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Fe superoxide dismutase
Anne-Frances Miller
in
Handbook of Metalloproteins
Edited by
Albrecht Messerschmidt, Robert Huber, Thomas Poulos and Karl Wieghardt
© John Wiley & Sons, Ltd, Chichester, 2001
Fe superoxide dismutase
Anne-Frances Miller
Chemistry Department, University of Kentucky, Lexington KY 40506-0055, USA
FU N CT I O N A L C L A S S
OCCURRENCE
Enzyme; E.C. 1.15.1.1; mononuclear non-heme; non-sulfur
Fe enzyme.
FeSOD is found in bacteria, especially the more primitive
ones, the chloroplasts of plants,1,2 a few protists3 and
possibly eukaryotes.4,5 The homologous MnSODs are
found in bacteria and mitochondria, and are believed to
protect DNA from endogenous oxidative stress, whereas
FeSOD may serve as a housekeeping enzyme and provide
Non-sulfur Fe enzyme known as superoxide dismutase,
SOD or SD. Catalyzes disproportionation of O2.2:
2O2.2+2H+ ! H2O2+O2.
3D Structure
Cartoon of the dimer structure of Fe3+SOD from E. coli, showing the Fe3+ as a large black sphere and the heavy atoms of
the ligands from Ca out in ball and stick format. Based on the coordinates of Lah et al., 1ISB,45 and produced using molscript.97
668
H A ND B OO K OF M ET A L LO PRO T E IN S
Fe-superoxide dismutase
resistance to environmental oxidative stress, by virtue of its
periplasmic location.6 Throughout this article, `SOD'
refers collectively to the closely related MnSODs and
FeSODs, but not the unrelated Cu, ZnSODs or NiSODs.
BIOLOGICAL FUNCTION
With catalases and peroxidases, SOD provides protection
against oxidative damage caused by the chemical progeny
of O2.2: H2O2 and OH..7±10 The regulatory role of NO
and O2.2's rapid reaction with NO raise the possibility that
SOD's activity may also have regulatory ramifications.
AMINO ACID SEQUENCE INFORMATION
FeSOD, MnSOD and Cu, Zn±SOD have been studied as
an example of parallel enzyme evolution.1 The amino
sequences of FeSODs and MnSODs are highly conserved,
especially with respect to residues in and around the active
site. Well over two hundred SOD amino acid sequences
have been reported as of March 2000 with the number
increasing every week. An up to date list is best obtained
via a BLAST search, which can be performed on-line at
http://www.ncbi.nlm.nih.gov:80/BLAST/. Because the
amino acid sequence between residues 143 and 157 is
`low in complexity' this may be excluded as a criterion for
homology by performing searches with the `filter on'.
Representative sequences are those of:
X
X
X
X
X
X
Escherichia coli, accession number gi.640114.11
Bordella pertussis, gi.586007.12
Nicotiana plumbaginifolia chloroplast, gi.170234.13
Helicobacter pylori, gi.4218197.14
Plasmodium falciparum, gi.4139318.15
Plectonema boryanum gi.1711435.16
PR O T E I N PR O D U C T I O N , PU R IF I C A T I O N
AND MOLECULAR CHARACTERIZATION
E. coli FeSOD is relatively easy to purify owing to its high
stability and orange color. The purification protocol of
Slykhouse and Fee17 works very well, though with a little
fine-tuning. After cell lysis in 0.1 M KCl and pH 7.4
phosphate buffer, DNA is removed by precipitation with
streptomycin sulfate or 30 mM MnCl2, and proteins less
stable than SOD are removed by denaturation at 60 8C for
3 min and centrifugation. Further substantial purification
is achieved by means of a 50% (NH4)2SO4 cut. At this
point, FeSOD is by far the majority species present if it was
expressed well. Pure FeSOD is obtained following anion
exchange chromatography on DE52 at pH 7.4 and, if
necessary, a flow-through column of CM52 at pH 5.5.
METAL CONTENT AND COFACTORS
Atomic absorption spectroscopy, as well as the X-ray
crystal structures, reveal that each SOD monomer contains
one metal ion and no other cofactors. Good preparations
of FeSOD contain one Fe per monomer but very often
when FeSOD is purified from bacteria grown in rich or illdefined medium SOD is found to contain a mixture of
metal ions including most importantly Mn2+ and Zn2+.
ACTIVITY TEST
By far the most common assay is the original one in which
the reduction of cytochrome c by O2.2 is monitored
spectroscopically and the activity of SOD is assessed based
on its ability to interfere with cytochrome c reduction.
Xanthine oxidase is used as a source of nM O2.2.18 A
variety of other assays exist19,20 and are useful under
different circumstances including in native electrophoresis
gels.21 Stopped-flow22 or pulse radiolytic assays23 yield the
kinetic constants kcat and KM.
S PE C T R O S C O PY
Fe3+SOD has been studied by UV/vis spectrophotometry
and the broad overlapping bands peaking at 350 nm
assigned to ligand-to-metal charge transfer transitions.17,24,25 The EPR spectrum has been described in
terms of D ˆ 21:7 cm21 and E=D ˆ 0:24:26±28 MoÈssbauer spectroscopy29 and EXAFs30,31 have also been
applied to both the oxidation states, and the reduced
state was studied by MCD.32 NMR spectroscopy was used
to observe the active site of Fe2+SOD from Methanobacterium thermoautotrophicum33 and E. coli,34,35 in which
the ligands' resonances have been assigned.36 Anion
binding has been studied by solvent proton magnetic
resonance dispersion.37 The results of these studies have
provided invaluable insights into the mechanism of
FeSOD, as discussed below.
T H E X - R A Y C R Y S T A L L O G R A PH I C
S TR U CT U R E A N D ME TA L S IT E O F Fe S O D
Crystallization of FeSOD
FeSOD has most commonly been crystallized from the
medium of pH 4.5±4.8 containing 40±55% (NH4)2SO4 or
35% methyl pentanediol and 20 mM CaCl238±41 or
50 mM phosphate buffer of pH 6.1 with 2.15 M
(NH4)2SO4.42 FeSODs from more exotic organisms have
been crystallized at higher pHs, including a pH 7.5
100 mM Tris and 23% PEG 6000 for Mycobacterium
HANDBOOK OF ME T A LL OP ROT E I NS
669
Fe-superoxide dismutase
a4
100
130
90
b1
190 150
b2
110
a5
a6
180
a7
b3
170
140 120
80
a3
a2
50
18 160
60
70
30
10
a1
40
Figure 1
Ribbon diagram of a monomer of E. coli Fe3+SOD showing the Fe3+ as a large black sphere and the heavy atoms of the ligands
from Ca out in ball and stick format. The orientation is the same as that of the monomer on the left side of the 3D Structure. Large
alphanumerics are used to label the different helices (a1, a2, a3¼) and b-strands (b1, b2, b3) according to Lah et al.45 Residue numbers are
indicated in smaller numbers. Based on the coordinates of Fe3+SOD published by Lah et al. (1ISB)45 and produced using molscript.97
tuberculosis,43 and pH 8.5 100 mM Tris 8% PEG 8000 for
Sulfolobus solfataricus.44
Overall description of the structure
FeSODs and MnSODs occur as dimers or tetramers of a
<22 kDa monomer whose fold is highly conserved (3D
670
H AN D B OOK OF M ETAL LOP RO TEI NS
Structure and Figure 1).38,39,43±49 The monomer is
composed of two domains, each of which contributes
two ligands to the single active site metal ion. The Nterminal domain is made up of two long helices separated
by a shorter and a more variable one (residues 1±80;
amino acid numbering of FeSOD from E. coli is used
throughout, except when referring to a MnSOD, in which
case the numbering of E. coli MnSOD is used). The first
Fe-superoxide dismutase
HO-
His73
Asp156His160
Fe
His26
Coordination of Fe3+ in Fe3+SOD at low pH as per the 1ISB coordinates of Lah et al.45 Metal ion-to-ligand distances in AÊ are
Ê resolution at R ˆ 0:18 : Fe3+-His26 N12 ˆ 2:16 A;
Fe3+-His73 N12 ˆ 2:05; Fe3+-Asp156
as follows and the crystal structure boasts 1.85 A
3+
3+
2+
2
Od1 ˆ 1:91; Fe -His160 N12 ˆ 2:07; Fe -O of OH ˆ 1:96: For Fe SOD the resolution is 1.80 AÊ at R ˆ 0:19 and the analogous
distances from Fe2+ are 2.18, 2.04, 1.94, 2.12, 2.04 AÊ, respectively.45 H atoms and backbone C 0 , O and N atoms are omitted for clarity.
Produced using molscript.97
Figure 2
helix contains a conserved kink which orients the
universally conserved Tyr34, His30 and ligand His26
into the active site. Ligand His73 derives from the second
long helix of the first domain.45 A linker of <10 variable
amino acids connects the two domains and the C-terminal
domain (residues 89±192), an open a/b structure, consists
of a three-stranded b-sheet flanked on both sides by a total
of four a-helices. The C-terminal domain supplies ligands
Asp156 and His160 from the C terminus of the b-sheet and
the loop that follows. The fifth ligand to Fe is a
coordinated solvent molecule believed to be OH2 in the
oxidized (Fe3+) state and H2O in the reduced (Fe2+) state
(Figure 2).49 The interface between the domains is
predominantly hydrophobic.45 The interface between the
subunits is highly conserved and generates symmetryrelated funnels that provide substrate access to the active
sites.45
Each monomer's metal ion and ligands are all inaccessible to the bulk solvent, sequestered behind Trp77, Tyr34
and His30 at the base of the funnel out to the bulk
solvent.45 The residues participating in the second coordination sphere are derived from both domains and even
include residues from the other monomer of the dimer
(Figure 3). Indeed, the funnel through which the substrate
is believed to access each active site is lined with residues
from both monomers.45
Thus, SOD displays a theme common among metalloenzymes, of metal binding at the interface between
domains or subunits. This device may allow protein
domains to fold around individual hydrophobic cores
before coordinating the metal ion with ligands distant in
the amino acid sequence. The metal ion can then be
sequestered between domains to obtain the low dielectric
environment needed for tight binding and activity, without
requiring a single hydrophobic core to swallow a metal
ion.
The metal ion coordination is approximately trigonally
bipyramidal with His26 and coordinated solvent as axial
ligands (Figure 2). Coordinated solvent is supported by an
extensive conserved hydrogen bond network including
Gln69, Tyr34, Trp122, Asn72, Asp156 and (via a solvent)
His30 and Tyr163B from the other subunit (Figure 3).45,50
There is a conserved Arg nearby51 and the active site is
surrounded by a shell of highly conserved predominantly
aromatic amino acids.39 These may serve to protect the
protein from radical inactivation by virtue of their
relatively long-lived free radical states, that may be stable
long enough to be rereduced in the next redox cycle of the
HANDBOOK OF ME T A LL OP ROT E I NS
671
Fe-superoxide dismutase
Glu159B
Figure 3 The active site including selected second sphere residues and commonly-reported hydrogen bonds. Residues identified by their
number alone are all from the same subunit, residues whose number includes `B' are contributed by the other subunit. H atoms and
backbone atoms are omitted for clarity. Fe3+SOD coordinates 1ISB were used45 and the figure was produced using molscript.97
metal ion instead of undergoing irreversible oxidative
chemistry.
F U N C T I O N A L A S PE C T S
Mechanism and inhibition
Fe-SOD from E. coli disproportionates O2.2 with saturation kinetics characterized by a turnover number of kcat ˆ
2:6 104 s21 and a KM of 80 mM for O2.2 at pH 8.4 and
25 8C,52 consistent with the observed second-order rate
constant of kcat =KM ˆ 3 108 M21 s21 :22,53 Pioneering
work demonstrated that at modest O2.2 concentrations
disproportionation occurs in two sequential steps (see
Equations (1) and (2))
3‡
2‡
O2
2 ‡ SOD…M † ! O2 ‡ SOD…M †
2‡
‡
3‡
O2
2 ‡ SOD…M † ‡ 2H ! H2 O2 ‡ SOD…M †
…1†
…2†
where SOD signifies the enzyme and M indicates the
bound iron or manganese ion, which cycles between its +3
and +2 oxidation states.23 The rate-limiting step is believed
to be dissociation of H2O2 and to involve H+ transfer, as it
is accelerated by weak acids.52 This is only one of several
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H AN D B OOK OF M ETAL LOP RO TEI NS
manifestations of the importance of protons to redox
catalysis.54
FeSOD is competitively inhibited by N32, which
coordinates directly to Fe3+ resulting in hexacoordinate
Fe3+.30,45 This suggests that the substrate binds directly
to Fe3+ and that substrate oxidation occurs by an innersphere mechanism (but see references 17, 55). F2 also
coordinates to Fe3+ as does OH2.17,24,30 Two F2 ions
can bind to Fe3+, consistent with one replacing the
coordinated solvent.17 Additional inhibitors do not
coordinate to Fe3+ but do compete with N32.52 This
and the temperature jump studies of N32 binding suggest
the existence of a prebinding site not on the metal ion
that nonetheless contributes either sequentially or spatially to N32 (and thus potentially substrate) binding.24
The KDs of N32 and F2 are both much lower than their
KIs suggesting much weaker interactions with the
reduced rather than the oxidized state. By contrast, the
non-coordinating inhibitors ClO42 and SCN2 have KIs
and KDs that are larger and comparable.52 Thus, an
outer-sphere binding site is inferred for the reduced state
that may be the same as the prebinding site inferred for
the oxidized state.
FeSOD is inactivated by H2O2 in a reaction that
involves an initial stoichiometric reduction of Fe followed
Fe-superoxide dismutase
by Fe release and a chemical modification of active site
residues including Trp.56±59
conserved among SODs and is expected to have a pK near
the pK of 9 that affects the catalytic activity.29,45,49,61±64
Protons involved in catalysis
pKs affecting the oxidized state
Two protons participate directly in SOD's reaction, in
Equation (2). It is likely that the first proton is derived
from the coordinated solvent, as the latter is expected to
have a much lower pK when Fe is oxidized than when it is
reduced.49,60 The second proton was assumed to be
derived from Tyr34 because this residue is universally
SOD activity decreases at increasing pH with a pK of <9
(at 25 8C). This is ascribed in part to the onset of
competitive inhibition caused by OH2 binding to Fe3+
for the following reasons. The pK affects KM but not kcat,
implying a greater significance to substrate binding than to
the rate-limiting chemical steps, and is associated with a
Figure 4
Signatures of the pK of Tyr34 in Fe2+SOD. The nuclear magnetic resonance chemical shifts of virtually every active site
resonance titrate with a pK of 8.5 in WT Fe2+SOD, but not in Y34F Fe2+SOD.35 The pH dependence of the Nd1 proton of His73 is shown
in WT (open circles) and Y34F (filled circles) Fe2+SOD. The same pK affects the solvent exchange rate of an active site hydrogen bond
network proton (the Nd1 proton of His160, which is hydrogen bonded to Glu159B),45 making this proton inaccessible to OH2. Log(basecatalyzed exchange) is expected to increase with pH linearly with a slope of 1.98 This behavior is indeed observed in Y34F Fe2+SOD.
However, ionization of Tyr34 at pH 8.5 interrupts this increase in WT Fe2+SOD.
HANDBOOK OF ME T A LL OP ROT E I NS
673
Fe-superoxide dismutase
decrease in the affinity of Fe3+SOD for azide.52 The latter
indicates that either a proton required for substrate analog
binding is lost at pH 9 or that substrate analog binding
occurs in competition with OH2 binding. Since the pK
coincides with a change in the EPR signal of Fe3+
suggesting a change in the coordination sphere24 and is
associated with expansion of the coordination sphere
based on EXAFS spectroscopy at a low temperature,30 it
is assigned to the coordination of OH2. (However the
active site geometry may not be the same at a low
temperature as at room temperature.17,55)
The phenolic H of neutral Tyr34 nonetheless does
appear to contribute a little to F2 binding at low pH, as the
KD of Y34F Fe3+SOD is higher than that of WT FeSOD (10
and 2 mM, respectively) and the same is true for Fe2+SOD
…KD ˆ 70 mM for Y34F and 40 mM for WT).69 By
contrast, the fact that Y34F Fe3+SOD's KD for N32 is 20
times smaller than that of WT probably reflects the relief of
interference between the three-atom substrate analog and
the OH of Tyr34.66 Crystal structures of FeSOD complexed with N32 indicate that N32 pushes Tyr34 out of its
native position.45 Thus, Tyr34 exploits both steric and
electrostatic mechanisms to enforce binding specificity for
small anions.
The pK affecting the reduced state
Fe2+SOD was predicted to also have a pK near 9 based on
elegant kinetic studies and the fact that a single proton is
taken up on reduction both above and below the oxidized
state pK.52 Anaerobic NMR pH titrations directly detected
this pK of 8:5 ^ 0:0735 and assigned it to Tyr34 based on
its complete absence from FeSOD in which Tyr34 had been
mutated to Phe, Y34F FeSOD (Figure 4).65
Given Tyr34's universal conservation among SODs and
its predicted role as a proton donor to substrate, it was
a big surprise that Y34F SOD retains at least 40% of
the normal wild-type (WT) activity in the standard
assay.50,65±67 However proton exchange measurements
demonstrated that upon ionization, Tyr342 bars OH2
from the active site. This implies that Tyr342 repels other
anions too,35 consistent with the increase in KM above
Tyr34's pK.52 Thus, Tyr342 shuts down SOD at high pH.
Arguably this would happen anyway when the concentration of OH2 became sufficient to outcompete substrate for
its binding site. However, Tyr34 is shown to help protect
FeSOD from inactivation by HO22,50,67 possibly by
denying HO22 access to the active site Fe2+ when the pH
rises sufficiently for H2O2 to dissociate significantly.
Substrate analog binding to Fe2+SOD via Tyr34
Tyr34 has also been shown to participate in substrate
binding and protonation in Fe2+SOD. In contrast to the
inner-sphere reaction implied for the oxidized state, MCD
studies at low temperatures indicated no change in the
coordination number for the reduced state in the presence
of 500 mM KN3 or concentrated NaF or NaOCN.32 NMR
spectroscopy indicates that F2 binds to Fe2+SOD as an
outer-sphere ligand,68 implying an outer-sphere mechanism
for substrate reduction. The low-pH KD is 40 mM but the
binding affinity decreases at high pH with a pK of 8.5.69
Thus, F2 binding either requires the phenolic proton of
Tyr34 or is prevented by Tyr342. Because Y34F Fe2+SOD
binds F2 in a pH independent manner, we conclude that
Tyr342 inhibits F2 (and substrate) binding.69
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H AN D B OOK OF M ETAL LOP RO TEI NS
Tyr34 as a possible proton shuttle
We have located the F2 binding site near residues 35±40 by
high-resolution NMR spectroscopy,68 although Tyr34
itself could not be observed in these experiments because
of paramagnetic relaxation of its resonances. In addition,
F2 binding raises the pK of Tyr34, suggesting that F2
hydrogen bonds with it or binds near it.69
The crystal structures of FeSOD and MnSOD contain a
solvent molecule in a position consistent with our
Ê from Tyr34's O and 3.5 AÊ from the Nd1
proposal, 3.3 A
45,70
of His30.
His30 is absolutely conserved in FeSODs
and MnSODs and the mutation of His30 in human
MnSOD decreases kcat and can increase KM71 (but see
reference 72). If F2 were to associate with Fe2+SOD in the
Ê from Fe2+.
place of this solvent molecule, it would be 7.5 A
However this distance should be regarded as a maximum,
and electron transfer could be facilitated by aromatic
residues His30 and Tyr34 which are 4.4 and 5.2 AÊ from
the ligands His160 and His73, respectively, and appear to
stack parallel with them (Figure 5). Thus, Tyr34 may begin
transferring a proton to substrate as part of substrate
binding. This would have the desirable effect of favoring
substrate reduction and could progress to full proton
transfer in the course of electron transfer to substrate. At
the same time, Tyr34's proton would be replaced by
proton transfer from the coordinated solvent, which
becomes increasingly acidic as Fe is oxidized. This
second part of the transfer could occur naturally via
the hydrogen bond network between Tyr34 and the
coordinated solvent.45,70
The high activity of Y34F FeSOD in the standard assay
argues that either the above proton relay is not necessary
or that a similarly effective mechanism for protonating
substrate and deprotonating the coordinated solvent exists
in Y34F SOD. Indeed, the Y34F mutation leaves more
space in the active site, results in significantly faster basecatalyzed proton exchange in and out of the active site at
high pH,65 and also allows greater substrate analog access
to the metal ion.66,67,69 Thus, at the low substrate
concentrations of the standard assay, proton transfer may
Fe-superoxide dismutase
Gln69
Tyr34
Wat414
His73
His30
Gln69
Tyr34
His73
Wat414
His160
His30
Figure 5
A proposed location of outer-sphere substrate binding to Fe2+SOD and a possible mechanism of proton transfer from
coordinated solvent via Tyr34, initiated upon substrate binding and progressing in conjunction with Fe2+(H2O) oxidation to Fe2+(OH2).
Dashed lines indicate hydrogen bonds involving coordinated solvent or the crystallographic solvent molecule whose position may be
indicative of an outer sphere substrate binding site. Heavy dashed lines indicate a possible proton transfer relay linking coordinated solvent
and outer sphere bound substrate. Coordinates of Fe2+ SOD were used (1ISA)45 and the figure was produced using molscript.97
be able to keep up with substrate binding, without Tyr34.
By contrast, stopped flow kinetics resolves kcat and kcat/KM
and demonstrates that kcat decreases by an order of
magnitude in human Y34F-MnSOD.50
Thus, Tyr34 functions as a selective barrier to the active
site that passes protons and bars HO22. Others have
proposed that Tyr34 mediates proton transfer from the
bulk solvent to the substrate in the active site,50 but we
propose that it shuttles protons between the bulk and
coordinated solvent or between the coordinated solvent
and substrate, in conjunction with outer-sphere electron
transfer.
Redox tuning: a model explaining SOD's metal ion
specificity
In addition to substrate binding and proton transfer,
electron transfer is central to redox catalysis. This is
inextricably linked to the identity of the metal ion, a
property of the enzyme that is so basic that it is usually
taken for granted. The metal ion determines what Ems are
possible, and the Em places a fundamental thermodynamic
limit on the chemistry the enzyme can perform.
Some SODs are active in the standard assay with either
Fe or Mn bound, and are called `cambialistic'.73±75
HANDBOOK OF ME T A LL OP ROT E I NS
675
Fe-superoxide dismutase
Table 1 Reduction midpoint potentials of analogous highspin Fe and Mn complexes in mV vs. NHE
Ligand
Fe complex
(H2O)6
EDTA
(Fe)SOD
(Mn)SOD
a
b
770
96
220a
2240a
Mn complex
Difference
1510
825
>900b
290b
740
730
.670
540
Vance and Miller.82
Vance, unpublished.
However, this is not the rule. It has been known for more
than 20 years that FeSOD protein ((Fe)SOD hereafter) can
be reconstituted with Mn (resulting in Mn(Fe)SOD), and
Fe can be incorporated into (Mn)SOD protein to make
Fe(Mn)SOD. Although both metal ions can perform SOD
chemistry and both proteins obviously can support it, the
majority of metal-exchanged SODs are not active in the
standard assay.76±79 Yamakura showed that Fe3+(Mn)SOD
displays a pK two pH units below that of native Fe3+SOD
Figure 6
and that Fe(Mn)SOD acquires a low but significant activity
below this pK.80,81 Whittaker and Whittaker found that
the mutation of Tyr34 to Phe raised the pH onset of
activity and proposed that different ligand basicities in the
(Fe)SOD and (Mn)SOD proteins are responsible for the
inactivity of Fe(Mn)SOD.67 We have demonstrated that
Fe(Mn)SOD and Mn(Fe)SOD have strongly shifted the
reduction midpoint potentials (Ems) that can simply and
fully account for their inactivities.82
Since SODs must both oxidize and reduce O2.2, their
Ems should be half way between the Ems of these two half
reactions, or approximately 360 mV vs. NHE, for optimal
turnover.83 Indeed, this appears to be the case, based on the
published Ems of FeSOD, MnSOD and Cu, ZnSOD, which
range from 190±420 mV.83,84 However, octahedral high
spin Mn tends to have a much higher 3+/2+ Em than
octahedral high spin Fe (Table 1). Thus, (Mn)SOD must
depress the Em of Mn much more than (Fe)SOD depresses
that of Fe. Given the structural similarity of Fe and Mn,
and the apparent similarities of the (Fe)SOD and (Mn)SOD
proteins, we proposed that the differences in Em tuning
A simple model that can explain the inactivities of Fe(Mn)SOD and Mn(Fe)SOD on the basis of Ems that are too low (or too
high) to support O2.2 oxidation (or reduction).82 Since high-spin Fe complexes have lower Ems than analogous high-spin Mn complexes,
yet the Ems of FeSOD and MnSOD are comparable, the MnSOD protein, (Mn)SOD, must depress the Em of Mn3+/2+ more than (Fe)SOD
protein depresses the Em of Fe3+/2+. If this difference persists in metal-exchanged SODs then Fe (the lower Em metal ion) bound in
(Mn)SOD protein, Fe(Mn)SOD, will be subjected to greater Em depression, and display a very low Em. Conversely, Mn will display an
unnaturally high Em in Mn(Fe)SOD. The reference compounds chosen are the hexaaquo complexes of Mn3+/2+ and Fe3+/2+. The Ems
obtained for E. coli FeSOD, MnSOD and Fe(Mn)SOD are indicated as well as a lower limit for the Em of Mn(Fe)SOD.82,99
676
H AN D B OOK OF M ETAL LOP RO TEI NS
Fe-superoxide dismutase
Asn72
Trp122
Gln69
Asp156
Tyr34
His73
Tyr76
His160
His31
His30
Figure 7
3+
His26
3+
Overlay of the active sites of Fe (Mn)SOD (grey Cs) and Fe SOD (white Cs) based on the A-chain coordinates of Edwards
et al. (1MMM)48 and the A chain coordinates of Lah et al. (1ISB).45 Coordinates were superimposed in Midas plus100 based on the Fe3+,
the coordinating O of Asp and the coordinating Ns of the His 0 . The rms difference between atomic coordinates was 0.2 AÊ. The figure was
produced using molscript.97
persist in the metal-exchanged SODs. Thus our model
predicted that the Em of Fe(Mn)SOD would be much lower
than those of FeSOD or MnSOD, and that the Em of
Mn(Fe)SOD would be much higher (Figure 6). Since the
potentials of Fe and Mn are different by a large amount
(Table 1), we predicted that the Em of Fe(Mn)SOD would
be below that required for it to oxidize O2.2, but that if
there were nothing else wrong with the active site, then
Fe(Mn)SOD should still be able to reduce O2.2. Similarly,
Mn(Fe)SOD could fail to turn over due to inability
to reduce O2.2 because of an Em higher than that of
O2.2/H2O2.82
Proof of the model
The Em of E. coli FeSOD is 220 mV vs. NHE based on
both ascending and descending titrations and two different
mediators82 but a significantly lower value is obtained
using a mediator cocktail. We have also evaluated the Em
of E. coli MnSOD to be 290 mV vs. NHE.85 However, the
Em of Fe bound to (Mn)SOD is 2240 mV, almost half a V
lower than Fe's Em in (Fe)SOD,82 and Mn(Fe)SOD's Em is
more than 900 mV, much higher than that of MnSOD.85
We have shown that metal-substituted SODs retain the
ability to bind substrate analogs and that their active sites
are similar to those of the native enzymes, ruling out gross
disruption as the cause of inactivity.86 Moreover, Fe(Mn)SOD retains ability to reduce substrate and therefore both
electron and proton transfer activity.82 Our model is
consistent with Fe(Mn)SOD's acquisition of activity at low
pH67,80,81 since protonation of an active site group is
expected to raise the Em. Thus, the catalytic inactivities of
Fe(Mn)SOD and Mn(Fe)SOD can be simply explained by
their very low and very high Ems, respectively.82
Mechanisms of Em tuning
The two SOD proteins' dramatically different Em tuning
indicates that despite their similar appearance in crystal
structures, the two proteins are very different indeed with
respect to their interactions with the metal ion. However,
differences between the active sites of FeSOD and MnSOD
are overshadowed by the similarities in the crystal
structures (Figure 7).39,45 In both, the same ligands
coordinate the metal ion in virtually superimposable
geometries. The second sphere contains few notable
differences.87,88 FeSODs have a Tyr at position 76 which
is occupied by Phe in MnSODs, but mutation of Tyr to Phe
in S. solfataricus FeSOD did not increase the Mnsupported activity.89 Mutation of the Glu that bridges
the dimer interface was found to confer higher Fe binding
on MnSOD, but no Fe-supported activity.90 Even the
HANDBOOK OF ME T A LL OP ROT E I NS
677
Fe-superoxide dismutase
computed electrostatic potentials of E. coli FeSOD and
Thermus thermophilus MnSOD do not differ substantially
at the active site (M Gunner, personal communication)
Thus, the two SOD proteins appear to use the same tools
to effect different redox tuning. Subtle biophysical
differences have been reported between E. coli FeSOD
and MnSOD but it is not known whether these differences
are general.76 The major difference is that the Gln
conserved at residue number 69 in FeSODs is absent
from MnSODs and replaced by a Gln conserved at position
146 instead.87 This Gln side chain is believed to hydrogen
bond with the same residues in both SODs.45,88
The appearance of similarity may be deceptive and
largely due to the relatively low resolution possible in
crystal structures of proteins, compared to the sensitivity of
metal ion electronics to relatively small perturbations in
placement, orientation and protonation of ligands. Many
of the SOD crystal structures published were obtained
using protein not fully metallated or containing a mixture
of metal ions. In these, the finer details of the active site
reflect the average of several different species and cannot
be interpreted in terms of aspects by which the individual
species might differ, including subtle positional and
hydrogen bonding differences. Indeed, spectroscopic
studies directly addressing the metal ion have revealed
significant differences between Fe coordinated to (Fe)SOD
and (Mn)SOD.86
FeSOD and Fe(Mn)SOD are significantly different with
respect to the effects of substrate analog binding and
protonation or OH2 binding equilibria, both of which
could be related to electron transfer.86 A pK near 8.4 is
associated with a dramatic change in Fe3+(Mn)SOD active
site geometry from weakly rhombic to a much more axial
structure.86 This is completely unlike the effect of
increasing pH on Fe3+SOD, which is to cause the closeto-rhombic EPR signal to approach the rhombic limit.24
Edwards et al. reported observing a non-native active site
geometry in the crystal structure of Fe3+(Mn)SOD.48
However, their crystallization pH of 8.5 was significantly
higher than the pHs used for the reference structures of
FeSOD39,45,48 and moreover roughly coincides with the pK
of 8.4 identified by EPR with a coordination geometry
change.86 Since the other published Fe3+SOD structures
represent the active site below its pK (where known) and
the Fe3+(Mn)SOD structure represents the active site at or
above its pK, it is not surprising that the sites in the
Fe3+(Mn)SOD structure display a second coordinated
solvent molecule but that none of the Fe3+SOD sites do.
The Edwards result indicates that the pK of Fe3+(Mn)SOD
corresponds to the coordination of a second OH2, like the
pK of Fe3+SOD.30
The very different EPR signatures associated with N32
and OH2 binding to Fe3+SOD vs. Fe3+(Mn)SOD67,86
underline the different behaviors of Fe3+ in these superficially similar sites, and may be related to the different
substrate analog binding modes observed crystallographi-
678
H AN D B OOK OF M ETAL LOP RO TEI NS
cally for N32 in Fe3+SOD and Mn3+SOD.45 Thus the large
Em differences are accompanied by large spectroscopic
and substrate analog binding mode differences that
are not easily explained based on the available crystal
structures.
The position of the active site Gln differs in
Fe2+SOD and Fe2+(Mn)SOD
The degree to which the ligands are protonated can exert a
very strong effect on the metal ion Em. In particular, the
ionizable proton of coordinated solvent greatly stabilizes
the electronic orbitals of the coordinated O and therefore
has a very large effect on its influence on the metal ion Em.
Based on computations, the Em of Mn could vary by 1.5 V
depending on whether the coordinated solvent is protonated or not.92 Thus, differences in the degree to which the
coordinated solvent is protonated, for example by different
hydrogen bonding, could explain the half V difference
between the Ems of metal ions bound to (Fe)SOD vs.
(Mn)SOD.
Gln69 (and Asp156) hydrogen bond directly to coordinated solvent. Asp156 is common to FeSODs and
MnSODs but the Gln comes from different directions,
with slightly different orientations and positions relative to
the metal ion.45,88 The side chain N is significantly closer
to the metal ion in Fe(Mn)SOD and MnSOD than it is in
FeSOD, and interacts much more strongly with the Fe2+ of
Fe2+(Mn)SOD than that of Fe2+SOD.54 Thus, a strong
contact between the Gln and the Fe correlates with a much
lower Em. The same correlation recurs in human
(Mn)SOD, in which Q143N MnSOD has a much higherthan-native Em, and weaker-than-native hydrogen bonding
between the side chain amide of Asn and Mn.93 Thus,
strong interaction between the side chain amide and the
metal ion depresses Em and destabilizes additional electron
density on the metal ion. It could do this by stabilizing the
coordinated OH2.54
Coordinated solvent as a device for Em tuning
Since the coordinated solvent is believed to be OH2 in the
oxidized state of both SODs, but to take up a proton upon
reduction, the acid dissociation constants of the coordinated solvent in the oxidized and reduced states (Kox and
Kred), contribute to the Em observed at a given pH (see
Equation (3))
Em ˆ EAH ‡
RT Kred ‡ ‰H‡ Š
ln
F
Kox ‡ ‰H‡ Š
…3†
where EAH is the potential relating the protonated oxidized
state to the protonated reduced state. Kred is relatively
unimportant since Kred ,, ‰H‡ Š;35,86 but if Kox differs
between FeSOD and Fe(Mn)SOD, it will contribute to the
Fe-superoxide dismutase
Figure 8
Cartoon depicting the derivation of Fe- and Mn-G77Q,Q146A-SOD.54 The amino acid sequence of MnSOD is colored purple
and the FeSOD sequence is in orange. Only the amino acids mutated are shown explicitly.
Em tuning difference. Thus, stabilization of OH2 could
lower Em via Kox. An implausibly large decrease in pKox of
8 pH units would be required to fully account for the 0.5 V
lower Em observed in Fe(Mn)SOD than in FeSOD.
However, modulation of pKs by 5 pH units is well
known in proteins and indicates that this mechanism
could be an important contributor to differential redox
tuning.
Since proteins achieve exquisite and dramatic control
over residue protonation states and pKs, coordinated
solvent molecules and hydrogen bonds between them and
protein residues offer a potentially general and powerful
mechanism by which proteins may tune the Ems of bound
metal ions. Thus, coordinated solvents may not only serve
to hold open labile coordination sites and supply the
protons whose transfer accompanies electron transfer, but
may also serve a general role as adapters between protein
and metal ion by which the protein's control over proton
density can be translated into control over metal ions' Ems,
and thus reactivity.
.7% WT FeSOD activity.54 This activity is well within
the range observed for cambialistic SODs. Consistent with
the greater Mn-supported than Fe-supported activity, the
active site remains spectroscopically much more similar to
that of (Mn)SOD than that of (Fe)SOD with either Fe or
Mn bound.54 That mutagenesis of the active site Gln does
not completely convert a MnSOD to a FeSOD (or vice
versa) is not surprising95 considering that Gln is only one
of the several amino acids that interacts with the metal ion.
Moreover, it is the side chain of Gln that is involved in
tuning metal ion reactivity, and this is positioned only
remotely by the peptide backbone, and much more
precisely by packing and hydrogen bond interactions
with residues that retain their MnSOD positions and
identities. Precise positioning of chemical functionalities
may account for many of the chemical differences observed
when a metal ion is bound to the two different proteins,
and be as important as the more often-cited conserved
differences between the identities of the active site amino
acids.
Mutation of the active site Gln
Relating proton transfer to redox tuning
Several groups have prepared mutants of the active site
Gln.54,94,95 Mutagenesis to the Glu enhanced Mn-binding
over Fe-binding but did not confer Mn-supported activity.94 In a more conservative strategy, Hiraoka et al.
mutated the Gln characteristic of FeSOD to Gly and
Ala141 to the Gln characteristic of MnSOD, in a
cambialistic SOD. The mutant displayed increased the
Mn-supported activity and decreased the Fe-supported
activity but remained cambialistic.95 The complementary
mutant has also been prepared (Figure 8). E. coli
G77Q,Q146A-(Mn)SOD lacking the Gln characteristic of
MnSOD and containing the Gln characteristic of FeSOD
instead retains 70% of WT Mn-supported activity in the
standard assay. However, in contrast to WT Fe(Mn)SOD
which has no detectable Fe-supported activity in the
standard assay, Fe-G77Q,Q146A-(Mn)SOD displays
Coupling between proton transfer and electron transfer is
an important mechanism of biological energy conservation. It is also a virtually ubiquitous feature of biological
redox catalysis, and is explicit in SOD. The ionizable
proton of the coordinated solvent and the protons in the
hydrogen bond network not only tune the Em of the metal
ion, they also participate in the reaction. Thus, SOD
proteins may bring about a balance between the two half
reactions of superoxide disproportionation by modulating
the pKs of the two protons involved. Since (lower
potential) Fe has a natural tendency to reduce substrates
whereas (higher potential) Mn has a natural tendency to
oxidize them, (Fe)SOD protein might promote substrate
oxidation at the expense of reduction by favoring proton
uptake by coordinated solvent. This is essentially the
opposite of what happens in O2 carrier proteins where Fe2+
HANDBOOK OF ME T A LL OP ROT E I NS
679
Fe-superoxide dismutase
Figure 9
An example of how different hydrogen bonding and proton flow related to electron transfer could favor different metal ion
oxidation states and half reactions in FeSOD vs. Fe(Mn)SOD. For example, the Gln N is closer to coordinated solvent in Fe(Mn)SOD than
it is to the O of Tyr34. However the reverse holds in FeSOD.45 Thus, in Fe(Mn)SOD hydrogen bonding from Gln may stabilize coordinated
OH2 more than in FeSOD, whereas in FeSOD Tyr- may be better stabilized (in agreement with the experiment),86 and coordinated solvent
may be more readily protonated, at the expense of Tyr34. Tyr34 of (Fe)MnSOD might be freer to donate a proton to incoming substrate,
favoring electron transfer from the metal ion onto substrate, whereas the proton flow onto coordinated solvent proposed for FeSOD would
favor the other half reaction in which substrate reduces the metal ion. While the above scheme is only a model, reversal of the direction of
proton flow, to substrate vs. to coordinated solvent, provides an alternate but equivalent perspective on the role of the coordinated
solvent's pKs in determining Em, given by Equation (3).
coordinates O2 to reversibly form a [Fe3+O22] state that is
stabilized by hydrogen bonding to O22. FeSOD may
provide a proton to coordinated OH2 instead, and thereby
favor electron transfer from O2.2 onto Fe3+, towards
coordinated solvent (Figure 9). (Mn)SOD might promote
substrate reduction by transferring a proton to the noncoordinating O of O2.2 simultaneously with electron
transfer (the pK of O2.2 is only 4.5) and by stabilizing
coordinated solvent as OH2 instead of protonating it.
Different hydrogen bonding to substrate is consistent with
the different modes of substrate analog binding observed in
FeSOD vs. Fe(Mn)SOD or MnSOD.30,45,48,86 Indeed, this
alternative use of active site protons to draw electron
transfer on to and off of substrate is equivalent to the
different stabilization of coordinated H2O/OH2 invoked
earlier to explain some of the difference in Em, and
constitutes an effective reversal of the active site proton
flow in one protein relative to the other.
C O N C L U D I N G R EM A R K S
In FeSOD as in many other redox-active enzymes,
substrate binding, proton transfer and electron transfer
are inextricably intertwined. Although FeSOD's overall
reaction is readily catalyzed by many simple inorganic
systems,96 FeSOD achieves the specificity characteristic of
enzymes via the protein's participation in and control over
substrate binding and protons. Our measurements of KDs
and pKs provide a necessary thermodynamic framework
for understanding FeSOD's mechanism while spectroscopic
studies permit interpretation in terms of conformational
changes and individual residues, complementing the
680
H AN D B OOK OF M ETAL LOP RO TEI NS
wealth of mechanistic, crystallographic and mutagenesisderived information available for this enzyme.
The apparent implausibility of Fe as an agent of
protection against oxidative stress is reconciled by an
outer-sphere mechanism for the second half reaction and
exclusion of HO22 from the active site Fe2+ by ionized
Tyr342. We propose that a conserved crystallographic
solvent approximately represents the outer sphere substrate binding site and that Tyr34 serves to shuttle a proton
from the coordinated solvent to the substrate in conjunction with its reduction in MnSOD. Gln also occupies a
pivotal position in FeSOD's active site, linking at least four
other amino acids to the coordinated solvent. We propose
that the differences between the strength of hydrogen
bonding to the coordinated solvent in the (Fe)SOD and
(Mn)SOD proteins goes a long way toward producing the
very large difference between the Ems achieved by these
two proteins. This in turn is sufficient to explain the two
proteins' intriguing metal ion specificities. Since coordinated solvent is not only crucial to Em tuning but also
proposed to be the source of one of the protons required to
generate the product, its pKs are crucial to activity. Protein
control over the degree to which coordinated solvent is
protonated, via hydrogen bonding and other proteindetermined interactions, may provide a general mechanism
for protein determination of metal ion Ems and reactivities.
By virtue of the very large range of Ems observed within
a conserved overall structural context, the FeSODs and
MnSODs provide an excellent system for understanding
redox tuning. The possibility of manipulating the Em at
will by amino acid and metal ion substitution opens the
way for designing metal ion sites to catalyze desired
chemistry.
Fe-superoxide dismutase
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