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Cite this: Phys.Chem.Chem.Phys.,
2014, 16, 14220
Reductive activation and structural rearrangement
in superoxide reductase: a combined infrared
spectroscopic and computational study†
M. Horch,*
a
A. F. Pinto,‡
b
T. Utesch,
a
M. A. Mroginski,
a
C. V. Roma
˜o,
b
M. Teixeira,
b
P. Hildebrandt
a
and I. Zebger*
a
Superoxide reductases (SOR) are a family of non-heme iron enzymes that limit oxidative stress by catalysing
the reduction of superoxide to hydrogen peroxide and, thus, represent model systems for the detoxification
of reactive oxygen species. In several enzymes of this type, reductive activation of theactivesiteinvolvesthe
reversible dissociation of a glutamate from the proposed substrate binding site at the iron. In this study we
have employed IR spectroscopic and theoretical methods to gain insights into redox-linked structural
changes of 1Fe-type superoxide reductases, focusing on the enzyme from the archaeon Ignicoccus
hospitalis. Guided by crystal structure data and complemented by spectra calculation for an active site
model, the main IR difference signals could be assigned. These signals reflect redox-induced structural
changes in the first coordination sphere of the iron centre, adjacent loop and helical regions, and more
remote b-sheets. By comparison with the spectra obtained for the E23A mutant of Ignicoccus hospitalis
SOR, it is shown that glutamate E23 dissociates reversibly from the ferrous iron during reductive activation
of the wild type enzyme. Moreover, this process is found to trigger a global conformational transition of the
protein that is strictly dependent on the presence of E23. Similar concerted structural changes can be
inferred from the IR spectra of related SORs such as that from Archaeoglobus fulgidus, indicating a
widespread mechanism. A possible functional role of this process in terms of synergistic effects during
reductive activation of the homotetrameric enzyme is proposed.
Introduction
In living systems, the superoxide anion radical, O
2
, and other
reactive oxygen species (ROS) are formed from molecular
oxygen by reactions with transition metals or radical species,
most notably via incomplete reduction of O
2
in the aerobic
membrane-bound electron transfer chain. These species may
have a beneficial role, e.g. in cell signalling, immune response,
and redox homeostasis. On the other hand, increased levels of
ROS severely harm cellular systems including DNA damage,
lipid peroxidation, oxidation of amino acids, and degradation
of protein metal centres. As a consequence, ROS have also been
associated with ageing and a wide range of diseases in humans
1
and, thus, biochemical reactions and detoxification of these
compounds are of general relevance.
To counteract oxidative stress, all known organisms harbour
molecular systems to detoxify ROS. In particular, the disproportiona-
tion of superoxide, yielding O
2
and H
2
O
2
, is catalysed by superoxide
dismutases (SODs),
2
which are almost ubiquitously present. Super-
oxide reductase (SOR) provides an alternative detoxification system
in anaerobic and microaerophilic bacteria or archaea (for recent
reviews, see ref. 3 and 4) as well as unicellular eukaryotes, such
as the human pathogenic protozoan Giardia intestinalis.
5
In contrast to SOD, superoxide reductase catalyses only the
reduction of superoxide:
3–10
SOR
red
+O
2
+2H
+
-SOR
ox
+H
2
O
2
i.e., the reductive part of the disproportionation reaction.
A detailed catalytic cycle,
3,9,10
based on an inner sphere
mechanism, has been proposed for this process mainly on
the basis of pulse radiolysis assays coupled to visible spectro-
scopy,
10–12
corroborated by crystallographic
9,13
and spectro-
scopic
14–17
data. Oxidized SOR is re-activated by cellular
reductases, such as NAD(P)H oxidoreductases, involving in
a
Technische Universita
¨t Berlin, Institut fu
¨r Chemie, Sekr. PC14,
Straße des 17. Juni 135, 10623 Berlin, Germany. E-mail: marius.horch@gmx.de,
b
Instituto de Tecnologia Quı
´mica e Biolo
´gica Anto
´nio Xavier,
Universidade Nova de Lisboa, Av. da Repu
´blica (EAN), P-2780-157 Oeiras,
Portugal
†Electronic supplementary information (ESI) available: Animated Fig. S1 and S2.
See DOI: 10.1039/c4cp00884g
‡Present Address: Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Scheeles Va
¨g 2, 17177 Stockholm, Sweden.
Received 28th February 2014,
Accepted 28th May 2014
DOI: 10.1039/c4cp00884g
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several cases rubredoxins as electron shuttles,
18–21
while H
2
O
2
may be further decomposed by catalase,
22
peroxiredoxin,
23
or
rubrerythrin.
24,25
In many cases, however, the physiological
electron donors are not known.
Superoxide reductases are non-heme iron metalloproteins,
which can be divided into two major classes according to
the number of metal centres: neelaredoxin (Nlr), denoted as
1Fe-SOR, and homodimeric desulfoferrodoxin (Dfx), or 2Fe-SOR,
both of which were first isolated from anaerobic sulphate
reducing bacteria.
26,27
In the ferrous state, the iron ion of the
catalytic site, named centre II in 2Fe-SORs, is pentacoordinated
in a square-pyramidal geometry by four equatorial histidine
imidazoles (one via the N
d
and three via the N
e
nitrogens) and
one apical cysteinyl sulphur donor (see Fig. 1(I)). For the inactive
ferric state, however, coordination of the iron is not uniform. For
SORs from Pyrococcus (P.)furiosus,
28
P. horikoshii (unpublished,
H. Yamamoto and N. Kunishima, PDB 2HVB), Desulfoarculus (D.)
baarsii,
29
and Treponema (T.)pallidum,
29
a glutamate carboxylate
was identified to bind to the sixth coordination site of the ferric
ion in a monodentate fashion, thereby forming an octahedral
geometry (as shown by X-ray crystallography for both Pyrococcus
enzymes and infrared (IR) spectroscopy for the remaining ones).
Although this residue is widely conserved, it is absent in several
1Fe-SORs, such as that from Nanoarchaeum equitans,
30
indicating
that the sixth coordination site of the ferric iron may be vacant or
occupied by a solvent molecule in some SORs. In 2Fe-SORs, there
is another iron site, named centre I, where the metal ion is
coordinated by four cysteines in a desulforedoxin-like, distorted
tetrahedral geometry.
31
Thefunctionofthiscentreissofar
unknown.
32
Methanoferrodoxin is a recently discovered type of
SOR proposed to contain another C-terminal domain harbouring
a[4Fe–4S]
2+/1+
cluster in addition to the common mononuclear
catalytic site.
33
IR difference spectroscopy provides information on structural
changes of individual amino acid side chains and local
perturbations of the peptide backbone in proteins by monitoring
spectral differences between two defined states of a chemical
species.
34–36
This technique is sensitive towards electrostatic inter-
actions, hydrogen bonding, and the protonation state of indivi-
dual titratable groups, thereby providing detailed insights into
bonding and non-bonding interactions complementary to crystal-
lographic data.
36
In contrast to the latter, these studies are
performed in solution and, thus, allow probing conformational
changes that might be obscured by crystal packing. In particular,
electrochemically triggered IR spectroscopy monitors structural
changes related to redox transitions, e.g. in metalloproteins.
29,34–37
In the present study, we have focused on the 1Fe-type SOR
from the hyperthermophilic, anaerobic archaeon Ignicoccus (I.)
hospitalis, using IR spectroscopic techniques complemented by
theoretical methods. A special interest in this enzyme comes
from the fact that it lacks a quasi conserved lysine residue that
has been proposed to stabilize and/or promote proton transfer
to a catalytic reaction intermediate.
3,4,11,38,39
Recently, a crystal
structure has been solved for the ferric homotetrameric enzyme
(see Fig. 1(I), PDB code 4BK8, P. Matias and T. Bandeiras,
personal communication). However, in the absence of a con-
clusive structure for the reduced state, it remained uncertain
whether the ferric iron glutamate ligand dissociates reversibly
upon reduction of this enzyme (E23 in I. hospitalis SOR; unless
otherwise stated, amino acid numbering for this enzyme will
be used). In previous studies, electrochemically triggered IR
difference spectroscopy was successfully applied to elucidate
this issue for 1Fe- and 2Fe-SORs from T. pallidum and
D. baarsii, respectively.
29
In the present work, we use this
technique to investigate reversible glutamate dissociation from
the metal centre of the I. hospitalis superoxide reductase by
monitoring redox-dependent perturbations of the E23 carboxylate
sidechainaswellasstructuralchangesofotheraminoacid
residues and the protein backbone around the active site. As
a reference, we also characterized the E23A mutant of the
Fig. 1 (I) Crystal structure of a single monomer of the I. hospitalis SOR. Essential amino acids (in one-letter code) and secondary structure elements
involved in the redox response of the enzyme are highlighted. 3
10
helices proximal and distal to E23 are marked with ‘prox’ and ‘dist’, respectively.
(II) Model compound of the SOR active site used for IR spectra calculations. All ligands are assigned to the respective coordinated amino acids using the
nomenclature of the I. hospitalis SOR. Both structures correspond to the ferric state.
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I. hospitalis SOR, where the active site glutamate is exchanged for
alanine, thus excluding any carboxylate binding to the iron. Since
the apical glutamate coordinates the iron ion of the 1Fe-type SOR
from Archaeoglobus (A.)fulgidus in a reversible manner (PDB
code 4BGL, P. Matias and T. Bandeiras, personal communication),
this enzyme was characterized for comparison as well.
Experimental procedures
Enzyme overexpression and purification
Recombinant I. hospitalis (wild type and E23A mutant) and
A. fulgidus 1Fe-SORs were overexpressed in Escherichia coli and
purified as previously described.
40,41
Sample preparation
Samples were concentrated to 2 mM and buffered at pH 7.5/pD
7.5 in an aqueous (H
2
O/D
2
O) solution of 20 mM Tris and 150 mM
NaCl using HCl/DCl for pH/pD adjustment. For measurements in
D
2
O, the pD was estimated according to pD = pH meter reading +
0.4.
42
To prevent back-exchange with atmospheric water vapour,
samples in deuterated solution were kept under Argon during
sample preparation. To ensure proper electron transfer in solution
sample preparations were incubated with a mixture of the following
redox mediators (40 mM each) for 30 min at 4 1C (potentials given
vs. standard hydrogen electrode): ferrocene (E
m
= 422 mV),
N,N-dimethyl-p-phenylenediamine (E
m
= 371 mV), 1,4-benzo-
quinone (E
m
=280mV),N,N,N0,N0-tetramethyl-p-phenylenediamine
(E
m
= 260 mV), 2,3,4,5-tetramethyl-p-phenylenediamine (E
m
=
230 mV), phenazine ethosulfate (E
m
= 55 mV), and duroquinone
(E
m
=5mV).
Spectroelectrochemistry
IR spectra were recorded with a spectral resolution of 4 cm
1
on
a Bruker IFS-66 v/S FTIR spectrometer equipped with a liquid
N
2
cooled MCT detector and a 5500 cm
1
low-pass filter.
400 scans per single channel spectrum were accumulated and
five equivalent difference spectra were averaged for each sample.
Samples were held in a temperature controlled (T=101C), optically
transparent thin-layer electrochemical (OTTLE) cell (VB10 mL)
equipped with CaF
2
windows, a Au mesh working electrode
(dB5mm), a Ag/AgCl reference electrode, and a Pt sheet as
counter electrode.
37
To prevent unspecific protein binding and
denaturation, the Au mesh was coated with a mixed self assembled
monolayer consisting of cysteamine and mercaptopropionic acid
for 30 min under Argon atmosphere prior to measurements.
Electrochemical control was accomplished using an Eco Chemie
B.V. Autolab PGStat12 potentiostat. Measurements were performed
under N
2
atmosphere. All spectra were processed using the Bruker
OPUS software version 5.5.
IR spectra calculation
IR spectra were computed for a model of the ferric SOR active site
(Fig. 1(II)), monoethanethiolato-monobutyrato-tri(4-ethylimidazole-
N1)-mono(4-ethylimidazole-N3)-iron(III), using density functional
theory (DFT). Initial geometric parameters of the complex were
derived from the crystal structure of the oxidized SOR from
I. hospitalis by extracting the active site and substituting the
–(NH–C
a
–(CQO))– peptide moieties with methyl groups (C
a
H
3
).
After adding protons according to neutral pH, geometry
optimization and spectra calculations for the model compound
were performed on the BP86 level of theory
43,44
in Gaussian 03,
using the 6-31g* and tzvp basis sets
45
for H, C, N, O, S atoms
and the Fe atom, respectively. Cartesian coordinates of the C
a
atoms of the coordinated amino acids were fixed in order to
compensate for the missing protein backbone rigidity, thereby
preserving a native-like framework for the active site geometry.
Calculated IR spectra were plotted using Gaussian-shaped
bands with a half-width at half height of 4 cm
1
.
Aliphatic index and hydrophobicity estimation
Both quantities were determined using the ProtParam tool as
implemented on the ExPASy – SIB Bioinformatics Resource
Portal (http://web.expasy.org/protparam). The aliphatic index
was calculated according to:
46
aliphatic index = X
A
+aX
V
+b(X
I
+X
L
)
where X
A
,X
V
,X
I
, and X
L
are the mole fractions (in %) of alanine,
valine, isoleucine, and leucine. Coefficients a= 2.9 and b=
3.9 represent the relative volumes of the valine and leucine/
isoleucine side chains, respectively, as compared to alanine.
Overall hydrophobicity was estimated according to the Grand
Average of Hydropathy (GRAVY), which was calculated as the
average of hydropathy values of all amino acids.
47
Calculation of protein packing density
Overall and local protein packing densities of the ferric
I. hospitalis SOR crystal structure were determined to a grid
distance of 0.01 Å by an improved Voronoi cell algorithm
48
as
implemented in the Voronoia software package
49
using standard
atomic radii and volumes.
50
Secondary structure evaluation
Localization and quantification of hydrogen bonds was accomplished
using the Define Secondary Structure of Proteins (DSSP) algorithm
51
as implemented in the program of the same name.
Determination of solvent accessibility
After protonating the crystal structure of the ferric I. hospitalis
SOR according to neutral pH, solvent accessibility was determined
by calculating the solvent accessible area (SAA) of each individual
atom using Visual Molecular Dynamics (VMD) 1.8.6.
52
An SAA map
was generated for the whole enzyme, assuming an average molecular
radius of 1.4 Å for water in the screening procedure.
Calculation of collective protein motions
Atom displacements were determined for the first 100 low-
frequency modes of the ferric I. hospitalis SOR using normal
mode analysis in concert with an all-atom elastic network
model, as implemented in the Normal Mode Analysis, Deformation
and Refinement (NOMAD-Ref) server.
53
Distance-dependent atomic
interactions were modelled from a global elastic constant of
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100 kcal mol
1
Å
2
by means of an exponential decay weighting
function, using a distance-weight and cut-off parameter of
5 and 10 Å, respectively.
Results and discussion
Analysis of iron ligand modes
To elucidate whether E23 binds reversibly to the ferric iron of
I. hospitalis SOR, we first focused on changes in the normal
modes of this amino acid. The symmetric and antisymmetric
stretching modes of the carboxylate side chain, which appear
at B1400 cm
1
[n
s
(COO)] and B1560 cm
1
[n
as
(COO)], respec-
tively,
54,55
are sensitive to bonding and non-bonding interactions,
such that anionic, protonated, H-bonded and different metal-
bound forms can be distinguished by vibrational spectroscopy.
56
Monodentate metal binding is generally indicated by an increase of
n
as
(COO) and a decrease of n
s
(COO) relative to the non-coordinated
state, leading to an increased frequency difference Dnbetween
these two vibrational modes.
54
Trace A of Fig. 2 shows the IR
difference spectrum [reduced–oxidized] of SOR from I. hospitalis,
where bands with a positive sign are related to the ferrous
form, while those with a negative sign are due to the ferric
state. Possible candidates for n
as
(COO) of E23 are detected at
1554/1546 cm
1
(/+), while bands at 1409/1386 cm
1
(+/)may
(partly) originate from the respective symmetric stretching mode.
Both difference signals are absent in the spectrum of the E23A
mutant protein (Fig. 2B), but observed for the wild type SOR from
A. fulgidus (Fig. 2C), supporting their assignment to binding/
dissociation related perturbations of the terminal COO
group of
E23. These findings are in line with previous studies comparing IR
spectroscopic data of D. baarsii WT SOR to the corresponding E47A
mutant.
29
Thus, the observed increase of Dnbetween n
as
(COO) and
n
s
(COO) upon oxidation indicates monodentate binding of E23 to
the ferric non-heme iron of SOR from I. hospitalis.
Redox-linked spectral changes of wild-type I. hospitalis SOR
(Fig. 2A) are not restricted to the carboxylate side chain of E23
but also include bands attributable to other amino acid residues
and the peptide backbone (Table 1). Some of these difference
signals are located in crowded regions of the spectrum such that
the pattern of conjugate positive and negative signals is partially
obscured by overlapping bands of opposite sign and different
amplitudes. These difference signals are well observed for the
I. hospitalis SOR wild type enzyme (Fig. 2A), but virtually absent
or less pronounced in the corresponding spectrum of the E23A
mutant (Fig. 2B), revealing a significant involvement of E23
in the redox-related structural changes of the wild type enzyme.
Fig. 2 IR difference spectra [reduced–oxidized] of SOR. Positive and negative bands correspond to the ferrous and ferric state of the enzyme,
respectively. Spectral regions of particular interest are highlighted in gray. Normal modes of important amino acids (in one-letter code) and the peptide
backbone are indicated. For a detailed band assignment and further information, see Table 1. (Left) IR difference spectra recorded from aqueous solutions
of (A) wild type SOR and (B) the E23A mutant protein from I. hospitalis as well as (C) the wild type SOR from A. fulgidus. (Right) IR difference spectra of the
wild type I. hospitalis SOR recorded in (D) H
2
Oand(E)D
2
O buffer.
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In particular, bands at 1595 (), 1280 (), 1233 (+), 1112 (+), and
1099 cm
1
() can be assigned to histidine side chain modes,
56–58
suggesting perturbations of the iron histidine ligands, H25, H50,
H56, and/or H112 (the only histidines of the I. hospitalis SOR, see
Table 1). Comparable features, as also observed in the difference
spectrum of the A. fulgidus SOR (Fig. 2C), have been monitored
previously during redox-dependent glutamate binding/dissociation
in SORs from T. pallidum and D. baarsii,
29
indicating that these
bands are general markers for changes in the coordination number
and geometry of the active site, caused by the binding/dissociation of
the glutamate (here E23). As a consequence, these bands are likely to
monitor the binding of other ligands as well and, thus, may provide
valuable insights into changes of the active site (electronic) structure
resulting from substrate binding and conversion.
Evaluation of vibrational markers of the first coordination
sphereoftheactivesiteFehasclearly shown reversible dissociation
of E23 from this metal ion. This is an important finding since this
process is essential to the enzyme’s catalytic function requiring a
vacant coordination site for substrate binding and interconversion.
Notably, glutamate displacement was not clearly observed in pre-
liminary crystal structure data of the reduced enzyme, possibly due
to crystal packing effects. Consequently, we have also turned our
attention to further redox-related structural changes of the enzyme
that might be restrained in the crystal state as well.
Redox-linked structural changes beyond the first coordination
sphere
IR difference spectroscopy also provides evidence that reductive
dissociation of E23 triggers extended structural changes of
I. hospitalis WT SOR, as proven by the absence of the corres-
ponding spectroscopic markers in the spectrum of the E23A
mutant (Fig. 2A and B). In this respect, the slightly H/D
sensitive difference signal at 1706/1697 cm
1
(+/) is assigned
to a mode dominated by the stretching of the carboxamide
group of an asparagine side chain
59–61
(Fig. 2A, D and E and
Table 1), most likely N110 (see Fig. 1(I)). This amino acid is also
present in the A. fulgidus SOR (N111) and, thus, a corre-
sponding difference signal at 1708/1697 cm
1
(+/) is observed
in the [red–ox] difference spectrum of this enzyme (Fig. 2C).
Since this mode exhibits a frequency close to that of the
backbone CQO stretching and NH bending (amide I) it may
have contributions from these latter vibrations, as demon-
strated for the model peptide Glu–Asn–Glu (for an animation,
see ESI,†S1). Consequently, this mode may serve as a probe for
changes of the N110 peptide bond and, thus, for a local
perturbation of the protein backbone. In I. hospitalis SOR,
N110 is located in a b-turn and adjacent to H112 and C109,
both of which are ligands to the iron (see Fig. 1(I)). Hence, it is
plausible to assume that the 1706/1697 cm
1
difference signal
(+/) reflects a redox-dependent rearrangement of the b-turn,
mediated by structural changes in H112 and C109 as discussed
above. This hypothesis is supported by a pronounced band at
1686 cm
1
(+), which is tentatively assigned to changes in the
amide I mode of this b-turn
62
(Fig. 2A). Interestingly, the
cysteinyl thiolate of the SOR active site is generally considered
to exert a strong trans influence on the opposite bond.
14,63,64
Therefore, assuming a mutual influence between two trans
oriented ligands,
65,66
probing of Fe–S stretching vibrations is
Table 1 Overview of essential vibrational modes of SOR that undergo redox-dependent changes in the enzymes from I. hospitalis (Ih)andA. fulgidus
(Af). WT and E23A refer to the wild type enzymes and the respective mutant protein from I. hospitalis. Amino acids are denoted by the one-letter code.
Band positions for the ferric and ferrous state are indicated by () and (+), respectively (n.a. – not assigned)
Assignment Protein Band position (H
2
O)/cm
1
Band position (D
2
O)/cm
1
Ref.
E, n
s
(COO) Ih WT 1386()/1409(+) 1386()/1409(+) 54–56
Af WT 1381()/1402(+) n.a.
E, n
as
(COO) Ih WT 1546(+)/1554() 1546(+)/1552() 54–56
Af WT 1554(+)/1569() n.a.
H, n(CN) Ih WT 1099()/1112(+) 1099()/1112(+) 56–58
Ih E23A 1097()/1115(+) 1098()/1113(+)
Af WT 1102() 1102()
H, d(CH + NH) + n(CN) Ih WT 1233(+) 1243(+) 56
H, n(CN + CC) or g
t
(CH
2
)Ih WT 1280() 1277()56
Ih E23A 1280() 1280()
Af WT 1271() n.a.
H, n(CQC) Ih WT 1595()/1580() 1595()/1586() 56–58
Ih E23A 1600()/1581() 1597()/1588()
Af WT 1599 1599
N, n(CQO) Ih WT 1697()/1706(+) 1690()/1702(+) 59–61
Af WT 1697()/1708(+) 1687()/1693(+)
N, g
r
(NH
2
)Ih WT 1132() 1044()83
Af WT 1149()o1000
Aliphatic amino acids, d
s
(CH
3
)Ih WT 1386() 1386()56
Helices, amide I Ih WT 1649() 1649()62
Ih E23A 1653() 1653()
Turn, amide I Ih WT 1686(+) 1666(+) 62
Ih E23A 1690(+) n.a.
Turn, amide II Ih WT 1501(+) 1409(+)
b-Sheets, amide I Ih WT 1635(+)/1627() 1635(+)/1627()62
Ih WT 1678()/1669(+) 1678()/1669(+)
Af WT 1639()/1627(+) 1639()/1627(+)
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a powerful means to assess changes in the binding of E23.
67
These normal modes are not directly accessible by mid-infrared
spectroscopy. However, the above mentioned local perturbations
in the b-turn encompassing C109 are an indirect indication for
structural changes around this cysteine and, thus, a probe for
alterations in the metal binding of E23 and related conforma-
tional transitions.
Further structural changes are indicated by a pronounced
amide I band at 1649 cm
1
(), which is assigned to a structural
reorganization in at least one of the three consecutive helices at
the N-terminus adjacent to E23 (Fig. 1(I)),
62
most likely related to
the binding/dissociation of this amino acid in the I. hospitalis
WT enzyme. Such structural changes are neither observed for the
SOR from A. fulgidus (Fig. 2C), whose N-terminus is shorter, nor
for those from T. pallidum and D. baarsii,
29
since this region
corresponds to the linker to the desulforedoxin-like domain in
these enzymes. The above findings show that dissociation of E23
involves extended structural changes in I. hospitalis WT SOR
especially affecting nearby helical regions as well as the b-turn in
trans position of the active site.
Another strong band, observed for the ferric I. hospitalis WT
SOR at 1386 cm
1
() (Fig. 2A), most likely originates from
the d
s
(CH
3
) modes of aliphatic amino acid side chains and
possibly, to a lesser extent, from the n
s
(COO) mode of E23 (vide
supra). Indeed, several hydrophobic amino acids are found near
the active site, all of which are located close to amino acids that
undergo structural reorganization upon the enzyme’s redox
transition. Hence, this band can be taken as a further indication
for a concerted structural change around the iron site in I. hospitalis
SOR due to reversible dissociation of E23. This rearrangement
appears to be a wide spread feature of SORs since similar
pronounced bands are also observed in the difference spectra
of the enzymes from T. pallidum and D. baarsii,
29
while the
A. fulgidus SOR exhibits less intense yet numerous features in
this spectral range (Fig. 2C). Such a concerted redox-dependent
structural change in SOR is also indicated by perturbations of
the peptide backbone, observed for all SORs characterized by IR
spectroscopy so far (this work and ref. 29).
Hydrogen–deuterium exchange of protein backbone protons
Additional experiments were performed in D
2
O (Fig. 2E and
Table 1) to promote band assignments. Due to limited protein
stability, proper evaluation of these experiments was only
possible for the I. hospitalis and A. fulgidus WT SORs.
Interestingly, large parts of I. hospitalis SOR appear to be
excluded from proton exchange under the present experimental
conditions, as indicated by an amide II/amide II’ ratio of almost
one (data not shown). In general, incomplete H/D exchange
may be ascribed to high protein packing densities, a large
number of hydrophobic amino acids and/or a significant content
of H-bonded amide protons involved in secondary structure
elements. Thus, evaluation of this observation may provide
valuable insights into structural aspects of the enzyme.
To estimate the contribution of hydrophobic amino acids to
the incomplete proton exchange, we determined the aliphatic
index and the overall hydrophobicity according to the Grand
Average of Hydropathy (GRAVY). Compared to other proteins,
68
the percentage of hydrophobic amino acids is only slightly
increased in this enzyme by 2.6% (see also Fig. 3(I)) and no
above-average values
46,47
were observed for the aliphatic index
(77) and the GRAVY (0.43). The impact of secondary structure
elements on H/D exchange properties can be assessed by
quantifying the amount of H-bonds per residue, which was
found to be slightly higher (0.69) compared to the average (0.63)
of globular proteins of this type and size,
69
in line with the
absence of large flexible regions (Fig. 3(II)). Most H-bonds are
located in the centre of the protein, consistent with a high
content of b-sheets in this region (Fig. 3(II)). For the packing
density, we found a low
70
average value of 0.688. However, local
densities strongly vary between 0.17 close to the surface and
0.92 in the core (Fig. 3(III)). Again, the latter region coincides
with the central b-sheet pattern and, thus, hindered H/D
exchange in I. hospitalis SOR is ascribed to limited solvent
accessibility in this highly dense structural motif. This conclusion
is in agreement with solvent accessibility calculations (Fig. 3(IV))
and previous observations for other proteins.
71
Notably, high
packing (and H-bond) densities, as observed in the b-sheet core
of the I. hospitalis SOR, have been described as a marker for rigid
domains and a prerequisite for non-local structural changes
70,72
like those observed in this study.
For I. hospitalis SOR in D
2
O, redox-dependent structural
changes are displayed in the [red–ox] difference spectrum in
trace E of Fig. 2. Although the iron site is solvent exposed, the
spectrum largely resembles that obtained in H
2
O, revealing a
few changes of prominent absorptions assigned to the amide I
and amide II modes. In particular, the dominating amide I
band at 1649 cm
1
(), ascribed to structural reorganization in
at least one of the three helices close to the active site, remains
unchanged in position and relative intensity in D
2
O. Consider-
ing the solvent exposed position of these secondary structure
motifs, hindered exchange of the corresponding amide protons
is most likely related to strong hydrogen bond interactions.
This suggests high rigidity of these helices, which in turn may
be able to transmit active site structural changes to other parts
of the protein (vide supra). The negative band at 1686 cm
1
(in H
2
O, Fig. 2D) is considerably down-shifted by 20 cm
1
in
D
2
O (trace E), supporting its assignment to the solvent exposed
b-turn including C109, N110, and H112 (see Fig. 1(I)). Consistently,
the positive band at 1501 cm
1
in H
2
O, shifted by 92 cm
1
in D
2
O,
likely results from changes in the amide II mode of this b-turn.
Additional, unchanged absorptions at 1635/1627 (+/) and 1678/
1669 cm
1
(/+) are ascribed to amide I modes
62
of H/D exchange-
resistant b-sheets (vide supra), as also observed for A. fulgidus SOR
(Fig. 2C). Redox dependent changes of these dense and rigid
structural motifs, remote from the active site, support the view of
a collective redox-dependent structural reorganization. Further
assignments are indicated in Fig. 2 and Table 1.
Extended redox-linked structural changes
IR difference spectra clearly indicate that SOR undergoes a
concerted structural change during reductive activation of the
active site. Global conformational changes of this type rely
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upon collective thermal motions that are well captured in one
or few low-frequency normal modes of the entire protein.
73
Normal mode analysis based on elastic network models
74
provides an efficient tool to gain insights into these large scale
movements.
75,76
Using this approach, we screened for normal
modes that include a considerable change of the O
E23
–Fe
distance in I. hospitalis SOR and, thus, may be involved in
redox-dependent domain movements with a possible impact on
enzyme activation. Accordingly, all modes fulfilling this requirement
match the structural changes probed by IR difference spectroscopy.
In particular, normal mode no. 62 (displayed in an animation
provided in the ESI,†S2) reveals considerable in-phase movements
of loops and helices close to the active site of all four subunits
accompanied by a rearrangement of the b-sheet core. As a
consequence, this mode may be essential for a guided structural
reorganization and promoting interactions of the individual
monomers during activation. Since the O
E23
–Fe stretching
approximates the reaction coordinate of the redox-linked struc-
tural transition, the associated domain movements also provide
a coarse-grained 3D illustration of the conformational changes
encoded in the IR difference spectrum.
Redox-linked perturbations and H/D exchange of coordinated
histidine ligands
H/D shifts have been evaluated for all major bands (see Fig. 2D
and E and Table 1). In contrast to expectations,
57,58
the histidine
side chain modes n(CN) at 1099/1112 cm
1
(/+) and n(CQC) at
1595 cm
1
() were found to remain largely unchanged in D
2
O
(Fig. 2D and E). Similar observations were made for the A. fulgidus
SOR (data not shown), the SOR from T. pallidum,andcentreIIof
the D. baarsii SOR,
29
pointing to a general feature of SOR active site
histidines. Since these amino acids are highly solvent exposed, this
observation cannot be related to a restricted accessibility.
To confirm band assignments and elucidate the intrinsic
H/D sensitivity of these modes for the metal-coordinated
histidines in SOR, we have carried out DFT calculations for
monoethane-thiolato-monobutyrato-tri(4-ethylimidazole-N1)-mono-
(4-ethylimidazole-N3)-iron(III)(1) (Fig. 1(II)), a model compound
designed to mimic essential features of the ferric active site (see
Experimental procedures). Spectral regions with essential histidine
side chain (imidazole) absorptions are shown in Fig. 4 while
selected vibrational modes are listed in Table 2.
Imidazole modes with major contributions from the n(CN)
coordinates are centred around 1100 cm
1
in 1, confirming the
assignment of experimental bands for the I. hospitalis SOR.
Here, the corresponding modes of H56, H25, H50, and H112
are observed at 1135, 1116, 1107, and 1082 cm
1
, respectively,
resulting in band separations of 9–25 cm
1
in the calculated
spectrum (left panel of Fig. 4). Thus, the well-defined difference
signal, observed at 1099/1112 cm
1
(/+) for I. hospitalis SOR, is
most likely related to redox-linked perturbations at the level
of a single histidine side chain. In agreement with the experi-
mental data, absorption bands with major contributions from
the imidazole n(CQC) modes of 1arise at 1596, 1591, 1586,
Fig. 3 Crystal structure of the homotetrameric SOR from I. hospitalis: (I) distribution of hydrophobic amino acids (blue). (II) Location of central b-sheets
(blue opaque) and other secondary structure elements (blue transparent) (III) regions exhibiting high (blue), medium (white), and low (red) packing
densities. (IV) Areas with low (blue), medium (white), and high (red) solvent accessibility.
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and 1565 cm
1
for H56, H50, H25, and H112, respectively (right
panel of Fig. 4).
Upon replacing the hydrogen atoms at the imidazole nitro-
gens (Fig. 5) by deuterium (1-D), the n(CN) modes of the N
d
-
protonated imidazoles show only a small (H56) or essentially no
change (H25, H50) whereas the N
e
-protonated tautomer (H112)
exhibits a considerable frequency upshift (left panel of Fig. 4
and Table 2). These results are in good qualitative agreement
with experimental data for 4-methylimidazole (4-MeIm).
57,77
Thus, solely based on these findings one might assign the H/D
insensitive 1099/1112 (/+) cm
1
signal in the experimental
spectrum of I. hospitalis WT SOR to the n(CN) containing modes
of the N
d
-protonated histidines H25, H50, and/or H56. However,
n(CQC) modes of both 1-D and (metal-bound) 4-MeIm
57,58
exhibit considerable H/D shifts, especially in case of N
d
-protonated
imidazole (right panel of Fig. 4). This observation is in contrast to
experimental SOR spectra, where these modes are merely changed in
D
2
O. Interestingly, the observed H/D insensitivity of both n(CN) and
Fig. 4 Calculated IR spectra of the SOR model (Fig. 1(II)) with neutral (A) N-protonated and (B) N-deuterated ethyl-imidazole ligands. The spectral region
of imidazole n(CN) and n(CQC) absorptions is enlarged in the left and right panel, respectively. For a better visualisation of the weak bands in the right
panel, the second derivatives of the spectra are included (a and b, dotted lines). IR bands are assigned to the corresponding histidine ligands of SOR using
the nomenclature of the enzyme from I. hospitalis.
Table 2 Comparison of experimental ligand vibrational modes of Ih SOR and the corresponding calculated frequencies for the cofactor model (see
Fig. 1). Both experimental and calculated IR bands are assigned according to the nomenclature of the I. hospitalis SOR. Amino acids are denoted by the
one-letter code. Band positions for the ferric and ferrous state are indicated by () and (+), respectively
Assignment Experiment/in silico I. hospitalis nomenclature Band position (H
2
O)/cm
1
Band position (D
2
O)/cm
1
H/D shift/cm
1
E, n
s
(COO) Experiment E23 1386()/1409(+) 1386()/1409(+) 0/0
In silico 1316/1350 1315/1350 1/0
E, n
as
(COO) Experiment E23 1546(+)/1554() 1546(+)/1552()0/2
In silico 1605 1604 1
H, n(CN) Experiment ? 1099()/1112(+) 1099()/1112(+) 0/0
In silico H25 1116 1117 +1
H50 1107 1107 0
H56 1135 1129 6
H112 1082 1094 +12
H, n(C=C) Experiment ? 1595()/1580() 1595()/1586() 0/+6
In silico H25 1586 1570 16
H50 1591 1576 15
H56 1596 1583 13
H112 1565 1559 6
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n(CQC) modes is a typical characteristic of anionic, deprotonated
imidazolate (Im
).
57
In view of this finding, a deprotonated histidine
(Fig. 5) at the ferric and ferrous active site may be an alternative
explanation for the lack of imidazole H/D shifts in I. hospitalis
WT and other SORs. Considering the solution pH of 7.5, this
would indicate a considerable decrease of the pK
a
for imidazolate
formation (pK
a
B14 in solution). Such an effect has been
previously observed for metal-bound imidazole and ascribed to
pronounced s-donation and the resulting withdrawal of electron
density from the non-coordinated imidazole nitrogen.
78,79
Albeit
weaker, this effect can be compared to the binding of a second
proton (Fig. 5), which lowers the pK
a
value to approximately 6 in
the imidazolium cation (ImH
2+
). Indeed, transition metal-
induced histidine ionization was previously claimed to be func-
tionally relevant
79
and proposed to occur in a few enzymes.
80–82
Consequently, the possible presence of a deprotonated histidine
at the SOR active site requires further investigation, since this
type of modification may have a considerable impact on electro-
nic structure and reactivity.
Conclusions
The redox-linked changes of the I. hospitalis SOR, as monitored
by IR difference spectroscopy, indicate the reversible dissociation of
glutamate E23 from the active site iron upon reductive activation,
thereby enabling substrate binding and transformation. This
change of the active site structure is linked to extended conforma-
tional perturbations of a nearby loop and especially helical regions.
In view of the compact protein structure, inter alia reflected by
restricted amide proton exchange, we conclude that the rearrange-
ment of these regions couples local changes of the active site to the
observed global movements of the b-sheet core. This interpretation
is supported by the analysis of low-frequency modes of the entire
enzyme, which are proposed to promote and guide the redox-
dependent structural transition. In view of the collective character
of these modes, the above mentioned structural changes might
integrate the conformational states of the individual subunits,
possibly constituting the basis for cooperativity in the enzymatic
reductive activation process. While this hypothesis requires further
experimental confirmation, the underlying structural transition
appears to be a general phenomenon for SORs as indicated by
similar observations in the IR difference spectra of enzymes from
A. fulgidus and other organisms. In contrast, these characteristic
spectroscopic signals are not observed for the E23A mutant from
I. hospitalis, demonstrating that the reduction-coupled E23
dissociation from the iron centre constitutes the trigger for protein
structure changes that are of potential functional relevance. This
latter process may be driven by the high electron density on the
ferrous iron, possibly further promoted by a deprotonated histidine
ligand.
Abbreviations
ROS Reactive oxygen species
SOD Superoxide dismutase
SOR Superoxide reductase
(FT)IR (Fourier transform) infrared
n
s
Symmetric stretching vibration
n
as
Antisymmetric stretching vibration
d
s
Symmetric bending vibration
g
t
Twisting vibration
g
r
Rocking vibration
DFT Density functional theory
GRAVY Grand average of hydropathy
DSSP Define secondary structure of proteins
SAA Solvent accessible area.
Acknowledgements
We would like to thank Dr Anto
´nio Baptista, ITQB, for helpful
discussions. We thank also Drs Tiago Bandeiras and Pedro Matias
for supplying prior to publication the coordinates for the I.
hospitalis and A. fulgidus SOR crystallographic structures. This work
was supported by the DFG (Cluster of Excellence ‘‘UniCat’’), the
program ‘‘Acço
˜es Integradas Luso-Alema
˜s/DAAD’’, Fundaça
˜oparaa
Cie
ˆncia e a Tecnologia (Portugal), grants PEst-OE/EQB/LA0004/
2011, and PTDC/BIA-PRO/111940/2009 (to CVR).
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