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Superoxide dismutases: active sites that save,
but a protein that kills
Anne-Frances Miller
Protection from oxidative damage is sufficiently important that
biology has evolved three independent enzymes for hastening
superoxide dismutation: the Cu- and Zn-containing superoxide
dismutases (Cu,Zn-SODs), the SODs that are specific for Fe or
Mn or function with either of the two (Fe-SODs, Mn-SODs or Fe/
Mn-SODs), and the SODs that use Ni (Ni-SODs). Despite the
overwhelming similarities between the active sites of Fe-SOD
and Mn-SOD, the mechanisms and redox tuning of these two
sites appear to incorporate crucial differences consistent with
the differences between Fe3þ/2þ and Mn3þ/2þ. Ni-SOD is
revealed by spectroscopy to employ completely different ligation
to that of the other SODs while nonetheless incorporating a
device also found in Cu,Zn-SOD. Finally, the protein of human
Cu,Zn-SOD appears to be an important contributor to the
development of amyotrophic lateral sclerosis, possibly because
of its propensity for extended b-sheet formation.
Addresses
Department of Chemistry, University of Kentucky, Lexington KY,
40506-0055, USA
e-mail: [email protected]
Current Opinion in Chemical Biology 2004, 8:162–168
This review comes from a themed issue on
Bioinorganic chemistry
Edited by Stephen J Lippard and Jeremy M Berg
1367-5931/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2004.02.011
Abbreviations
ALS amyotrophic lateral sclerosis
SOD superoxide dismutase
XAS X-ray absorption spectroscopy
Introduction
Disproportionation of superoxide (O2 ) is an exceptionally simple reaction, yet it incorporates central themes
of redox catalysis in general, including the coupling
between protons and electrons, which is a potent device
for conservation, transduction and utilization of energy.
The overall disproportionation: 2O2 þ 2Hþ $ O2 þ
H2 O2 is accomplished by Fe-, Mn- and Cu,Zn-superoxide
dismutases (SODs) in two steps, which are both first-order
with respect to O2 (reviewed in [1]):
Ezox þ O2 þ Hþ $ Ezred ðHþ Þ þ O2
þ
Ezred ðH Þ þ O2
þ
þ H $ Ezox þ H2 O2
Current Opinion in Chemical Biology 2004, 8:162–168
(1a)
(1b)
where Ezox and Ezred denote the Cu2þZn2þ and
CuþZn2þ, Fe3þ and Fe2þ, Mn3þ and Mn2þ, Ni3þ and
Ni2þ states of the Cu,Zn-, Fe-, Mn- and Ni-SODs,
respectively, and the acquisition of a redox-coupled proton is indicated by the appended ‘(Hþ)’. (Proton uptake
coupled to enzyme reduction is presumed but has not
been demonstrated experimentally for Ni-SOD.) Oxidation of O2 does not involve proton transfer at neutral pH
(HO2 $ O2 þ Hþ has a pK of 4.5 [2]). Thus, this half
of the reaction of O2 explicitly requires only substrate
binding and electron transfer, and it is favorable. By
contrast, pure reduction of O2 is extremely unfavorable.
However, by supplying even only one of the protons
required for formation of H2O2, the enzyme can greatly
promote and accelerate reduction of O2 [2]. Thus, one
theme evident among SODs is that the favorable first
half-reaction is coupled to uptake of one proton, which in
turn can facilitate substrate reduction (and binding,
below). Excess energy available from the first half reaction is thereby applied to the second.
An additional barrier to spontaneous disproportionation of
superoxide at neutral pH is the difficulty of overcoming
electrostatic repulsion between two O2 anions. SODs
circumvent this difficulty in two ways: first, Fe-, Mn- and
Cu,Zn-SODs react with only one molecule of O2 at a
time; and second, they exploit electrostatic interactions
with the negative charge of O2 to favor specific binding
of substrate but not products. Structurally characterized
SODs have so far been found to have net positive electrostatic potential near the active site, caused by the metal
centre itself. In Cu,Zn-, Fe- and Mn-SODs, the charge on
the metal centre is not fully neutralized by its ligands
(although second-sphere residues tend to compensate).
Net positive charge probably contributes to substrate
binding, and these SODs all bind not only O2 , but also
a series of other small anions. By contrast, each of the two
products of disproportionation is neutral and therefore
would not be bound by this mechanism. Thus, the change
in charge associated with conversion of substrate to product is exploited by the mechanism of substrate binding.
The SOD ping-pong mechanism requires that O2 bind
to two different versions of the same active site. However
the active site’s electrostatic attraction for incoming O2 is preserved upon metal ion reduction by the coupled
uptake of a proton (above). Thus, although the charge
distribution is changed, the total charge is not, and substrate binding should still be favored to a similar extent, if
in a different mode. Although the three kinds of SODs are
completely different in their protein structure and metal
ion content (Figure 1), their evolution to address the same
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Superoxide dismutases: active sites that save, but a protein that kills Miller 163
Figure 1
Fe-SOD and Mn-SOD
Cu,Zn-SOD
Current Opinion in Chemical Biology
Comparison of the protein folds of Mn- or Fe-SODs (left) and Cu,Zn-SODs (right). Fe- and Mn-SODs have monomers of 200 amino acids and occur
as dimers or tetramers. The magenta ball depicts the active site Fe or Mn ion of the colored monomer (coordinates 1ISB.pdb for Fe-SOD). Cu,ZnSODs have monomers of 150 amino acids and mostly occur as dimers. The Cu ion is in green and the Zn in black for the colored monomer
(coordinates 2SOD.pdb). The Zn loop and the electrostatic loop of Cu,Zn-SOD make up what appear in the figure to be the upper and lower lips of the
metal-containing active site. The structure of Ni-SOD has been solved too, but could not yet be made public (see [55]).
challenge has produced common as well as different
features.
Fe-SOD
The active site contains a single Fe ion coordinated in a
trigonal bipyramid by three histidines, an Asp and a
molecule of OH/H2O that is supported by a conserved
hydrogen bond network including Gln69 and Tyr34
(numbering of Escherichia coli, Figure 2).
Substrate analogues have been shown to bind differently
to the two oxidation states, forming a six-coordinate
complex of the oxidized state but leaving a five-coordinate active site in the reduced state [4]. However,
substrate binding is almost certainly coupled to transfer
of electron density, as NO binding produces a state best
described as formal Fe3þ-NO [5]. In Fe-SOD’s first
half-reaction, the flow of electrons would be the reverse,
with the Fe3þ-O2 adduct dissociating as in reversible
O2-binding systems to yield O2 þ Fe2þ. Proton uptake
coupled to metal ion reduction was demonstrated in the
classic paper by Bull and Fee [6]. Recent experimental [7]
and computational [8] studies indicate that the coordinated OH becomes protonated to H2O upon Fe reduction (Protein(Fe3þ-OH) $ Protein(Fe2þ-OH2)). An
‘extra’ proton is thus available to aid in substrate binding
to the reduced state. Because the reduced metal ion itself
is less positively charged and less disposed to coordinate
O2 (below) an outer sphere mechanism is attractive
instead of the inner-sphere mechanism proposed for the
first half reaction. Studies of both NO binding and N3 www.sciencedirect.com
binding demonstrate the existence of two similar but
distinct substrate analogue-bound configurations, whose
populations are modulated by the second sphere [5,9].
Perhaps they represent occupancy of the substrate binding sites that are normally employed in the two different
oxidation states.
Substrate binding has been difficult to elucidate unambiguously in X-ray crystal structures because it has been
difficult to obtain full occupancy of the substrate analogue,
the metal ion is subject to reduction in the X-ray beam,
and the substrate analogues are very small molecules
whose electron density can be difficult to distinguish from
that of a coordinated solvent, next to a relatively electrondense metal ion. However, recent successful resonance
Raman of the N3 complex of Fe3þ-SOD and variable
temperature-variable field magnetic circular dichroism in
conjunction with computations support the crystallographically determined six-coordinate N3 complex [9].
In addition, better resolution is being attained in some
of the recent crystal structures, such as the 1.6 Å structure
of Fe-SOD from Thermosynechococcus elongatus [10].
Mn-SOD
The obvious similarities between Fe- and Mn-SODs
[1,4] belie crucial differences between the events underlying the mechanism [4,11,12]. Whereas Fe3þ-SOD
coordinates substrate analogues to form a six-coordinate
complex, substrate analogue binding to Mn3þ-SOD
appears to involve only partial, weak or transient coordination of the anion to Mn3þ (although at low temperature or
Current Opinion in Chemical Biology 2004, 8:162–168
164 Bioinorganic chemistry
Figure 2
Q146
N80
W128
H80
Y34
H71
H81
HO-
H63
R143
H2O
Mn
D167
H2O
H48
D83
Cu
H171
D124
H30
Zn
H26
H46
H120
Mn-SOD (or Fe-SOD)
Cu,Zn-SOD
Current Opinion in Chemical Biology
Comparison of the active site of Mn- or Fe-SODs (left) with that of oxidized Cu,Zn-SODs (right), including important hydrogen bonding residues and
residues mentioned in the text. For Mn- or Fe-SOD, the magenta ball depicts the Mn3þ/2þ or Fe3þ/2þ ion and the water molecule to its left may indicate
a substrate-binding site [3]. The amino acid residues are numbered according to E. coli Mn-SOD and the coordinates used were 1D5N.pdb. In E. coli
Fe-SOD, the active site Gln is Q69. For Cu,Zn-SOD, the green ball depicts the Cu2þ ion and the black ball depicts the Zn2þ ion. The amino acid residues
are numbered according to human cytoplasmic Cu,Zn-SOD (SOD1) although the coordinates used were those of the bovine enzyme, 2SOD.pdb. The
water molecule above and to the right of the Cu2þ ion represents the presumed substrate binding site (outer-sphere binding via R143 and H63 is
envisioned for the reduced state). Solid lines indicate bonds and dashed lines indicate hydrogen bonds.
in the Y34F mutant, a dominant six-coordinate complex is
formed) [13]. Similarly, Fe3þ-SOD has a pKa of 8.5
assigned to coordination of OH to Fe3þ but Mn3þ-SOD’s
active site pK of 9.5 has been assigned to deprotonation
of Tyr34, based on the pH dependence of the 13 Cz NMR
signal of Tyr34 [12]. These differences are consistent
with the different electronic configurations of Fe and Mn
in each oxidation state [11,12]. Thus, Mn3þ, a d4 ion,
would loose the benefit of Jahn-Teller stabilization upon
formation of a six-coordinate complex with OH, other
substrate analogues, or O2 , whereas Fe3þ is a d5 ion with
no such cost. Conversely Fe2þ has some preference for
retention of an unsaturated coordination sphere, whereas
Mn2þ prefers octahedral hexa-coordination. Thus, differences persist but are less pronounced in the reduced
states. The Fe2þ of Fe2þ-SOD does not appear to coordinate substrate analogues, but the Mn2þ of Mn2þ-SOD
does [14,15]. However, Mn3þ-SOD has been shown to
take up a proton upon reduction, similar to Fe-SOD [7]
and the active site pK of the reduced state has been
assigned to Tyr34 but also affects the metal site [12],
as in Fe2þ-SOD [16]. The greater tendency of Fe3þ than
Mn3þ to coordinate OH can explain the much lower pK
(and lower activity at higher pH) of the Fe-containing
versions of metal-specific and cambialistic SODs (most
recently [17], discussed in [4,18,19]).
Current Opinion in Chemical Biology 2004, 8:162–168
Thus, the protein features that would optimally complement the electronic configuration and intrinsic reduction
potential of Mn are not expected to provide nearly as good
support for Fe, and it is not surprising that metal ion
substitution produces altered substrate analogue affinities, shifted pKs and very different reduction midpoint
potentials, all of which could contribute to the observed
decreases in activities. While the overall electronic configuration and coordination geometry seems to be conserved when one of Fe or Mn is bound in the active sites
of proteins with different metal ion specificities for activity [11], high field EPR and optical band maxima demonstrate that subtle differences in the geometry of the Mn2þ
or Fe3þ ion in the different active sites correlate with
activity [19], as does the EPR spectral change associated
with high pH for bound Fe3þ [20]. Such spectroscopic
differences have been produced by perturbations even
beyond the second sphere, such as mutations that affect
the hydrogen bonding network [19,21,22] or ionization
of Tyr34 [12], and distinguish the Fe- or Mn-SODs of
different species [15].
The active site of Mn-SOD also appears to depress the
reduction midpoint potential of the bound metal ion
much more than does the active site of Fe-SOD
[23,24], as is to be expected since intrinsic potential of
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Superoxide dismutases: active sites that save, but a protein that kills Miller 165
high-spin Mn3þ/2þ is much higher than that of high spin
Fe3þ/2þ. There is evidence that this reflects, at least in
part, the extent to which the different sites stabilize
coordinated OH versus coordinated H2O: because the
former is associated with the 3þ state for both Fe and Mn,
its relative stabilization would lower the reduction potential [25,26]. Thus, the Mn-SOD protein, which appears
to donate a stronger hydrogen bond to coordinated solvent [25], would be expected to lower the reduction
potential more, as observed, and mutation of the hydrogen-bond-donating Gln to hydrogen-bond-accepting Glu
is associated with a large increase in reduction potential
[26]. Similarly, mutation of a nearby residue that partially
disrupts the hydrogen bond network increases Fe-based
activity at the expense of Mn-based activity in a SOD
protein that supports both, and also appears to raise the
metal ion reduction potential, as appropriate for Fe [19].
Moreover, insofar as the strength (and polarity) of the
hydrogen bond to coordinated solvent would also modulate
the ligand strength of coordinated solvent, it could produce
the observed effects on the EPR signal too [19].
Another area of progress has been characterization of the
peroxide-inhibited complex of Mn-SOD. Silverman’s
team have found that mutations affecting residues that
hydrogen bond with the conserved active site Gln146, or
His30 in the substrate access channel, result in very longlived inhibited complexes that accumulate to up to 20
times higher levels than in WT Mn-SOD [27]. By contrast, a mutant Mn-SOD displaying decreased formation
of the inhibited complex has anti-proliferative properties
and results in slower-growing tumors [28]. The optical
signature of the peroxide-inhibited complex is very similar to that of the six-coordinate N3 complex of Mn3þSOD [27]. Thus, the inhibited complex could represent a
deviation from normal turnover, if after productive O2 coordination to Mn2þ to form a six-coordinate complex,
electron transfer from Mn2þ were accompanied by proton
transfer to the terminal O to form a hydroperoxide adduct.
This could explain the observed six-coordinate Mn3þ
complex, whereas on-pathway catalysis might be more
likely to involve proton transfer to the proximal O, which
would prompt dissociation of nascent HO2– in conjunction with Mn2þ oxidation to the (favored) five-coordinate
Mn3þ complex.
Cu,Zn-SOD
‘Erythrocuprein’ was the first of the SODs to be identified
as such [29]. The active site of the oxidized form of
Cu,Zn-SOD has a Cu2þ ion coordinated in a distorted
square pyramid by a solvent molecule and four histidines,
one of which is also a ligand to a roughly tetrahedral Zn2þ
coordinated by two other His and an Asp (Figure 2;
[30,31]). Thus the active site is completely unrelated to
that of Fe-SOD and Mn-SOD, and different mechanisms
are found to accomplish many of the elements of superoxide disproportionation. A series of small anions are
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attracted by local positive charge, bind directly to Cu2þ
in place of the solvent molecule, and extend in the
direction of the conserved Arg143 (reviewed by [31]).
Upon reduction, Cu is thought to release the His63 ligand
it shared with the Zn2þ ion, as well as departing O2, to
become trigonal and roughly planar. The active site
electrostatics are conserved overall by uptake of a proton
upon reduction, as in Fe-SOD and Mn-SOD, but in this
case the His63 released by Cu appears to take up a proton
at the now free Ne, on the basis of NMR observation of a
proton at that position [32].
This device also serves to make the redox-coupled proton available to promote reduction of O2 by Cuþ. In the
second half reaction, O2 is believed to bind to the
protein via the conserved Arg143 and the Ne of His63.
Upon (partial) protonation by these groups, O2 would
oxidize the Cu, which as Cu2þ would recapture its ligand
His63, completing displacement of the Ne Hþ presumably transferred to nascent product (reviewed in [1]). It
is my hope that more experiments will be done to define
the site and mode of substrate binding to the reduced
state.
Finally, Cu,Zn-SOD has been the basis for protein engineering to produce a monomeric version and to increase
activity by modifying the electrostatic environment of the
active site (reviewed in [33]). Cu,Zn-SOD also remains
prominent as a model system for developments in the
application of NMR spectroscopy to paramagnetic systems [34].
Recent research on Cu,Zn-SOD has been propelled by
questions as to the relationship between CuZn-SOD and
amyotrophic lateral sclerosis (ALS, reviewed in [35]).
From the first, it was evident that mutants of CuZnSOD gained some property that was associated with
development of ALS, and dominated over coexpressed
WT-Cu,Zn-SOD (reviewed in [35,36]). However, a bewildering variety of mutants, approximately 90 as of 2003
[35], are all associated with familial ALS. Two possibilities
dominate current thinking, and are potentially related to
one another. One model is that mutant CuZn-SODs have
higher-than-WT oxidative activity that contributes to
oxidative damage [31,37,38,39], and appears to be stimulated by physiological bicarbonate concentrations ([40],
but see [41]). Indeed levels of oxidative stress appear
higher in the presence of ALS-associated Cu,Zn-SOD
mutants [39] and there is good evidence of production
of CO3 – ([42] and references therein), which is sufficiently long-lived to diffuse away and initiate oxidative
damage to other molecules nearby. However the majority
of ALS-associated mutants have not been shown to have
significantly higher-than-WT peroxidase activity [41].
Instead, there is growing support for the notion that many
ALS-associated SOD mutants are less stable [33,35] and
Current Opinion in Chemical Biology 2004, 8:162–168
166 Bioinorganic chemistry
more prone to formation of aggregates and fibrils [43],
which could saturate the cell’s chaperone [44,45] and
proteasome resources [36] or inhibit axonal transport
machinery [46,47]. In vitro, WT-Cu,Zn-SOD becomes
modified by CO3 at its surface Trp32, and cross-linked
([42] and references therein). Cu,Zn-SODs with mutations that abrogate metal ion binding are much less stable
and form amyloid-like filaments [43,48], and in mutant
and WT-Cu,Zn-SODs that retain bound Cu, oxidative
damage to active site residues causes metal ion release
[49]. Even metal-depleted WT-Cu,Zn-SOD has been
shown to form filaments based on association of the b
sheets of individual SOD monomers [30]. Thus metaldepleted and mutant Cu,Zn-SODs have a much higher
tendency to aggregate, even without oxidative cross-linking, and oxidative damage leads to metal depletion.
Finally, Tobisawa et al. have shown, for a set of four
Cu,Zn-SOD mutants comprising mutants with disrupted
active sites as well as mutants that retain close-to WT
catalytic activity, that the mutants but not the WT form
aggregates when they are either expressed at a high level,
or proteasome activity is inhibited [47]. Moreover,
mutant SOD aggregation was accompanied by defective
microtubule assembly, and mis-localization of mitochondria and proteins [47]. These results provide attractive
rationales for the special vulnerability of motor neurons,
as even slightly defective localization and transport
mechanisms would be particularly damaging since many
proteins must be moved over 1 m distances to reach nerve
termini [46]. The delayed onset of symptoms associated
with ALS could reflect age-related decline of proteasome
activity and thus loss of ability to prevent accumulation of
aggregates [47,50].
Ni-SOD contains one Ni per monomer [54] but there are
conflicting reports as to whether the enzyme is tetrameric
or hexameric. X-ray absorption spectroscopy (XAS) indicates penta-coordinate Ni3þ coordinated by multiple S
donors and N/O ligands [56]. However, there are only
two Cys per monomer and the one Met has been shown
not to be a ligand, since mutation to Leu does not disrupt
activity or the spectral signatures [57]. Thus, a dinuclear,
Ni2 site was proposed, most reasonably, consistent with
the Ni3þ EPR signal intensity corresponding to 0.5 Ni3þ
per monomer (one Ni3þ per Ni2 cluster and one such
cluster per protein dimer) [54,56]. Well-resolved 14N
superhyperfine structure on the EPR signal further indicates an axial N donor [54,56]. Mutation of the only His
(His1) to Gln decreases the activity 100-fold, and causes
the enzyme to be largely reduced as-isolated, indicating
an increase in reduction potential [57]. The resting Ni3þ
state is proposed to alternate with a Ni2þ state upon
reaction with O2 [56], and the dithionite-reduced
enzyme displays exclusively four-coordinated Ni2þ,
indicating that the Ni3þ site releases a ligand upon
reduction (reminiscent of Cu,Zn-SOD). On the basis
of sulfur XAS, Szilagyi et al. have proposed that a terminal
thiolate ligand takes up a proton upon Ni3þ reduction by
peroxide [58], analogous to proton uptake by a ligand in
Fe- and Mn-SOD. Alternatively, it is possible that the
dissociating N donor could become protonated upon its
release from Ni and thus accomplish redox-coupled
proton uptake in a fashion analogous to that observed
in Cu,Zn-SOD [56]. The forthcoming crystal structures
will provide exciting complements to the spectroscopic
information.
Conclusions
Thus, the two models should probably be viewed as
interrelated with the roles of cause and effect blurring
and varying in different mutants, and even WT-Cu,ZnSOD could contribute to sporadic ALS [30,40]. There is
conflicting evidence as to the importance of Zn-depletion
[35,48]. However Cu,Zn-SOD’s association with familial
ALS could derive from a tendency of b sheets to form
extended arrays [51]. This could happen in Cu,Zn-SOD if
the sheet edges normally protected by the Zn-binding
and electrostatic loops are unmasked upon metal ion
depletion or loss of loop structure [30] (see Figure 1).
There is also recent evidence that Cu,Zn-SOD’s localization to mitochondria could play an important role in the
aetiology of ALS [52,53].
Progress continues apace for all three types of SOD.
However, the directions this is taking are strikingly
different. For the Fe-, Mn- and Fe/Mn-SODs, spectroscopic methods are advancing our ability to understand
electronic bases for reactivity and how these vary from
enzyme to enzyme. For the Cu,Zn-SODs there is great
excitement in understanding this enzyme’s link to ALS.
Research on Ni-SOD has jumped forward with new
spectroscopic results and most exciting crystal structures.
Ni-SOD
References and recommended reading
Several Streptomyces species have been shown to produce a
Ni-dependent SOD that is completely unrelated to the
better-known Cu,Zn-, Fe- and Mn-SODs [54]. Crystal
structures have been solved but (alas) were not yet public
at the time of this writing [55]. However, a substantial
amount of spectroscopic evidence provides important
insights into how the active site Ni ions function.
Papers of particular interest, published within the annual period of
review, have been highlighted as:
Current Opinion in Chemical Biology 2004, 8:162–168
Acknowledgements
I offer my belated thanks to Dr JA Fee for generosity to me at the beginning
of my independent career. Work on SOD in my laboratory is currently funded
by NSF MCB0129599 and the Kentucky science and engineering fund
KSEF172.
of special interest
of outstanding interest
1.
Miller A-F: Superoxide Processing. In Coordination Chemistry
in the Biosphere and Geosphere. Edited by Que L Jr, Tolman W:
Oxford, Amsterdam, New York and Tokyo: Pergamon;
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Superoxide dismutases: active sites that save, but a protein that kills Miller 167
2.
Sawyer DT, Valentine JS: How super is superoxide?
Acc Chem Res 1981, 14:393-400.
3.
Borgstahl GEO, Pokross M, Chehab R, Sekher A, Snell EH:
Cryo-trapping the six-coordinate, distorted-octahedral active
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A thorough review of the Fe-SOD literature up to 2000.
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exploring the geometric and electronic structures of the
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A long-overdue investigation of Fe-SOD’s reactivity with NO, which is a
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alone and coupled to proton transfer, from an expert. This paper duplicates trends observed among different Fe- and Mn-SODs and validates
the expected link between observed rate constants and the reduction
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9.
Xie J, Yikilmaz E, Miller A-F, Brunold TC: Second-sphere
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10. Kerfeld CA, Yoshida S, Tran KT, Yeates TO, Cascio D,
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12. Maliekal J, Karapetian A, Vance C, Yikilmaz E, Wu Q, Jackson T,
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those of Fe-superoxide dismutase. J Am Chem Soc 2002,
124:15064-15075.
Despite the overwhelming appearance of similarity between the active sites
of Fe-SOD and MnSOD, the events responsible for the oxidized state pK
are not the same. The mechanistic significance of this finding is discussed.
13. Whittaker MM, Whittaker JW: Low-temperature
thermochromism marks a change in coordination for the metal
ion in manganese superoxide dismutase. Biochemistry 1996,
35:6762-6770.
14. Whittaker JW, Whittaker MM: Active site spectral studies on
manganese superoxide dismutase. J Am Chem Soc 1991,
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15. Un S, Dorlet P, Voyard G, Tabares LC, Cortez N: High-field EPR
characterization of manganese reconstituted superoxide
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cambialistic, manganese-containing enzyme. J Bacteriol 2003,
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18. Edwards RA, Whittaker MM, Whittaker JW, Baker EN,
Jameson GB: Outer sphere mutations perturb metal reactivity in
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A thoughtful comparison of several mutants addressing the Gln that
hydrogen bonds to coordinated solvent, as well as the ‘gateway’ Tyr.
19. Yamakura F, Sugio S, Hiraoka BY, Ohmori D, Yokota T:
Pronounced conversion of the metal-specific activity of
superoxide dismutase from Porphyromonas gingivalis by the
mutation of a single amino acid (Gly155Thr) located apart from
the active site. Biochemistry 2003, 42:10790-10799.
A lesson in the subtlety of proteins, and enzyme active sites. A remote
mutation indirectly modifies the strengths of hydrogen bonds and thus
achieves a 20-fold effect on activity. This most recent contribution from
one of the founders of the field combines biochemical, spectroscopic and
structural data.
20. Vance CK, Miller A-F: Spectroscopic comparisons of the pH
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