The role of electrostatics in siderophore recognition by the immunoprotein siderocalin

Article abstract of DOI:10.1021/ja8074665

Iron is required for virulence of most bacterial pathogens, many of which rely on siderophores, small-molecule chelators, to scavenge iron in mammalian hosts. As an immune response, the human protein Siderocalin binds both apo and ferric siderophores in o

Full text of DOI:10.1021/ja8074665

Published on Web 11/19/2008
The Role of Electrostatics in Siderophore Recognition by the
Immunoprotein Siderocalin1
Trisha M. Hoette,† Rebecca J. Abergel,† Jide Xu,† Roland K. Strong,‡ and
Kenneth N. Raymond*,†
Department of Chemistry, UniVersity of California, Berkeley, California 94720-1460, and DiVision of
Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
Received September 19, 2008; E-mail: raymond@socrates.berkeley.edu
Abstract: Iron is required for virulence of most bacterial pathogens, many of which rely on siderophores,
small-molecule chelators, to scavenge iron in mammalian hosts. As an immune response, the human protein
Siderocalin binds both apo and ferric siderophores in order to intercept delivery of iron to the bacterium,
impeding virulence. The introduction of steric clashes into the siderophore structure is an important
mechanism of evading sequestration. However, in the absence of steric incompatibilities, electrostatic
interactions determine siderophore strength of binding by Siderocalin. By using a series of isosteric
enterobactin analogues, the contribution of electrostatic interactions, including both charge-charge and
cation-π, to the recognition of 2,3-catecholate siderophores has been deconvoluted. The analogues used
in the study incorporate a systematic combination of 2,3-catecholamide (CAM) and N-hydroxypyridinonate
(1,2-HOPO) binding units on a tris(2-aminoethyl)amine (tren) backbone, [tren(CAM)m(1,2-HOPO)n, where
m ) 0, 1, 2, or 3 and n ) 3 - m]. The shape complementarity of the synthetic analogue series was
determined through small-molecule crystallography, and the binding interactions were investigated through
a fluorescence-based binding assay. These results were modeled and correlated through ab initio
calculations of the electrostatic properties of the binding units. Although all the analogues are accommodated
in the binding pocket of Siderocalin, the ferric complexes incorporating decreasing numbers of CAM units
are bound with decreasing affinities (Kd ) >600, 43, 0.8, and 0.3 nM for m ) 0-3). These results elucidate
the role of electrostatics in the mechanism of siderophore recognition by Siderocalin.
Introduction
bin, another 0.4 g is engaged in other cellular functions, and
the small remaining portion of iron is bound by transport and
Iron has a range of biologically relevant oxidation states,
including Fe(II), Fe(III), and Fe(IV), and is therefore a platform
for electron transfer, redox chemistry, and oxygen transport in
living systems.2 However, the availability of ferric ion is limited
by its insolubility in aqueous, pH-neutral environments,3 and
the presence of ferrous ion poses the risk of the production of
destructive oxygen radicals.4 In response to these environmental
pressures for tight regulation, both mammals and microorgan-
isms have developed efficient and competitive systems to
acquire, transport, and store iron.3,5,6
storage proteins such as extracellular transferrins and intracel-
lular ferritin.7,8 With nearly all the iron confined to such proteins,
the concentration of “free” extracellular iron is estimated at 10-24
M.9 This iron containment strategy serves multiple physiological
functions: first, to solubilize ferric ions which would otherwise
be unavailable in a biological environment; second, to prevent
ferrous ions from participating in unregulated production of
reactive oxygen species (ROS) via the Fenton reaction; and
finally, to act as a first line of defense against microbial infection
by withholding iron.
Like mammals, microorganisms have developed effective
systems to secure their iron supply. Many bacteria synthesize
and secrete low-molecular-weight, highly iron-selective chelators
called siderophores to collect iron from the extracellular
environment.8 There are hundreds of known siderophore
structures with a variety of denticities, chelating units, and
backbone scaffolds (Figure 1). Usually bacteria synthesize more
than one type of siderophore (often a catecholate and a
hydroxamate or citrate) by taking advantage of this modular
Of the 3-4 g of iron in the average human body, ap-
proximately 2.5 g participates in oxygen transport in hemoglo-
† University of California, Berkeley.
‡ Fred Hutchinson Cancer Research Center.
(1) Paper #84 in the series Coordination Chemistry of Microbial Iron
Transport. For previous paper, see: Abergel, R. J.; Clifton, M. C.;
Pizarro, J. C.; Warner, J. A.; Shuh, D. K.; Strong, R. K.; Raymond,
K. N. J. Am. Chem. Soc. 2008, 130, 11524–11534.
(2) Iron MetabolismsInorganic Biochemistry and Regulatory Metabolism;
Ferreira, G. C., Moura, J. J. C., Franco, R., Eds.; Wiley-VCH:
Weinheim, 1999.
(3) Raymond, K. N.; Dertz, E. A. In Iron Transport in Bacteria; Crosa,
J. H., Mey, A. R., Payne, S. M., Eds.; ASM Press: Washington, DC,
2004; pp 3-17.
(7) Gordeuk, V. R.; Bacon, B. R.; Brittenham, G. M. Annu. ReV. Nutr.
1987, 7, 485–508.
(4) Pierre, J. L.; Fontecave, M.; Crichton, R. R. Biometals 2002, 15, 341–
346.
(8) Molecular and Cellular Iron Transport; Templeton, D. M., Ed.; Marcel
Dekker, Inc.: New York, 2002.
(5) Hentze, M. W.; Muckenthaler, M. U.; Andrews, N. C. Cell 2004, 117,
285–297.
(9) Raymond, K. N.; Dertz, E. A.; Kim, S. S. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 3584–3588.
(6) Kontoghiorghes, G. J.; Weinberg, E. D. Blood ReV. 1995, 9, 33–45.
9
17584 J. AM. CHEM. SOC. 2008, 130, 17584–17592
10.1021/ja8074665 CCC: $40.75
2008 American Chemical Society

Electrostatic Recognition by Siderocalin
A R T I C L E S
Figure 1. Selected siderophore structures, illustrating a variety of backbones and chelating moieties. Iron-binding atoms are shown in red. High-affinity,
archetypal siderophores enterobactin and bacillibactin, from Gram-negative and Gram-positive bacteria, respectively, are sequestered by the immune system
protein Siderocalin (Scn), while aerobactin (Escherichia coli), salmochelin-S4 (Salmonella enterica), petrobactin (Bacillus anthracis), and alcaligin (Bordetella
pertussis) are not bound by Scn and confer virulence in the mammalian host.
Figure 2. Enterobactin bound within the ꢀ-barrel of Scn (left). A space-filling, close-up view of the calyx showing positively charged residues in blue that
shape the binding pocket (center). Cationic residues K125, K134, and R81 create a cyclically permuted cation-π interaction with enterobactin (right).
ligand assembly. In addition, most microorganisms are able to
utilize xenosiderophores, expressing receptors for siderophores
that the microbe does not itself produce.10,11 The use of
divergent, multi-siderophore systems by successful pathogens
has proven to be an advantage against a number of environ-
mental pressures, including the bacteriostatic action of the
immune system in a mammalian host.
While containment of iron in a mammalian host is important
as a preliminary protection against infection, many pathogens
can overcome this limitation by producing siderophores, such
as enterobactin and bacillibactin, that have a strong kinetic and
thermodynamic advantage for removal of iron from transferrin.12
In response to this action, the mammalian innate immune system
produces Siderocalin (Scn, see Figure 2, also known as lipocalin
2, NGAL, uterocalin, and 24p3), a 20 kDa lipocalin that binds
both ferric and apo siderophores, thereby preventing iron
delivery.13-16 The calyx (or binding site) of Scn is shallow and
lined with positive residues (K125, K134, and R81) that promote
the binding of a number of different siderophores, including
enterobactin and bacillibactin, which have the highest known
affinities for iron. The recognition and the sub-nanomolar
dissociation constant for the Scn/ferric enterobactin interaction
are mediated by electrostatic bonds (including both Coulombic
and cation-π) in the absence of good shape complementarity,
atypical for tight protein/small-molecule binding. Nonetheless,
recent work has shown that the most successful (and deadly)
pathogens evade the host immune system by utilizing sidero-
phores that are not recognized by Scn.
There are several examples in the literature that describe the
substrate shape limitations of the Scn calyx. Successful patho-
gens such as Bacillus anthracis and Salmonella enterica use
these shape constraints to their advantage by producing the
“stealth siderophores”, salmochelin17,18 and petrobactin,15 (Fig-
ure 1), which are not recognized because of gross steric
incompatibilities with the rigid calyx. Hydroxamate-based
(10) Brickman, T. J.; Anderson, M. T.; Armstrong, S. K. Biometals 2007,
20, 303–322.
(14) Goetz, D. H.; Holmes, M. A.; Borregaard, N.; Bluhm, M. E.; Raymond,
K. N.; Strong, R. K. Mol. Cell 2002, 10, 1033–1043.
(15) Abergel, R. J.; Wilson, M. K.; Arceneaux, J. E. L.; Hoette, T. M.;
Strong, R. K.; Byers, B. R.; Raymond, K. N. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 18499–18503.
(16) Holmes, M. A.; Paulsene, W.; Xu, J.; Ratledge, C.; Strong, R. K.
Structure 2005, 13, 29–41.
(11) Matzanke, B. F.; Bohnke, R.; Mollmann, U.; Reissbrodt, R.; Schun-
emann, V.; Trautwein, A. X. Biometals 1997, 10, 193–203.
(12) Carrano, C. J.; Raymond, K. N. J. Am. Chem. Soc. 1979, 101, 5401–
5404.
(13) Flo, T. H.; Smith, K. D.; Sato, S.; Rodriguez, D. J.; Holmes, M. A.;
Strong, R. K.; Akira, S.; Aderem, A. Nature 2004, 432, 917–921.
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A R T I C L E S
Hoette et al.
Figure 3. Isosteric enterobactin analogues (1-4) and larger Me-3,2-HOPO analogues (5-7) used to study the influence of the electrostatic recognition of
siderophores by Scn. The bidentate CAM and 1,2-HOPO groups have the same steric profile but pose different electrostatic interactions, whereas the Me-
3,2-HOPO unit is similar electronically to the 1,2-HOPO unit but incorporates added steric bulk. Iron-binding atoms shown in red.
siderophores, which form neutral complexes, are another class
of siderophores not bound by Scn.14 Pathogens such as
Escherichia coli and Bordetella pertussis, both of which can
utilize enterobactin, instead rely on the hydroxamate sidero-
phores, aerobactin19 and alcaligin,20 respectively (Figure 1), for
virulence in a mammalian host.21 While these systems similarly
demonstrate that siderophore-mediated iron acquisition in
pathogenic bacteria is closely associated with successful evasion
of Scn, it raises the question: Do both steric and electronic
features of siderophores contribute to Siderocalin recognition?
The work presented here aims to determine the relative
contributions of the electrostatic components, both Coulombic
and cation-π, to siderophore recognition through the use of
an isosteric series of tris(2-aminoethyl)amine (tren)-based en-
terobactin analogues (tren(CAM)m(1,2-HOPO)n, 1-4, Figure 3).
The analogues incorporate a systematic combination of cat-
echolamide (CAM) and 1,2-hydroxypyridinone (1,2-HOPO)
metal-binding units. The isosteric nature of the ferric complexes
was verified though small-molecule X-ray crystallography.22 The
sequestration by Scn of the apo, FeIII and VIV complex analogues
was evaluated using fluorescence spectroscopy. A series of ab
initio calculations were performed to study cation binding to
the π face of the aromatic units. The Scn interactions with a
larger analogue series (5-7) incorporating N-methyl-3,2-hy-
droxypyridinone binding units (Me-3,2-HOPO, Figure 3) were
inspected as a parallel control for the isosteric 1,2-HOPO series.
These studies demonstrate the importance of cation-π interac-
tions to recognition, which, in concert with steric constraints
imposed by the relatively rigid calyx, enables Scn to target
unmodified catecholate siderophores but restricts binding of
modified, virulence-associated “stealth” siderophores.
Results
Ligand and Metal Complex Synthesis. We have previously
shown that ferric tren(CAM)3, a well-studied synthetic analogue
of enterobactin, binds to Scn with an affinity comparable to
that of the ferric complex of the natural siderophore.18 This and
other studies indicated that the backbone is not an important
part of recognition by Scn.18 Syntheses of tren(CAM)3,23
25
tren(1,2-HOPO)3,24 and tren(Me-3,2-HOPO)3 have been re-
ported previously. The synthesis of the mixed binding unit
analogues (Scheme 1) is based on methods previously described
in the literature for multiple substitution of tren.18,26 Slow
addition of a stoichiometric (2:1) amount of thiazolide-activated
benzyl-protected CAM, 1,2-HOPO, or Me-3,2-HOPO to the
commercially available tren scaffold afforded the bis-substituted
tren intermediate (8,-10). A third binding unit was subsequently
coupled, yielding the benzyl-protected ligands (11-14). The
ligands were deprotected using acidic conditions (1:1 HCl:
HOAc) and recrystallized from methanol and diethyl ether to
afford the HCl salt of the ligands (2, 3 and 5, 6) in good yields.
The iron complexes of all ligands were either prepared in situ
(for fluorescence studies) or isolated (for crystallization). One
(17) Fischbach, M. A.; Lin, H. N.; Zhou, L.; Yu, Y.; Abergel, R. J.; Liu,
D. R.; Raymond, K. N.; Wanner, B. L.; Strong, R. K.; Walsh, C. T.;
Aderem, A.; Smith, K. D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
16502–16507.
(18) Abergel, R. J.; Moore, E. G.; Strong, R. K.; Raymond, K. N. J. Am.
Chem. Soc. 2006, 128, 10998–10999.
(19) Wooldridge, K. G.; Williams, P. H. FEMS Microbiol. ReV. 1993, 12,
325–348.
(23) Rodgers, S. J.; Lee, C. W.; Ng, C. Y.; Raymond, K. N. Inorg. Chem.
1987, 26, 1622–1625.
(20) Brickman, T. J.; Armstrong, S. K. Infect. Immun. 2007, 75, 5305–
5312.
(24) Xu, J.; Churchill, D. G.; Botta, M.; Raymond, K. N. Inorg. Chem.
2004, 43, 5492–5494.
(21) Williams, P. H.; Warner, P. J. Infect. Immun. 1980, 29, 411–416.
(22) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields,
G. P.; Taylor, R.; Towler, M.; van de Streek, J. J. Appl. Crystallogr.
2006, 39, 453–457.
(25) Xu, J.; O’Sullivan, B.; Raymond, K. N. Inorg. Chem. 2002, 41, 6731–
6742.
(26) Cohen, S. M.; Raymond, K. N. Inorg. Chem. 2000, 39, 3624–3631.
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A R T I C L E S
Scheme 1. Synthesis of Tren-Based Enterobactin Analogues with Mixed Binding Unitsa
a Reagents and conditions: (i) 0.5 equiv of tris(2-aminoethyl)amine, CH2Cl2; (ii) 1 equiv of Bn-1,2-HOPO-thiaz, Bn-Me-3,2-HOPO-thiaz, or Bn2CAM-
thiaz, CH2Cl2; (iii) 1:1 HCl:AcOH; (iv) Fe(acac)3, KOH, MeOH, under N2.
Figure 4. Structural diagrams (ORTEP), top and side views, of [FeIIItren(CAM)2(1,2-HOPO)]2- (A), [FeIIItren(CAM)(1,2-HOPO)2]- (B), and [FeIIItren(1,2-
HOPO)3]0 (C). Solvent molecules, counterions, and hydrogens are omitted for clarity (50% probability ellipsoids). Top and side views of MERCURY
overlaid structures of [FeIIItren(CAM)3]3- and [FeIIItren(1,2-HOPO)3]0 (D) with a room-mean-square deviation of 0.476 Å for the 34 atom pairs.
equivalent of Fe(acac)3 was added to each of the ligands in
methanol, followed by a stoichiometric amount of KOH to
neutralize the HCl salt and deprotonate the ligand. In order to
attain the complexes as solids, the metal complexes were
recrystallized from diethyl ether. Potassium counterions were
associated with the negatively charged complexes.
ferric tren(CAM)3 complex has been previously described,27 the
solid-state characterization of the dianion, monoanion, and
neutral ferric complexes is described here (Figure 4).
All three complexes, 2K+[FeIII(2)]2-, K+[FeIII(3)]-, and
[FeIII(4)]0, crystallize in the monoclinic space group P21/c (Table
S1, Supporting Information), while 3K+[FeIII(1)]3- crystallizes
in the cubic space group P213 (published structure). As described
for [FeIII(1)]3- and [VIV(enterobactin)]2-, the amide protons in
these structures are directed inward and participate in a
Solid-State Structures of [FeIII(tren(CAM)m(1,2-HOPO)n)]m–
and Structural Overlay of Isosteric Series. In order to
demonstrate that the ligand series proposed would test only the
electronic recognition of the ligands and their complexes and
not their steric interaction, the X-ray structures of the ferric
complexes were compared. While the structure of the trianionic
(27) Stack, T. D. P.; Karpishin, T. B.; Raymond, K. N. J. Am. Chem. Soc.
1992, 114, 1512–1514.
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Figure 5. Fluorescence quenching titrations measuring the affinity of Scn for isosteric analogues 1-4 in the FeIII (top left), VIV (top right), and apo (bottom
left) forms, as well binding curves with the FeIII complexes of mixed CAM ligands incorporating a bulkier Me-3,2-HOPO (bottom right). Experiments were
performed with 100 nM Scn samples buffered at pH 7.4 with TBS. An excitation wavelength of 281 nm was used, and emission was monitored at 340 nm.
The symbols represent the experimental fluorescence data (tren(CAM)m(HOPO)n: b, m ) 3, n ) 0; 2, m ) 2, n ) 1; 9, m ) 1, n ) 2; f, m ) 0, n ) 3),
and the calculated fits are shown as lines.
hydrogen-bonding network with the o-hydroxyls typical of these
types of complexes.27,28 The potassium counterions are either
coordinated to an amide carbonyl or situated under the iron
center interacting with the catecholate m-hydroxyls. There is
no evidence of K+-π interactions in these small-molecule
structures.
The tren-based ligands are achiral and therefore afford a
racemic mixture of complexes (∆ and Λ). Two molecules of
the same metal center chirality were used for the structural
overlay. Since the backbone is not important to Scn recognition,
only the binding units including the amides were used in the
shape comparison. The ferric catecholamide units and the ferric
hydroxypyridinone amide units of [Fe-tren(CAM)3]3- and [Fe-
tren(1,2-HOPO)3]0 were overlaid and distance minimized,22
yielding a root-mean-square deviation for the 34 atom pairs of
0.476 Å (Figure 4). This shows that the complexes have virtually
identical spatial arrangements, and Scn recognition should
therefore differ only due to the electrostatic nature of the
complexes.
Fluorescence Binding Assay. The affinities of Scn for the
analogues were determined by a fluorescence-based binding
assay as previously described.14 A 100 nM solution of Scn was
titrated with between 2 and 10 equiv of substrate, depending
on the affinity. The binding curves were fit by nonlinear least-
squares refinement and the resulting dissociation constants
calculated.29 Four sets of substrates were used in this study:
FeIII and VIV complexes of the tren(CAM)m(1,2-HOPO)n series
were used for direct comparison of complex charge; apo ligands
were used to explore the recognition of the aromatic units; and
ferric complexes of a series of ligands incorporating the
structurally similar Me-3,2-HOPO unit (Figure 3) rather than
the 1,2-HOPO unit were studied, combining both steric and
electronic perturbations as a parallel control for the isosteric
1,2-HOPO series. Binding results are shown in Figure 5 and
Table 1. Crystallographic analyses of complexes between Scn
and the isosteric 1,2-HOPO series show that, in general, these
compounds bind in the calyx in the same orientation as
enterobactin (Clifton et al., manuscript in preparation), making
them ideal compounds for isolating the relative effects of
cation-π versus Coulombic bonds.
A decrease of affinity was observed within the ligand series
for both the ferric and vanadium complexes with decreasing
charge. In a comparison of a ferric complex to the vanadium
complex with the same ligand, there is a decrease in affinity
for every pair of substrates. In addition, the charge-matched
complexes are bound with comparable Kd’s for the pairs of
complexes. The more highly charged complexes (3- and 2-)
are recognized with fairly strong affinities with only slight
charge effect. The complexes of lesser charge (1-, neutral, and
1+) experience a more dramatic decrease in recognition
compared to larger anions, such that binding was barely
detectable under the experimental conditions.
The apo ligands were also tested for Scn binding. The charges
on the ligands were estimated from the previously determined
protonation constants for tren(CAM)3 and tren(1,2-HOPO)3.
Since the 1,2-HOPO unit is a relatively acidic binding unit (in
(28) Karpishin, T. B.; Dewey, T. M.; Raymond, K. N. J. Am. Chem. Soc.
1993, 115, 1842–1851.
(29) Kuzmic, P. Anal. Biochem. 1996, 237, 260–273.
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A R T I C L E S
Table 1. Scn Affinities for Tren-Based Analogues and Their Metal Complexesa
Kd
FeIII
VIV
no metal
trenCAM3
0.3 ( 0.1 nM18
0.8 ( 0.3 nM
0.12 ( 0.01 µM
43 ( 2 nM
0.15 ( 0.02 µM
>0.6 µM
>0.6 µM
1.3 ( 0.3 nM
40 ( 9 nM
n.d.
0.22 ( 0.07 µM
n.d.
1.2 ( 0.2 nM
1.4 ( 0.3 nM
n.d.
0.14 ( 0.01 µM
n.d.
trenCAM2(1,2-HOPO)
trenCAM2(Me-3,2-HOPO)
trenCAM(1,2-HOPO)2
trenCAM(Me-3,2-HOPO)2
tren(1,2-HOPO)3
0.30 ( 0.03 µM
n.d.
0.25 ( 0.03 µM
n.d.
tren(Me-3,2-HOPO)3
a Dissociation constants are reported in units of either nM or µM depending on the magnitude in order to maintain consistent significant figures. See
Supporting Information (Figure S1) for a bar graph representation of Scn dissociation constants.
Figure 6. Schematic of the quadrupole moment of benzene with negative density above and below the ring represented in red (A). Representative input
structure for calculations of cation-π interactions energies with the model aromatic units; as shown here, the cation (in pink) was fixed at 2.47 Å above the
centroid of the M(1,2-HOPO) aromatic (B). Computational studies describe the electrostatic interactions of metal-binding units of siderophores with positive
residues of the calyx. The bottom panel includes quadrupole moments (calculated at the level RHF/6-311G** of theory), cation-π interaction energies
(calculated at MP2/6-311++G** the level of theory), molecular structures, and electrostatic potentials (with a range of -0.18 (red) to +0.18 kcal/mol
(blue)) for the four model aromatic units used in this study.
tren(1,2-HOPO)3, pKaHOPO ) 3.60, 4.32, 5.6230), the hydroxyl
Ab Initio Calculations of Quadrupole Moments and
will be deprotonated in our experimental conditions (pH 7.4).
For the more basic CAM unit (in tren(CAM)3, pKaCAM ) 6.71,
8.61, 8.75, 11.3, 12.1, 12.923), only one of the meta oxygens is
deprotonated at pH 7.4; therefore, the overall ligand charge at
physiological pH is 1-. The fluorescence binding data indicated
that Scn affinity decreased with increasing 1,2-HOPO content,
in that tren(CAM)3 is recognized with sub-nanomolar affinity
while tren(1,2-HOPO)3 is bound in a micromolar affinity range.
Relative to the 1,2-HOPO series, the Me-3,2-HOPO
analogue series provides a platform for comparison of the
influence of shape versus charge. An additional methyl group
functionalizes the N group on the binding unit, placing more
steric demand on the bottom side of the binding unit. The
addition of both one and two Me-3,2-HOPO units decreases
the affinity 3 orders of magnitude compared to that of the
parent tris-catecholate. The complex with three Me-3,2-
HOPO units is not bound.
Cation-π Interaction Energies. Two sets of ab initio calcula-
tions were performed to examine the interactions of the π
surfaces of a siderophore with cationic residues in the calyx of
Scn. The methyl amide bidentate units of CAM and 1,2-HOPO
were used as a simplified model (Figure 6). In order to mimic
coordination to a metal center, the 1,2-HOPO and CAM units
were coordinated to alkaline metal ions, sodium and calcium,
respectively, to form the neutral metal complexes. The free
ligand forms of the bidentate units were also studied. These
four molecules were geometry optimized, and the quadrupole
tensor components were determined at the RHF/6-311G** level
of theory.31
Next, the cation interaction energies with the π surfaces of
the four aromatic units were determined at the MP2/6-
311++G** level of theory, and the basis set superposition error
was corrected for with the counterpoise method.31 A cation was
placed above the centroid of the aromatic at 2.47 Å, as described
(30) Jocher, C. J.; Moore, E. G.; Xu, J.; Avedano, S.; Botta, M.; Aime, S.;
(31) Frisch, M. J.; et al. Gaussian 03, Revision C.02; Gaussian, Inc.:
Raymond, K. N. Inorg. Chem. 2007, 46, 9182–9191.
Wallingford, CT, 2004.
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in the literature32 (Figure 6B). In these calculations, a sodium
ion was used as a non-polarizable model cation, as is common
practice in these types of calculations.32,33 Since these molecules
have alternate binding sites for the cation (for example, the
amide carbonyl), the geometry was fixed with the cation above
the centroid of the previously optimized π system. In these
calculations, a purely electrostatic model was employed and,
therefore, did not account for the influence of polarizability in
the cation-π system. Nonetheless, this model has proven
suitable for comparison of cation-π interactions within an
aromatic series.32
while a Coulombic attraction provides a strong interaction
between two charged groups, the cation-π interaction creates
a specific interaction dependent upon geometric constraints. The
calculated quadrupole moments and cation-π interaction ener-
gies for the 2,3-catecholamide structures support the experi-
mental observation that, in general, the CAM-containing ligands
have stronger binding affinities with Scn than the 1,2-HOPO
ligands. This becomes most apparent when comparing the
binding trend for the apo ligands. While the tris-substituted 1,2-
HOPO ligand, with an overall 3- charge, binds weakly to Scn,
the tris-substituted CAM ligand has an overall 1- charge and
is bound much more tightly. Thus, it appears that the cation-π
interaction of the 2,3-catecholamide is more important to binding
than the Coulombic attraction between the anionic substrate and
the cationic calyx. The recognition of apo catecholate sidero-
phores is biologically relevant, since it has previously been
demonstrated that both apo bacillibactin and apo enterobactin
are bound with high affinity by Scn.15,18
Overall Recognition Mechanism. This study has focused on
the importance of the electronic character of a siderophore to
Scn recognition, demonstrating that the electrostatic nature,
including the quadrupole of aromatics and the overall charge
of the substrate, is important to selectivity. Whereas previous
studies have shown that large adducts also affect selectivity by
introducing intolerable steric clashes, here we show that even
the N-methyl substituent on the Me-3,2-HOPO unit is not
accommodated. It has been shown previously that neither the
backbone nor the metal, nor the chirality at the metal center, is
important for recognition by Scn.15,18 The features of the metal-
binding units seem to single-handedly influence whether Scn
binds a substrate.
The anisotropy of the tensor components of the four
molecules used in these calculations was calculated as Θzz
)
Qzz - 0.5(Qxx + Qyy). The quadrupole moments calculated for
the neutral molecules were within the range of values reported
for similarly functionalized aromatics.33 Both CAM molecules
were found to have more negative Θzz values than their 1,2-
HOPO counterparts. In comparing the protonated molecules to
the metal complexes, both CAM and 1,2-HOPO experienced a
more negative Θzz upon metal binding (Figure 6). Calculated
cation interaction energies with these metal-binding units
confirmed that, as Θzz becomes more negative, the binding
energy increases.
Discussion
Recognition by Coulombic Interactions. A comparison of the
dissociation constants of the FeIII complexes and the VIV
complexes provides direct evidence of the effect of substrate
charge on recognition. Previous work has shown that the Scn
recognition of [VIV(enterobactin)]2- is slightly weaker than for
18
[FeIII(enterobactin)]3-
.
The present study expands on this
One of the most remarkable features of Scn is that it can
bind a number of substrates of differing structures while
maintaining an affinity comparable to that of cognate sidero-
phore receptors, in order to act effectively as a bacteriostatic
agent. The recognition mechanism relies on interactions between
the protein and key substructures of the substrate rather than
global recognition of the entire ligand. This recognition
degeneracy may also enable the protein to participate in
moonlighting functions. Scn has been implicated in several other
physiological events unrelated to bacteremia, such as iron
trafficking, tissue development, and apoptosis.37-39 Recognition
mechanisms that broaden the specificity of Scn-ligand interac-
tions may, therefore, extend to as-yet uncharacterized endog-
enous ligands that mediate pleiotropic functions of Scn.
result, demonstrating the generality of the charge effect on Scn
recognition. Since there is a significant difference in binding
between the neutral and monoanionic complexes compared to
the diaionic and trianionic complexes, it appears that Scn favors
at least dianionic substrates for strong recognition. In addition,
FeIII and VIV complexes of the same charge (different ligands),
for example [FeIII(tren(CAM)(1,2-HOPO)2)]- and [VIV(tren-
(CAM)2(1,2-HOPO))]-, have the same binding affinity within
experimental error.
The catecholate binding unit, when compared to the hydrox-
amate, has enhanced stability with iron that increases from the
bidentate to tetradentate to hexadentate complexes. For example,
while the tris-hydroxamate desferrioxamine B has a pM value
of 26.6, the tris-catecholate enterobactin has a pM value of 35.3
(where pM ) -log[FeIII] at [L] ) 10 µmol L-1 and [FeIII] ) 1
µmol L-1),34 making it a far superior ligand for iron binding.3
Catecholate complexes of ferric ion are highly anionic due to
the dianionic bidentate unit.35 Therefore, Scn impedes iron
scavenging via catecholate siderophores by recognizing these
anionic substrates through Coulombic electrostatic binding
interactions.
Conclusion
Scn has an affinity for ferric enterobactin similar to that of
the siderophore’s bacterial membrane receptor FepA, making
it an effective competitor as part of an immune response against
bacterial iron theft. Scn also binds ferric bacillibactin with equal
affinity. Bacillibactin and enterobactin are the archetypal sid-
erophores of Gram-positive and Gram-negative bacteria, re-
spectively. Both are tripodal 2,3-catecholates based on trilactone
backbones, which provides these compounds unrivaled affinities
for ferric ion. But, because of Scn, neither siderophore
contributes to virulence.
Recognition by Cation-π Interactions. A cation-π interac-
tion has a distance dependence of 1/rn, where n < 2, similar to
the 1/r dependence in a Coulombic interaction.36 However,
(32) Mecozzi, S.; West, A. P.; Dougherty, D. A. J. Am. Chem. Soc. 1996,
118, 2307–2308.
(33) Tran, D.; Beg, S.; Clements, A.; Lewis, M. Chem. Phys. Lett. 2006,
425, 347–352.
(36) Dougherty, D. A. Science 1996, 271, 163–168.
(37) Devireddy, L. R.; Gazin, C.; Zhu, X. C.; Green, M. R. Cell 2005,
123, 1293–1305.
(34) Pecoraro, V. L.; Weitl, F. L.; Raymond, K. N. J. Am. Chem. Soc.
1981, 103, 5133–5140.
(38) Kaplan, J. Cell 2002, 111, 603–606.
(35) Karpishin, T. B.; Gebhard, M. S.; Solomon, E. I.; Raymond, K. N.
J. Am. Chem. Soc. 1991, 113, 2977–2984.
(39) Schmidt-Ott, K. M.; Mori, K.; Li, J. Y.; Kalandadze, A.; Cohen, D. J.;
Devarajan, P.; Barasch, J. J. Am. Soc. Nephrol. 2007, 18, 407–413.
9
17590 J. AM. CHEM. SOC. VOL. 130, NO. 51, 2008

Electrostatic Recognition by Siderocalin
A R T I C L E S
129.43, 129.12, 129.08, 129.06, 128.83, 128.67, 128.14, 127.70,
124.79, 123.72, 123.17, 117.11, 105.29, 53.99, 53.69, 52.19, 38.15,
37.62 ppm. (+)FABMS (MH+): calcd, 1006; obsd 1006.
On the basis of this study, we conclude that hydroxamates
do not bind to Scn, in part due to the formation of neutral ferric
complexes and the lack of aromatic binding units, the hallmark
structural unit recognized by Scn. Hydroxamates, while having
a lower affinity for iron compared to catecholate siderophores,
may be the workhorse siderophores for many organisms because
they are not impeded by Scn. Many microorganisms rely on a
dual siderophore system for iron acquisition, often including a
catecholate and a hydroxamate. While this strategy offers several
advantages, including the ability to adapt to varying degrees of
environmental stress, one reason to employ a hydroxamate
siderophore in a mammalian host is to ensure virulence in the
presence of the bacteriostatic action of the immune system.
Tren(CAM)2(1,2-HOPO) (2). Bn-tren(CAM)2(1,2-HOPO) (11)
(2.580 g, 2.565 mmol) was dissolved in 1:1 HCl:CH3COOH. The
mixture was stirred overnight and the solution evaporated to dryness.
The product was recrystallized by dissolving the crude residue in
minimum amount of CH3OH and dripped slowly into 50 mL of
stirring ether in a polypropylene tube. The slurry was then
centrifuged and the ether decanted to obtain the deprotected ligand
1
2 as a white solid. Yield: 68%. Mp: 152-155 °C. H NMR (300
MHz, DMSO): δ 9.12 (m, 3H), 7.39 (dd, J ) 6.9, 2.1 Hz, 1H),
7.33 (d, J ) 7.2 Hz, 2H), 6.94 (d, J ) 6.9 Hz, 2H), 6.70 (t, J ) 7.8
Hz, 2H), 6.59 (dd, J ) 9, 1.5 Hz, 1H), 6.40 (dd, J ) 6.9, 1.5 Hz,
1H), 3.73 (m, 6H), 3.37 (m, 6H) ppm. 13C NMR (75 MHz, DMSO-
d6): δ 170.13, 157.48, 149.31, 146.20, 137.18, 119.09, 118.16,
117.71, 115.07, 104.38, 51.51, 33.85 ppm. (+)FABMS (MH+):
calcd, 556; obsd, 556. Anal. Calcd (found) for C26H29N5O9 ·HCl ·H2O:
C, 49.64 (49.86); H, 5.13 (5.44); N, 11.13 (10.84).
Experimental Section
General. Unless otherwise noted, starting materials were ob-
tained from commercial suppliers and were used without further
purification. Flash silica gel chromatography was performed using
Merck silica gel (40-7 mesh). Microanalyses were performed by
the Microanalytical Services Laboratory, College of Chemistry,
University of California, Berkeley. Mass spectra were recorded at
the Mass Spectrometry Laboratory, College of Chemistry, Univer-
sity of California, Berkeley. Melting points were taken on a Bu¨chi
melting apparatus and are uncorrected. All 1H NMR and 13C NMR
spectra were recorded on AV-300 or AVB-400 Bruker FT
spectrometers unless otherwise noted. Tren(CAM)3 (1),23 tren(1,2-
HOPO)3 (4),24 tren(Me-3,2-HOPO)3 (7),25 Bn2-2,3-CAM-thiaz,26
Bn-1,2-HOPO-thiaz,24 Bn-Me-3,2-HOPO-thiaz,40 and [FeIIItren-
[FeIIItren(CAM)2(1,2-HOPO)]2- ([FeIII(2)]2-). Tren(CAM)2(1,2-
HOPO) (2) (24.3 mg, 0.041 mmol) was dissolved in methanol (10
mL) and degassed. Fe(acac)3 (14.5 mg, 1 equiv) was dissolved in
methanol (5 mL) and added to the ligand solution, followed by 3
equiv of KOH (0.095 M in MeOH). The solution was degassed
and stirred under N2 overnight. The iron complex solution was
condensed (not to dryness) and precipitated with ether. The purple
solid was filtered. Yield: 20.1 mg (81 %). Mp: 300 °C. (-)ESMS
(M - H-): calcd, 607; obsd, 607. Anal. Calcd (found) for
[C26H24N5O9Fe]2-2K+ ·3H2O: C, 42.29 (42.08); H, 4.10 (4.19); N,
9.48 (9.12).
27
(CAM)3]3- were prepared according to published procedures.
Tren(CAM)(1,2-HOPO)2 (3). The bis-substituted intermediate
Syntheses. The syntheses of all of the mixed ligands followed
the same process, as illustrated in Scheme 1. The synthetic
procedure of tren(CAM)2(1,2-HOPO) is fully described, as well as
1
9 was confirmed by H NMR (300 MHz, CDCl3): δ 7.42 (t-br,
2H), 7.24 (m-br, 2H), 7.12 (t, J ) 6.9 Hz, 2H), 6.42 (d, J ) 9 Hz,
2H), 6.10 (d, J ) 6.6 Hz 2H, ArH), 5.21 (s, 4H, CH2), 3.15 (m,
6H), 2.29 (m, 4H), 2.04 (m, 2H) ppm.
the synthesis of the iron complex [FeIIItren(CAM)2(1,2-HOPO)]2-
,
as representative examples for the syntheses all of the other
intermediates, ligands, and iron complexes. Complete characteriza-
tions for all new compounds are included.
1
Yield of 12 (clear oil): 59%. H NMR (300 MHz, CDCl3): δ
8.08 (t, J ) 6.3 Hz, 1H), 7.47-6.93 (m, 24H), 6.85 (t, J ) 8.1 Hz,
2H), 6.58 (d, J ) 9.3 Hz, 2H), 6.08 (d, J ) 6.6 Hz, 2H), 5.30 (s,
4H), 5.13 (s, 2H), 4.90 (s, 2H), 3.14 (m, 4H), 3.05 (m, 2H), 2.42
(m, 4H), 2.15 (m, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): δ
170.10, 160.69, 157.49, 146.22, 141.46, 137.22, 130.56, 129.43,
129.12, 129.08, 129.06, 128.83, 128.67, 128.14, 127.70, 120.05,
118.15, 117.70, 115.03, 104.38, 51.07, 33.89 ppm. (+)FABMS
(MLi+): calcd, 923; obsd, 923.
Bn-tren(CAM)2(1,2-HOPO) (11). 2,3-Benzyloxybenzoylmer-
captothiazoline (Bn2-2,3-CAM-thiaz) (1.334 g, 3.062 mmol, 2
equiv) was dissolved in chloroform (150 mL) and added dropwise
via capillary over 4 days to tris(2-aminoethyl)amine (0.229 mL,
1.531 mmol, 1 equiv) in dichloromethane (50 mL). The resulting
mixture was evaporated to dryness, and the presence of the bis-
substituted intermediate 8 was confirmed by proton NMR, at which
point the crude material was carried into the next step without
further purification. 1H NMR (300 MHz, CDCl3): δ 7.92 (t-br, 2H),
7.59-7.09 (m, 22H), 7.07 (d, J ) 8.1 Hz, 4H), 5.10 (s, 4H, CH2),
5.02 (s, 4H, CH2), 3.18 (q, J ) 6.3 Hz, 4H, CH2), 2.45 (t, J ) 6
Hz, 2H, CH2), 2.35 (t, J ) 6.6 Hz, 6H, CH2), 1.86 (s, 2H, NH2)
ppm.
1
Yield of 3 (white powder), 67%. Mp: 161-163 °C. H NMR
(300 MHz, DMSO): δ 9.15 (m, 3H), 7.40 (m, 3H), 6.95 (d, J )
6.9 Hz, 1H), 6.70 (t, J ) 7.8 Hz, 1H), 6.60 (dd, J ) 7.5, 1.8 Hz,
2H), 6.43 (dd, J ) 5.4, 1.5 Hz, 2H), 3.70 (m, 6H), 3.45 (m, 6H)
ppm. 13C NMR (75 MHz, DMSO-d6): δ 161.40, 158.19, 150.04,
146.92, 142.16, 137.92, 105.08, 51.70, 34.61 ppm. (+)FABMS
(MH+): calcd, 557; obsd, 557. Anal. Calcd (found) for
C25H28N6O9 ·HCl·H2O: C, 47.65 (47.83); H, 4.96 (5.16); N, 13.30
(12.85).
The bis-substituted intermediate 8 (0.990 g, 1.271 mmol) was
dissolved in CH2Cl2 and added to N-benzyloxypyridin-2-one-6-
mercaptothiazoline (Bn-1,2-HOPO-thiaz) (0.440 g, 1.271 mmol, 1
equiv) in CH2Cl2. The reaction mixture was stirred until the starting
material was consumed (the solution went from yellow to clear).
The reaction mixture was extracted with 1 M NaOH three times,
dried over Na2SO4, and condensed. The resulting residue was
applied to a silica column, and product was eluted with 2:98
CH3OH:CH2Cl2. The fractions containing the protected ligand 11
were combined, and the solvent was evaporated to obtain the
Yield of [FeIII(3)]- (bluish purple solid): 70 %. Mp: >300 °C.
(-)ESMS (M - H): calcd, 608; obsd, 608. Anal. Calcd (found)
for [C25H24N6O9Fe]-K+ ·2H2O·CH3OH: C, 43.65 (43.49); H, 4.50
(4.19); N, 11.75 (11.35).
Tren(CAM)2(Me-3,2-HOPO) (5). Yield of 13 (clear oil): 77%.
1H NMR (300 MHz, CDCl3): δ 7.84 (t-br, 2H), 7.79 (t-br, 1H),
7.64 (t, J ) 4.8 Hz, 2H), 7.08-7.50 (m, 29H), 6.99 (d, J ) 7.2
Hz, 1H), 6.64 (d, J ) 7.2 Hz, 1H), 5.29 (s, 2H), 5.14 (s, 4H), 5.04
(s, 4H), 3.52 (s, 3H), 3.16 (q-br, J ) 6 Hz), 3.10 (t, J ) 4.0 Hz,
2H), 2.31 (s-br, 6H) ppm. 13C NMR (300MHz, CDCl3): δ 164.8,
162.9, 159.0, 151.3, 146.2, 145.7, 136.2, 136.0, 131.8, 130.5, 128.4,
128.2, 127.8, 127.3, 127.2, 123.9, 122.5, 116.3, 104.3, 75.8, 74.2,
70.7, 52.1, 51.7, 45.7, 37.2, 36.8 ppm. (+)FABMS: m/z 1020.5
(MH+).
1
product as clear oil. Yield: 62%. H NMR (300 MHz, CDCl3): δ
7.82 (t-br, 2H), 7.65 (t-br, 1H), 7.32-6.98 (m, 32H), 6.53 (d, J )
7.8 Hz, 1H), 6.11 (d, J ) 5.1 Hz, 1H), 5.33 (s, 2H), 5.09 (s, 4H),
4.99 (s, 4H), 3.12 (m, 6H), 2.48 (t-br, 2H), 2.27 (t, J ) 6.3 Hz,
4H) ppm. 13C NMR (75 MHz, CDCl3): δ 165.92, 161.26, 158.99,
152.04, 146.94, 143.92, 138.29, 136.93, 136.80, 134.42, 130.56,
1
Yield of 5 (white powder): 73%. Mp: 115-118 °C. H NMR
(40) Xu, J.; Franklin, S. J.; Whisenhunt, D. W.; Raymond, K. N. J. Am.
Chem. Soc. 1995, 117, 7245–7246.
(400 MHz, DMSO-d6): δ 12.15 (s-br, 2H), 11.35 (s-br, 1H), 10.06
9
J. AM. CHEM. SOC. VOL. 130, NO. 51, 2008 17591

A R T I C L E S
Hoette et al.
(s-br, 1H), 9.26 (s-br, 1H), 9.01 (t-br, 2H), 8.68 (t-br, 1H), 7.27 (d,
J ) 7.6 Hz, 2H), 7.14 (d, J ) 7.2 Hz, 1H), 6.92 (d, J ) 7.6 Hz,
2H), 6.68 (t, J ) 8.0 Hz, 2H), 6.45 (d, J ) 7.2 Hz, 1H), 3.72 (m-
br, 6H), 3.46 (m 9H) ppm. 13C NMR (400 MHz, DMSO-d6): δ
172.0, 170.1, 166.1, 158.1, 149.4, 147.5, 146.2, 127.7, 119.1, 118.1,
117.7, 117.0, 115.0, 102,9, 51.6, 45.4, 36.9, 33.8, 21.1 ppm.
(+)FABMS: m/z 570.5 (MH+). Anal. Calcd (found) for
C27H31N5O9 ·2H2O (FW 605.59): C, 53.55 (53.28); H, 5.83 (5.95);
N, 11.56 (11.21).
Yield of [FeIII(5)]2- (reddish purple solid): 80%. Mp: >300 °C.
(-)FABMS: m/z 621 [(HM)-]. Anal. Calcd (found) for
K2[FeC27H26N5O9]·3H2O (FW 752.61): C, 43.09 (42.78); H, 4.29
(4.12); N, 9.31 (9.01).
µM in 5% DMSO, aqueous buffer (pH 7.4). Apo ligand solutions
were prepared analogously without the addition of a metal salt.
Structure Solution and Refinement. X-ray data sets were
collected either at the Advanced Light Source ([FeIIItren(CAM)2(1,2-
HOPO)]2- and [FeIIItren(CAM)(1,2-HOPO)2]-) or on a SMART
diffractometer ([FeIIItren(1,2-HOPO)3]0). The data sets were col-
lected, solved, and refined as previously described.41-45 Structural
overlays were calculated in MERCURY.22
[FeIIItren(CAM)2(1,2-HOPO)]2-
. Crystals of ferric tren-
(CAM)2(1,2-HOPO) were grown from a solution of complex in
DMF diffused with a mixture of diethyl ether and DMF. The
complex crystallized as pink plates. Two potassium counterions, a
DMF molecule, and four waters (three of which were disordered)
were found in the asymmetric unit.
Tren(CAM)(Me-3,2-HOPO)2 (6). Yield of 14 (clear oil): 65%.
1H NMR (400 MHz, CDCl3): δ 1.64 (s, 6H), 3.08 (s, br, 4H), 3.10
(s, br, 4H), 3.58 (s, br, 2H), 3.61 (s, br, 2H) 5.16 (s, 4H), 5.61 (s,
4H), 6.54 (d, J ) 7.2 Hz, 2H), 7.06 (d, J ) 7.2 Hz, 2H), 7.29-7.43
(m, 20H), 7.61 (d, J ) 8.4 Hz, 1H), 7.82 (d, J ) 8.4 Hz, 1H), 8.24
(m, br, 3H) ppm. (+)FABMS: m/z 946.0 (MH+). Anal. Calcd
(found) for C55H56N6O9: C, 69.90 (70.17); H, 5.97 (6.22); N, 8.89
(8.75).
[FeIIItren(CAM)(1,2-HOPO)2]-. Crystals of ferric tren(CAM)(1,2-
HOPO)2 were grown from a solution of complex in DMF diffused
with a mixture of diethyl ether and DMF. The complex crystallized
as blue rhombohedral plates. A potassium counterion and two
disordered DMF molecules were found in the asymmetric unit.
[FeIIItren(1,2-HOPO)3]0. Crystals of ferric tren(1,2-HOPO)3
were grow from a saturated solution of complex in dichloromethane
by evaporation. The complex crystallized as bluish plates. Two
disordered dichloromethane molecules were found in the asym-
metric unit.
1
Yield of 6 (off white solid): 88%. Mp: 103-105 °C. H NMR
(500 MHz, DMSO-d6): δ 3.46 (br, 12H), 3.70 (s, br, 6H), 6.44 (d,
J ) 7.0 Hz, 2H), 6.68 (t, J ) 8.0 Hz, 1H), 6.93 (d, J ) 7.5 Hz,
1H), 7.14 (d, J ) 7 Hz, 2H), 7.26 (d, J ) 7.5 Hz, 1H), 8.67 (br,
2H), 8.98 (br, 1H), 9.91 (br, 1H), 12.13 (br, 1H) ppm. 13C NMR
(500 MHz, DMSO-d6): δ 34.03, 36.90, 51.51, 102.81, 115.01,
116.88, 117.70, 118.09, 127.69, 146.18, 147.54, 149.34, 158.05,
166.08, 170.17 ppm. (+)FABMS: m/z 585 (MH+). Anal. Calcd
(found) for C27H32N6O9 ·0.5HCl ·1MeOH ·3H2O: C, 47.56 (47.89);
H, 6.06 (6.02); N, 11.88 (11.81).
Computational Methods. Computational studies were conducted
at the Molecular Graphics and Computation Facility, College of
Chemistry, University of California, Berkeley. To determine the
quadrupole moments, Θzz, the aromatic structures were geometry
optimized and characterized via frequency calculations at the RHF/
6-311G** level of theory in the Gaussian 0331 package. To
determine the aromatic-cation interaction energies, the aromatic
structures, the sodium cation, and the aromatic-cation complexes
were characterized via a frequency calculation at the MP2/6-
311++G** level of theory and the aromatic-cation interaction
energies corrected for basis set superposition error with the
counterpoise method in the Gaussian 0331 package. In the
aromatic-cation calculations, the sodium ion was fixed at a distance
of 2.47 Å above the centroid of the aromatic unit.
Yield of [FeIII(6)]- (red solid): 85%. Mp: >300 °C. (-)ESMS:
m/z 636.1 ([M
+
2H]-). Anal. Calcd (found) for
C27H28FeKN6O9 ·2HCl·0.5H2O·MeOH: C, 39.00 (39.01); H, 4.09
(3.99); N, 9.75 (9.59). UV-vis: λ ) 526 nm (ꢀ ) 3500 M-1 cm-1),
413 nm (ꢀ ) 5000 M-1 cm-1). IR: ν 1637 m, 1551 s, 1529 s,
1457 m, 1294 m, 1240 s, 1225 s, 1159 w, 743 m, 717 m cm-1
.
Fluorescence Quenching Binding Assay. Fluorescence quench-
ing of recombinant Scn was measured on either a Jobin Yvon
fluoroLOG-3 fluorimeter or a Cary Eclipse fluorescence spectro-
photometer. A 5 nm slit band-pass for excitation and a 10 nm slit
band-pass for emission were used with a high-voltage detector. An
excitation wavelength of λexc ) 281 nm was used, and emission
was collected at λem ) 340 nm. Measurements were made at a
protein concentration of 100 nM in buffered aqueous solutions, plus
32 µg/mL ubiquitin (Sigma) and 5% DMSO. Fluorescence values
were corrected for dilution upon addition of substrate. Fluorescence
data were analyzed by nonlinear regression analysis of fluorescence
response versus substrate concentration using a one-site binding
model as implemented in DYNAFIT.29 All standard binding
experiments were done at pH 7.4 using TBS aqueous buffer. Control
experiments were performed to ensure the stability of the protein
at experimental conditions, including the dilution and the addition
of DMSO and ubiquitin.
Acknowledgment. We thank Géza Szigethy for assistance with
the X-ray structure determinations, Dr. Kathy Durkin and Dr. Jamin
Krinsky for assistance with calculations, and Dr. Matt Clifton and
Dr. Anna Zawadzka for helpful discussions. This work was
supported by NIH (Grant AI117448).
Supporting Information Available: Three X-ray crystal-
lographic files in CIF format; crystallographic data table and a
fluorescence binding figure; complete ref 31. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA8074665
(41) Szigethy, G.; Xu, J.; Gorden, A. E. V.; Teat, S. J.; Shuh, D. K.;
Raymond, K. N. Eur. J. Inorg. Chem. 2008, 2143–2147.
(42) SHELXTL (V.5.10), Crystal Structure Determination Package; Bruker
Analytical X-ray Systems, Inc.: Madison, WI, 1997.
(43) XPREP (V.6.12), part of SHELXTL Crystal Structure Determination
Package; Bruker Analytical X-ray Systems, Inc.: Madison, WI, 2001.
(44) SAINT, SAX Area-Detector Integration Program, V.6.40; Bruker
Analytical X-ray Systems, Inc.: Madison, WI, 2003.
(45) SADABS, Bruker Nonius Area Detector Scaling and Absorption V.2.05;
Bruker Analytical X-ray Systems, Inc.: Madison, WI, 2003.
Substrate Solutions Preparation. Metal complex solutions were
freshly prepared in situ. An aliquot of a DMSO stock solution of
the free ligand (4 mM, 25 µL) and metal salt (1 equiv) were
combined, vigorously shaken, and diluted with TBS buffer to form
the metal complex at a concentration of 0.1 mM. The solutions
were equilibrated for 1 h and diluted to a final concentration of 6
9
17592 J. AM. CHEM. SOC. VOL. 130, NO. 51, 2008