How does single oxygen atom addition affect the properties of an Fe-Nitrile hydratase analogue? The compensatory role of the unmodified thiolate

Article abstract of DOI:10.1021/ja062706k

Nitrile hydratase (NHase) is one of a growing number of enzymes shown to contain posttranslationally modified cysteine sulfenic acids (Cys-SOH). Cysteine sulfenic acids have been shown to play diverse roles in cellular processes, including transcriptional regulation, signal transduction, and the regulation of oxygen metabolism and oxidative stress responses. The function of the cysteine sulfenic acid coordinated to the iron active site of NHase is unknown. Herein we report the first example of a sulfenate-ligated iron complex, [Fe ^III(ADIT)(ADIT-O)]⁺ (5), and compare its electronic and magnetic properties with those of structurally related complexes in which the sulfur oxidation state and protonation state have been systematically altered. Oxygen atom addition was found to decrease the unmodified thiolate Fe-S bond length and blue-shift the ligand-to-metal charge-transfer band (without loss of intensity). S K-edge X-ray absorption spectroscopy and density functional theory calculations show that, although the modified RS-O^- fragment is incapable of forming a π bond with the Fe^III center, the unmodified thiolate compensates for this loss of π bonding by increasing its covalent bond strength. The redox potential shifts only slightly (75 mV), and the magnetic properties are not affected (the S = 1/2 spin state is maintained). The coordinated sulfenate S-O bond is activated and fairly polarized (S ⁺-O^-). Addition of strong acids at low temperatures results in the reversible protonation of sulfenate-ligated 5. An X-ray structure demonstrates that Zn²⁺ binds to the sulfenate oxygen to afford [Fe^III(ADIT)(ADIT-O-ZnCl₃)] (6). The coordination of ZnCl₃- to the RS-O^- unit causes the covalent overlap with the unmodified thiolate to increase further. A possible catalytic role for the unmodified NHase thiolate, involving its ability to tune the electronics in response to protonation of the sulfenate (RS-O^-) oxygen and/or substrate binding, is discussed.

Full text of DOI:10.1021/ja062706k

Published on Web 08/05/2006
How Does Single Oxygen Atom Addition Affect the Properties
of an Fe-Nitrile Hydratase Analogue? The Compensatory
Role of the Unmodified Thiolate
Priscilla Lugo-Mas,† Abhishek Dey,‡ Liang Xu,† Steven D. Davin,† Jason Benedict,†
Werner Kaminsky,†,# Keith O. Hodgson,*,‡,§ Britt Hedman,*,§
Edward I. Solomon,*,‡ and Julie A. Kovacs*,†
Contribution from the Department of Chemistry, UniVersity of Washington, Box 351700,
Seattle, Washington 98195-1700, Department of Chemistry, Stanford UniVersity,
Stanford, California 94305, and Stanford Synchrotron Radiation Laboratory, SLAC,
Stanford UniVersity, Menlo Park, California 94025
Received April 18, 2006; E-mail: kovacs@chem.washington.edu
Abstract: Nitrile hydratase (NHase) is one of a growing number of enzymes shown to contain post-
translationally modified cysteine sulfenic acids (Cys-SOH). Cysteine sulfenic acids have been shown to
play diverse roles in cellular processes, including transcriptional regulation, signal transduction, and the
regulation of oxygen metabolism and oxidative stress responses. The function of the cysteine sulfenic acid
coordinated to the iron active site of NHase is unknown. Herein we report the first example of a sulfenate-
ligated iron complex, [FeIII(ADIT)(ADIT-O)]+ (5), and compare its electronic and magnetic properties with
those of structurally related complexes in which the sulfur oxidation state and protonation state have been
systematically altered. Oxygen atom addition was found to decrease the unmodified thiolate Fe-S bond
length and blue-shift the ligand-to-metal charge-transfer band (without loss of intensity). S K-edge X-ray
absorption spectroscopy and density functional theory calculations show that, although the modified RS-
O- fragment is incapable of forming a π bond with the FeIII center, the unmodified thiolate compensates for
this loss of π bonding by increasing its covalent bond strength. The redox potential shifts only slightly (75
1
mV), and the magnetic properties are not affected (the S ) /2 spin state is maintained). The coordinated
sulfenate S-O bond is activated and fairly polarized (S+-O-). Addition of strong acids at low temperatures
results in the reversible protonation of sulfenate-ligated 5. An X-ray structure demonstrates that Zn2+ binds
to the sulfenate oxygen to afford [FeIII(ADIT)(ADIT-O-ZnCl3)] (6). The coordination of ZnCl3- to the RS-O-
unit causes the covalent overlap with the unmodified thiolate to increase further. A possible catalytic role
for the unmodified NHase thiolate, involving its ability to “tune” the electronics in response to protonation
of the sulfenate (RS-O-) oxygen and/or substrate binding, is discussed.
Nitrile hydratases (NHases) are non-heme iron enzymes that
convert nitriles to less toxic amides cleanly, and rapidly, under
mild conditions.1-16 It is unusual for a hydrolytic metalloenzyme
to incorporate iron, as opposed to zinc,17,18 presumably because,
unlike other metal ions, Zn2+ is not complicated by redox
chemistry. Iron, on the other hand, can promote unwanted side
reactions with dioxygen (i.e., Fenton chemistry involving OH•
radicals) upon reduction to the Fe2+ oxidation state.19,20 The
† University of Washington.
‡ Stanford University.
§ Stanford Synchrotron Radiation Laboratory.
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# University of Washington staff crystallographer.
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J. AM. CHEM. SOC. 2006, 128, 11211-11221
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A R T I C L E S
Lugo-Mas et al.
density from the metal ion. Metal ion Lewis acidity would play
an important role in the mechanism of nitrile hydrolysis if nitrile
binding to the metal ion were involved. The mechanism by
which nitriles are hydrolyzed by NHase is unknown, and nitriles
are extremely resistant to hydrolysis. However, rates of hydration
have been shown to increase dramatically in the presence of a
Lewis acidic metal ion capable of coordinating nitriles.
A number of structural models for Fe-NHase and Co-
NHase containing oxidized thiolate ligands have been re-
ported,31-36 and in one case sulfur oxygenation was shown to
influence the pKa of a coordinated water molecule.37 Very few
models containing singly oxygenated sulfurs are available, and
none of these contain iron.38-40 Only one model has been
reported containing both a singly oxygenated and a doubly
oxygenated thiolate sulfur in the same molecule.40 The selective
incorporation of an odd number of oxygens is nontrivial, since
sulfenates (RS-O(H)) are more reactive than thiolates and readily
disproportionate to sulfinates (RSO2-) and thiolates. Very little
is known about how the electronic structure and, more impor-
tantly, Fe-S bonding within NHase (or model compounds
thereof) respond to oxidation and/or protonation of the coor-
dinated sulfur ligands, and how this might affect reactivity.
Herein, the synthesis and characterization of a series of
structurally related Fe-NHase model complexes is described,
in which the oxidation and protonation states of one of the
thiolates are systematically altered. Comparison of the electronic,
magnetic, redox, bonding, and reactivity properties of this series
reveals how oxo atom addition and protonation affects the metal
ion Lewis acidity. The implications of these findings with
respect to how post-translational modification might influence
function in NHase will also be discussed. Prior to the work
reported herein, there were no examples of sulfenate-ligated iron
complexes.
Figure 1. Ball-and-stick diagram of the active site of nitrile hydratase
(NHase) drawn using PDB crystallographic coordinates (2AHJ) and Spartan
(Wavefunction).
NHase iron site is, however, redox inactive and stabilized in
the 3+ oxidation state. The stabilization of Fe3+ is accomplished
by placing the iron in an electron-rich environment consisting
of five anionic ligandsstwo deprotonated peptide amides and
three cysteinates (Figure 1). Two of the three coordinated
cysteinate sulfurs are oxidized (post-translationally modified)
in NHase s one to a sulfenic acid (114Cys-S-(OH)) and the other
to a sulfinate 112CysSO2- 3,21 The third cysteinate sulfur, which
.
is trans to the inhibitor/substrate binding site and less accessible
to solvent, remains unmodified.3,21-23 34S-sensitive vibrations,
attributed to the doubly and singly oxygenated νSdO stretches,
are seen in the FT-IR spectra of NHase (from Rhodococcus
N-771)24 at 1140, 1030, and 910 cm-1, which shift slightly upon
deuteration, providing evidence that at least one of the sulfenate
or sulfinate oxygens is either protonated or H-bonded, perhaps
to the conserved Arg56 and/or Arg141 residues. One would expect
a metal-bound sulfenate oxygen (RS-(OH)) to be highly acidic.
However, sulfur K-edge X-ray absorption spectroscopy (XAS)
studies suggest that the sulfenic acid residue is protonated.25
The sulfinate (RSO2-) has been shown to remain unprotonated.25
The enzyme becomes inactive when a second oxygen atom is
added to the sulfenate 114Cys-S-(OH), implying that the singly
oxygenated sulfur plays a specific role in catalysis.26 For this
reason, the studies herein focus on the effect of single oxygen
atom addition on properties likely to influence catalytic activity.
Cysteine sulfenic acids have been shown to play diverse roles
in cellular processes, including signal transduction, oxygen
metabolism and oxidative stress response, and transcriptional
regulation.27 Catalytically active Cys-SOH are found in NADH
peroxidase,28 NADH oxidase,29 and peroxyredoxins.30 Oxygen
atom addition to a coordinated thiolate sulfur would be expected
to create a more Lewis acidic metal site by withdrawing electron
Experimental Section
General Methods. All reactions were performed under an atmo-
sphere of dinitrogen in a glovebox, or using standard Schlenk
techniques, or using a custom-made solution cell equipped with a
threaded glass connector sized to fit a dip probe. Reagents purchased
from commercial vendors were of the highest purity available and used
without further purification. Toluene, pentane, Et2O, and MeCN were
rigorously degassed and purified using solvent purification columns
housed in a custom stainless steel cabinet, dispensed via a stainless
steel Schlenk-line (GlassContour). Methanol (MeOH) and ethanol
(EtOH) were distilled from magnesium methoxide or ethoxide. 1H NMR
spectra were recorded on Bruker AV 301, Bruker AV 500, or Bruker
DRX 499 FT-NMR spectrometers and are referenced to an external
standard of tetramethylsilane (TMS) (paramagnetic compounds) or to
residual protio-solvent (diamagnetic compounds). EPR spectra were
(21) Murakami, T.; Nojiri, M.; Nakayama, H.; Odaka, M.; Yohda, M.; Dohmae,
Na.; Takio, K.; Nagamune, T.; Endo, I. Protein Sci. 2000, 9, 1024-1030.
(22) Nojiri, M.; Yohda, M.; Odaka, M.; Matsushita, Y.; Tsujimura, M.; Yoshida,
T.; Dohmae, N.; Takio, K.; Endo, I. J. Biochem. (Tokyo) 1999, 125, 696-
704.
(23) Tsujimura, M.; Dohmae, N.; Odaka, M.; Chijimatsu, M.; Takio, K.; Yohda,
M.; Hoshino, M.; Nagashima, S.; Endo, I. J. Biol. Chem. 1997, 272, 29454-
29459.
(31) Mascharak, P. K. Coord. Chem. 2002, 225, 201-214.
(32) Kobayashi, M.; Shimizu, S. Eur. J. Biochem. 1999, 261, 1-9.
(33) Boone, A. J.; Chang, C. H.; Greene, S. N.; Herz, T.; Richards, N. G. J.
Coord. Chem. ReV. 2003, 238, 291-314.
(24) Noguchi, T.; Nojiri, M.; Takei, K.; Odaka, M.; Kamiya, N. Biochemistry
2003, 42, 11642-11650.
(25) Dey, A.; Chow, M.; Taniguchi, K.; Lugo-Mas, P.; Davin, S. D.; Maeda,
M.; Kovacs, J. A.; Odaka, M.; Hedman, B.; Hodgson, K. O.; Solomon, E.
I. J. Am. Chem. Soc. 2006, 128, 533-541.
(34) Mascharak, P. K.; Harrop, T. C. Acc. Chem. Res. 2004, 37, 253-260.
(35) Grapperhaus, C. A.; Patra, A. K.; Mashuta, M. S. Inorg. Chem. 2002, 41,
1039-1041.
(26) Tsujimura, M.; Odaka, M.; Nakayama, H.; Dohmae, N.; Koshino, H.;
Asami, T.; Hoshino, M.; Takio, K.; Yoshida, S.; Maeda, M.; Endo, I. J.
Am. Chem. Soc. 2003, 125, 11532-11538.
(36) Bourles, E.; Alves de Sousa, R.; Galardon, E.; Giorgi, M.; Artaud, I. Angew.
Chem., Int. Ed. 2005, 44, 2-5.
(37) Tyler, L. A.; Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K. Inorg.
Chem. 2003, 42, 5751-5761.
(27) Claiborne, A.; Yeh, J. I.; Mallet, C.; Luba, J.; Crane, E. J.; Charrier, V.;
Parsonage, D. Biochemistry 1999, 38, 15407-15416.
(38) Heinrich, L.; Mary-Verla, A.; Li, Y.; Vaissermann, J.; Chottard, J.-C. Eur.
J. Inorg. Chem. 2001, 2203-2206.
(28) Yeh, J. I.; Claiborne, A.; Hol, W. G. I. Biochemistry 1996, 35, 9951-
9957.
(39) Galvez, C.; Ho, D. G.; Azpd, A.; Selke, M. J. Am. Chem. Soc. 2001, 123,
3381-3382.
(29) Ahmed, S. A.; Claiborne, A. J. Biol. Chem. 1989, 264, 19864-19870.
(30) Chae, H. Z.; Robison, K.; Poole, L. B.; Church, G.; Storz, G.; Rhee, S. G.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7017-7021.
(40) Kung, I. Y.; Schweitzer, D.; Shearer, J.; Taylor, W. D.; Jackson, H. L.;
Lovell, S.; Kovacs, J. A. J. Am. Chem. Soc. 2000, 122, 8299-8300.
9
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Compensatory Role of Thiolate in NHase
A R T I C L E S
recorded on a Bruker EPX CW-EPR spectrometer operating at X-band
frequency at 7 K. IR spectra were recorded on a Perkin-Elmer 1700
FT-IR spectrometer as KBr pellets. Cyclic voltammograms were
for 10 min and then stored in a freezer overnight. Filtration of the
reaction mixture afforded 7 (0.10 g, 0.18 mmol, 56%) as a midnight-
blue crystalline solid. IR (KBr pellet), ν (cm-1): 1624 (imine), 893
(SdO) (Figure S-2, Supporting Information). Electronic absorption, λmax
(ꢀ): in CH3CN, 487 (674), 608 (1350) nm. Solution magnetic moment
(298 K; MeCN): µeff ) 1.83 µB. Epc(MeCN) ) -990 mV vs SCE.
Anal. Calcd for FeC14H30Cl3N4OS2Zn: C, 29.9; H, 5.40; N, 9.97.
Found: C, 29.5; H, 5.22; N, 9.93.
Protonation of [FeIII(ADIT)(ADIT-O)](Cl)‚MeCN (5b). Addition
of 1.07 equiv of HBF4‚Et2O (1.8 µL, 0.01 mmol) to 5b (5.7 mg, 0.01
mmol) in MeCN at -40 °C causes a color change from grape-purple
to cobalt-blue, and λmax red-shifts from 575 to 622 nm in the electronic
absorption spectrum. EPR (MeOH/EtOH glass (9:1), 7 K): g1 ) 2.24,
g2 ) 2.15, g3 ) 1.97.
recorded in MeCN (100 mM Bun N(PF6) solutions) on a PAR 273
4
potentiostat utilizing a glassy carbon working electrode, a platinum
auxiliary electrode, and an SCE reference electrode. Magnetic moments
(solution state) were obtained using the Evans’ method as modified
for superconducting solenoids.41,42 Temperatures were obtained using
Van Geet’s method.43 Solid-state magnetic measurements were obtained
with polycrystalline samples in gel-caps using a Quantum Design
MPMS S5 SQUID magnetometer. Ambient temperature electronic
absorption spectra were recorded on a Hewlett-Packard model 8450
spectrometer, interfaced to an IBM personal computer (PC). Low-
temperature electronic absorption spectra were recorded using a Varian
Cary 50 spectrophotometer equipped with a fiber optic cable connected
to a “dip” ATR probe (C-technologies), with a custom-built two-neck
solution sample holder equipped with a threaded glass connector (sized
to fit the dip probe). High-temperature electronic absorption spectra
were recorded on a Hewlett-Packard model 8453 spectrometer with a
Lauda/Brinkmann circulator, model K-2/R, interfaced to an IBM PC.
N-Sulfonyloxaziridine (4)44 and thiolate-ligated [FeIII(ADIT)2]Cl (3)45
were synthesized as previously described.
Preparation of [FeIII(ADIT)(ADIT-O)](Cl)‚MeCN‚PhS(O)2NH2
(5a). To a stirred solution of [FeIII(ADIT)2]Cl (3, 0.50 g, 1.2 mmol) in
MeOH (20 mL) at -35 °C was added dropwise a pre-cooled (-35 °C)
solution of N-sulfonyloxaziridine (4) (0.43 g, 1.67 mmol) in MeOH
(15 mL). The resulting reaction mixture was allowed to stir for 10 min
and then stored in a freezer overnight. The volume of the solution was
then reduced to dryness under vacuum. The remaining solids were
dissolved in MeCN (∼10 mL) and filtered to remove insoluble
impurities. The volume of the filtrate was then reduced to ∼2 mL,
layered with 20 mL of an Et2O/pentane solution (2:1), and cooled to
-35 °C overnight to afford 5a (0.32 g, 0.51 mmol, 42%) as a grape-
purple crystalline solid. ESI-MS: calcd for [FeC14H30N4OS2]+, 390.4;
found, 390.3. Electronic absorption, λmax (ꢀ): in CH3CN, 319 (5120),
575 (1690) nm; in MeOH, 319 (3950), 574 (1220) nm; in H2O, 318
(5450), 572 (1620) nm.
Protonation of [FeIII(ADIT)2]Cl (3). Addition of 3.5 equiv of HBF4‚
Et2O (5.0 µL, 0.04 mmol) to 1 (4.2 mg, 0.01 mmol) in MeCN at -40
°C causes a color change from green to turquoise, and λmax blue-shifts
from 718 to 640 nm in the electronic absorption spectrum. EPR (MeCN/
toluene glass (1:1), 7 K): g1 ) 2.22, g2 ) 2.15, g3 ) 1.97.
XAS Sample Preparation. All three model complexes, FeIII(ADIT)2
(3), FeIII(ADIT)(ADIT-O) (5b), and FeIII(ADIT)(ADIT-O-ZnCl3) (6)
were ground into a fine powder, dispersed as thinly as possible on
sulfur-free Mylar tape in a dry, anaerobic glovebox (N2) atmosphere,
and mounted across the window of an aluminum plate. This procedure
has been verified to minimize self-absorption effects. A 6.35 µm
polypropylene film window protected the solid samples from exposure
to air during transfer from the glovebox to the experimental sample
chamber.
Data Collection and Reduction. XAS data were measured at the
Stanford Synchrotron Radiation Laboratory using the 54-pole wiggler
beam line 6-2. Details of the experimental configuration for low-energy
studies have been described in an earlier publication.46 The data
reduction and error analysis follow the same method discussed in earlier
publications except that, for pre-edge subtraction, the PYSPLINE
program (written by A. Tenderholt, Stanford University) was used
instead of the standard splining program in EXAFSPAK.47
Fitting Procedure. Pre-edge features were fit by pseudo-Voigt line
shapes (sums of Lorentzian and Gaussian functions). This line shape
is appropriate as the experimental features are expected to be a
convolution of a Lorentzian transition envelope and a Gaussian line
shape imposed by the spectrometer optics.48,49 A fixed 1:1 ratio of
Lorentzian to Gaussian contribution successfully reproduced the pre-
edge features. The rising edge was also fit with pseudo-Voigt line
shapes. Good fits reproduce the data and its second derivative using a
minimum number of peaks. The intensity of a pre-edge feature (peak
area) is the sum of the intensities of all the pseudo-Voigt peaks that
successfully fit the feature in a given fit. The reported intensity values
for the proteins are an average of all good pre-edge fits.
Computational Details. All calculations were performed on dual-
CPU Pentium Xeon 2.8 GHz workstations using the Amsterdam Density
Functional (ADF) program, version 2004.01, developed by Baerends
et al.,50,51 and with the Gaussian 03 package.52 A triple-ú Slater-type
orbital basis set (ADF basis set TZP) with a single polarization function
at the local density approximation of Vosko, Wilk, and Nusair,53 with
nonlocal gradient corrections described by Becke54 and Perdew,55 was
Preparation of Benzenesulfonamide-Free [FeIII(ADIT)(ADIT-O)]-
(Cl)‚MeCN (5b). Addition of 1.0 equiv, as opposed to 1.4 equiv,
of N-sulfonyloxaziridine (4, 0.14 g, 0.55 mmol) to 3 (0.25 g, 0.55 mmol)
under the conditions described above afforded, upon crystallization from
MeCN/pentane/Et2O (1:4:6), microcrystalline samples of benzenesul-
fonamide-free 5b (0.11 g, 0.24 mmol, 39%) which lacked the νS(dO)
2
stretches due to cocrystallized benzenesulfonamide in the infrared
spectra. IR (KBr pellet), ν (cm-1): 1631 (imine), 931 (SdO) (Figure
S-1, Supporting Information). Solution magnetic moment (303 K;
MeCN): µeff ) 2.33 µB. E1/2(MeCN) ) -945 mV vs SCE. EPR
(MeOH/EtOH glass (9:1), 7 K): g1 ) 2.20, g2 ) 2.16, g3 ) 1.98. Anal.
Calcd for FeC16H33ClN5OS2: C, 41.2; H, 7.12; N, 15.0. Found: C,
40.7; H, 6.94; N, 14.5. Sulfenate-ligated 5b does not appear to react
with nitriles (MeCN, isobutyronitrile). Addition of nitriles to 5b in the
presence of H2O, under basic (pH ) 8), acidic (pH ) 4.8), and neutral
conditions, followed by stirring for 12-72 h at elevated (60 °C),
ambient, and low (4 °C) temperatures, resulted in no reaction, as
indicated by the absence of hydrolyzed amide or amine products in
the 1H NMR of organics obtained via metal ion removal on a silica gel
column.
(46) Hedman, B.; Frank, P.; Gheller, S. F.; Roe, A. L.; Newton, W. E.; Hodgson,
K. O. J. Am. Chem. Soc. 1988, 110, 3798-3805.
Preparation of [FeIII(ADIT)(ADIT-O-ZnCl3)] (6). To a stirred
solution of 5b (0.15 g, 0.32 mmol) in CH3CN (10 mL) at -35 °C was
added dropwise a pre-cooled (-35 °C) solution of ZnCl2 (0.08 g, 0.59
mmol) in CH3CN. The resulting reaction mixture was allowed to stir
(47) Shadle, S. E.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Inorg. Chem.
1994, 33, 4235-4244.
(48) Agarwal, B. K. X-ray Spectroscopy; Springer-Verlag: Berlin, 1979; p 276.
(49) Tyson, T. A.; Roe, A. L.; Frank, P.; Hodgson, K. O.; Hedman, B. Phys.
ReV. B 1989, 39A, 6305-6315.
(50) Baerends, E. J.; Ellis, D. E.; Roos, P. Chem. Phys. 1973, 2, 41-51.
(51) te Velde, G.; Baerends, E. J. Int. J. Comput. Phys. 1992, 99, 84-98.
(52) Frisch, M. J.; et al. Gaussian 03, Revision C.02; Gaussian, Inc.: Walling-
ford, CT, 2004.
(41) Evans, D. A. J. Chem. Soc. 1959, 2003-2005.
(42) Live, D. H.; Chan, S. I. Anal. Chem. 1970, 42, 791-792.
(43) Van Geet, A. L. Anal. Chem. 1968, 40, 2227-2229.
(44) Sandrinelli, F.; Perrio, S.; Beslin, P. J. Org. Chem. 1997, 62, 8626-8627.
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(53) Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200-1211.
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9
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A R T I C L E S
Lugo-Mas et al.
Table 1. Crystal Data for
Table 2. Comparison of Selected Metrical Parameters for the
[FeIII(ADIT)(ADIT-O)](Cl)‚MeCN‚PhS(O)2NH2 (5a) and
[FeIII(ADIT)(ADIT-O-ZnCl3)] (6)
Cations of [FeIII((ADIT)2]Cl (3),
[FeIII(ADIT)(ADIT-O)]Cl‚H2NS(O)2Ph‚MeCN (5a), and
[FeIII(ADIT)(ADIT-OZnCl3)] (6)a
5a
6
3
5a
6
formula
MW (g/mol)
temp (K)
unit cella
space group
a (Å)
C22H40ClFeN6O3S3
624.08
130(2)
monoclinic
P21/c
13.5500(8)
9.9180(6)
25.0040(19)
90
117.413(2)
90
2982.9(3)
4
1.390
0.0557
C14H30FeN4O Cl3S2Zn
Fe(1)-S(1)
Fe(1)-S(2)
Fe(1)-N(1)
Fe(1)-N(2)
Fe(1)-N(3)
Fe(1)-N(4)
S(1)-O(1)
Zn-O(1)
2.200(1)
2.207(1)
2.043(3)
1.938(2)
2.059(3)
1.938(2)
2.255(2)
2.169(2)
2.043(4)
1.931 (5)
2.019(4)
1.922(5)
1.550(4)
2.255(1)
2.161(1)
2.029(3)
1.945(4)
2.022(3)
1.938(3)
1.565(3)
1.978(3)
2.223(1)
2.289(1)
2.255(1)
562.11
130(2)
orthorhombic
Pbca
15.4610(5)
14.4680(6)
20.5360(11)
90
90
90
4593.7(3)
8
1.626
b (Å)
c (Å)
R (°)
Zn-Cl(1)
â (°)
Zn-Cl(2)
Zn-Cl(3)
γ (°)
V (Å3)
Z
Fe-S(1)-O(1)
Fe-S(1)-C(4)
O(1)-S(1)-C(4)
S(1)-O(1)-Zn
S(1)-Fe-N(1)
S(2)-Fe-N(3)
109.1(2)
97.6(2)
105.9(2)
105.8(1)
99.1(2)
102.3(2)
124.1(1)
168.1(1)
169.7(1)
σ
calc (mg/m3)
99.1(1)
Rb
0.0466
0.0600
1.001
Rw
GOF
0.0940
0.999
167.9(1)
168.9(1)
168.9(2)
170.9(2)
a In all cases, Mo KR (λ ) 0.71070 Å) radiation. b R ) ∑||Fo| - |Fc||/
∑|Fo|; Rw ) [∑w(|Fo| - |Fc|)2/∑wFo2]1/2, where w-1 ) [σ2count + (0.05F2)2]/
4F2.
a Bond lengths and bond angles are given in angstroms and degrees,
respectively.
employed. The molecular orbitals were plotted using Molden version
5.1 and gOpenmol version 2.2.
SCE), and intense green in color (λmax ) 718 (2500) nm). A
low spin state is unexpected for non-heme iron, especially when
ligated by π-donors such as RS-, which tend to decrease 10Dq.57
Addition of 1.4 equiv of tert-butyl N-sulfonyloxaziridine (4) to
a MeOH solution of 3 causes the solution color to change from
green to grape-purple, and results in the addition of a single
oxygen atom to the coordination sphere, as determined by ESI-
MS. The oxidant in this reaction, 4, has been used previously
to oxygenate free thiols.44 The structure of the product of
reaction 1, [FeIII(ADIT)(ADIT-O)]+ (5), was determined by
X-ray Crystallographic Structure Determination. A purple crystal
plate, 0.48 × 0.19 × 0.05 mm, of 5a was mounted on a glass capillary
with epoxy. Data were collected at -143 °C. The crystal-to-detector
distance was set to 30 mm, and exposure time was 70 s per degree for
all data sets with a scan width of 1°. The data collection was 97.7%
complete to 23.25° in θ. A total of 7755 partial and complete reflections
were collected, covering the indices h ) -14 to 15, k ) -9 to 10, l
) -27 to 27. Of those, 4170 reflections were symmetry independent,
and the Rint ) 0.1133 indicated that the data was fair. Indexing and
unit cell refinements indicated a monoclinic P lattice in the space group
P21/c (No. 14).
A black prism, 0.24 × 0.15 × 0.10 mm, of 6 was submerged in
mineral oil, placed on a glass capillary, and mounted over a stream of
cold nitrogen gas (-143(2) °C). The crystal-to-detector distance was
set to 30 mm, and exposure time was 20 s per degree for all data sets
with a scan width of 1°. The data collection was 87.1% complete to
27.49° in θ. A total of 8692 reflections were collected, covering the
indices h ) -19 to 19, k ) -14 to 14, l ) -26 to 26. Of those, 4595
reflections were symmetry independent, and the Rint ) 0.1032 indicated
that the data was fair. Indexing and unit cell refinements indicated an
orthorhombic P lattice in the space group Pbca (No. 61).
The data for both 5a and 6 were integrated and scaled using Denzo-
hkl-SCALEPACK, and an absorption correction was performed using
SORTAV. Solution by direct methods (SIR97) produced a complete
heavy-atom phasing model consistent with the proposed structures. All
non-hydrogen atoms were refined anisotropically by full-matrix least-
squares methods, while all hydrogen atoms were then located using a
riding model. Crystal data for 5a and 6 are presented in Table 1.
Selected bond distances and angles are assembled in Table 2 (below).
X-ray crystallography and found to contain a singly oxygenated
thiolate sulfur (i.e., a sulfenate) coordinated to the metal ion.
As shown in the ORTEP of Figure 2, the iron of [FeIII(ADIT)-
(ADIT-O)]+ (5) is six-coordinate and ligated by one thiolate
sulfur (S(2)), two imine nitrogens (N(2) and N(4)), and two
amine nitrogens (N(1) and N(3)), in addition to the singly
oxygenated sulfenate sulfur (S(1)). Key bond distances and
angles of mono-thiolate/sulfenate-ligated 5a are compared with
those of di-thiolate-ligated 3 in Table 2. The sulfenate Fe-
S(1) bond length in 5a is 0.052 Å longer than both of the Fe-S
thiolate bonds in 3, showing that the interaction between the
metal and oxygenated sulfur weakens slightly as a result of
oxygenation. The unmodified iron thiolate Fe-S(2) bond, on
the other hand, is 0.038 Å shorter in 5a relative to that in the
parent unoxygenated compound 3. The Fe-N(3) and Fe-N(4)
distances in 5a also shrink (by 0.04 and 0.02 Å, respectively)
as a result of oxygen atom addition, consistent with an increase
in Lewis acidity of the metal ion (Table 2). The unmodified
thiolate Fe-S(2) bond is 0.086 Å shorter than the oxygenated
Results and Discussion
Synthesis and Structure of Singly Oxygenated [FeIII-
(ADIT)(ADIT-O)]+ (5). The thiolate-ligated iron complex,
[FeIII(ADIT)2]+ (3), described by our group prior to the NHase
crystal structure,3 models the electronic and magnetic properties
of the NHase non-heme iron site.45,56 Like the NHase active
site,7,11 complex 3 is low-spin (S ) 1/2) over a wide temperature
range, stabilized in the 3+ oxidation state (E1/2 ) -1.01 V vs
(56) Jackson, H. L.; Shoner, S. C.; Rittenberg, D.; Cowen, J. A.; Lovell, S.;
(57) Kennepohl, P.; Neese, F.; Schweitzer, D.; Jackson, H. L.; Kovacs, J. A.;
Barnhart, D.; Kovacs, J. A. Inorg. Chem. 2001, 40, 1646-1653.
Solomon, E. I. Inorg. Chem. 2005, 44, 1826-1836.
9
11214 J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006

Compensatory Role of Thiolate in NHase
A R T I C L E S
Figure 2. ORTEP diagram of the cation of [FeIII(ADIT)(ADIT-O)]Cl‚
MeCN‚PhS(O)2NH2 (5a), showing atom labeling scheme. All hydrogen
atoms have been omitted for clarity.
sulfenate Fe-S(1) bond of 5a (Table 2). This implies that the
iron-thiolate Fe-S(2) bond becomes stronger as a result of
single oxygen atom addition to the other sulfur S(1) (Figure 2).
Sulfur K-Edge X-ray Absorption Spectroscopy (XAS) and
Density Functional Theory (DFT) Calculations. To under-
stand the origin of the observed decrease in the unmodified
Fe-S bond length (Fe-S(2)) resulting from oxygen atom
addition to 3, sulfur K-edge XAS data was obtained and DFT
calculations were performed. S K-edge XAS allows one to
directly probe the covalency of metal-thiolate (M-S) bonds
and has proven useful in establishing how these properties
contribute to the function of both metalloenzymes and model
compounds.25,58-65 Although the main feature at the sulfur
K-edge is the sulfur 1sf4p transition, transitions between S1s
and any unoccupied orbital that has S3p character, including the
σ*(C-S)and σ*(Fe-S) (with eg* Fe3d character), gain intensity,
because the 1sf3p transition is also electric dipole allowed.
The intensity of these transitions is directly proportional to the
extent of S3p mixing, as reflected by the covalency parameter
(R2) in eq 2,66 where I(S1sfFe3d) is the intensity of the pre-
Figure 3. Sulfur K-edge XAS spectra for [FeIII(ADIT)2]+ (3) (in blue),
[FeIII(ADIT)(ADIT-O)]+ (5b) (in red), and [FeIII(ADIT)(ADIT-O-ZnCl3)]
(6) (in green).
(see Experimental section). As shown in Figure 3, the S K-edge
XAS spectra for [FeIII(ADIT)2]+ (3) and sulfonamide-free [FeIII-
(ADIT)(ADIT-O)]+ (5b) contain three distinct transition
envelopes s one between 2469 and 2471 eV, assigned to
S1sfFe3d pre-edge transitions (Table 3), one at 2473 eV,
assigned to a thiolate (RS-) S1sfσ*(C-S) transition, and one
at 2475 eV, assigned to sulfenate (RS-O-) S1sfσ*(C-S) and
S1sfσ*(S-O) transitions. The oxidized sulfenate S1sfσ*(C-
S) transition (at 2475 eV) is shifted 2 eV above that of the
unmodified thiolate (RSu-) ligand, reflecting the stabilization
of the RS-O- ligand’s S1s orbital. For both 3 and 5b, the
unmodified thiolate’s S1sfFe3d pre-edge region consists of five
transitions s one involving a S1sft2 (singly occupied dπ low-
spin Fe(III) orbital) transition (â-spin) and four involving S1sfe
(empty dσ low-spin Fe(III) orbitals) transitions (R- and â-spin).
The integrated intensities (I, Table 3) of the pre-edge thiolate
S1sft2 transition increase from 0.35 in 3 to 0.47 in 5b. This
corresponds to two Fe-S thiolate bonds, each with 10.5% S3p
character in 3, and one thiolate Fe-Su bond (Figure 4), with
27% S3p character in 5b. From the integrated intensities of the
e-feature, it is apparent that the σ-contribution to the iron thiolate
bonding also increases from 30.5% per Fe-S bond in 3 to 45%
per Fe-Su bond in 5b. This provides quantitative experimental
evidence for an increase in the covalency of the thiolate Fe-Su
bond (Figure 4) upon oxygen atom addition to the other sulfur
(Sm).
I(S1sfFe3d) ) R2 S1s|r|S3p
(2)
edge feature, S1s|r|S3p is the intensity of a pure ligand-based
1sf3p, transition which depends on the Zeff of the sulfur atom,
and R2 is the covalency parameter as defined by Ψ* ) (1 -
R2)1/2|Fe3d + R|S3p . Since the co-crystallized benzenesulfon-
amide of 5a complicated the S K-edge spectrum, a method of
generating the sulfonamide-free derivative 5b was developed
(58) Szilagyi, R. K.; Bryngelson, P. A.; Maroney, M. J.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 3018-3019.
(59) George, S. D.; Metz, M.; Szilagyi, R. K.; Wang, H.; Cramer, S. P.; Lu, Y.;
Tolman, W. B.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem.
Soc. 2001, 123, 5757-5767.
(60) Szilagyi, R. K.; Lim, B. S.; Glaser, T.; Holm, R. H.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 2003, 125, 9158-9169.
(61) Anxolabe´he`re-Mallart, E.; Glaser, T.; Frank, P.; Aliverti, A.; Zanetti, G.;
Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 2001,
123, 5444-5452.
(62) Dey, A.; Glaser, T.; Moura, J. J. G.; Holm, R. H.; Hedman, B.; Hodgson,
K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 16868-16878.
(63) Glaser, T.; Bertini, I.; Moura, J. J. G.; Hedman, B.; Hodgson, K. O.;
Solomon, E. I. J. Am. Chem. Soc. 2001, 123, 4859-4860.
(64) Solomon, E. I.; Hedman, B.; Hodgson, K. O.; Dey, A.; Szilagyi, R. K.
Coord. Chem. ReV. 2005, 249, 97-129.
(65) Glaser, T.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Acc. Chem. Res.
2000, 33, 859-868.
(66) Neese, F.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Inorg. Chem. 1999,
38, 4854-4860.
DFT calculations nicely reproduce the increase in the Fe-
Su bond covalency observed by S K-edge XAS, and the DFT-
optimized distances and angles (Table 4) are in good agreement
with the crystallographically determined metrical parameters
(Table 2).67 As shown in the MO diagram of Figure 5, the t2
hole (singly occupied molecular orbital, SOMO) for 3 lies in
the π*(Fe-S) dxy orbital and possesses 24% S3p character (12%
2
2
2
on each thiolate sulfur). The e set of σ*(Fe-S) dx -y and dz
orbitals possess 28% and 6% S3p character, respectively. The t2
hole for 5 also lies in the dxy orbital, which is predominantly
(67) The Fe-N bonds are an exception, since they are longer (0.03-0.06 Å)
than the crystallographically observed distances.
9
J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006 11215

A R T I C L E S
Lugo-Mas et al.
total %S3p
Table 3. Fitted Sulfur K-Edge XAS Pre-edge Positions and Intensities
t2
e
energy
I
% S3p
energy
I
% S3p
energy
I
% S3p
3
5b
6
2469.9
2469.8
2469.5
0.35 ( 0.06
0.47 ( 0.01
0.51 ( 0.01
21 ( 6
27 ( 1
30 ( 1
2470.5
2470.8
2470.5
0.84 ( 0.06
0.62 ( 0.01
0.57 ( 0.01
50 ( 6
36 ( 1
34 ( 2
2471.4
2471.6
2471.5
0.16 ( 0.05
0.15 ( 0.06
0.21 ( 0.03
11 ( 5
9 ( 5
12 ( 2
61
45
46
Table 4. DFT-Optimized Bond Lengths (Å) and Angles (deg)
Fe−Su
Fe−Sm
Fe−Nu
Fe−Nm
Fe−Nax
C−Su−Fe
C−Sm−Fe
[FeIII(ADIT)2]+ (3)
2.21
2.19
2.20
2.19
2.21
2.31
2.29
2.27
2.14
2.06
2.05
2.08
2.14
2.13
2.12
2.06
1.95
1.93
1.94
1.94
98
99
99
99
98
95
96
[FeIII(ADIT)(ADIT-O)]+ (5)
[FeIII(ADIT)(ADIT-O-ZnCl3)] (6)
[FeIII(ADIT)(ADIT-OH)]2+ (5-H+)
100
π-antibonding with respect to the unmodified Fe-Su bond
(Figure 4) and possesses 23% S3p character. The calculated
increase in Fe-Su bond covalency (from 12% per bond in 3 to
23% per bond in 5b) upon oxygen atom addition is thus in good
agreement with S K-edge data (from 10.5% per bond in 3 to
27% per bond in 5; Table 3). Only one Fe-S bond is included
in the total π-covalency of 5 because the modified RS-O-
fragment only interacts with the e-set of d orbitals and has no
interaction with the dxy π-orbital. This is because its π-symmetry
2
2
orbital is involved in the S-O σ-bond (Figure 6). The Fe dx -y
orbital of 5 is antibonding with respect the RS-O- ligand and
has significant contribution from the RS-O- oxygen. This is
because the S3p orbital, which is involved in σ*-overlap with
Figure 4. Schematic representation of [FeIII(ADIT)(ADIT-O)]+ (5), show-
ing the modified (Sm) and unmodified (Su) thiolates.
2
2
the Fe dx -y , is also involved in π*-overlap with one of the
O2p orbitals (Figure 6). This S-O π-bond does not contribute
to the S-O bond strength, since both the bonding (π) and
antibonding (π*) orbitals are occupied. However the S-O π*-
orbital has 50% S3p character (Figure 6, HOMO) and thus can
making it more Lewis acidic. Decreases in the Fe-N bond
lengths observed in the X-ray crystal structure (Table 2) support
this. One could conceivably determine the extent to which the
shift in electron density toward the oxygen is offset by an
increased π-back-donation from the unmodified thiolate sulfur
(Su; Figure 4) by examining the electronic absorption spectrum
of 5 and comparing it with that of 3. The electronic absorption
spectra of dithiolate-ligated low-spin FeIII complexes, such as
3, have been shown to be dominated by an intense πSfFe dxy
charge-transfer band.57 If the metal ion Lewis acidity were to
increase upon oxygen atom addition, then one would expect
the πSfFe dxy transition to red-shift, since the metal ion orbitals
would drop in energy toward the thiolate sulfur orbitals. The
oxygenated sulfur would not contribute to the spectrum, since
these orbitals would drop well below those of the metal ion. If
the remaining thiolate donor RSu- were to back-donate exactly
the same amount of electron density removed, then the πSfFe
2
2
reasonably overlap in a σ-fashion with the metal dx -y . The
resulting MO represents the â LUMO of 5 shown in Figure 5.
Although the S3p coefficient of this donor orbital is less than
that of a RS- donor orbital, its π*-interaction with the O2p
elevates its energy relative to that of a RS- donor orbital (by
∼0.5 eV), favoring interaction with the Fe3d orbital.
Overall, the %S3p character and its energy distribution
obtained from DFT calculations agree well with the experimental
data, illustrating the compensatory role of the unmodified
thiolate. The XAS data show, however, that the %S3p character
in the t2 hole of 3 (10.5%) is in fact less than half of that for 5b
(27%) per thiolate Fe-S bond. This overcompensation by the
unmodified thiolate is not reproduced in the DFT calculations,
which is consistent with the fact that these calculations
underestimate the shortening of the Fe-S bond (0.04 Å in X-ray
vs 0.02 Å in DFT).
-
dxy CT band would not shift. If, on the other hand, the RSu
thiolate overcompensates by π-back-bonding more electron
density than was removed, then the πSfFe dxy CT band would
blue-shift. The electronic absorption spectrum in Figure 7
demonstrates that the latter occurs. The blue-shift caused by
oxygen atom addition is similar in direction to that induced by
H-bonding between the thiolates of 3 and protic solvents;56
however, it is considerably larger in magnitude for oxygen atom
addition (3463.7 cm-1 (9.9 kcal/mol) versus H-bonding (502.4
cm-1 (1.44 kcal/mol); Table 5).56
Changes to the Electronic Structure Upon Single Oxygen
Atom Addition to [FeIII(ADIT)2]+ (3). The dramatic color
change (from green to grape-purple) associated with oxygen
atom addition to 3 suggested that sulfur oxidation caused fairly
major changes to the metal complex’s electronic structure.
Notable changes to the Fe-S bonding were detected by sulfur
K-edge XAS and X-ray crystallography, and DFT calculations
predicted this. Although π-interactions are lost between the
oxygenated sulfur (Sm; Figure 4) and the metal ion, the
unmodified thiolate (Su) more than compensates for this by
increasing its covalent bond strength. This is due in part to the
fact that the oxygen shifts electron density away from the metal,
Redox and Magnetic Properties. On the basis of the S
K-edge XAS, electronic absorption, and X-ray crystallographic
data, which show that π-back-donation from the unmodified
-
thiolate RSu increases in response to oxygen atom addition,
9
11216 J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006

Compensatory Role of Thiolate in NHase
A R T I C L E S
Figure 6. Schematic representation of the donor MOs of the oxidized RS-
O- (in blue) and unmodified RS- (in black) ligands.
Figure 5. Energy level diagrams of 3, 5, and 6 (contours not shown). Only
â unoccupied orbitals are shown. The %S3p character per S atom is given
next to the MOs. The green triangles represent low-lying unoccupied Cd
N π*-orbitals. The energies are referenced to the C-H bond energies of
the CH3 ligand substituent.
one would not expect the redox potential to shift dramatically.
One would also expect the low-spin state to be maintained, given
that the covalent Fe-S π-interaction increases upon oxygen
atom addition. As shown by the redox potential obtained from
the CV of Figure 8, the oxygenated sulfenate/thiolate ligand
stabilizes iron in the 3+ oxidation state, as does the dithiolate
ligand of 3 (E1/2 ) -1.01 vs SCE).45,56 Although there is a
slight cathodic shift upon oxygen atom addition, the magnitude
of this shift (75 mV ) 1.73 kcal/mol) is less than one might
have anticipated, had there not been a second unmodified
thiolate that compensates for the removal of electron density.
Figure 7. Comparison of the electronic absorption spectra of [FeIII-
(ADIT)2]+ (3) versus [FeIII(ADIT)(ADIT-O)]+ (5a) in MeCN solution at
298 K.
-
Table 5. Shift in the Lowest Energy LMCT Band Induced by
Chemical Modification of FesSR or FesS(R)dO Bonds
In other words, the unmodified thiolate RSu acts as an
electronic buffer in maintaining a relatively constant Lewis
acidity at the metal ion.
∆E (LMCT transition)
As shown by the inverse magnetic susceptibility versus
temperature plot in Figure 9, 5b obeys the Curie law and
chemical
LMCT shift
direction
1
modification
in cm-
in kcal/mol
1
maintains the S ) /2 spin state (µeff ) 1.88 µB) seen with 3
O-atom addition to 3
H-bonding to 3
blue-shift
blue-shift
blue-shift
red-shift
red-shift
3464
502
1697
1314
998
9.91
1.44
4.85
3.76
2.85
(µeff ) 1.85 µB)45 over the temperature range 5-300 K. The
low-temperature (7 K) EPR spectrum of 5b (Figure 10) also
H+ addition to 3
H+ addition to 5
ZnCl2 addition to 5
1
supports an S ) /2 ground-spin-state assignment. Dithiolate-
ligated 3 displays a similar, albeit slightly less rhombic, EPR
spectrum with g ) 2.17, 2.11, 1.99. Thus, single oxygen atom
addition to 3 does not significantly alter its magnetic properties.
Structural and Reactivity Properties of the Metal-Bound
Sulfenate. Singly oxygenated sulfenates are typically un-
stable68,69 unless coordinated to a transition metal.27,39,68,70-74
DFT calculations described earlier indicated that the S-O bond
of 5 is single and significantly polarized. This is supported by
the X-ray structure. The S(1)-O(1) distance in 5a (Table 2)
falls at the long end of the reported range (1.510-1.551
(69) Goto, K.; Holler, M.; Okazaki, R. J. Am. Chem. Soc. 1997, 119, 1460-
1461.
(70) Grapperhaus, C. A.; Darensbourg, M. Y. Acc. Chem. Res. 1998, 31, 451-
(68) Allison, W. S. Acc. Chem. Res. 1976, 9, 293-299.
459.
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J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006 11217

A R T I C L E S
Lugo-Mas et al.
Figure 8. Cyclic voltammogram of [FeIII(ADIT)(ADIT-O)]+ (5b) in MeCN,
with Bu4N(PF6) supporting electrolyte (0.100 M), over the range from -200
to -1400 mV vs SCE.
Figure 9. Inverse molar magnetic susceptibility 1/øm vs temperature (T)
plot for [FeIII(ADIT)(ADIT-O)]+ (5b) fit to an S ) /2 spin state.
1
Figure 10. X-band EPR spectra of [FeIII(ADIT)(ADIT-O)]+ (5a) (a) versus
protonated [FeIII(ADIT)(ADIT-OH)]2+ (b) in MeOH/EtOH glass at 7 K,
and [FeIII(ADIT)2]+ (3) (c) versus protonated [FeIII(ADIT)(ADIT-H)]2+ (d)
in MeCN/toluene glass at 7 K.
Å),39,70-74 and as shown by the angles about S(1), the
coordinated sulfenate sulfur possesses trigonal pyramidal ge-
ometry. Although organic sulfenic acids are unstable, the S-OH
bond in the few reported examples is significantly longer
(range: 1.620-1.679 Å) than the S(1)-O(1) bond in 5a,
demonstrating that the Fe-S(R)-O- bond is less activated than
a protonated RS-OH.69,75,76
Proton Addition. Addition of 1.07 equiv of HBF4 to 5b at
-40 °C in MeCN (reaction 3) causes the color to change from
grape-purple to cobalt-blue, and the πSfFe dxy CT band red-
Figure 11. Electronic absorption spectrum showing the spectral changes
which take place upon protonation of [FeIII(ADIT)(ADIT-O)]+ (5) with 1.07
equiv of HBF4 (added in 0.12 aliquots) in MeCN at -40 °C.
shifts by 47 nm (3.76 kcal/mol; Table 5) with very little loss of
intensity (Figure 11). This spectral change is reversed upon the
addition of 1.02 equiv of Et3N (Figure S-3, Supporting Informa-
tion). Warming to ambient temperature results in irreversible
bleaching of color. The thermal instability of 5-H+ is consistent
with the behavior of organic sulfenic acids.69,77-79 The direction
of the shift induced by proton addition to 5 is opposite to that
caused by oxygenation of 3, and the magnitude is much less
dramatic (Table 5). Given that the energy of the πSfFe dxy
CT band was shown to reflect the Fe-S π-bond strength (vide
supra), the relative energies of the CT band in 5-H+ versus 3
versus 5 (16 077 cm-1 (5-H+) vs 13 928 cm-1 (3) vs 17 391
cm-1 (5)) imply that, although the thiolate Fe-S(2) π-bond of
5 weakens as a result of protonation, it remains stronger than it
(71) Adzamili, I. K.; Libson, K.; Lydon, J. D.; Elder, R. C.; Deutsch, E. Inorg.
Chem. 1979, 18, 303-311.
(72) Buonomo, R. M.; Font, I.; Maguire, M. J.; Reibenspies, J. H.; Tuntulani,
T.; Darensbourg, M. Y. J. Am. Chem. Soc. 1995, 117, 963-973.
(73) Font, I.; Buonomo, R.; Reibenspies, J. H.; Darensbourg, M. Y. Inorg. Chem.
1993, 32, 5897-5898.
(74) Cornman, C. R.; Stautler, T. C.; Boyle, P. D. J. Am. Chem. Soc. 1997,
119, 5986-5987.
(77) Davis, F. A.; Billmers, R. L. J. Org. Chem. 1985, 50, 2593-2595.
(78) Bachi, M. D.; Gross, A. J. Org. Chem. 1982, 47, 897-898.
(79) Chou, T. S.; Burgtorf, J. R.; Ellis, A. L.; Lammert, S. R.; Kukolja, S. P. J.
Am. Chem. Soc. 1974, 96, 1609-1610.
(75) Ishii, A.; Komiya, K.; Nakayama, J. J. Am. Chem. Soc. 1996, 118, 12836-
12837.
(76) Tripost, R.; Belaj, F.; Nachbaur, E. Z. Naturforsch. 1993, 48b, 1212-1222.
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11218 J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006

Compensatory Role of Thiolate in NHase
A R T I C L E S
Figure 12. Electronic absorption spectrum showing the spectral changes
which take place upon protonation of of [FeIII(ADIT)2]+ (3) with 4.0 equiv
of HBF4 in MeCN at -40 °C.
Figure 13. Electronic absorption spectrum showing the spectral changes
which take place upon the addition of 1.8 equiv of ZnCl2 to [FeIII(ADIT)-
(ADIT-O)]Cl (5) in MeCN at 25 °C.
was in dithiolate-ligated 3. This would appear to rule out
protonation at the unmodified thiolate sulfur. DFT calculations
show that the unmodified Fe-Su thiolate bond becomes even
more covalent upon protonation of the RS-O- oxygen (Figure
5) and that the %S3p character increases from 54% in 5 to 63%
in 5-H+. This suggests, as one would anticipate, that the RS-
OH ligand is a poorer donor than a RS-O-.
In contrast to the behavior of 5, addition of HBF4 to dithiolate-
ligated [FeIII(ADIT)2]+ (3) at -40 °C (reaction 4) causes the
ligand-to-metal charge-transfer (LMCT) band to blue-shift (by
78 nm ) 4.85 kcal/mol; Figure 12, Table 5), as opposed to
red-shifting. The solution color changes from intense green to
Figure 14. ORTEP diagram of [FeIII(ADIT)(ADIT-O-ZnCl3)] (6), showing
the atom labeling scheme. All hydrogen atoms have been omitted for clarity.
consistent with proton addition to the oxygen of 5, as opposed
to one of the nitrogens or sulfurs.81
Addition of Lewis Acidic ZnCl2. Since protonated [FeIII-
(ADIT)(ADIT-OH)]2+ (5-H+) is thermally unstable and could
not be isolated, we decided to probe the reactivity of [FeIII-
(ADIT)(ADIT-O)]+ (5) with Lewis acids more likely to afford
a stable derivative. Addition of 1.8 equiv of ZnCl2 to [FeIII-
(ADIT)(ADIT-O)]Cl in MeCN causes the solution color to
change from grape-purple to midnight-blue and the LMCT band
to red-shift from 575 to 610 nm (Figure 13, Table 5). The
direction of this shift is identical to that observed upon proton
addition to 5, but the magnitude of this shift is smaller (1314.1
cm-1 for H+ vs 997.9 cm-1 for ZnCl2). The product of this
reaction, [FeIII(ADIT)(ADIT-O-ZnCl3)] (6), is stable at ambient
temperature and crystallized readily from MeCN (Figure 14).
The sulfur K-edge XAS spectrum of 6 (Figure 3) shows an
unmodified thiolate S1sft2 pre-edge transition at 2469.5 eV,
the integrated intensity of which is consistent with a slight
increase in the π-covalency of the Fe-Su bond (from 27% to
30% S3p character; Table 3) upon the addition of an electrophile
to the sulfenate oxygen. DFT calculations replicate this increased
covalency (Figure 5).
intense turquoise. The fact that the direction of this shift is
opposite to that seen with 5 implies that the protonation site is
different in 3 versus 5. Strong acids (HCl, HBF4) are required
to protonate both 3 and 5. Addition of weaker acids, such as
HOAc, results in no reaction. More equivalents of HBF4 are
required to completely protonate 3 (4.0 equiv; as determined
via a UV/vis monitored titration) versus 5 (1.1 equiv), however,
suggesting that the protonation site in 3 is more acidic than
that in 5. Furthermore, protonated [FeIII(ADIT)(ADIT-H)]2+ (3-
H+) can only be generated in aprotic solvents at -40 °C
(solution bleaching is observed in protic solvents), whereas
protonated [FeIII(ADIT)(ADIT-OH)]2+ (5-H+) is stable in both
protic and aprotic solvents, as long as the temperature is below
-40 °C. Again, this suggests that the protonation site is different
in 3 versus 5. As shown by the X-band EPR signal of Figure
10, the magnetic properties of 5 are not dramatically perturbed
as a result of protonation, and the S )1/2 low-spin state is
maintained. The signal does become slightly more rhombic (the
g-spread, ∆g ) gmax - gmin, increases from 0.22 to 0.27),
however, indicating that the coordination sphere has been subtly
altered. ENDOR studies aimed at locating this proton are
underway.80 X-ray absorption spectroscopic (XAS) data are most
As shown in the ORTEP diagram (Figure 14), the Lewis
acidic Zn2+ ion coordinates to the sulfenate oxygen of 6. This
lends support to the possibility that protons add to the sulfenate
-
oxygen as well, although the steric requirements for ZnCl3
(80) McNaughton, R.; Lugo-Mas, P.; Kovacs, J. A.; Hoffman, B., manuscript
in preparation.
(81) Slonkina, E.; Dey, A.; Lugo-Mas, P.; Kovacs, J. A.; Solomon, E. I.;
Hodgson, K. O.; Hedman, B., manuscript in preparation.
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J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006 11219

A R T I C L E S
Lugo-Mas et al.
Figure 16. Comparison of the iron coordination environment of 5 with
that of the NHase active site.
that the metal ion is coordinatively saturated, it has been
suggested, on the basis of the demonstrated nitrile hydrolysis
chemistry promoted by six-coordinate, sulfenate-ligated [CoIII(L-
N2SOSO)(tBuNC)2]-, that nitrile hydrolysis occurs at the NHase
sulfenate sulfur as opposed to the metal.38 The analogous doubly
oxygenated sulfinate derivative, [CoIII(L-N2SO2SO2)(tBuNC)2]-,
does not hydrolyze nitriles,38 again suggesting that reactivity is
due to the specific properties of the singly oxygenated sulfenate.
Implications Regarding NHase Function. The results herein
demonstrate that the unoxidized thiolate ligand plays the role
of an “electronic buffer” in a series of model complexes, each
of which contains at least one unmodified (i.e., unoxidized,
deprotonated) thiolate ligand. The covalency of the Fe-Su bond
increases as the other thiolate (Sm) becomes a poorer donor upon
oxidation and electrophile addition. DFT calculations reproduce
this compensating effect and also show that this effect originates
from the fact that both thiolates compete for the same unoc-
cupied or partially occupied 3d orbitals on the metal. Thus, it
seems that the modification of one thiolate donor is an efficient
way to control the interaction between the metal ion and the
other thiolate donor. As shown in Figure 16, the topology of 5
(left) matches that of the NHase active site (right) in that it has
one unmodified RS- donor and one RS-O(H) donor in the same
plane as those derived from 109Cys and 114Cys. In addition, both
sites contain a set of two strong N-σ donors. In contrast to the
complexes studied here, however, the NHase unmodified thiolate
Figure 15. Electronic absorption spectrum, showing that PEt3 (100 equiv;
220 mL of a 1 M solution in THF)) reacts with [FeIII(ADIT)(ADIT-O)]+
(5) in MeCN at 60 °C, over the course of 12 h, to afford [FeIII(ADIT)2]+
(3).
might preclude binding to the inner coordination sphere N- or
S-atoms. The Cl- counterion present in 5a binds to the Zn2+
ion of 6 to complete its tetrahedral coordination sphere. Zinc
(ZnCl3-) binding does not noticeably affect the Fe-S(1) or Fe-
S(2) bond lengths; however, it does slightly activate the S-O
bond, as indicated by its slightly longer bond length (Table 2)
and lower νSdO stretching frequency (893 cm-1 (Figure S-2,
Supporting Information) in 6 vs 930 cm-1 in 5b). As expected,
the magnetic properties are not perturbed by Zn2+ ion addition,
and the S )1/2 spin state is maintained (Figure S-5, Supporting
Information). ZnCl2 addition to [FeIII(ADIT)(ADIT-O)]+ (5) is
reversible and highly solvent-dependent. In MeCN, the equi-
librium favors neutral [FeIII(ADIT)(ADIT-O-ZnCl3)] (6), and
only 1.8 equiv of ZnCl2 is required for complete conversion.
In MeOH, on the other hand, the equilibrium favors monoca-
tionic [FeIII(ADIT)(ADIT-O)]+ (5), and 400 equiv of ZnCl2 is
required to completely convert 5 to 6. Dissolution of crystalline
samples of 6, pre-isolated from MeCN, into either H2O or
MeOH results in its complete conversion to 5.
Oxygen Atom Abstraction. The oxygen atom of 5 can be
removed, albeit under forcing conditions, via the addition of
PEt3 at elevated temperatures. This reaction was monitored by
electronic absorption spectroscopy, where [FeIII(ADIT)(ADIT-
O)]+ (5) was shown to convert to [FeIII(ADIT)2]+ (3) upon the
addition of PEt3, as shown in Figure 15. Triethylphosphine oxide
(Et3PdO) is detected in this reaction by mass spectrometry. The
reactive nature of the coordinated sulfenate in 5 fits with the
established reactivity patterns of organic sulfenates.27,68,69
Sulfenates are particularly reactive and prone to both electro-
philic and nucleophilic attack.82,83 The slow reaction times and
harsh conditions required to abstract the oxygen atom from 5
demonstrate, however, that once coordinated to Fe3+, the
sulfenate is less reactive.
(
109Cys) is positioned trans relative to the exchangeable sixth
coordination site. On the basis of the results described herein,
it seems likely that the oxidation/protonation/H-bonding state
of the iron-bound cysteine sulfenic acid provides a mechanism
for tuning the donor strength of the unmodified thiolate, which
in turn would affect the pKa, and lability of a trans-bound H2O.
Summary and Conclusions. In summary, the synthesis and
structure of the first oxidized sulfenate (RS-O-)-ligated iron
complex are described. Its redox, electronic, magnetic, reactivity,
and bonding properties were examined and compared with those
of its reduced dithiolate precursor. The unmodified thiolate
ligand (RSu-) was shown to overcompensate for the lost Fe-
Sm π-bonding and shift in electron density toward the added
oxygen atom by increasing its π-back-donation to the metal and
forming a stronger covalent Fe-Su bond. In other words, the
unmodified thiolate ligand acts as a buffer in modulating the
electron density at the metal ion. This electronic buffering effect
maintains a relatively constant redox potential, stabilizing iron
in the 3+ oxidation state, even in the presence of an oxidized
sulfenate ligand. With a hydrolytic metalloenzyme such as
Sulfenate-ligated 5 does not appear to react with nitriles
(MeCN, isobutyronitrile), even at elevated temperatures and/or
extreme pH conditions. Although this is not surprising, given
(82) Sandrinelli, F.; Perrio, S.; Averbuch-Pouchot, M.-T. Org. Lett 2002, 4,
3619-3622.
(83) Farmer, P. J.; Verpeaux, J.-N.; Amatore, C.; Darensbourg, M. Y.; Musie,
G. J. Am. Chem. Soc. 1994, 116, 9355-9356.
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11220 J. AM. CHEM. SOC. VOL. 128, NO. 34, 2006

Compensatory Role of Thiolate in NHase
A R T I C L E S
NHase, this would be important to avoid radical damage caused
by O2-induced Fenton chemistry. The unmodified cysteinate
appears to play a protectiVe role in that it prevents a potentially
redox-active metal ion from attaining a potentially damaging
(Fe2+) oxidation state. It is also possible that the unmodified
cysteinate plays a role in modulating the pKa, lability, and
nucleophilicity of the trans H2O via its compensatory response
to changes in the protonation state of the sulfenate (RS-O-)
oxygen.
ment of Energy, Office of Biological and Environmental
Research. P.L.-M. gratefully acknowledges support by an NIH
pre-doctoral minority fellowship (F31 GM73583-01). We thank
Daniel Gamelin for helpful discussion, and Dan Patel, Nick
Norberg, Jim Roe, and Terry Kitagawa for experimental
assistance.
Supporting Information Available: Crystallographic data, in
CIF format, for 5a and 6; IR spectra of [FeIII(ADIT)(ADIT-
O)](Cl)‚MeCN (5b) and [FeIII(ADIT)(ADIT-O-ZnCl3)] (6),
magnetic data for 6, titration monitored by UV/vis, showing
that [FeIII(ADIT)(ADIT-OH)]2+ can be reversibly deprotonated,
all optimized coordinates and MO diagram of (ADIT-OH) ligand
systems, and the complete ref 52. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This research was supported by NIH
Grants GM45881 (J.A.K.), NIH-GM 40392 (E.I.S.), and RR-
01209 (K.O.H.). SSRL operations are supported by the Depart-
ment of Energy, Office of Basic Energy Sciences. The SSRL
Structural Molecular Biology Program is supported by the
National Institutes of Health, National Center for Research
Resources, Biomedical Technology Program, and the Depart-
JA062706K
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