A synthetic analogue of the active site of Fe-containing nitrile hydratase with carboxamido N and thiolato S as donors: Synthesis, structure, and reactivities

Article abstract of DOI:10.1021/ja001253v

As part of our work on models of the iron(III) site of Fe-containing nitrile hydratase, a designed ligand PyPSH₄ with two carboxamide and two thiolate donor groups has been synthesized. Reaction of (Et₄N)[FeCl₄] with the d

Full text of DOI:10.1021/ja001253v

J. Am. Chem. Soc. 2001, 123, 3247-3259
3247
A Synthetic Analogue of the Active Site of Fe-Containing Nitrile
Hydratase with Carboxamido N and Thiolato S as Donors: Synthesis,
Structure, and Reactivities
Juan C. Noveron, Marilyn M. Olmstead, and Pradip K. Mascharak*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of California,
Santa Cruz, California 95064, and, Department of Chemistry, UniVersity of California,
DaVis, California 95616
ReceiVed April 10, 2000. ReVised Manuscript ReceiVed September 4, 2000
Abstract: As part of our work on models of the iron(III) site of Fe-containing nitrile hydratase, a designed
ligand PyPSH4 with two carboxamide and two thiolate donor groups has been synthesized. Reaction of (Et4N)-
[FeCl4] with the deprotonated form of the ligand in DMF affords the mononuclear iron(III) complex (Et4N)-
[FeIII(PyPS)] (1) in high yield. The iron(III) center is in a trigonal bipyramidal geometry with two deprotonated
carboxamido nitrogens, one pyridine nitrogen, and two thiolato sulfurs as donors. Complex 1 is stable in
water and binds a variety of Lewis bases at the sixth site at low temperature to afford green solutions with a
band around 700 nm. The iron(III) centers in these six-coordinate species are low-spin and exhibit EPR spectra
much like the enzyme. The pKa of the water molecule in [FeIII(PyPS)(H2O)]- is 6.3 ( 0.4. The iron(III) site
in 1 with ligated carboxamido nitrogens and thiolato sulfurs does not show any affinity toward nitriles. It thus
appears that at physiological pH, a metal-bound hydroxide promotes hydration of nitriles nested in close
proximity of the iron center in the enzyme. Redox measurements demonstrate that the carboxamido nitrogens
prefer Fe(III) to Fe(II) centers. This fact explains the absence of any redox behavior at the iron site in nitrile
hydratase. Upon exposure to limited amount of dioxygen, 1 is converted to the bis-sulfinic species. The structure
of the more stable O-bonded sulfinato complex (Et4N)[FeIII(PyP{SO2}2)] (2) has been determined. Six-
coordinated low-spin cyanide adducts of the S-bonded and the O-bonded sulfinato complexes, namely, Na2-
[FeIII(PyP{SO2}2)(CN)] (4) and (Et4N)2[FeIII(PyP{SO2}2)(CN)] (5), afford green solutions in water and other
solvents. The iron(II) complex (Et4N)2[FeII(PyPS)] (3) has also been isolated and structurally characterized.
Introduction
environmental remediation of nitriles.15-17 NHases consist of
R and â subunits and exist as Râ 46 kDa heterodimers or (Râ)2
92 kDa tetramers. The R subunit contains either a low-spin (S
) 1/2) non-heme iron(III) center18,19 or a non-corrinoid cobalt-
(III) center,20-22 depending on the bacterial source, and these
M(III) centers have been shown to be the active site for
hydrolysis of nitriles. Unlike other non-heme iron centers in
biology,23,24 the iron(III) center of the NHase does not participate
in redox reaction(s) and acts as a Lewis acid. The iron- and
cobalt-containing NHases (Fe-NHase and Co-NHase, here-
after) possess almost identical sequence homology at the loci
of their active sites, and they hydrolyze nitriles at similar rates.
These observations have led to the suggestion that the structures
The microbial enzyme Nitrile Hydratase (NHase) catalyzes
the hydrolysis of a wide variety of nitriles (RCN) to the
corresponding amides (RCONH2).1-5 In recent years, NHases
from several microorganisms (and related mutants) have been
employed as biocatalysts in industrial production of acrylamide
in kiloton quantities6-10 and in enantioselective syntheses of
other carboxamides.11-14 NHases have also been used in
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10.1021/ja001253v CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/04/2001

3248 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
NoVeron et al.
The donor set of the iron(III) site of the Fe-NHases, namely,
carboxamido N, sulfenato (RSO-) and sulfinato (RSO2-), is
unprecedented. Coordination of deprotonated carboxamido N
has only been discovered recently in the nitrogenase P cluster35
while coordination by the modified cysteines residues (RSO-,
RSO2-) through the sulfur atoms has not been observed in any
other metalloenzyme. Thus far, the mechanism underlying the
biogenesis of the modified cysteines remains elusive. Proposed
suggestions for such posttranslational modification of cysteines
include (a) involvement of oxidative enzymes such as cysteine
dioxygenases,36,37 (b) reaction with peroxynitrite anion derived
from NO,38-40 and (c) generation of iron-peroxides from the
reaction between oxygen and the iron center of NHase such as
the case with the biogenesis of topa quinone.41,42 The effect(s)
of the modified Cys-S centers on the overall properties of the
iron(III) site is an open question in the chemistry of the NHase
at this time.
Figure 1. Structure of the NO-bound iron site of Rhodococcus sp.
N-771 (ref 29).
There are several postulated mechanisms for nitrile hydrolysis
by NHases.3,28 The first one involves displacement of the metal-
bound water molecule by the nitrile substrate, followed by
hydrolysis of the transient metal-bound nitrile species with the
eventual rearrangement and release of the amide product (an
inner-sphere mechanism). The second mechanism postulates a
direct attack of the metal-bound hydroxide on the nitrile group
of the substrate with eventual rearrangement to the correspond-
ing amide product (an outer-sphere mechanism). The third
mechanism involves activation of a water molecule in close
proximity to the metal center via deprotonation by the metal-
bound hydroxide. The hydroxide ion generated in such process
ultimately causes hydrolysis of nitriles (also an outer-sphere
mechanism). To our knowledge, there has not been any report
that confirms any one of these mechanisms of nitrile hydrolysis
by NHases.
The extent to which the unusual coordination structure of
the iron site of the Fe-NHase dictates its ability to hydrolyze
nitriles is an important question. To address this question, one
needs to determine the intrinsic properties of the iron(III) site
via studies on suitably designed model complexes that mimic
the coordination structure of the iron(III) center of the Fe-
NHases. In such pursuit, Kovacs and co-workers have synthe-
sized a five-coordinated iron(III) complex with N3S2 donor set,
[FeIIIS2Me2N3(Pr,Pr)]PF6 (structure i), that mimics some features
of the active site of the Fe-NHase.43,44 For example,
[FeIIIS2Me2N3(Pr,Pr)]+ reacts with NO to form a diamagnetic
photolabile iron-nitrosyl complex. This result is noteworthy
since the reactivity of the model complex demonstrates that NO
can bind to an iron(III) site and the resultant species could be
photolabile. The iron(III) center of [FeIIIS2Me2N3(Pr,Pr)]+ also
binds azide (N3-), a competetive inhibitor of NHase, in a
reversible manner. This model is, however, not a good structural
of the metal sites in the two types of NHases as well as their
mode of catalysis are similar in nature. There is, however, an
interesting difference between the two varieties of NHases; the
activity of the Fe-NHases is regulated by photoreversible
binding of exogenous nitric oxide (NO) to the iron center.25-27
Such a regulatory role of NO in a non-heme iron enzyme is
quite unique and is not observed with the Co-NHases.
Recent crystallographic studies on the Fe-NHase from
Rhodococcus sp. R312 have revealed that the Fe(III) center is
ligated to two deprotonated carboxamido nitrogens and three
sulfur donors situated in the highly conserved -C-S-L-C-S-C-
motif of the R subunit.28 A more precise (higher resolution)
structure of the dark-adapted Fe-NHase isolated from another
Rhodococcus species (N771)29 further reveals that two of the
ligated Cys-S residues are posttranslationally modified to the
sulfinic(Cys-SO2) and sulfenic (Cys-SO) forms and a molecule
NO is coordinated at the solvent-exposed site of the metal
(Figure 1). The iron-bound NO molecule is released upon
illumination, and the active form of the NHase is generated.25-27
The N2S3 protein donor set around the iron(III) center is
conserved in the active form of the enzyme.18,19,30-33 Results
of spectroscopic studies on the active form of the Fe-NHase
from Rhodococcus sp. R312 also suggest that a water molecule
occupies the site of NO, hence conferring a six-coordinate iron-
(III) site with pseudooctahedral geometry. Recent electron-
nuclear double resonance (ENDOR) studies34 however indicate
that the iron(III) site may contain a coordinated hydroxide at
the optimum pH (pH 7.3) of the enzyme.34
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I. FEBS Lett. 1995, 358, 9.
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Nagashima, S.; Yohda, M.; Nagamune, T.; Inoue, Y.; Endo, I. J. Am. Chem.
Soc. 1997, 119, 3785.
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Lindqvist, Y. Structure 1997, 5, 691.
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K.; Odaka, M.; Yohda, M.; Kamiya, N.; Endo, I. Nat. Struct. Biol. 1998, 5,
347.
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Johnson, M. K.; Rees, D. C. Biochemistry 1997, 36, 1181.
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K. Biochem. Biophys. Res. Commun. 1990, 168, 473.
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Duong, D. J.; Kindt, J. T.; Nelson, M. J. Biochemistry 1996, 35, 10078.
(31) Brennan, B. A.; Cummings, J. G.; Chase, B.; Turner, I. M., Jr.;
Nelson, M. J. Biochemistry 1996, 35, 10068.
(32) Jin, H.; Turner, I. M., Jr.; Nelson, M. J.; Gurbiel, R. J.; Doan, P.
E.; Hoffman, B. M. J. Am. Chem. Soc. 1993, 115, 5290.
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J. A.; Cummings, J. G.; Nelson, M. J. J. Am. Chem. Soc. 1998, 120, 9237.
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Soc. 1996, 118, 7014.
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(40) DeMaster, E. G.; Quast, B. J.; Redfern, B.; Nagasawa, H. T.
Biochemistry 1995, 34, 11494.
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Loehr, J. J. Biol. Chem. 1996, 271, 4718.
(42) Cai, D.; Klinman, J. P. Biochemistry 1994, 33, 7647.
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A.; Kovacs, J. A. J. Am. Chem. Soc. 1998, 120, 5691.
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A. J. Am. Chem. Soc. 1998, 120, 10996.

Model of the Iron(III) Site of Nitrile Hydratase
J. Am. Chem. Soc., Vol. 123, No. 14, 2001 3249
mimic since it does not contain any deprotonated carboxamido
N in the coordination sphere of iron. Very recently, Chottard
and co-workers have reported an iron(III) complex that contains
two carboxamido N and three thiolato S donor sets.45 In this
As part of our continuing effort toward elucidation of the
intrinsic chemistry of the M(III) sites of the NHases, we have
recently synthesized a designed pentadentate ligand N,N′-bis-
(2-mercaptophenyl)pyridine-2,6-dicarboxamide (PyPSH4, struc-
ture vi, H’s are the dissociable carboxamide and thiol protons)
that contains two carboxamide and two thiol groups. In a recent
communication, we have reported the syntheses and structures
of two cobalt (III) complexes of PyPSH4.51 One of these
complexes, namely, [CoIII(PyPS)(CN)]2-, gives rise to [CoIII-
(PyPS)(OH)]2- in basic solution. This cobalt(III) species with
N3S2O coordination catalyzes a variety of nitriles under mild
conditions (T < 50 °C, pH 9). This success prompted us to
examine its coordination chemistry with iron. In this paper we
report the synthesis, structure, and reactivity of the iron(III)
complex with PyPSH4, namely (Et4N)[FeIII(PyPS)] (1). Complex
model complex ((Et4N)2[FeIII(L-O2)], structure ii), one of the
thiolato S donors is in the sulfinic form and is bonded to iron
through one of the oxygen atoms of the -SO2 unit. Although
this model is a very close structural analogue of the iron site in
3
Fe-NHase, its physical parameters (S ) /2 and λmax ) 425
nm) are quite different from those of the iron site of the enzyme
1
(S ) /2, λmax ) 700 nm). This is surprising in view of the
structural similarities that exist between the biological iron(III)
site and this model complex. In a recent article, Artaud and
co-workers have reported an iron(III) complex (Et4N)2[FeIII-
(N2S2)Cl] (structure iii) which contains two carboxamido N and
two thiolato S donors around the iron center.46 The spectroscopic
3
parameters for this model complex (S ) /2, λmax ) 470 nm)
are also very different from those of Fe-NHase. Clearly, more
model studies are required to establish the underlying principles
that correlate the structural features with spectroscopic param-
eters and reactivities.
For some time, we have been involved in syntheses of model
complexes that mimic the structural features of the Fe- and
Co-NHases.47-51 In such pursuit, we have reported for the first
time the synthesis, structure, and redox properties of iron(III)
and cobalt(III) complexes with ligands containing carboxamido
N and thiolato S donors.48,50 The redox properties of the low-
1
spin (S ) /2) complex (Et4N)[FeIII(PyPepS)2]48 (structure iv)
demonstrate that ligation of carboxamido N to iron provides
significant stabilization to the +3 oxidation state. This could
explain the stability of the iron(III) site (and lack of redox
activity) in Fe-NHase. Interestingly, both [FeIII(PyPepS)2]- and
[CoIII(PyPepS)2]- can readily be converted to the corresponding
sulfinato species by reaction with H2O2 or O2, and the sulfinato
species [FeIII(PyPepSO2)2]- (structure v) exhibits an electronic
absorption spectrum in water which is practically identical to
the absorption spectrum of the Fe-NHase.49,50 We have also
shown that such oxidation can only take place when carboxa-
mido nitrogens are present in the coordination sphere of the
metal centers.47 It is therefore evident that studies on model
complexes such as ours (and those from others) do provide
valuable insight into the intrinsic properties of the biological
M(III) sites in NHases.
1 contains a coordinatively unsaturated iron(III) center in a N3S2
coordination sphere. The iron center reversibly binds a variety
of Lewis bases such as pyridine, N-methylimidazole, cyanide,
thiolates, methanol, and water in a solvent- and temperature-
dependent fashion, and such binding modulates the spin state
of the iron(III) center. We report here for the first time, the pKa
of a water molecule bound to an iron(III) center with carboxa-
mido N and thiolato S donors. We also report that complex 1
reacts with dioxygen at room temperature to afford the corre-
sponding sulfinato species which exists as S-bonded and
O-bonded forms. The structure of the O-bonded sulfinato
complex (Et4N)[FeIII(PyP{SO2}2]-(2) is reported in this paper.
In addition, we have included the synthesis and structure of the
iron(II) complex (Et4N)2[FeII(PyPS)] (3). And finally, the
spectroscopic properties of the various iron complexes have been
compared with those of the iron site in Fe-NHase to assess
the success of this modeling work.
(45) Heinrich, L.; Li, Y.; Vaussermann, J.; Chottard, G.; Chottard, J. C.
Angew. Chem., Int. Ed. 1999, 38, 3526.
(46) Artaud, I.; Chatel, S.; Chauvin, A. S.; Bonnet, D.; Kopf, M. A.;
Leduc, P. Coord. Chem. ReV. 1999, 190-192, 577.
(47) Noveron, J. C.; Herradora, R.; Olmstead, M. M.; Mascharak, P. K.
Inorg. Chim. Acta 1999, 285, 269.
(48) Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K. Inorg. Chem.
1998, 37, 1138.
(49) Tyler, L. A.; Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K.
Inorg. Chem. 1999, 38, 616.
(50) Tyler, L. A.; Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K.
Inorg. Chem. 2000, 39, 357.
Experimental Section
Preparation of Compounds. 2,6-Pyridinedicarbonyl dichloride,
2-aminothiophenol, sodium hydride, triethylamine, triphenylmethanol,
triethylsilane, tetraethylammonium cyanide, and trifluoroacetic acid
were procured from Aldrich Chemical Co. and used without further
purification. Small amounts (5 mL) of 98% hydrogen peroxide were
prepared from 30% aqueous solution of H2O2 (Fisher Scientific) by
following the standard procedure.52 All of the solvents were purified
by standard procedures. Standard Schlenk techniques were used during
(51) Noveron, J. C.; Olmstead, M. M.; Mascharak, P. K. J. Am. Chem.
Soc. 1999, 121, 3553.

3250 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
NoVeron et al.
all syntheses to avoid exposure to dioxygen. Elemental analyses were
performed by Atlantic Microlab Inc.
a red-brown color developed. The solution was then filtered, and the
filtrate was concentrated to ∼10 mL. Diffusion of diethyl ether to this
solution afforded large red crystals after 48 h. Yield: 0.265 g (42%).
Anal. Calcd for C27H31N4S2O6Fe: C, 51.68; H, 4.98; N, 8.93. Found:
C, 51.85; H, 4.68; N, 8.60. Selected IR bands (KBr pellet, cm-1) 2966
(m), 1618 (vs, νCO), 1558 (vs), 1568 (vs), 1366 (m), 1263 (m), 1158
(m), 1070 (s, νSO2), 1026 (m), 804 (s). Absorption spectrum in DMF
(λmax, nm (ꢀ M-1 cm-1)) 480 (3400), 360 (5500) nm.
Trytilated Aminothiophenol. To a solution of triphenylmethanol
(15.0 g, 57.6 mmol) in 100 mL of trifluoroacetic acid (TFA) was added
a solution of 2-aminothiophenol (7.21 g, 57.6 mmol) in 10 mL of
methylene chloride. The reaction was stirred for 1 h, and then the TFA
was removed by vacuum distillation. Next, the dry residue was added
to a saturated solution of aqueous NaHCO3 and allowed to stir for 1 h.
The mixture was then extracted with 150 mL of chloroform, and the
organic layer was collected and dried with anhydrous MgSO4. The
solution was filtrated, and the chloroform was removed by vacuum
distillation. The solid residue thus obtained was recrystallized from
(Et4N)2[FeII(PyPS)] (3). A batch of 0.101 g (4.22 mmol) of NaH
was added to a cold (4 °C) solution of PyPSH4 (0.400 g, 1.05 mmol)
in 15 mL of degassed DMF, and the mixture was stirred for 15 min. A
solution of (NEt4)2[FeCl4] (0.345 g, 1.05 mmol) in 5 mL of DMF was
then added to it dropwise, and the mixture was stirred for 1 h at room
temperature during which a brown color developed. The reaction
mixture was concentrated to ∼5 mL, and 10 mL of degassed acetonitrile
was added to it. Diffusion of diethyl ether to the resulting solution
afforded large blocks of 3 after 48 h. Yield: 0.265 g (65%). Anal.
Calcd for C35H51N5S2O2Fe: C, 60.59; H, 7.41; N, 10.09. Found: C,
60.85; H, 7.70; N, 10.15. Selected IR bands (KBr pellet, cm-1) 2976
(m), 1603 (m) 1572 (vs, νCO), 1558 (vs), 1450 (s), 1364 (m), 1260
(m), 1172 (m), 1056 (m), 1028 (m), 1000 (m), 772 (s), 757 (s).
Absorption spectrum in DMF (λmax, nm (ꢀ M-1 cm-1)) 680 (sh), 330
(6800).
Na2[FeIII(PyP{SO2}2)(CN)] (4). A solution of 55 mg (0.35 mmol)
of (Et4N)(CN) in 1 mL of acetonitrile was slowly added to a slurry of
200 mg (0.35 mmol) of (Et4N)[FeIII(PyPS)] in 10 mL of acetonitrile.
The reaction mixture turned homogeneous almost immediately. It was
then cooled to -40 °C in a slush bath, and a batch of 150 µL (6.0
mmol) of freshly prepared 98% H2O2 was added. The bright green
mixture was stirred for 30 min at -40 °C. Addition of a solution of
108 mg (0.80 mmol) of NaClO4 in 1 mL of acetonitrile to this reaction
mixture afforded a green precipitate. It was filtered, washed three times
with acetonitrile, and dried under vacuo. Yield: 150 mg (75%). Anal.
Calcd for C20H11N4S2O6Na2Fe: C, 42.19; H, 1.95; N, 9.84. Found: C,
42.30; H, 1.81; N, 9.60. Selected IR bands (KBr pellet, cm-1) 2208
(m, νCN), 1626 (vs, νCO), 1460, 1438, 1363, 1219, 1186 (s, νSO2), 1137,
762, 617. Absorption spectrum in DMF (λmax, nm (ꢀ M-1 cm-1)) 640
(1350), 470 (sh, 1600).
(Et4N)2[FeIII(PyP{SO2}2)(CN)] (5). To a solution of 80 mg (0.14
mmol) of (Et4N)[FeIII(PyP{SO2}2)] in 40 mL of acetone was added
with stirring a solution of (Et4N)(CN) (22 mg, 0.14 mmol) in 1 mL of
acetonitrile. The color of the initial red solution turned green. A green
precipitate formed within 10 min. The solid was filtered, washed twice
with acetone, and dried in vacuo. Yield: 36 mg (40%). Anal. Calcd
for C36H51N6S2O6Fe: C, 55.17; H, 6.56; N, 10.72. Found: C, 54.95;
H, 6.42; N, 10.91. Selected IR bands (KBr pellet, cm-1) 2117 (m, νCN),
1604 (vs, νCO), 1460, 1456, 1356, 1262, 1030 (s, νSO2), 799. Absorption
spectrum in DMF (λmax, nm (ꢀ M-1 cm-1)) 660 (1300), 425 (sh, 1800).
Studies on Binding of Donors at the Sixth Site of Iron in 1. (a)
Water. Binding studies were performed with freshly prepared solutions
of 1 in degassed acetone:water (30:70) mixture. Changes in the
electronic absorption spectrum due to binding of water at low
temperatures were monitored with the aid of a custom-designed low-
temperature optical Dewar filled with the appropriate cooling bath.
Approximately two minutes were necessary for the temperature to
equilibrate in each case. An OMEGA temperature probe was used to
monitor the temperature. The spectra were recorded with a diode-array
Polytec PI UV-vis instrument. The pH of the solution mixtures was
measured with a Beckman 200 pH meter. In a typical experiment, an
aliquot (50 µL) of a 0.18 M solution of 1 in DMF was added to a
Tris-buffer solution (2 mM) in acetone:water (30:70) mixture with its
pH previously determined at 25 °C and -30 °C. The solution was then
placed in a 10 mm quartz cell that fits on the custom-designed Dewar
apparatus, and the absorption spectrum was recorded until the sample
temperature reached -30 °C (temperature at which no further change
in the spectrum was observed). Since the samples started freezing
around -35 °C, the spectra were obtained at -30 °C. The spectra of
the water adduct [FeIII(PyPS)(H2O)]- at different pH values were used
to determine the pKa of the water bound to the iron(III) center. That
the buffer components do not coordinate to the iron(III) center of 1 in
these experiments is evident from the fact that the electronic absorption
1
methanol and dried under vacuum for 24 h. Yield: 16.5 g (78%). H
NMR spectrum (CDCl3, 250 MHz) δ from TMS: 3.63 (s, NH2), 6.46
(dd, 2H), 7.04 (t, 2H), 7.37 (dd, 6H, Try CH’s), 7.25 (m, 9H, Try CH’s).
13C NMR spectrum (CDCl3, 62.5 MHz) δ 70.8, 105.2, 115.0, 116.2,
117.9, 126.6, 127.5, 129.9, 137.8, 144.3, 151.3. Selected IR bands (KBr
pellet, cm-1) 3462 (m), 3368 (m), 3053 (m), 1606 (s), 1477 (s), 1444
(s), 1310 (m), 743 (s), 701 (s).
PyPS-Try. A solution of trytilated aminothiophenol (7.21 g, 19.6
mmol) and triethylamine (3.47 g, 34.3 mmol) in 40 mL of chloroform
was added dropwise to a solution of 2,6-pyridinedicarbonyl dichloride
(2.0 g, 9.81 mmol) and triethylamine (3.47 g, 34.3 mmol) in 40 mL of
chloroform. The mixture was allowed to react for 2 days at room
temperature during which a portion of PyPS-Try (protected ligand vi)
precipitated out of solution. The mixture was concentrated to 15 mL,
and the precipitate was vacuum filtrated and washed with cold methanol.
1
Yield: 6.02 g (71%). H NMR spectrum (CDCl3, 250 MHz) δ from
TMS: 6.93 (t, 6H), 6.75 (t, 12H), 7.09 (d, 12H), 7.52 (dd, 4H), 8.03
(t, 1H) 8.21 (d, 2H), 8.67 (d, 2H), 10.66 (s, 2H, amide NH). 13C NMR
spectrum (CDCl3, 62.5 MHz) δ 71.8, 119.7, 121.5, 123.8, 124.7, 126.8,
127.3, 129.5, 131.4, 138.3, 142.4, 143.2, 148.8, 161.0, 179.9. Selected
IR bands (KBr pellet, cm-1) 3300 (m, NH), 3015 (m), 1683 (vs, νCO),
1575 (vs), 1438 (s), 753 (s), 700 (s).
PyPSH4 (vi). A batch of triethylsilane (2.68 g, 23.1 mmol) was added
to a solution of PyPS-Try (5 g, 5.77 mmol) in 13 mL of TFA and 6
mL of dichloromethane, and the mixture was stirred for 15 min. The
reaction mixture was then concentrated to half the original volume,
and the resulting precipitate was filtered. Following removal of TFA
and other volatiles in vacuo, the residue was washed three times with
diethyl ether and collected by filtration. Yield: 2.05 g (94%). 1H NMR
spectrum (CDCl3, 250 MHz) δ from TMS: 3.39 (s, 2H, SH), 7.12 (t,
2H), 7.36 (t, 2H), 7.56 (d, 2H), 8.19 (t, 1H) 8.36 (d, 2H), 8.53 (d, 2H),
10.49 (s, 2H, amide NH). 13C NMR spectrum (CDCl3, 62.5 MHz) δ
118.9, 122.0, 125.2, 125.8, 129.0, 134.6, 137.7, 139.7, 148.9, 161.2.
Selected IR bands (KBr pellet, cm-1) 3300 (m, NH), 2525 (w, SH),
1684 (vs, ν CO), 1580 (vs), 1522 (s), 1445 (m), 1311 (m), 749 (m).
(Et4N)[FeIII(PyPS)] (1). A batch of 0.101 g (4.22 mmol) of NaH
was slowly added to a cold (4 °C) solution of PyPSH4 (0.400 g, 1.05
mmol) in 15 mL of degassed DMF, and the mixture was stirred for 15
min. Next, the light orange solution was chilled to -40 °C, and a
solution of (Et4N)[FeCl4] (0.345 g, 1.05 mmol) in 5 mL of DMF was
slowly added to it. The mixture was allowed to stir for 1 h at room
temperature during which a blue-green color developed. The reaction
mixture was concentrated to ∼10 mL, and a batch of 10 mL of degassed
acetonitrile was added. Slow diffusion of diethyl ether to this solution
afforded large blocks of 1 after 48 h. The crystalline product was washed
with methanol and vacuum-dried. Yield: 0.265 g (45%). Anal. Calcd
for C27H31N4S2O2Fe: C, 57.55; H, 5.54; N, 9.94. Found: C, 57.35; H,
5.61; N, 9.93. Selected IR bands (KBr pellet, cm-1) 2982 (m), 1632
(vs, νCO), 1617 (vs), 1589 (vs), 1568 (m), 1449 (s), 1351 (m), 1270
(m), 1140 (m), 1061 (m), 1026 (m), 964 (m), 774 (s), 746 (s).
Absorption spectrum in DMF (λmax, nm (ꢀ M-1 cm-1)) 650 (3700),
540 (3600), 420 (4400), 320 (11 000).
(Et4N)[FeIII(PyP{SO2}2)] (2). A slurry of 1 (0.400 g, 1.05 mmol)
in 40 mL of anhydrous acetone was placed in a stoppered flask of 100
mL volume, and 4 cm3 of pure dioxygen was introduced into the flask.
The mixture was stirred for 3 days at room temperature during which
(52) Brauer, G. Handbook of PreparatiVe Inorganic Chemistry; Academic
Press: New York, 1963; pp 140-142.

Model of the Iron(III) Site of Nitrile Hydratase
J. Am. Chem. Soc., Vol. 123, No. 14, 2001 3251
spectrum of 1 in pure water is identical to that in 2 mM tris buffer at
all temperatures. To correct for changes in absorbance values due to
deposition of water vapor on the cold window and uncertainties in
attaining the right pH in buffers containing 30% acetone, three
measurements were performed at each pH, and the average values were
used to plot the pH versus absorbance curve and calculate the pKa of
the bound water molecule in [FeIII(PyPS)(H2O)]-.
(b) Methanol. An aliquot (50 µL) of a 0.18 M solution of 1 in DMF
was added to 2 mL of methanol at room temperature. The solution
was then placed in the 10 mm quartz cell that fits on the custom-
designed Dewar apparatus, and the electronic spectrum was recorded
when no further change in the spectrum was observed. Binding of
methanol was complete at -60 °C.
(c) Pyridine. At room temperature, an aliquot (50 µL) of a 0.18 M
solution of 1 in DMF was added to a mixture of 0.4 mL of acetone
and 0.6 mL of pyridine. This mixture was then placed in the low-
temperature cell. The electronic spectrum was recorded when no further
change in the spectrum was observed. For pyridine, complete binding
to 1 occurred at -70 °C.
Table 1. Summary of Crystal Data and Intensity Collection and
Structure Refinement Parameters for (Et4N)[FeIII(PyPS)] (1),
(Et4N)[FeIII(PyP{SO2}2)] (2), and (Et4N)2[FeII(PyPS)] (3)
complex 1
complex 2
complex 3
formula
(mol wt)
cryst color, habit
T, K
cryst system
space group
a, Å
b, Å
c, Å
R, deg
â, deg
C27H31FeN4O2S2
563.53
dark green block
140 (2)
monoclinic
P21/c
14.910 (5)
18.369 (4)
19.222 (4)
90
91.12 (2)
90
C27H31FeN4O6S2
627.53
red needle
90 (2)
monoclinic
P21/n
7.7471 (4)
21.4308 (12)
17.0590 (9)
90
C35H51FeN5O2S2
693.78
brown needle
90 (2)
monoclinic
P21/n
9.5604 (5)
15.5545 (8)
23.6924 (13)
90
92.9000 (10)
90
2828.6 (3)
4
90.6610 (10)
90
3523.0 (3)
4
γ, deg
V, Å3
5264 (2)
8
Z
dcalcd, g cm-3
abs coeff, µ, mm-1
GOFa on F2
R1,b %
1.422
0.764
1.009
5.27
1.474
0.729
0.872
4.15
1.308
0.585
0.885
5.41
(d) N-Methylimidazole. At room temperature, an aliquot (50 µL)
of a 0.18 M solution of 1 in DMF was added to a mixture of 1 mL of
acetone and 70 µL of N-methylimidazole. This mixture was then placed
in the low-temperature cell. The electronic spectrum was recorded when
no further change in the spectrum was observed (-70 °C).
(e) PhS-. At room temperature, an aliquot (50 µL) of a 0.18 M
solution of 1 in DMF was added to a mixture of 1 mL of acetone and
20 equiv of (Et4N)(SPh). This mixture was then placed in the low-
temperature cell. The electronic spectrum was recorded when no further
change in the spectrum was observed (-70 °C).
wR2,c %
9.83
9.10
11.77
a GOF ) {∑[w(Fo2 - Fc2)2]/(M-N)}1/2 (M ) no. of reflections, N )
no. of parameters refined). b R1 ) ∑||Fo - Fc||/∑|Fo|. c wR2 )
{∑[w(Fo - Fc2)2]/∑[w(Fo )2]}1/2
.
2
2
1000 system (at 90 K). Mo KR radiation (λ ) 0.71073 Å) was used
for diffraction, and the data were solved by direct methods (SHELXS-
97, Sheldrick, 1990). Only random fluctuations of <1% in the intensities
of two standard reflections were observed during data collection for
all the three complexes. Hydrogen atoms bonded to carbon were added
geometrically and refined with the use of a riding model.
Machine parameters, crystal data, and data collection parameters are
summerized in Table 1. Selected bond distances and angles are listed
in Table 2. The rest of the crystallographic data has been submitted as
Supporting Information.
(f) Cyanide (CN-). At room temperature, an aliquot (50 µL) of a
0.18 M solution of 1 in DMF was diluted in 2 mL of thoroughly
degassed acetonitrile, and to it was added 1 equiv of (Et4N)(CN). The
absorption spectrum of the cyanide-adduct was recorded in the same
low-temperature cell. In acetonitrile, complete binding of CN- was
noted at -10 °C.
Determination of Thermodynamic Parameters of Binding of
Water and Pyridine to 1. Binding studies were performed using freshly
prepared solutions of 1 in aqueous acetone or in acetone/pyridine
mixture. The van’t Hoff equation was employed for the determination
of the thermodynamic parameter ∆H, the enthalpy of formation of the
six-coordinate adducts [FeIII(PyPS)(B)]- (B ) H2O or py). The
equilibrium constants were determined by monitoring the changes in
the electronic absortion spectra at four different temperatures. Solutions
of 6.9 mM of 1 were used in combination with appropriate concentra-
tions of water or pyridine in acetone so that 30-90% of adducts were
present at the four temperatures. The distinct low-temperature values
were attained by the use of appropiate low-temperature slush baths.
Spectral data at 420 nm was collected to calculate ∆H since it displayed
the largest change in absorbance. The extinction coefficient of 1 at
420 nm was calculated at 22 °C (4400 M-1 cm-1), -20 °C (4800 M-1
cm-1), -40 °C (5000 M-1 cm-1), and -75 °C (5150 M-1 cm-1). These
values were used to determine the concentration of 1 in the samples
for these experiments. The equilibrium constants were calculated using
eqs 1 and 2. The ∆H values for water and pyridine are -25.9 and
-21.3 kcal mol-1, respectively.
Other Physical Measurements. The electronic absorption spectra
were measured on a Perkin-Elmer Lambda 9 spectrophotometer. A
Perkin-Elmer 1600 FTIR spectrophotometer was used to monitor the
1
infrared (IR) spectra. H and 13C NMR spectra were recorded on a
Bruker 250 machine. EPR spectra were monitored at X-band frequen-
cies by using a Bruker ESP-300 spectrometer. Room-temperature
magnetic measurements on solid samples were made with a Johnson-
Matthey magnetic susceptibility balance. Electrochemical measurements
were performed with standard Princeton Applied Research instrumenta-
tion and a Pt inlay electrode. Potentials were measured at 25 °C versus
an aqueous saturated calomel electrode (SCE) as reference. The pKa
of the bound water in [FeIII(PyPS)(H2O)]- was determined by fitting
the plot of pH versus absorbance at 420 nm with the aid of MATLAB
package (see Supporting Information) and by using the function A )
fHA aHA + fB- aB- where A is total absorbance, aHA and aB- are the
absorbances of the protonated and deprotonated species at 420 nm
respectively, and
1
1
fHA
)
, fB- )
[H+]
Ka
Ka
+ 1
+ 1
[H+]
A - ꢀ1[1]o
ꢀ1‚B - ꢀ1
{(Et4N)[FeIII(PyPS)(B)]} ) [1‚B] )
(1)
(2)
Results and Discussion
{[1‚B]}
Keq
)
Syntheses of the Complexes. In a series of papers published
from our laboratory, we have described the syntheses and
structures of iron(III) (and iron(II)) and cobalt(III) complexes
that contain carboxamido N coordination along with other donor
sets.53,54,58-63 The discovery of the highly unprecedented co-
ordination sphere at the iron and cobalt centers in NHases has
prompted us to further explore the coordination chemistry of
these two metals with new ligands with carboxamide and thiolate
groups.48-51 In the present study, we have utilized the designed
ligand PyPSH4 which comprises two carboxamide and two
{[1]o - [1‚B]}{[B]o - [1‚B]}
X-ray Data Collection and Structure Solution and Refinement.
Dark green blocks of 1 were obtained by slow diffusion of diethyl
ether into a DMF:acetonitrile (50:50 v/v) solution of 1. Slow evaporation
of an acetone solution of 2 afforded red needles which were suitable
for diffraction studies. Brown needles of 3 were grown by slow diffusion
of diethyl ether into an acetonitrile solution of the complex. Diffraction
data for 1 were collected on a Siemens R3m/V machine (at 140 K),
while data for complexes 2 and 3 were collected on a Bruker SMART

3252 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
NoVeron et al.
Table 2. Selected Bond Distances (Å) and Angles (deg) for 1-3
(Et4N)[FeIII(PyPS)] (1)
Bond Distances
S(1)-C(1)
S(2)-C(19)
O(1)-C(7)
O(2)-C(13)
N(1)-C(6)
Fe(1)-N(1)
Fe(1)-N(2)
Fe(1)-N(3)
Fe(1)-S(1)
Fe(1)-S(2)
2.035 (3)
2.099 (3)
2.041 (3)
2.3147 (13)
2.3104 (13)
1.770 (4)
1.773 (4)
1.237 (5)
1.242 (5)
1.423 (5)
N(1)-C(7)
N(2)-C(8)
N(3)-C(13)
N(3)-C(14)
C(17)-C(18)
1.355 (5)
1.324 (5)
1.354 (5)
1.408 (5)
1.383 (7)
Bond Angles
N(1)-Fe(1)-N(3)
150.28 (14)
N(2)-Fe(1)-S(2)
S(2)-Fe(1)-S(1)
C(2)-C(1)-S(1)
C(1)-C(6)-N(1)
O(1)-C(7)-C(8)
C(15)-C(14)-N(3)
126.21 (10)
N(3)-Fe(1)-N(2)
N(3)-Fe(1)-S(2)
N(1)-Fe(1)-S(1)
N(1)-Fe(1)-N(2)
N(1)-Fe(1)-S(2)
75.01 (14)
83.95 (10)
84.17 (10)
75.28 (14)
114.33 (11)
109.53 (5)
120.7 (3)
115.2 (4)
120.2 (4)
124.0 (4)
(Et4N)[FeIII(PyP{SO2}2)] (2)
Bond Distances
Fe-O(1)
1.9112 (14)
1.9127 (15)
2.0201 (17)
2.0708 (17)
2.0175 (17)
S(1)-O(1)
1.5633 (16)
1.4788 (16)
1.798 (2)
O(3)-C(7)
O(4)-C(13)
N(3)-C(13)
N(1)-C(7)
C(12)-C(13)
1.229 (2)
Fe-O(6)
Fe-N(1)
Fe-N(2)
Fe-N(3)
S(1)-O(2)
S(1)-C(1)
S(2)-O(5A)
S(2)-C(19)
1.223 (3)
1.360 (3)
1.364 (2)
1.514 (3)
1.431 (2)
1.809 (2)
Bond Angles
O(1)-Fe-O(6)
106.24 (7)
O(5A)-S(2)-O(6)
O(2)-S(1)-O(1)
O(2)-S(1)-C(1)
O(1)-S(1)-C(1)
S(2)-O(6)-Fe
107.68 (11)
O(1)-Fe-N(1)
O(1)-Fe-N(3)
O(6)-Fe-N(3)
O(6)-Fe-N(2)
O(6)-Fe-N(1)
O(1)-Fe-N(2)
N(3)-Fe-N(1)
N(3)-Fe-N(2)
94.81 (6)
101.89 (6)
93.13 (7)
125.75 (7)
104.33 (7)
127.96 (7)
151.43 (7)
75.42 (7)
108.85 (9)
104.73 (9)
103.12 (9)
124.31 (9)
118.43 (12)
118.43 (14)
121.30 (19)
120.6 (2)
C(6)-N(1)-Fe
C(8)-N(2)-Fe
O(4)-C(13)-C(12)
C(15)-C(16)-C(17)
(Et4N)2[FeII(PyPS)] (3)
Bond Distances
Fe-N(1)
2.160(2)
2.129(2)
2.179(2)
2.4094(8)
2.3783(8)
S(1)-C(1)
1.778(3)
1.768(3)
1.249(3)
1.242(3)
1.407(4)
N(1)-C(7)
N(2)-C(8)
N(3)-C(13)
N(3)-C(14)
C(17)-C(18)
1.326(4)
Fe-N(2)
Fe-N(3)
Fe-S(1)
Fe-S(2)
S(2)-C(19)
O(1)-C(7)
O(2)-C(13)
N(1)-C(6)
1.332(4)
1.344(4)
1.389(4)
1.373(5)
Bond Angles
N(2)-Fe-N(1)
N(2)-Fe-N(3)
N(1)-Fe-N(3)
N(2)-Fe-S(2)
73.07(9)
73.33(9)
146.34(10)
128.44(77)
N(1)-Fe-S(2)
N(3)-Fe-S(2)
N(1)-Fe-S(1)
S(2)-Fe-S(1)
120.79(7)
79.85(7)
80.00(8)
101.05(3)
C(2)-C(1)-S(1)
N(1)-C(6)-C(1)
O(1)-C(7)-C(8)
N(3)-C(14)-C(15)
120.2(3)
116.0(3)
118.1(3)
125.9(3)
thiolate groups. The fully deprotonated PyPS4- ligand binds
iron(III) in DMF at low temperature to form [FeIII(PyPS)]-
without any complication arising out of reduction of the iron
center and formation of disulfide.48,56,57 This behavior is in
contrast to the reaction of iron(III) with the corresponding Schiff
base ligand 2,6-bis[1-methyl-2-(2-thiolophenyl)-2-azaethene]-
pyridine55 which only gives rise to the iron(II) complex. It is
evident that coordination of the carboxamido nitrogens provides
stability to iron(III) centers and prohibits reduction in case of
PyPS.4- This is further supported by the fact that when one
of iron(II) and disulfides.62,63 The fully deprotonated PyPS4-
,
however, affords the iron(II) complex [FeII(PyPS)]2- when one
starts with an iron(II) source.
Solutions of 1 and 3 are sensitive to dioxygen. However, when
a solution of 1 is stirred with controlled amount (∼12 equiv)
of dioxygen in dry acetone, 1 is smoothly converted into the
sulfinato species 2 in which both thiolato groups are converted
into sulfinato groups. To date, we have only been able to isolate
the O-bonded isomer of 2. In absence of H-bonding interactions
that involve the sulfinic O atoms (like in the enzyme), the
oxophilic nature of the iron(III) center and strain in the five-
membered chelate rings of 1 both prefer formation of the
O-bonded isomer upon oxidation. It is quite possible that the
2-
uses only 2 equiv of NaH, reaction of PyPSH2 (only the
thiolate groups are deprotonated) with iron(III) leads to autore-
dox reaction resulting in the formation of polymeric products
(53) Brown, S. J.; Olmstead, M. M.; Mascharak, P. K. Inorg. Chem.
1990, 29, 3229.
(54) Tan, J. D.; Hudson, S. E.; Brown, S. J.; Olmstead, M. M.;
Mascharak, P. K. J. Am. Chem. Soc. 1992, 114, 3841.
(55) Chavez, F. A.; Rowland, J. M.; Olmstead, M. M.; Mascharak, P.
K. J. Am. Chem. Soc. 1998, 120, 9015.
(56) Chavez, F. A.; Olmstead, M. M.; Mascharak, P. K. Inorg. Chem.
1997, 36, 6323.
(57) Chavez, F. A.; Olmstead, M. M.; Mascharak, P. K. Inorg. Chem.
1996, 35, 1410.
(58) Marlin, D. S.; Olmstead, M. M.; Mascharak, P. K. Inorg. Chem.
1999, 38, 3258.
(59) Marlin, D. S.; Olmstead, M. M.; Mascharak, P. K. Inorg. Chim.
Acta 2000, 297, 106.
(60) Marlin, D. S.; Mascharak, P. K. Chem. Soc. ReV. 2000, 29, 69.
(61) Lindoy, L. F.; Busch, D. H. Inorg. Chem. 1974, 13, 2494.
(62) Holm, R. H.; Ibers, J. A. In Iron-Sulfur Proteins; Lovenberg, W.,
Ed.; Academic Press: New York, 1997; Vol. III, Chapter 7.
(63) Ellis, K. J.; Lappin, A. G.; McAuley, A. J. Chem. Soc., Dalton Trans.
1975, 1931.

Model of the Iron(III) Site of Nitrile Hydratase
J. Am. Chem. Soc., Vol. 123, No. 14, 2001 3253
Figure 3. Thermal ellipsoid plot (probability level 50%) of [FeIII-
(PyP{SO2}2)]-, the anion of complex 2. H atoms are omitted for the
sake of clarity.
Figure 2. Thermal ellipsoid plot (probability level 50%) of [FeIII(PyPS)]-,
the anion of complex 1. H atoms are omitted for the sake of clarity.
O-bonded isomer of 2 is more stable than the S-bonded one.
Further support for this statement comes from the successful
isolation of the O-bonded sulfinato species (Et4N)2[FeIII(L-O2)]
(structure ii) by Chottard and co-workers.45
[FeIIIS2Me2N3(Pr,Pr)]+ (structure i) from Kovacs’ group however
exhibits a shorter Fe(III)-Sthio distance (2.13 Å) presumably
due to constraints in the ligand frame.43 Presence of four sets
of five-membered rings in the coordination sphere of iron in 1
gives rise to smaller bond angles such as S(1)-Fe-S(2)
(109.53° instead of 120°) and N(1)-Fe-N(3) (150.28° instead
of 180°). The same fact opens up both S(1)-Fe-N(2) and
S(2)-Fe-N(2) angles and allows further coordination to
generate six-coordinate complexes (vide infra).
Structure of (Et4N)[FeIII(PyP{SO2}2] (2). The structure of
the anion of 2 (Figure 3) clearly demonstrates that the two-
coordinated thiolato sulfurs are converted to sulfinato groups
and the sulfinato groups are coordinated to iron through one of
the oxygen atoms. The coordination geometry around iron is
still distorted trigonal bipyramidal and the sulfinato groups reside
in the basal plane. The binding mode of the sulfinato groups in
1 resemble that observed in Chottard’s complex ii.45 In both
cases, changes from S-coordination to O-coordination occurs
upon oxidation of the ligated thiolato sulfurs. It is however
interesting to note that oxidation of the six-coordinated iron-
(III) complex [FeIII(PyPepS)2]- (structure iv) affords the S-
bonded sulfinato species [FeIII(PyPepSO2)2]- (structure v)
without isomerization to the O-bonded species.49
Conversion of 1 into 2 does not bring any significant change
in the average Fe(III)-Namido distances. In complex 2, the
average Fe(III)-Namido distance is 2.02 Å. The average Fe(III)-
SO2 (SO2 denotes O-bonded sulfinato group) bond distance
(1.91 Å) in 2 compares well with the only other known Fe-
(III)-SO2 distance (2.00 Å) reported for complex [FeIII(L-O2)]-
(structure ii).45 The bond angles of the sulfinate groups (like
O(1)-S(1)-O(2) ) 108.85°) indicates sp3 hybridized sulfur
atoms in 2.
Structure of (Et4N)2[FeII(PyPS)] (3). The structure of the
anion of this iron(II) complex is shown in Figure 4 while
selected bond distances and angles are collected in Table 2.
The iron(II) center in the anion of 3 is ligated to the tetraanionic
ligand PyPS4- in a distorted trigonal bipyramidal fashion. The
N3S2 coordination sphere is similar to that observed in the
corresponding iron(III) complex 1 except for the fact that the
bond distances are somewhat longer.in case of 3. For example,
the average Fe(II)-Namido and Fe(II)-Sthio bond distances in 3
are 2.16 and 2.38 Å respectively. Overall, the Fe(II)-Namido
Structure of (Et4N)[FeIII(PyPS)] (1). The structure of 1
consists of discrete cations and monomeric anions (shown in
Figure 2) and is devoid of any solvent molecules of crystal-
lization. The tetraanionic pentadentate ligand PyPS4- is coor-
dinated to the iron(III) center in a distorted trigonal bipyramidal
fashion to give rise to a N3S2 coordination sphere. There is no
interaction between the iron(III) centers (minimum Fe-Fe
distance ) 8 Å, Figure S1, Supporting Information). This is in
contrast to the structure of the cobalt(III) complex of PyPS4-
which is dimeric.51 In the dimeric [Co2(PyPS)2]2- anion, the
sixth site of each cobalt is ligated to one of the bound thiolato
sulfurs of the other [CoIII(PyPS)]- moiety. The propensity of
the low-spin Co(III) complexes for octahedral coordination is
most possibly responsible for this difference. Close examination
of the two structures also reveals that in the dimeric cobalt(III)
complex, the PyPS4- ligand frame is wrapped around the two
cobalt centers in identical helical configurations. The asymmetric
unit of 1 on the other hand, contains two [FeIII(PyPS)]- anions
with opposite configurations pertaining to the two possible
helical orientations the ligand can adopt at the iron(III) center
(Figure S1). Results of solution magnetic measurements and
EPR experiments at low temperatures (down to 4 K) indicate
that the anions of 1 remain mononuclear at all temperatures.
The reason for the different ways of ligation of the PyPS4-
ligand frame around cobalt(III) and iron(III) remains unclear at
this time.
In complex 1, the average Fe(III)-Namido distance is 2.04 Å
(Table 2) and compares well with the corresponding distance
reported for NHase, 2.07 Å (average value).29 This value is
somewhat longer compared to the Fe(III)-Namido distances
observed in other carboxamido complexes of trivalent iron46,63,64
including Chottard’s complex ii.45 The average Fe(III)-Sthio
(thio ) thiolate) distance in 1 (2.31 Å) also compares well with
the corresponding average Fe(III)-Sthio distance reported for
NHase (2.32 Å)29 and for other complexes with Fe(III)-Sthio
coordination (2.25-2.30 Å).46,48,65-67 The model complex
(64) Ray, M.; Ghosh, D.; Shirin, Z.; Mukherjee, R. Inorg. Chem. 1997,
36, 3568.
(65) Hsu, H.; Koch, S. A.; Popescu, C. V.; Munk, E. J. Am. Chem. Soc.
1997, 119, 8371.
(66) Millar, M.; Lee, J. F.; Koch, S. A.; Fikar, R. Inorg. Chem. 1982,
21, 4105.
(67) Shoner, S. C.; Barnhart, D.; Kovacs, J. A. Inorg. Chem. 1995, 34,
4517.

3254 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
NoVeron et al.
[FeIII(N2S2)Cl]2- (structure iii) are also red and exhibit absorp-
tions at 475 (in acetonitrile) and 500 nm (in dichloromethane),
respectively.45,46 Nevertheless it is interesting to note that binding
of azide (N3-) to [FeIIIS2Me2N3(Pr,Pr)]- results in a green
solution (absorption maxima at 708 and 460 nm). Since binding
of many Lewis bases to the iron(III) center of 1 also gives rise
to green solutions with absorptions around 700 nm, it appears
that the iron site in the enzyme most possibly is six-coordinate.
The iron(III) center in 1 is high-spin and exhibits a magnetic
moment of 6.1 µB in the polycrystalline state and in DMF
solution at 300 K. It also exhibits a broad EPR spectrum with
g ) 8.32, 5.19, 4.14, and 2.11 at 4 K in DMF glass. The
spectrum, typical of high-spin iron(III) in distorted crystal field,
arises from transitions associated with all three Kramers’
doublets.68,69 The spin states of iron(III) centers in complexes
with N,S coordination show wide variabilities and the reason-
(s) for such behavior is not clear at the present time. For
example, despite strong chelation effect of the ligand frames, 1
contains a high-spin iron(III) center while Chottard’s model
Figure 4. Thermal ellipsoid plot (probability level 50%) of [FeII-
(PyPS)]2-, the anion of complex 3. H atoms are omitted for the sake
of clarity.
3
complex NEt4[FeIII(L-O2)] exhibits intermediate spin (S ) /2)
at all temperatures. The iron(III) center in Kovacs’ pentacoor-
dinate complex [FeIIIS2Me2N3(Pr,Pr)]PF6 is predominantly low-
1
5
spin (S ) /2) below 50 K while the high-spin state (S ) /2)
gets significantly populated above 150 K. The complex
[FeIII(N2S2)Cl]2- has intermediate spin (S ) /2) at low tem-
3
peratures. All these complexes are pentacoordinate and contain
N,S coordination spheres. Wieghardt and co-workers have
reported two octahedral iron(III) complexes of macrocyclic N3S3
ligands which show spin equilibrium between high-spin (S )
5/2) and low-spin (S ) 1/2) forms.70 In contrast, a few octahedral
iron(III) complexes with N, S coordination have also been
reported that exist in low-spin (S ) 1/2) forms at all temperatures.
These include [FeIII(ADIT)2]+,67 [Fe(L)2]+ (L ) 2-aminoeth-
ylthiosalicylideneimine),71 [FeIII(PyArS)2]+,47 [FeIII(PyRS)2]+,47
[FeIII(PypepS)2]-,48 and [FeIII(PypepSO2)2]-.49 Collectively,
these examples indicate that six-coordinate iron(III) complexes
with N,S coordination are mostly low-spin. This hypothesis is
further supported by the fact that both 1 and [FeIIIS2Me2N3(Pr,-
Pr)]PF6 afford low-spin adducts with CN- (vide infra) and NO,
respectively. Again, these results corroborate the proposition
that the biological iron site is six-coordinate.18,32
Figure 5. Electronic absorption spectra of (Et4N)[FeIII(PyPS)] (complex
1, dotted line) in DMF and (Et4N)[FeIII(PyPS)(py)] (solid line) in
acetone/py (2:3) mixture. The temperatures of measurements are
indicated. See Experimental Section for detail.
distance of 3 is considerably longer than that noted for other
iron(II) complexes with ligated carboxamido nitrogens.53
Properties of (Et4N)[FeIII(PyPS)] (1). Coordination of the
deprotonated carboxamido nitrogens to the iron(III) center of 1
is evidenced by the red-shift of the carbonyl stretching frequency
(νco) from 1680 cm-1 in free ligand to 1632 cm-1 in the
complex. Similar red shift has been noted for (Et4N)[FeIII-
(PyPepS)2]48 and other iron(III) complexes with coordinated
carboxamido nitrogens.63 The electronic absorption spectrum
of 1 in solvents such as DMF and acetone consists of three
strong bands with maxima at 650, 540, and 420 nm (Figure 5).
These absorptions arise from thiolate-to-iron(III) charge-transfer
transitions and are not present in the absorption spectrum of
the iron(II) complex 3. The overlapping bands at 650 and 540
nm are responsible for the blue-green color of 1. Binding of
ligands such as pyridine to the iron(III) center of 1 (vide infra)
results in green solutions which exhibit absorption spectra much
like NHase (Figure 5). It is important to note that to date, no
pentacoordinate iron(III) complex with N,S coordination has
afforded an absorption spectrum which resembles the absorption
spectrum of NHase (broad bands at 700 and 420 nm) that gives
the enzyme its characteristic green color. For instance, the
pentacoordinate iron(III) complex [FeIIIS2Me2N3(Pr,Pr)]PF6 (struc-
ture i) is red and exhibits band maxima at 450 and 380 nm.43
The other two model complexes [FeIII(L-O2)]- (structure ii) and
Coordination of the carboxamido nitrogens provides stability
to the +3 oxidation state of iron in 1. This is evidenced by the
reduction potential of 1. In DMF, 1 exhibits a reversible cyclic
voltammogram (Figure 6) with half wave potential (E1/2) of
-0.65 V (vs SCE). A E1/2 value of -0.27 V (vs SCE) has been
reported for [FeIIIS2N3(Pr,Pr)]PF6, a complex with no carboxa-
mido nitrogen in the coordination sphere.43 We attribute the
greater stability of the iron(III) center in 1 due to the coordinated
carboxamido nitrogens. The reduction potential of the iron(III)
center of the NHase from Rhodococcus sp. R312 in the presence
of butyric acid has recently been measured.46 The E1/2 value
(-0.48 V vs SCE) suggests that the biological iron(III) site is
not easily reduced. It is quite evident that extra stability due to
coordination of the carboxamido nitrogens prevents redox
processes at the iron(III) site in NHase and allows it to act more
like a Lewis acid. The reversibility of the voltammogram in
Figure 6 first prompted us to synthesize the iron(II) complex 3.
(68) Drago, R. In Physical Methods for Chemists, 2nd ed.; Saunders
College Publishing: Ft. Worth, 1977; pp 583-586.
(69) Oosterhuis, W. T. Struct. Bonding (Berlin) 1974, 20, 59.
(70) Beissel, T.; Burger, K. S.; Voigt, G.; Wieghardt, K.; Butzlaff, C.;
Trautwein, A. X. Inorg. Chem. 1993, 32, 124.
(71) Fallon, G. D.; Gatehouse, B. M. J. Chem. Soc., Dalton Trans. 1975,
1344.

Model of the Iron(III) Site of Nitrile Hydratase
J. Am. Chem. Soc., Vol. 123, No. 14, 2001 3255
Reactivity of (Et4N)[FeIII(PyPS)] Toward a Sixth Ligand.
The trigonal bipyramidal complex 1 comprises an open angle
(N(2)-Fe-S(2)) of 127.34° and is expected to bind a sixth
ligand. We discovered that ligands with good Lewis base
strength binds to the iron(III) center of 1 in a temperature- and
solvent-dependent fashion. Ligands such as pyridine, N-meth-
ylimidazole, methanol, aryl thiolates, methoxide, and water all
bind to 1 at low temperatures (T < 0 °C) to generate
six-coordinate species. Binding of CN- occurs at room tem-
perature, and the cyanide adduct (νCN ) 2024 cm-1, g ) 2.29,
2.10, 1.97, Figure 7) can be isolated from acetonitrile solution
in high yield. Although we do not have a structure of (Et4N)2-
[FeIII(PyPS)(CN)] as of yet, the corresponding cobalt(III) species
has been structurally characterized.51 The spectroscopic param-
eters of these six-coordinate adducts indicate a general structure
(structure vii) that includes two carboxamido nitrogens coor-
Figure 6. Cyclic voltammograms of 5mM solutions of (Et4N)[FeIII-
(PyPS)] (1) (top) and (Et4N)[FeIII(PyP{SO2}2)] (2) (bottom) in DMF
(0.1 M (Et4N)(ClO4); Pt electrode; 100 mV/s scan speed). Potentials
are indicated vs aqueous SCE.
Incidentally, 3 is the first example of an iron(II) species with
ligated carboxamido N and thiolato S donors.
Properties of (Et4N)[FeIII(PyP{SO2}2)](2). The infrared
dinated in the basal plane of iron and two coordinated thiolato
S donors cis to each other. One of the thiolato S occupies an
axial position, trans to the incoming ligand L. These structural
features are similar to those observed for the iron center in
NHase (Figure 1) and hence the properties of these adducts
deserve attention.
spectrum of 2 shows a moderately strong band at 1070 cm-1
,
corresponding to the S-O stretching (νSO2) of the O-bonded
sulfinato groups. This value compares well with the νSO2 (1040
cm-1) noted for the model complex NEt4[FeIII(L-O2)] (structure
ii).45 Complex 2 also exhibits strong νCO stretching at 1618
cm-1, a value that confirms the presence of coordinated
carboxamido groups. A solution of 2 in DMF displays bands at
480 and 360 nm. Overall, this electronic absorption spectrum
resembles that of the two pentacoordinate model complexes
[FeIII(L-O2)] (450 and 380 nm) and [FeIII(N2S2)Cl] (500 and
390 nm) with coordinated carboxamido nitrogens.45,46
Both the magnetic moment at 300 K (5.8 µB, polycrystalline
sample) and the EPR spectrum (g ) 4.27, DMF glass, 77 K) of
2 indicate that the iron(III) center exists in the high-spin state.
The reduction potential of 2 in DMF (-0.36 V vs SCE, Figure
6) demonstrates that conversion of the bound thiolato groups
to the O-bonded sulfinato groups reduces the stability of the
iron(III) center. It will be interesting to study the effect of the
posttranslational modifications of the bound Cys-residues on
the overall redox potential of the iron(III) site in NHase.
Properties of (Et4N)2[FeII(PyPS)] (3). The infrared spectrum
of 3 exhibits a strong νCO stretching at 1572 cm-1 for the bound
carboxamide groups. As discussed in our previous papers, νCO
stretching frequencies of iron(II)-amides are always lower than
those of the corresponding iron(III)-amides due to greater
contribution of the imminolate (-O-CdN-) tautomeric form
of the bound peptide moiety.53,63 Complexes 1 and 3 are no
exception in this regard. The iron(II) center in 3 exists in high-
spin state as evidenced by the magnetic moment of the complex
(4.9 µB, polycrystalline sample) at 300 K. Upon exposure to
dioxygen, 3 in DMF is readily converted into 1. The oxidation
can also be achieved by ferrocenium salts.
In the present study, binding of ligands at the sixth site of
the iron(III) center in 1 has been studied at different temperatures
and in different solvents to explore the basic chemistry
associated with an iron(III) center with ligated carboxamido N
and thiolato S donors. To begin with, we found that binding of
ligands is dependent on both the solvent and the temperature
of binding. For example, when 1 is dissolved in methanol and
cooled, the absorption spectrum changes slowly and finally at
-60 °C, affords the methanol adduct [Fe(PyPS)(HOMe)]-
which exhibits bands at 790 and 550 nm. This six-coordinate
species is low-spin and displays a rhombic EPR signal with g
) 2.23, 2.20, and 1.93. It is important to note that [Fe(PyPS)-
(HOMe)]- with a N,S,O coordination sphere around iron(III)
exhibits spectral properties that are similar to that of the iron-
(III) center of NHase. Similar experiments with solutions of 1
in acetone and different ligands show that complete binding of
L occurs for N-methylimidazole at -60 °C, for pyridine at -60
°C, and for PhS- at -30 °C. In all cases, the six-coordinate
adducts exhibit low-spin EPR spectra (Figure 7) and have one
strong absorption band near 700 nm (Table 3, Figure S2,
Supporting Information). Clearly, the iron(III) center in 1 with
ligated carboxamido N and thiolato S donors binds to a wide
variety of neutral and anionic ligands with varying affinities. It
is, however, interesting to note that the iron(III) center of 1
shows no affinity to nitriles even at -100 °C. Kovacs and co-
workers have reported that the iron(III) center in [FeIIIS2N3-
(Pr,Pr)]PF6 binds to azide (N3-) and NO at low temperatures

3256 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
NoVeron et al.
hydration, namely, hydration by an iron(III)-bound hydroxide
(outer-sphere mechanism), we have studied the binding of water
at the sixth site of iron(III) in 1 and determined the pKa of such
a bound water molecule. Early EPR18 and ENDOR32 data
indicated that the active site of NHase contains a water molecule
bound to the apical site of the iron(III) center. On the basis of
the structures of other hydrolases, it has also been proposed
that the bound water at the metal center is in the hydroxide
form and there is one ENDOR work which supports the presence
of a hydroxide group bonded to the apical site of the metal center
in NHase.34 We discovered that complex 1 binds water at low
temperatures and results in the formation of [FeIII(PyPS)(H2O)]-,
a reaction that can be followed by monitoring changes in the
absorption spectrum of 1 in an acetone:water (30:70) mixture
(Figure 8). As the temperature of the solution is decreased,
changes in the absorption spectrum are noticed with the
appearance of two isosbestic points at 590 and 790 nm. No
change is observed beyond -30 °C when the formation of
[FeIII(PyPS)(H2O)]- becomes complete (acetone does not bind
to the iron(III) center of 1 even at -94 °C, its freezing point).
These changes are completely reversible and as one expects,
is sensitive to the pH of the solution. The plot of the absorbance
of the 420 nm band of the final spectra of such solutions of 1
at -30 °C versus the pH values (Figure S3, Supporting
Information) affords an apparent value of 6.3 ( 0.4 for the pKa
of the bound water molecule in [FeIII(PyPS)(H2O)]- at -30 °C.
We have previously reported that the pKa of the bound water
in [CoIII(PyPS)(H2O)]- at room temperature is 8.3.51 Replace-
ment of cobalt(III) with iron(III) in [MIII(PyPS)(H2O)]- appar-
ently increases the acidity of the bound water molecule. The
pKa of water bound to other iron(III) complexes have been
reported. For example, in [FeIII(3,4-TDTA)(H2O)]- (TDTA is
a tetraanionic ligand with N2O4 donor set), the pKa of the bound
water is 8.2.72
In the present study, the hydroxide-bound species [MIII-
(PyPS)(OH)]2- has been generated in solution by adjusting the
pH of a solution of [FeIII(PyPS)(H2O)]- (in aqueous acetone)
to 10. The same species is formed when (Me4N)(OH) is added
to a solution of 1 in DMF/acetonitrile mixture. Binding of
hydroxide ion to the iron(III) center of 1 results in low-spin
[MIII(PyPS)(OH)]2- which exhibits a strong EPR signal with
g-values ) 2.22, 2.12, 1.99. The EPR spectrum (Figure 7) is
comparable to the EPR spectrum of NHase (g ) 2.21, 2.14,
1.95). These results support the notion that the water molecule
bound at the iron center of NHase could exist as hydroxide at
the functional pH of the enzyme (∼7.5), and hence such an
iron center could hydrolyze substrates much like other known
hydrolases.
Thermodynamics of Binding of Water and Pyridine to
(Et4N)[FeIII(PyPS)]. These two ligands were chosen since they
do not pose any effect due to charge and offer different donor
atom (O vs N) to the iron(III) center of 1. The van’t Hoff plots
for the binding of these two ligands (Figure S4) were used to
calculate the enthalpy of formation (∆H) of the adducts
[FeIII(PyPS)(H2O)]- and [FeIII(PyPS)(py)]-. The ∆H value for
the water adduct (-25.9 kcal mol-1) is higher than that of the
pyridine adduct (-21.3 kcal mol-1), a fact that supports the
higher affinity of the iron(III) center toward water, an O-donor.
As mentioned before, binding of water is complete at -30 °C
while for pyridine, complete binding is noted only at -70 °C.
These results clearly indicate that the iron(III) center in 1 with
coordinated carboxamido N and thiolato S donors prefers H2O
Figure 7. Top panel: X-band EPR spectrum of [Fe(PyPS)]- at 4 K.
Bottom panel: X-band EPR spectra of (a) [FeIII(PyPS)(OH)]2-, (b) [FeIII-
(PyPS)(N-MeIm)]-, (c) [FeIII(PyPS)(SPh)]2- and (d) [FeIII(PyPS)(CN)]2-
at 80 K. See experimental details for generation of these adducts.
Sample concentration: 3 mM in all cases. The g values are tabulated
in Table 3. Spectrometer settings: microwave frequency, 9.43 GHz;
microwave power, 10 mW; modulation frequency, 100 kHz; modulation
amplitude, 2G.
but has no affinity toward nitriles.43 Therefore, it seems unlikely
that the mechanism of catalysis by NHase involves binding of
the nitrile substrates to the iron(III) center (inner-sphere mech-
anism).
Formation of (Et4N)[FeIII(PyPS)(H2O)] and its Reactivity.
To investigate the other suggested mechanism for nitrile
(72) Sanchiz, J.; Dominguez, S.; Mederos, A.; Brito, F.; Arrieta, J. M.
Inorg. Chem. 1997, 36, 4108.

Model of the Iron(III) Site of Nitrile Hydratase
J. Am. Chem. Soc., Vol. 123, No. 14, 2001 3257
)
max (ꢀ M-1 cm-1 g valuesb
Table 3. Electronic Absorption and EPR Data for the Six-coordinate Complexes
a
λ
(Et4N)[FeIII(PyPS)(MeOH)]
(Et4N)2[FeIII(PyPS)(OH)]
(Et4N)[FeIII(PyPS)(N-MeIm)]
(Et4N)2[FeIII(PyPS)(SPh)]
(Et4N)2[FeIII(PyPS)(CN)]
(Et4N)2[FeIII(PyPSO2)(CN)]
(Et4N)2[FeIII(PyPSO2)(CN)]
790 (950), 550 (3800), 410 (8500)
790 (700), 595 (3 900), 410 (7600)
910 (1600), 700 (1800), 420 (6500)
640 (3400), 540 (4400)
910 (1800), 620 (2400)
640 (1350), 470 (1600)
2.23 2.20 1.93c
2.31 2.12 1.93d
2.32 2.15 1.93e
2.27 2.13 1.96f
2.30 2.11 1.96g
2.27 2.19 1.94h
2.20 1.93h
660 (1200), 425 (1800)
a See Experimental Section for details. b Spectra recorded at 77 K at X-band frequencies. Spectrometer settings: microwave frequency, 9.2 GHz;
microwave power, 25 mW; modulation frequency, 100 kHz; modulation amplitude, 2 G. c MeOH glass of 1. d [FeIII(PyPS)(OH)]2- was generated
as follows. 0.8 equiv of Me4NOH was mixed with 1 equiv of 1 at 0 °C in a CH3CN:DMF mixture and then frozen. e 1 equiv of N-MeIm added to
1 in DMF and frozen. f 1 equiv of (Et4N)(SPh) added to 1 in DMF and frozen. g 1 equiv of (Et4N)(CN) to 1 in DMF and frozen. h DMF glass.
Scheme 1
Affinity of iron(III) centers toward anionic oxygens could be
the driving force for this rearrangement. It is interesting to note
that oxidation of (Et4N)[FeIII(PyPepS)2] (structure iv) to (Et4N)-
Figure 8. Top: Changes in the electronic absorption spectrum of a
[FeIII(PyPepSO2)2] does not allow such rearrangement. We
attribute this difference in behavior to the coordinative saturation
in (Et4N)[FeIII(PyPepS)2]. For the rearrangement to take place,
the O atom of the coordinated S-bonded sulfinato group should
be temporarily ligated to the iron(III) center while the Fe-S
bond starts to break (Scheme 1). Since (Et4N)[FeIII(PyPepS)2].
does not have any such temporary site, oxidation of this complex
results in pure (Et4N)[FeIII(PyPepSO2)2], the S-bonded sulfinato
complex. In case of 1, the iron(III) center is coordinatively
unsaturated, and this fact allows temporary binding of the
O-atom of the bound -SO2 group to iron, and hence both the
sulfinato groups sequentially isomerize to the O-bonded form
(thermodynamically more stable) to afford 2 as the final product.
The reaction of 1 with dioxygen has been followed by
electrospray mass spectrometry. As the solution of 1 in acetone
is exposed to dioxygen, all intermediate oxygenated species with
one, two, and three O atoms are observed along with
[FeIII(PyP{SO2}2)]- (2) in the reaction mixture. It thus appears
that the oxygenation reaction proceeds via a persulfoxidic
intermediate which can give rise to products with bound
sulfenato groups (i.e., products with one or three O atoms).73-75
At the end of 48 h, only peaks corresponding to 2 are observed
in the mass spectrum.
0.35 mM solution of [FeIII(PyPS)]- in acetone:water (30:70) mixture
(pH 5.5) with temperature. At -30 °C, the final spectrum of the water
adduct [FeIII(PyPS)(H2O)]- was recorded after 10 min.
over N-donors. Indeed, we did not notice any binding of nitriles
to the iron(III) center of 1. The suggestion that nitriles displace
water bound to the iron(III) site in NHase and then are
hydrolyzed thus appears to be unlikely.
Reactivity of (Et4N)[FeIII(PyPS)] with Oxygen (O2). One
unique feature of the unprecedented coordination structure of
the iron site in NHase is the presence of one sulfenato and one
sulfinato group bound to the iron through the S centers. The
oxidant responsible for the posttranslational modifications of
the bound Cys-S donors at the active site of the enzyme is
unknown at the present time. In the past, we have reported that
iron(III) and cobalt(III) complexes that contain carboxamido N
and thiolato S donors undergo oxidation of the ligated thiolato
sulfurs to sufinates (-SO2) when reacted with H2O2.49,50 In this
work we show that the iron(III) complex 1 reacts readily with
dioxygen to afford the corresponding sulfinato species [FeIII-
(PyP{SO2}2)]- (2) in which both the bound thiolato sulfurs are
converted to sulfinato groups. To our knowledge, complex 1 is
the first example of a model complex of NHase that shows this
reactivity with dioxygen. Although Chottard and co-workers
have reported a sulfinato species (Et4N)2[FeIII(L-O2)] (structure
ii), the corresponding thiolato complex is not reported, and the
oxidant actually responsible for the thiolato f sulfinato
transformation has not been identified.45
Addition of dilute hydrochloric acid to a solution of 2 in
acetone removes the sulfinato ligand from iron as evidenced
by the rapid loss of color. The precipitated ligand exhibits a
single peak in the mass spectrum, corresponding to PyP-
In contrast to the complex (Et4N)[FeIII(PyPepSO2)2] (structure
v), where the sulfinato groups are coordinated to the iron(III)
center via the sulfur atoms,49 the sulfinato moieties of 2 undergo
a structural rearrangement that results in the coordination of
the oxygen atom of the sulfinato groups to the iron(III) center.
(73) Grapperhaus, C. A.; Darensbourg, M. Y.; Sumner, L. W.; Russell,
D. H. J. Am. Chem. Soc. 1996, 118, 1791.
(74) Farmer, P. J.; Solouki, T.; Soma, T.; Russell, D. H.; Darensbourg,
M. Y. Inorg. Chem. 1993, 32, 4171.
(75) Farmer, P. J.; Solouki, T.; Mills, D. K.; Soma, T.; Russell, D. H.;
Reibenspies, J. H.; Darensbourg, M. Y. J. Am. Chem. Soc. 1992, 114, 4601.

3258 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
NoVeron et al.
Figure 9. Electronic absorption spectra of Na2[FeIII(PyP{SO2)2})(CN)] (4) (solid line) and (Et4N)2[FeIII(PyP{SO2)2})(CN)] (5) (broken line) in
DMF (sample concentration: 0.4 mM).
{SO2}2H4, and its IR spectrum displays strong νNH at 3271 cm-1
,
2.20, 1.93). Successful isolation of 4 and 5 provides support to
our hypothesis that oxygenation of coordinatively saturated and
kinetically inert iron(III) complexes gives rise to S-bonded
sulfinato species.
νCO at 1656 cm-1, and νSO2 at 1121 and 1082 cm-1. The
deprotonated form of this ligand readily affords the iron(III)
complex 2 in DMF. It thus appears that the preferred mode of
binding of the sulfinato groups of [PyP{SO2}2]4- to iron(III) is
through the O (not the S) atoms.
Summary and Conclusions
Reactivity of (Et4N)[FeIII(PyPS)(CN)] with H2O2. In the
previous section, we have hypothesized that the rearrangement
of the S-bonded sulfinato complex to the O-bonded species can
occur when the iron(III) center is not coordinatively saturated
(Scheme 1). If this is true, then one expects to synthesize the
S-bonded sulfinato complex via oxygenation of a coordinatively
saturated iron(III) complex of PyPSH4. Since we had access to
such a complex, namely, [FeIII(PyPS)(CN)]2-, we studied the
reactions of [FeIII(PyPS)(CN)]2- with O2 and H2O2. Although
both reactions afforded the same results, the reaction with
dioxygen was very slow. The reaction of [FeIII(PyPS)(CN)]2-
with H2O2 is, however, rapid even at subzero temperature and
hence we have studied this reaction in detail. Reaction of H2O2
with [FeIII(PyPS)(CN)]2- in acetonitrile at -20 °C affords
[FeIII(PyP{SO2}2)(CN)]2- which has been isolated as the sodium
salt and characterized by spectroscopic techniques, elemental
analysis, and electrospray mass spectrometry. That the S atom
of the sulfinato groups are bonded to the Fe(III) center in Na2-
[FeIII(PyP{SO2}2)(CN)] (4) is evident from the similarities in
spectroscopic parameters of this complex with those of the
structurally characterized S-bonded sulfinato complex Na[FeIII-
(PyPepSO2)2].49 For example, the IR spectrum of 4 displays
strong νSO2 at 1186 cm-1, while Na[FeIII(PyPepSO2)2] exhibits
The following are the principal results and conclusions of
this investigation.
(i) The iron(III) and iron(II) complexes of a designed
pentadentate ligand PyPSH4 with two carboxamide and two
thiolate groups have been synthesized and structurally character-
ized. These complexes add to the very short list of iron
complexes that contain an iron(III) center ligated to deprotonated
carboxamido nitrogens and thiolato sulfurs and are good
structural models for the iron(III) site in Fe-NHase.
(ii) The redox potential of the iron(III) center in (Et4N)[FeIII-
(PyPS)] (1) (-0.65 V versus SCE in DMF) indicates that the
carboxamido nitrogens provide significant stability to the +3
oxidation state of iron76 and this in turn indicates that such
stabilization could account for the absence of any redox activity
of the iron site in NHase.
(iii) The coordinatively unsaturated iron(III) site in 1 binds
various Lewis bases to give six-coordinate low-spin iron(III)
species which afford green solution in water and DMF. Ligands
such as methanol, water, pyridine, N-MeIm, and PhS- bind at
subzero temperature, while CN- binds at room temperature.
These results suggest that the low-spin green iron center in the
enzyme is most probably six-coordinate.
(iv) The pKa of the bound water molecule in [FeIII(PyPS)-
(H2O)]- has been determined by low-temperature spectropho-
tometry. The pKa value (6.3 ( 0.4) indicates that at physiological
pH, the water at the iron(III) site in NHase could exist as
hydroxide (as suggested by ENDOR studies on the enzyme).
The pKa of the bound water in the corresponding cobalt(III)
complex [CoIII(PyPS)(H2O)]- is 8.3, a fact that suggests that
the iron-bound water is more acidic.
(v) The iron(III) center in 1 does not show any affinity toward
nitriles even at low temperature. This behavior is observed with
other model complexes. The enthalpy of formation of [FeIII-
(PyPS)(H2O)]- (-25.9 kcal mol-1) also indicates strong binding
ν
SO2 at 1184 cm-1. Dark-green solutions of 4 in water and DMF
exhibit a moderately strong band with maximum at 670 and
640 nm, respectively. The EPR spectrum of 4 in DMF glass (g
) 2.27, 2.19, 1.94) indicates the presence of a low-spin iron-
(III) center (Figure S5, Supporting Information).
The spectral characteristics of the S-bonded sulfinato complex
4 are distinct from those of the O-bonded sulfinato complex
(Et4N)2[FeIII(PyP{SO2}2)(CN)] (5) which has been synthesized
by the addition of CN- to complex 2 in acetone. For example,
the IR spectrum of 5 displays νSO2 at 1030 cm-1, a value close
to the νSO2 of 2 (1070 cm-1), a O-bonded sulfinato complex.
Also, in DMF, 5 exhibits an absorption band with maximum at
660 nm (Figure 9). The iron(III) center in 5 is also low-spin
and gives rise to an axial EPR spectrum in DMF glass (g )
(76) No oxidation wave corresponding to iron(III)/(IV) couple has been
observed with 1 in any solvent up to +1.5 V (vs SCE).

Model of the Iron(III) Site of Nitrile Hydratase
J. Am. Chem. Soc., Vol. 123, No. 14, 2001 3259
by water and hence replacement of water (or hydroxide) at the
active site of NHase by nitriles appears unlikely.
funded in part by the NSF Instrumentation Grant CHE-9808259.
We thank Dr. Robert Goldbeck and Dr. Trevor Swartz for
experimental assistance.
(vi) Exposure of solutions of 1 to dioxygen affords the
O-bonded sulfinato complex (Et4N)[FeIII(PyP{SO2}2)] (2). This
reaction mimics the posttranslational modification of the bound
Cys-S centers of the active site of NHase. Conversion of similar
thiolato complex (Et4N)[FeIII(PyPepS)2] to the S-bonded sulfi-
nato complex (Et4N)[FeIII(PyPepSO2)2] indicates that the S-
bonded iron(III) sulfinato species rearrange to the corresponding
O-bonded species when the iron(III) center is coordinatively
unsaturated (like (Et4N)[FeIII(PyPS)]). This is further supported
by the fact that the coordinatively saturated complex [FeIII-
(PyPS)(CN)]2- affords the S-bonded sulfinato complex
[FeIII(PyP{SO2}2)(CN)]2- (5).
Supporting Information Available: Crystal structure of
complex 1 (Figure S1), electronic absorption spectrum of (Et4N)-
[FeIII(PyPS)(N-MeIm)] in acetone at -70 °C (Figure S2), spectra
of solutions of 1 in acetone:water mixtures at -30 °C vs the
pH values (Figure S3), van’t Hoff plots for binding of water
and pyridine to the iron(III) center in 1 (Figure S4), EPR
spectrum of complex 4 in DMF glass (Figure S5), crystal
structure data for 1-3 including atomic coordinates and isotropic
thermal parameters, bond distances and angles, anisotropic
thermal parameters, and H-atom coordinates (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. Financial support from NSF (CHE-
9818492) and NIH (GM 61636) is gratefully acknowledged.
J.C.N. received support from the NIH-IMSD Grant
1R25GM58903. The Bruker SMART 1000 diffractometer was
JA001253V