H69 H75 H119 C116 K48 A.fulgidus E47 H49 H69 H75 H119 C116 K48 1Fe-SOR/Class III

T.pallidum E48 H50 H70 H76 H122 C119 K49 Rd 1Fe-SOR uncultured bacterium - H53 H83 H89 H137 C134 K52

Table 2

Table 3 – Spectroscopic and redox properties of SOR’s catalytic center

Oxidized λmax (nm) Eº (mV) center II pKa T1- Fe 3+ "hydroperoxo" λmax (nm)

K1(T1) K2(T2) (neutral

pH)

References

Low pH High pH (x 109 M-1s-1) (s-1) 2Fe-SOR

A.fulgidus 630 540 365 8.5 580 0.6 57 [57] D.vulgaris 647 560 250 ─ 590 1.5 40 [52]

D.vulgaris E47A ─ ─ ─ ─ 600 1.5 65 [52] D.vulgaris K48A ─ ─ ─ ─ 600 0.21 25 [52]

D.baarsii 644(pH 7.6) ─ 350 (pH 6-9) 9 610 1.1 500 [5, 30] D.baarsii E47A 580(pH 7.6) ─ 520 (pH5.5-6.5) 6.6 630 1.2 440 [5, 30] D.baarsii K48A 635(pH 7.6) ─ 520 (pH5.5-6.5) 7.6 600 0.38 300 [5, 30]

1Fe-SOR

A.fulgidus 666 590 250 9.6 620 1.2 ~400 [41] A.fulgidus E12V 670 590 302 6.3 620 0.22 ~400 [41]

N.equitans 655 550 350 6.5 590 1 <10 [22] T.pallidum 650 560 ~200 6 610 0.6 4800 [68]

T.pallidum E48A 650 560 ─ 6 ~600 0.6 2080 [47] D.gigas 666 590 190 >9 ─ ─ ─ [9]

Table 3

A

B

C

His114

His16

His47

His41

Glu14

Cys111

Cys30

Cys13

Cys13

Cys30

Cys29

Cys10 Glu14

His114

His16

His47

His41

Cys111

Pinto et al, Figure 1

Figure 1

Pyrococcus furiosus

:--------------------------MISETIRSGDWKG-----------EKHVPVIEYER------EGELVKVKVQVGK

Desulfovibrio gigas

:-------------------------MKMCDMFQTADWKT-----------EKHVPAIECDDAV---AADAFFPVTVSLGK

Archaeoglobus fulgidus

:----------------------------MELFQTADWKK-----------EKHVPVIEVLRA-----EGGVVEVKVSVGK

Nanoarchaeum equitans

:------------------------------MIKT-EYN------------PKHSPIIEIEK------EGELYKITIEVGK

Thermofilum pendens

:-----------------------MPKKFGDLIYTPETASGEAISKV----ETHTPRIEAPDSV---KAGEPFYVKIYVG-

Methanosarcina acetivorans

:----------------------MMGKKMAEEKINKPADPNNLTDGE----KKHIPIINVPETI---VAGEPFDVTVEVG-

Clostridium phytofermentans :---MTKEQKFFIC-ETCGNIIGMIEDKGVPVVCCGKKMTELVANTSDGAQEKHVPVVEVKDN----------LVYVSVG-

Desulfovibrio vulgaris

:---MPNQYEIYKC-IHCGNIVEVLHAGGGDLVCCGEPMKLMKEGTSDGAKEKHVPVIEKTAN----------GYKVTVG-

Desulfovibrio desulfuricans

:---MPKHLEVYKC-THCGNIVEVLHGGGAELVCCGEPMKHMVEGSTDGAMEKHVPVIEKVDG----------GYLIKVG-

Desulfoarculus Baarsii

:---MPERLQVYKC-EVCGNIVEVLNGGIGELVCCNQDMKLMSENTVDAAKEKHVPVIEKIDG----------GYKVKVG-

Geobacter uraniireducens

:---MAKNLEIYKC-ESCGNIIEILHSGPGDLVCCGSPMQLQVENTVDASREKHLPVLEKANG----------SVTVKVG-


:---MTEVMQVYKC-MVCGNIVEVVHAGRGQLVCCGQPMKLMEVKTTDEGKEKHVPVIEREGN----------KVYVKVG-

Treponema pallidum

:---MGRELSFFLQKESAGFFLGMDAPAGSSVACGSEVLRAVPVGTVDAAKEKHIPVVEVHGH----------EVKVKVG-

uncultured bacterium

:MKGLVCKVCGYVALDGNKERCPVCRSKNVFEEKEDAYKMPDFKAASDETEKKHIPSFMLMSECSLIPDTGCVDVHVKIG-

Pyrococcus furiosus

:IPHPNTTEHHIRYIELYFLPEGENFVYQVGRVEFTAHGESVNGPNTSDVYTEPIAYFVLKTKKKGKLYALSYCNIHGLWE

Desulfovibrio gigas

:IAHPNTTEHHIRWIRCYFKPEGDKFSYEVGSFEFTAHGECAKGPNEGPVYTNHTVTFQLKIKTPGVLVASSFCNIHGLWE


:IPHPNTTEHHIAWIELVFQPEGSKFPYVVGRAEFAAHGASVDGPNTSGVYTDPVAVFAFKAEKSGKLTAFSYCNIHGLWM


:VKHPNEPSHHIQWVDLYFEPEG-KEPTHIARIEFKAHGEYNN-------YTEPKAIVYAKLEGKGKLIAISYCTLHGLWK

Thermofilum pendens

:-PHPNTLQHSIRWIEVYFEEEGRPFNPVMLSRIHLEP-----------ELVEPEVTLKLVLKKSGVIYALEYCNLHGVWE


:IPHVMEEKHHIEWIELYLNDKKIRRAELSLENKKAEA-------------TFTVEADKSLAGKESKLRALENCNIHGLWE

Clostridium phytofermentans :VVHPMLEEHSIQWVYLRTNQGGHRKSLAPGS--------------------EPKVVFALTEGEE-AIEVFEYCNLHGLWK


:VAHPMEEKHWIEWIELVADGVSYKKFLKPGD--------------------APEAEFCIKADK---VVAREYCNLHGHWK


:VPHPMEEKHWIEWIELLADGRSYTKFLKPGD--------------------APEAFFAIDASK---VTAREYCNLHGHWK


:VAHPMEEKHYIQWIELLADDKCYTQFLKPGQ--------------------APEAVFLIEAAK---VVAREYCNIHGHWK


:VPHPMEEQHYIEWIEVIADGTVYRQALKPGD--------------------APEATFPITAGS---ITVREYCSLHGQLS


:VAHPMEEQHYIEWIEVIDDGCVHRKQLKPGD--------------------EPKAEFTVMSDR---VSARAYCNIHGLWQ

Treponema pallidum

:VAHPMTPEHYIAWVCLKTRKGIQLKELPVDG--------------------APEVTFALTADDQ-VLEAYEFCNLHGVWS


:ILHPTLPEHHITGIAFYIDNKFVENIMLESD-------------------INPAAVIHLNGSTKGRVQVIENCNIHGKWF

Pyrococcus furiosus

:EVTLE--

:124

Desulfovibrio gigas

:SKAVALK

:130


:EATLSLE

:120


:EKEL---

:109

Thermofilum pendens

:RKQVKVQ

:125


:FMTIKMS

:126

Clostridium phytofermentans :VL-----

:128


:EA-----

:126


:EN-----

:126


:EN-----

:126


:IG-----

:126


:G------

:125

Treponema pallidum

:K------

:128


:EVEVK--

:147

**

**

**


Figure 2

0.1

NlrAf

NlrNeq

NlrMa

NlrTp

RdNlr

DfxDv

DfxGu

DfxCp

ClassIII Tp

ClassIII Fm

ClassIII Eb

Class III

Nlr

–1F

e S

OR

NlrPfur

NlrDg

DfxAf

DfxDb

DfxDd

Dfx – 2Fe SORs

0.1

NlrAf

NlrNeq

NlrMa

NlrTp

RdNlr

DfxDv

DfxGu

DfxCp

ClassIII Tp

ClassIII Fm

ClassIII Eb

Class III

Nlr

–1F

e S

OR

NlrPfur

NlrDg

DfxAf

DfxDb

DfxDd

Dfx – 2Fe SORs


Figure 3

300 400 500 600 700 8000

10

20

30

40

Wavelength (nm)

ε m

M-1 c

m-1

500 600 700 800 9000

1

2

3

4

Wavelength (nm)

ε m

M-1 c

m-1

6 7 8 9 10 11 120.04

0.05

0.06

0.07

0.08

Abs

560

nm

pH

300 400 500 600 700 8000

4

8

12

16

20

24

ε m

M-1 c

m-1

Wavelength (nm)

300 400 500 600 700 8000

2

4

6

ε m

M-1 c

m-1

Wavelength (nm)

a

b

c

pH 7

pH 11


Figure 4

T1

T2

Final

Fe3+

N

N

N

S

N

HOO

Fe3+

N

N

N

S

N

HO

Fe3+

N

N

N

S

N

O

O CGlu

0

1

2

3

4

εmM

-1cm

-1

500 550 600 650 700 7500

1

2

3

4

Wavelength (nm)

εmM

-1cm

-1

0

1

2

3

4

εmM

-1cm

-1

T1

T2

Final

Fe3+

N

N

N

S

N

HOO

Fe3+

N

N

N

S

N

HO

Fe3+

N

N

N

S

N

O

O CGlu

0

1

2

3

4

εmM

-1cm

-1

500 550 600 650 700 7500

1

2

3

4

Wavelength (nm)

εmM

-1cm

-1

0

1

2

3

4

εmM

-1cm

-1


Figure 5


k3

T1

T2

Final

O2- + H+

k1

H+

H2O2

Fe3+

His

His

His

Cys

His

HOO

Fe3+

His

His

His

Cys

His

HO

H2O2

H+

k2´

k2

Fe2+

His

His

Cys

His His

OH-/OH2

Fe3+

His

His

His

Cys

His

Glu

O

O C

CellularReductants

Fe3+

His

His

His

Cys

His

H2OpKa = 6.1

Fe3+

His

His

His

Cys

His

HO

pKa= 9.6

Fe3+

His

His

His

Cys

His

O

O P

OH

OH

H2PO4-

T2P

Glu

Glu

Glu

H2PO4-

Fe-SOD

H2O

Fe2+

Asp

HisHis

His

OH

Fe3+

Asp

HisHis

His

H2O

OH

Fe3+

Asp

HisHis

His

HO

O2-

O2-

H2O2

O2

pKa ≈≈≈≈ 9

Inactive

H2 O

Fe2+

Asp

HisHis

His

T yrOH

Inactive

pKa ≈≈≈≈ 9

TyrO-

Fe-SOR

Fe2+

His

His

His

S-Cys

His

H2O

Fe3+

His

His

His

S-Cys

His

O2-

O2

O2-

O2-

H2O2

O2

OH

Fe3+

His

His

His

S-Cys

His

pKa ≈≈≈≈ 6

?

O2-

H2O2

Fe-SOD

H2O

Fe2+

Asp

HisHis

His

OH

Fe3+

Asp

HisHis

His

H2O

OH

Fe3+

Asp

HisHis

His

HO

O2-

O2-

H2O2

O2

pKa ≈≈≈≈ 9

Inactive

H2 O

Fe2+

Asp

HisHis

His

T yrOH

Inactive

pKa ≈≈≈≈ 9

TyrO-

Fe-SOR

Fe2+

His

His

His

S-Cys

His

H2O

Fe3+

His

His

His

S-Cys

His

O2-

O2

O2-

O2-

H2O2

O2

OH

Fe3+

His

His

His

S-Cys

His

pKa ≈≈≈≈ 6

?

O2-

H2O2


Figure 7

Reductive elimination of superoxide: Structure and mechanism of superoxide reductases

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