Water affects the stereochemistry and dioxygen reactivity of carboxylate-rich diiron(II) models for the diiron centers in dioxygen-dependent non-heme enzymes

Article abstract of DOI:10.1021/ja0512531

Carboxylate-bridged high-spin diiron(II) complexes with distinctive electronic transitions were prepared by using 4-cyanopyridine (4-NCC ₅H₄N) ligands to shift the charge-transfer bands to the visible region of the absorption spectrum. This property facilitated quantitation of water-dependent equilibria in the carboxylate-rich diiron(II) complex, [Fe₂(μ-O₂CAr^Tol) ₄(4-NCC₅H₄N)₂] (1), where ^-O₂CAr^Tol is 2,6-di-(p-tolyl)benzoate. Addition of water to 1 reversibly shifts two of the bridging carboxylate ligands to chelating terminal coordination positions, converting the structure from a paddlewheel to a windmill geometry and generating [Fe₂(μ-O ₂CAr^Tol)₂(O₂CAr^Tol) ₂(4-NCC₅H₄N)₂(H₂O) ₂] (3). This process is temperature dependent in solution, rendering the system thermochromic. Quantitative treatment of the temperature-dependent spectroscopic changes over the temperature range from 188 to 298 K in CH ₂Cl₂ afforded thermodynamic parameters for the interconversion of 1 and 3. Stopped flow kinetic studies revealed that water reacts with the diiron(II) center ca. 1000 time faster than dioxygen and that the water-containing diiron(II) complex reacts with dioxygen ca. 10 times faster than anhydrous analogue 1. Addition of {H(OEt₂)₂} {BAr′₄}, where BAr′₄- is tetrakis(3,5- di(trifluoromethyl)phenyl)borate, to 1 converts it to [Fe₂(μ- O₂CAr^Tol)₃(4-NCC₅H ₄N)₂]-(BAr′₄) (5), which was also structurally characterized. Moessbauer spectroscopic investigations of solid samples of 1, 3, and 5, in conjunction with several literature values for high-spin iron(II) complexes in an oxygen-rich coordination environment, establish a correlation between isomer shift, coordination number, and N/O composition. The products of oxygenating 1 in CH₂Cl₂ were identified crystallographically to be [Fe₂(μ-OH) ₂(μ-O₂CAr^Tol)₂(O ₂CAr^Tol)₂(4-NCC₅H₄N) ₂]·2(HO₂CAr^Tol) (6) and [Fe ₆(μ-O)₂(μ-OH)₄(μ-O₂CAr ^Tol)₆(4-NCC₅H₄N)₄Cl ₂] (7).

Full text of DOI:10.1021/ja0512531

Published on Web 05/21/2005
Water Affects the Stereochemistry and Dioxygen Reactivity of
Carboxylate-Rich Diiron(II) Models for the Diiron Centers in
Dioxygen-Dependent Non-Heme Enzymes
Sungho Yoon and Stephen J. Lippard*
Contribution from the Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received February 28, 2005; E-mail: lippard@mit.edu
Abstract: Carboxylate-bridged high-spin diiron(II) complexes with distinctive electronic transitions were
5 4
prepared by using 4-cyanopyridine (4-NCC H N) ligands to shift the charge-transfer bands to the visible
region of the absorption spectrum. This property facilitated quantitation of water-dependent equilibria in
Tol
-
Tol
the carboxylate-rich diiron(II) complex, [Fe
2
2 4 5 4 2 2
(µ-O CAr ) (4-NCC H N) ] (1), where O CAr is 2,6-di-(p-
tolyl)benzoate. Addition of water to 1 reversibly shifts two of the bridging carboxylate ligands to chelating
terminal coordination positions, converting the structure from a paddlewheel to a windmill geometry and
Tol
Tol
generating [Fe
in solution, rendering the system thermochromic. Quantitative treatment of the temperature-dependent
spectroscopic changes over the temperature range from 188 to 298 K in CH Cl afforded thermodynamic
2
(µ-O
2 2 2 2 5 4 2 2 2
CAr ) (O CAr ) (4-NCC H N) (H O) ] (3). This process is temperature dependent
2
2
parameters for the interconversion of 1 and 3. Stopped flow kinetic studies revealed that water reacts with
the diiron(II) center ca. 1000 time faster than dioxygen and that the water-containing diiron(II) complex
reacts with dioxygen ca. 10 times faster than anhydrous analogue 1. Addition of {H(OEt
2
)
2
}{BAr′ }, where
4
-
2 3 5 4 2
(µ-O ) (4-NCC H N) ]-
CArTol
BAr4′ is tetrakis(3,5-di(trifluoromethyl)phenyl)borate, to 1 converts it to [Fe
2
(
BAr′ ) (5), which was also structurally characterized. M o¨ ssbauer spectroscopic investigations of solid
4
samples of 1, 3, and 5, in conjunction with several literature values for high-spin iron(II) complexes in an
oxygen-rich coordination environment, establish a correlation between isomer shift, coordination number,
and N/O composition. The products of oxygenating 1 in CH
2
Cl
2
were identified crystallographically to be
Tol
Tol
CArTol) (6) and [Fe
Tol
[
Fe
2
2 2
(µ-OH) (µ-O CAr
N)
2 2
) (O CAr
)
2
(4-NCC
5
H
4
N)
2
]‚2(HO
2
6 2 4 2 6
(µ-O) (µ-OH) (µ-O CAr ) (4-
NCC H
5 4
4
Cl ] (7).
2
Introduction
subtle differences among the crystallographically characterized
enzyme active sites, including the binding modes of the
carboxylates and the existence of variable numbers of water
molecules. These distinctions and the surrounding polypeptide
matrix alter the chemical properties of the core to achieve the
physiologically defined enzyme functions. Understanding how
these shared carboxylate-bridged diiron(II) units work, how the
variable surroundings differentiate their chemical properties, and
the manner in which their dioxygen-activation mechanisms relate
to one another represents a challenging and active area of
research in bioinorganic chemistry.
Carboxylate-bridged diiron units constitute a commonly
occurring element in a growing number of dioxygen-dependent
metalloenzymes.1,2 Important members of this class include the
3
-5
R2 subunit of Class I ribonucleotide reductases (RNR-R2),
6,7
soluble methane monooxygenase (sMMO), fatty acid desatu-
9
8,9
10
rase (∆ D), and toluene monooxygenase (ToMO). Although
these metalloproteins share a common structural motif, their
diiron centers are tuned to achieve diverse functions. There are
(
1) Holm, R. H.; Kennepohl, P.; Solomon, E. I. Chem. ReV. 1996, 96, 2239-
2
314.
(
2) Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S.-K.;
Considerable effort has been expended to synthesize and study
diiron complexes as analogues of the enzyme active sites in
order to enrich our understanding of the chemistry of these
Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y.-S.; Zhou, J. Chem. ReV.
2
000, 100, 235-349.
(
(
3) Stubbe, J.; van der Donk, W. A. Chem. ReV. 1998, 98, 705-762.
4) Logan, D. T.; Su, X.-D.; A° berg, A.; Regnstr o¨ m, K.; Hajdu, J.; Eklund, H.;
Nordlund, P. Structure 1996, 4, 1053-1064.
1
1
systems at the molecular level. A major goal is to reproduce
the architecture of the enzyme active sites using carboxylate-
based ligand frameworks and then to investigate the oxygenation
of the resulting carboxylate-rich diiron(II) complexes. Two
features that, until now, have best been appreciated mainly
(
(
(
(
(
5) Bollinger, J. M., Jr.; Edmondson, D. E.; Huynh, B. H.; Filley, J.; Norton,
J. R.; Stubbe, J. Science 1991, 253, 292-298.
6) Merkx, M.; Kopp, D. A.; Sazinsky, M. H.; Blazyk, J. L.; M u¨ ller, J.; Lippard,
S. J. Angew. Chem., Int. Ed. 2001, 40, 2782-2807.
7) Elango, N.; Radhakrishnan, R.; Froland, W. A.; Wallar, B. J.; Earhart, C.
A.; Lipscomb, J. D.; Ohlendorf, D. H. Protein Sci. 1997, 6, 556-568.
8) Lindqvist, Y.; Huang, W.; Schneider, G.; Shanklin, J. EMBO J. 1996, 15,
6
4
081-4092.
through direct studies of the biomolecules themselves and by
9) Broadwater, J. A.; Ai, J.; Loehr, T. M.; Sanders-Loehr, J. S.; Fox, B. G.
Biochemistry 1998, 37, 14664-14671.
(
10) Sazinsky, M. H.; Bard, J.; Di Donato, A.; Lippard, S. J. J. Biol. Chem.
(11) Du Bois, J.; Mizoguchi, T. J.; Lippard, S. J. Coord. Chem. ReV. 2000,
200-202, 443-485.
2
004, 279, 30610, and refs. cited therein.
8386
9
J. AM. CHEM. SOC. 2005, 127, 8386-8397
10.1021/ja0512531 CCC: $30.25 © 2005 American Chemical Society

Dioxygen-Dependent Non-Heme Enzymes
A R T I C L E S
theoretical methods12 are the effects of water molecules in the
diiron coordination environment and the influence of the
surrounding polypeptide matrix. The importance of certain
amino acid residues in the surrounding polypeptide can be
probed by selective mutagenesis.13,14 The effect of water on the
oxygenation cycle has mainly been a focus of computational
studies, however. In recently published DFT calculations the
coordinated H2O molecule, used as a hydrogen-bond donor, was
considered to be a key component in forming reactive inter-
lations were carried out under nitrogen in an MBraun glovebox. FT-
IR spectra were recorded with a Thermo Nicolet Avatar 360 spectrom-
eter. UV-vis spectra were obtained on a Hewlett-Packard 8453 diode
array spectrophotometer. During variable temperature UV-vis studies,
the temperature was controlled with an Oxford ITC 601 cryostat.
[
Fe
of [Fe
100 mL) was added solid 4-NCC
2
(µ-O
2
CArTol)
CArTol)
(O
4
(4-NCC
5
H
4
N)
(THF)
N (263 mg, 2.53 mmol), and the
2
] (1). To a light yellow solution
CArTol)
] (1.85 g, 1.26 mmol) in CH Cl
2
(µ-O
2
2
2
2
2
2
2
(
5
H
4
dark red pink solution was stirred for 20 min. The solution was filtered
through Celite, and pentane was layered on top. Dichroic red pink
blocks of 1 (1.44 g, 75%) formed overnight. The best crystals for X-ray
diffraction study were obtained by recrystallization from THF/pentane.
1
5
mediate(s) upon oxygenation of the diiron(II) site. The
participation of water in the oxygenation chemistry of the diiron
protein core has also has been invoked by 18O2 studies in which
-1
FT-IR (KBr, cm ) 3050 (w), 3021 (w), 2918 (w), 2862 (w), 2236 (w,
the labeled oxygen atom is not fully incorporated into the
ν
C≡N), 1609 (s), 1551 (w), 1513 (w), 1493 (w), 1440 (m), 1404 (m),
1384 (s), 1303 (w), 1215 (w), 1188 (w), 1109 (w), 1065 (w), 843 (w),
14 (s), 789 (m), 762 (w), 727 (w), 706 (m), 580 (w), 558 (w), 526
m). Anal. Calcd for 1‚0.5(CH Cl ), isolated from methylene chloride,
products.16
8
Recently, we have begun to address the role that coordinated
water might play in the oxygenation cycle of carboxylate-rich
diiron sites in metalloenzymes by using small molecule synthetic
mimics. The reaction between water and carboxylate-rich
diiron(II) complexes has been qualitatively investigated and
iron(II) complexes with varying numbers of water ligands
isolated, revealing that the nature of the isolated carboxylate-
bridged diiron(II) complexes is quite responsive to water present
in the reaction mixture.17,18 Herein we report our efforts to treat
the water-dependent equilibria quantitatively and to address the
effects of water on the rate of oxygenation of the diiron(II)
carboxylate complexes. These studies were facilitated by the
introduction of 4-cyanopyridine (4-NCC5H4N) as a ligand,
which shifts the metal-to-ligand charge-transfer bands of the
starting diiron(II) compounds into the visible region of the
spectrum. In addition, a correlation between isomer shifts in
the M o¨ ssbauer spectra and coordination number for high-spin
iron(II) carboxylate complexes was discovered through com-
parison of the new diiron(II) complexes with related ones in
the literature.
(
2
2
C
103
H93Fe
N
2 4
O
8
Cl: C, 73.93; H, 4.95; N, 3.57. Found: C, 74.44; H,
5
.10; N, 3.40.
Fe (µ-O ] (2). To a rapidly stirred solution
of [Fe (THF) ] (762 mg, 51.0 mmol) in
2 5 4
CH Cl (50 mL) was added solid 4-NCC H N (106 mg, 102 mmol),
4F-Ph
[
2
2
CAr
)
4
(4-NCC
5
H
4
N)
2
CAr4F-Ph
4F-Ph
2
(µ-O
2
)
2
(O
2
CAr
)
2
2
2
yielding a red solution. A red precipitate of 2 (512 mg, 65%) formed
immediately and was isolated by filtration and washed with pentane.
2 2
The solid (20.3 mg) was dissolved in CH Cl (10 mL), and exposure
of the solution to pentane vapor diffusion yielded dichroic yellow-red
-
1
blocks, suitable for X-ray crystallography. FT-IR (KBr, cm ) 3056
w), 2239 (w, νC≡N), 1610 (s), 1569 (w), 1551 (w), 1509 (s), 1452 (w),
(
1
404 (m), 1382 (s), 1299 (w), 1220 (s), 1159 (m), 1095 (w), 844 (m),
8
32 (m), 818 (m), 793 (w), 773 (w), 727 (w), 706 (w), 582 (w), 556
52 8 2 4 8
(m), 532 (w). Anal. Calcd for C88H F Fe N O : C, 67.88; H, 3.37; N,
3
.60. Found: C, 67.55; H, 3.45; N, 3.72.
CArTol
CArTol
[
Fe
2
(µ-O
2
) (O
2 2
)
2
(4-NCC
5
H
4
N)
2
(OH
N)
2
)
2
] (3). To a stirred
Tol
yellow solution of [Fe
2
(µ-O
2
CAr
)
4
(4-NCC
5
H
4
2
] (49.5 mg, 29.8
µmol) in THF (1 mL) was added slowly H O (7 µL) under nitrogen.
2
Dichroic yellow-red needle crystals of 3 (20.3 mg, 44%), suitable for
X-ray crystallography, were obtained by vapor diffusion of pentanes
-
1
into the solution. FT-IR (KBr, cm ) 3653 (w), 3423 (w, br), 2918
w), 2235 (w, νC≡N), 1609 (s), 1515 (s), 1452 (s), 1411 (m), 1381 (s),
217 (w), 1190 (w), 1109 (w), 860 (w), 846 (w), 818 (s), 797 (m), 790
m), 781 (m), 766 (w), 734 (w), 713 (w), 586 (w), 559 (m), 540 (w),
21 (w), 454 (w). Anal. Calcd for C96 10: C, 73.85; H, 5.16;
N, 3.59. Found: C, 73.85; H, 5.48; N, 3.81.
Fe (µ-O (O (4-NCC
CAr4F-Ph) CAr4F-Ph
pound 4 was synthesized as described for 3 and identified by X-ray
crystallography (yield 58%). FT-IR (KBr, cm ) 3655 (w), 3420 (w,
br), 3054 (w), 2244 (w, νC≡N), 1604 (s), 1510 (s), 1451 (s), 1409 (m),
1380 (s), 1301 (w), 1224 (s), 1160 (m), 1093 (w), 1011 (w), 852 (m),
833 (s), 814 (s), 807 (s), 789 (m), 773 (m), 733 (w), 712 (m), 701 (w),
(
1
(
5
Experimental Section
5 4
General Considerations. All reagents, including 4-NCC H N, were
obtained from commercial suppliers and used as received unless
otherwise noted. Dichloromethane, pentane, toluene, and THF were
saturated with argon and purified by passage through activated Al O
2 3
columns under argon.19 Dioxygen (99.994%, BOC Gases) was dried
by passing the gas stream through Drierite. The compounds
2 4
H80Fe N O
[
2
2
2
2
)
2
5 4 2 2 2
H N) (OH ) ] (4). Com-
-
1
Tol
Tol
20
-
Tol
[
Fe
2
(µ-O
2
CAr
)
2
(O
[Fe
2
CAr
)
2
(THF)
2
], where
O
2
CAr is 2,6-di-(p-
2
1,22
CAr4F-Ph
4F-Ph
20
tolyl)benzoate,
2
(µ-O
2
)
2
(CAr
)
2
(THF)
and {H(OEt ) }-
2 2
2
], where
-
4F-Ph
21,22
O
2
CAr
is 2,6-di-(p-fluorophenyl)benzoate,
BAr′}, where BAr′4 is tetrakis(3,5-di(trifluoromethyl)phenyl)bo-
2
3
-
556 (m), 527 (m), 464 (w). Anal. Calcd for C88
5.04; H, 3.47; N, 3.45. Found: C, 65.55; H, 3.57; N, 3.70.
Fe (µ-O (4-NCC N) ][BAr′] (5). To a stirred red-pink
CArTol
solution of [Fe (µ-O (4-NCC
CH Cl (3 mL) was added 1 equiv of {H(OEt
56 8 2 4
H F Fe N O10: C,
{
4
6
rate, were prepared as described in the literature. Air-sensitive manipu-
[
2
2
)
3
5
H
4
2
4
Tol
CAr
)
4
H
5 4
N)
2
] (14.5 mg, 8.7 µmol) in
}{BAr′}, yielding a
(
12) Gherman, B. F.; Baik, M.-H.; Lippard, S. J.; Friesner, R. A. J. Am. Chem.
Soc. 2004, 126, 2978-2990.
2
2
2
2
2 2
)
4
(
13) Skulan, A. J.; Brunold, T. C.; Baldwin, J.; Saleh, L.; Bollinger, J. M., Jr;
Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 8842-8855.
yellow solution under anaerobic conditions. The solution was filtered
through Celite, and pentane was diffused into it. Yellow-red blocks of
(
(
14) Fox, B. G.; Lyle, K. S.; Rogge, C. E. Acc. Chem. Res. 2004, 37, 421-429.
15) Baik, M.-H.; Newcomb, M.; Friesner, R. A.; Lippard, S. J. Chem. ReV.
5
(12.3 mg, 68%), suitable for X-ray crystallography, formed overnight.
2
003, 103, 2385-2419.
-
1
FT-IR (KBr, cm ) 3051 (w), 2920 (w), 2237 (νC≡N), 1614 (m), 1585
(
16) Moe, L. A.; Hu, Z.; Deng, D.; Austin, R. N.; Groves, J. T.; Fox, B. G.
Biochemistry 2004, 43, 15688-15701.
(s), 1514 (s), 1493 (s), 1436 (w), 1405 (m), 1385 (m), 1271 (m), 1219
(
(
(
17) Yoon, S.; Kelly, A. E.; Lippard, S. J. Polyhedron 2004, 23, 2805-2812.
18) Yoon, S.; Lippard, S. J. J. Am. Chem. Soc. 2004, 126, 16692-16693.
19) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers,
F. J. Organometallics 1996, 15, 1518-1520.
(
w), 1030 (w), 832 (s), 807 (s), 791 (m), 636 (w), 586 (m), 560 (w),
528 (w). Anal. Calcd for 5‚0.5(CH Cl
C, 60.63; H, 3.41; N, 2.63. Found: C, 60.74; H, 3.56; N, 3.02.
2
2 2 4 6
) or C107.5H72BClF24Fe N O :
(
(
20) Lee, D.; Lippard, S. J. Inorg. Chem. 2002, 41, 2704-2719.
21) Du, C.-J. F.; Hart, H.; Daniel Ng, K.-K. J. Org. Chem. 1986, 51, 3162-
[Fe
2
Tol
(µ-OH)
2
(µ-O
CAr ) (6) and [Fe (µ-O)
7). A pink-red solution of 1 (15 mg, 0.017 mmol) in CH
2
CArTol
(µ-OH)
)
2
(O
2
CArTol
(µ-O CAr
)
2
(4-NCC
5
H
4
N)
2
2
]‚2(HO -
3
165.
Tol
6
2
4
2
6
) (4-NCC
5
H
4
N) (Cl) ]
4 2
(
22) Chen, C.-T.; Siegel, J. S. J. Am. Chem. Soc. 1994, 116, 5959-5960.
(
2
Cl
2
(10 mL)
(
23) Brookhart, M.; Grant, B.; Volpe, A. F., Jr. Organometallics 1992, 11, 3920-
3
922.
was saturated with dry dioxygen by bubbling over a period of 10 min,
J. AM. CHEM. SOC. VOL. 127, NO. 23, 2005 8387
9

A R T I C L E S
Yoon and Lippard
Table 1. Selected Bond Lengths (Å) for 1-4a
57
Fe M o1 ssbauer Spectroscopy. Zero-field M o¨ ssbauer spectra were
5
7
recorded on an MS1 spectrometer (WEB Research Co.) with a Co
source in a Rh matrix maintained at room temperature in the
Massachusetts Institute of Technology Department of Chemistry
Instrument Facility (MIT DCIF). Solid samples were prepared by
suspending ca. 30 mg of the powdered solids in Apeizon N grease and
packing the mixture into a nylon sample holder. Data were collected
at 4.2 K, and the isomer shift (δ) values are reported with respect to
natural iron foil that was used for velocity calibration at room
temperature. The spectra were fit to Lorentzian lines by using the
WMOSS plot and fit program.28
1
2
3
4
Resonance Raman Spectroscopy. Resonance Raman spectra were
obtained by using an Ar ion laser with excitation provided at 514
Fe1‚‚‚Fe2
Fe1-O1A
Fe1-O1B
Fe1-O2B
Fe1-O1C
Fe1-O1D
2.7754(9)
2.032(3)
2.133(3)
2.7415(16)
2.036(5)
2.120(5)
4.4658(13)
2.0285(13)
2.1475(13)
2.3278(14)
2.0871(13)
4.4019(10)
2.0485(15)
2.1565(15)
2.3352(15)
2.0714(14)
+
nm. A monochromator (1200 grooves/nm grating) with an entrance
slit of 10 µm and a TE-CCD-1100-PB-VISAR detector cooled to 243
K with a circulating water bath was employed in a standard back-
scattering configuration. Data were collected at 296 K in dichloro-
methane using an NMR tube. Each sample concentration was 6 mM.
Measurements were made on more than one freshly prepared sample
and in triplicate to ensure the authenticity of the results. The
2.016(3)
2.132(3)
2.091(4)
2.040(5)
2.112(5)
2.086(7)
b
Fe1-L1
2.1505(16)
2.1686(15)
2.1412(18)
2.1552(18)
Fe1-O1W
Fe2-O2A
Fe2-O2B
Fe2-O2C
Fe2-O2D
2.131(3)
2.010(3)
2.145(3)
2.004(3)
2.100(4)
2.057(5)
2.082(5)
2.064(5)
2.070(5)
2.097(7)
-
1
dichloromethane bands at 1156, 897, 704, and 286 cm were used as
an internal calibration standard. Data were processed using WinSpec
b
Fe2-L2
3
.2.1 (Princeton Instruments, Inc.) and were further manipulated by
Kaleidagraph.
a
Numbers in parentheses are estimated standard deviations in the last
b
significant figures. L indicates the aromatic ring nitrogen atom in
Stopped-Flow Kinetics Experiments. All ambient-pressure kinetics
4
-cyanopyridine. Atoms are labeled as indicated in Figures 1 and 2.
experiments were carried out by using a Canterbury stopped-flow SF-
4
0 and MG-6000 rapid diode array system (Hi-Tech Scientific), fitted
with stainless steel flow lines and an argon-purged anaerobic attachment.
Teflon-lined stainless steel flow lines, adopted in the previous generation
of stopped-flow instruments, retain air and water, causing premature
oxidation or aquation of air-sensitive solutions. The instrument was
resulting in a yellow-green solution. The solution was filtered through
Celite, and pentane was diffused into it. Light green blocks of 6 and
yellow-brown blocks of 7 were obtained after several days. Although
the structures of 6 and 7 could be determined by X-ray crystallography,
bulk samples could not be obtained.
X-ray Crystallographic Studies. Single crystals were mounted at
room temperature on the tips of quartz fibers, coated with Paratone-N
oil, and cooled under a stream of cold nitrogen. Intensity data were
collected on a Bruker (formerly Siemens) APEX CCD diffractometer
running the SMART software package, with Mo KR radiation (λ )
therefore rebuilt to avoid these problems. Solutions of 1 in CH
2 2
Cl
(ca. 0.385 mM) were prepared in a glovebox under a nitrogen
atmosphere and stored in a gastight syringe prior to loading into the
stopped-flow apparatus. A saturated solution of dioxygen was prepared
by bubbling the gas through CH
flask closed with a septum and maintained at 293 K. The saturated
CH Cl solution of dioxygen containing 2 mM water was prepared by
2 2
Cl for 20 min in a round-bottomed
2
2
0
.71073 Å). Data collection and reduction protocols are described in
2
4
bubbling the dioxygen gas into a solution of that composition.
detail elsewhere. The structures were solved by both direct and
Patterson methods and refined on F by using the SHELXTL soft-
ware package. Empirical absorption corrections were applied with
SADABS, part of the SHELXTL program package, and the structures
2
Results and Discussion
2
5
26
In contrast to the relatively rich optical absorption spectra
afforded by low-spin iron(II) complexes, high-spin iron(II)
analogues with carboxylate-rich coordination environments are
typically colorless, making it difficult to apply standard UV-
vis spectroscopic methods to follow their chemistry. For
example, the charge-transfer band in the visible region of the
spectrum are well documented for low-spin iron(II) complexes
27
were checked for higher symmetry by the program PLATON. All
non-hydrogen atoms were refined anisotropically. In general, hydrogen
atoms were assigned idealized positions and given thermal parameters
equivalent to 1.2 (all other hydrogen atoms) times the thermal parameter
of the atom to which they were attached. Hydrogen atoms of the
bridging hydroxides and water molecules were located on difference
electron density maps. In the structure of 1, four THF molecules were
assigned in the lattice and refined isotropically. The structure of 2
2
9,30
having pyridine ligands.
On the other hand, most high-spin
iron(II) complexes are colorless or at best light yellow.2
In our previous studies, the detection of water-dependent
interconversions in carboxylate-rich diiron(II) complexes was
0,31-36
contains four CH
one BAr4′ anion was assigned to two positions, each with a half
2
Cl
2
molecules in the lattice. In the structure of 5,
-
occupancy factor and severe disorder. Four CH
found in the lattice of compound 6. There were six CH
2
Cl
2
molecules were
Cl molecules
2
2
achieved by using solid-state methods, chiefly X-ray crystal-
in the lattice of compound 7. Data collection and experimental details
for the complexes are summarized in Table S1 (Supporting Information)
and relevant interatomic bond lengths and angles for 1-4 are listed in
Tables S2 and S3. Selected geometric details for complexes 1-4 are
reported in Table 1.
17,18
lograpy.
The inability to monitor these reactions in fluid
solution by spectroscopy prohibited their definitive assignment
as equilibrium phenomena. To treat the water-dependent reac-
tions in a quantitative manner, assign equilibrium states, and
address the effects of water on the oxygenation of high-spin
diiron(II) complexes, an optical probe was highly desired. Just
such a probe was discovered with the introduction of 4-cyano-
pyridine as a ligand.
(
(
(
(
24) Kuzelka, J.; Mukhopadhyay, S.; Spingler, B.; Lippard, S. J. Inorg. Chem.
2
004, 43, 1751-1761.
25) Sheldrick, G. M. Program Library for Structure Solution and Molecular
Graphics: Version 6.2, Bruker AXS: Madison, WI, 2000.
26) Sheldrick, G. M. SADABS: Area-Detector Absorption Correction; Uni-
versity of G o¨ ttingen: G o¨ ttingen, Germany, 2001.
27) Spek, A. L. PLATON, A Multipurpose Crystallographic Tool; Utrecht
University: Utrecht, The Netherlands, 2000.
(28) Kent, T. A. WMOSS. M o¨ ssbauer Spectral Analysis Software; Version 2.5,
Minneapolis, MN, 1998.
8388 J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005

Dioxygen-Dependent Non-Heme Enzymes
A R T I C L E S
Scheme 1
Synthesis and Structural Characterization of Diiron(II)
S1 display the structure and Tables 1 and S2 list selected
interatomic distances and angles. Two crystallographically
inequivalent square-pyramidal iron(II) centers, separated by
2.7754(9) Å, are linked by four bridging carboxylate ligands.
The 4-NCC5H4N ligands coordinate to the iron(II) sites in axial
positions through pockets that are generated by the four bulky
carboxylate ligands. Most of geometric features of 1 are the
same as in previously characterized paddle-wheel complexes
Tol
Complexes [Fe2(µ-O2CAr )4(4-NCC5H4N)2] (1) and [Fe2-
4
F-Ph
(
µ-O2CAr
)4(4-NCC5H4N)2] (2). A clue for how to access
high-spin diiron(II) carboxylate complexes with visible absorp-
tion bands came from the electronic spectrum of the [Fe2(µ-
Tol
t
20,37
O2CAr )4(4- BuC5H4N)2],
which has a shoulder at 370 nm
-
1
-1
with ꢀ ) 1400 cm M , thought to be a charge-transfer
transition from Fe(II) to a π* orbital of the pyridine ligand
Tol
t
37
Tol
(
MLCT). If this assignment is correct, we reasoned that, by
[Fe2(µ-O2CAr )4(4- BuC5H4N)2] and [Fe2(µ-O2CAr )4-
p-OMe
38
p-OMe
lowering the energy of the acceptor orbital, it should be possible
to narrow the energy gap between the states involved in this
transition. This hypothesis was tested by synthesizing 4-NCC5H4N
(BA
)2], where BA
is 4-methoxylbenzylamine.
A powdered sample of 1 is stable in the solid state in air for
over 2 months, judging by the maintenance of its distinctive
color.
R
t
analogues of [Fe2(µ-O2CAr )4(4- BuC5H4N)2], anticipating a red
shift of the electronic transition. Upon addition of 2 equiv of
Treatment of a light yellow CH2Cl2 solution of [Fe2(µ-
Tol
4F-Ph
4F-Ph
4
-NCC5H4N to a light yellow solution of [Fe2(µ-O2CAr )2-
O2CAr
)2(O2CAr
)2(THF)2] with 2 equiv of 4-NCC5H4N
Tol
4F-Ph
(O2CAr )2(THF)2] in CH2Cl2, the color instantaneously turned
afforded the red diiron(II) complex [Fe2(µ-O2CAr
)4(4-
to intense red-pink, affording the desired diiron(II) complex
NCC5H4N)2] (2) (Scheme 1), which is the structural analogue
of 1 (Figure S2). Selected interatomic distances and angles are
listed in Tables 1 and S2. Since 2 is not very soluble in most
solvents, further studies were performed mainly with compound
Tol
[Fe2(µ-O2CAr )4(4-NCC5H4N)2] (1) (Scheme 1). When isolated
from solution, however, the crystals developed fissures, probably
due to loss of solvent molecule(s) in the lattice. Figures 1 and
1
.
(
29) Monat, J. E.; McCusker, J. K. J. Am. Chem. Soc. 2000, 122, 4092-4097.
30) Roelfes, G.; Vrajmasu, V.; Chen, K.; Ho, R. Y. N.; Rohde, J.-U.; Zondervan,
C.; la Crois, R. M.; Schudde, E. P.; Lutz, M.; Spek, A. L.; Hage, R.; Feringa,
B. L.; M u¨ nck, E.; Que, L., Jr. Inorg. Chem. 2003, 42, 2639-2653.
31) Lee, D.; Lippard, S. J. Inorg. Chim. Acta 2002, 341, 1-11.
Syntheses and Structural Characterization of Water-
(
Tol
Containing Diiron(II) Complexes [Fe2(µ-O2CAr )2(O2-
CAr )2(4-NCC5H4N)2(OH2)2] (3) and [Fe2(µ-O2CAr
(O2CAr
CH2Cl2 or THF solution containing a measured amount of water
resulted in an instantaneous color change from red-pink to light
yellow-red. Out of these solutions crystallized compound 3
following vapor diffusion of pentane into it. The structure of 3
is displayed in Figures 2 and S3, with interatomic distances
Tol
4F-Ph
)-
(
(
4
F-Ph
32) Hagadorn, J. R.; Que, L., Jr.; Tolman, W. B. J. Am. Chem. Soc. 1998, 120,
)2(4-NCC5H4N)2(OH2)2] (4). Addition of 1 to a
1
3531-13532.
(
(
33) Lee, D.; DuBois, J. L.; Pierce, B.; Hedman, B.; Hodgson, K. O.; Hendrich,
M. P.; Lippard, S. J. Inorg. Chem. 2002, 41, 3172-3182.
34) Lee, D.; Hung, P.-L.; Spingler, B.; Lippard, S. J. Inorg. Chem. 2002, 41,
5
21-531.
(
35) Lee, D.; Lippard, S. J. Inorg. Chem. 2002, 41, 827-837.
36) Lee, D.; Pierce, B.; Krebs, C.; Hendrich, M. P.; Huynh, B. H.; Lippard, S.
J. J. Am. Chem. Soc. 2002, 124, 3993-4007.
(
(
37) Lee, D.; Du Bois, J.; Petasis, D.; Hendrich, M. P.; Krebs, C.; Huynh, B.
H.; Lippard, S. J. J. Am. Chem. Soc. 1999, 121, 9893-9894.
(38) Yoon, S.; Lippard, S. J. Inorg. Chem. 2003, 42, 8606-8608.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005 8389

A R T I C L E S
Yoon and Lippard
Figure 1. Top: ORTEP diagram of [Fe2(µ-O2CArTol)4(4-NCC5H4N)2] (1)
Figure 2. Top: ORTEP diagram of [Fe2(µ-O2CArTol)2(O2CArTol)2(4-
NCC5H4N)2(OH2)2] (3) showing 50% probability thermal ellipsoids for all
non-hydrogen atoms. Bottom: Structure with the aromatic rings of
showing 50% probability thermal ellipsoids for all non-hydrogen atoms.
Tol
-
Bottom: Structure with the aromatic rings of Ar CO2 ligands omitted
for clarity.
Tol
-
Ar CO2 ligands omitted for clarity and depicting H-bonding interactions
dotted lines).
(
and angles listed in Tables 1 and S3. Two distorted octahedral
iron(II) centers are bridged by two carboxylate ligands with an
Fe‚‚‚Fe separation of 4.4658(13) Å. This value is 1.69 Å longer
than the Fe‚‚‚Fe separation of 2.7754(9) Å in 1 and arises from
water-induced shifting of two carboxylate ligands from a
bridging to a terminal chelating mode. The reaction typifies how
the binding of water in carboxylate-bridged diiron(II) complexes
can induce a dramatic structural change. The nonbridging
coordination sites are occupied by 4-NCC5H4N, water, and the
bidentate terminal carboxylate ligands. The assignment of water
as the ligand rather than hydroxide is based on the Fe-O(aqua)
distance, 2.1686(15) Å, the location and refinement of the
associated hydrogen atoms in the X-ray structure determination
by X-ray crystallography, the large unit cell volume and severe
disorder in the BAr′4 counterion prohibited satisfactory re-
-
finement. Drawings of 5 are depicted in Figure S5. The two
pseudo-tetrahedral iron(II) sites with NO3 coordination geom-
etries are bridged by three carboxylates at an Fe‚‚‚Fe distance
of 3.11 Å. Two 4-NCC5H4N ligands coordinate to the iron(II)
sites through the pockets generated by the three bulky carbox-
ylate ligands.
M o1 ssbauer Spectroscopy. Correlation of Isomer Shift with
Core Composition. Zero-field M o¨ ssbauer spectra of solid
samples of 1, 2, 3, and 5 were recorded at 4.2 K in the absence
of an external field. These spectra are displayed in Figures S6
and S7 and M o¨ ssbauer parameters are listed in Table 2, which
also includes results for related carboxylate-rich iron(II) com-
plexes and enzymes of interest. The M o¨ ssbauer spectra of both
(O-H, 0.71 and 0.86 Å), and charge considerations. Inter- and
intramolecular hydrogen bonding interactions between the metal-
bound water molecules and the carboxylate ligand are character-
ized by short O-H‚‚‚O distances, ranging from 2.731 Å to 2.755
Å (Figure 2, bottom). The intermolecular hydrogen bonds may
contribute to the observed hardness of the crystals. When 3 is
dissolved in anhydrous CH2Cl2, a red-pink color is obtained
instead of red-yellow, indicating that the coordinated water
ligands observed in the solid state are readily displaced from
the first coordination sphere. Compound 4 is essentially iso-
structural with 3 (Figure S4 and Table S3).
1
and 2 exhibit slightly asymmetric quadrupole doublets,
probably reflecting the inequivalence of the two iron atoms in
the crystal lattice. By contrast, the iron centers of compound 3
are crystallographically equivalent, resulting in spectra that are
symmetric. The isomer shifts of these compounds compare well
with those of other high-spin iron(II) complexes having oxygen-
rich coordination environments, indicating that compounds 1-3
3
9
and 5 have high spin S ) 2 ground states.
Synthesis and Characterization of [Fe2(µ-O2CArTol)3(4-
One obvious trend in the data of Table 2 is that iron(II) sites
with coordination number 4 tend to have lower isomer shifts
NCC5H4N)2](BAr′) (5) Upon Reaction of Compound 1 with
4
+
-1
H . When compound 1 was titrated with a CH2Cl2 solution of
(1.0-1.1 mm s ) compared to coordinatively saturated iron(II)
2
3
-1
[
H(OEt2)2]{BAr′}, the intense red-pink color disappeared
centers (∼1.3 mm s ). A possible explanation for this trend is
4
following addition of ca. 1.2 equiv of the acid, generating a
yellow solution. Although the structure of 5 could be determined
(39) Tshuva, E. Y.; Lippard, S. J. Chem. ReV. 2004, 104, 987-1012.
8390 J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005

Dioxygen-Dependent Non-Heme Enzymes
A R T I C L E S
Table 2. Zero-Field M o¨ ssbauer Parameters at 4.2 K for 1-3, 5, Carboxylate-Rich Iron(II) Complexes, and Related Enzymes
coordination geometry
δ
(mm s-1)
∆E
Q
(mm s-1)
ref
a
[
5
[
[
Fe(O2CAr4F-Ph)2(Hdmpz)2]
Td, N2O2
1.04(2)
1.04(2)
1.08(2)
1.05(2)
1.23(2)
1.10(2)
1.11(2)
1.12(2)
1.12(2)
1.13(2)
1.19(2)
1.19(2)
1.26(2)
1.27(2)
1.26(2)
1.27(2)
1.29(2)
1.31(2)
1.35(2)
1.30(2)
1.17(2)
3.19(2)
2.85(2)
2.46(2)
2.18(2)
2.80(2)
3.52(2)
3.02(2)
3.00(2)
3.05(2)
2.91(2)
2.90(2)
2.99(2)
2.90(2)
3.00(2)
2.96(2)
2.90(2)
3.09(2)
2.86(2)
3.26(2)
3.53(2)
2.35(2)
2.87e
i
h
31
31
Td, NO3
Td, N2O2
Td, NO3
Fe(O2CArTol)2(1-BnIm)2]
Fe2(µ-O2CAr )3(O2CArTol)(2,6-lutidine)2]
Tol
site 1
site 2
b
Tbp, O5
[
1
2
Fe(O2CArTol)2(BPTA)2]
Tbp, N3O2
31
h
h
20
31
20
35
20
j
j
j
h
k
c
Sp, NO4
Sp, NO4
Sp, NO4
Tbp, N2O3
Tbp, NO4
Tbp, NO4
Tbp, O5
Tol
t
[
[
[
[
[
[
[
[
Fe2(µ-O2CAr )4(4- BuC5H4N)2]
4
-tBuPh
Fe(O2CAr
)2(2,2′-bipy)2]
Tol
Tol
Fe2(µ-O2CAr )2(O2CAr )2(C6H5N)2]
Tol
Tol
Fe2(µ-O2CAr )2(O2CAr )2(Bn2en)2]
Tol
Tol
Fe2(µ-O2CAr )2(O2CAr )2(THF)2]
4
4
4
-tBuPh
-tBuPh
-tBuPh
d
Fe4(µ-OH)4(µ-O2CAr
Fe4(µ-OH)4(µ-O2CAr
Fe4(µ-OH)4(µ-O2CAr
)2(µ-OTf)2(C6H5N)4]
Oh, NO5
t
)2(µ-OTf)2(4- BuC5H4N)4]
Oh, NO5
Oh, NO5
Oh, NO5
Oh, O6
Oh, O6
Oh, O6
)2(µ-OTf)2(3-FC6H4N)4]
3
Tol
[
[
[
Fe2(µ-OH2)2(µ-O2CAr ) 2(O2CArTol)2(THF)2]
4
F-Ph
4F-Ph
Fe2(µ-OH2)2(µ-O2CAr
)(O2CAr
)3(THF)2(OH2)]
18
i
Tol
Fe4(µ3-OCH3)4(O2CAr )4(HOCH3)6]
site 1
site 2
Tbp, O5
e
MMOH
1.30
l
f,g
f,g
1.3
2.4-3.1
m
n
o
RNR-R2
∆
1.26
3.13
9D
1.30g
3.04-3.36g
a
Tetrahedral. b Trigonal bipyramidal. Square pyramidal. d Octahedral. e Methylococcus capsulatus (Bath). f Methylosinus trichosporium OB3b. Two
c
g
h
i
j
k
quadrupole doublets. This work. Supporting Information. Lee, D.; Sorace, L.; Caneschi, A.; Lippard, S. J. Inorg. Chem. 2001, 40, 6774. Kelly, A. E.;
l
Lippard, S. J. Unpublished result. Liu, K. E; Valentine, A. M.; Wang, D.; Huynh, B. H.; Edmondson, D. E.; Salifoglou, A.; Lippard, S. J. J. Am. Chem.
Soc. 1995, 117, 10174. m Pulver, S. C.; Froland, W. A.; Fox, B. G.; Lipscomb, J. D.; Solomon, E. I. J. Am. Chem. Soc. 1993, 115, 12409. Pulver, S. C.;
n
o
Tong, W. H.; Bollinger, M. J., Jr.; Stubbe, J.; Solomon, E. I. J. Am. Chem. Soc. 1995, 117, 12664. Fox, B. G.; Shanklin, J.; Somerville, C.; M u¨ nck, E.;
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2486.
the following.40 The isomer shift is defined by the expression
in eq 1, where S and A are two different chemical environments
termination (Figure S8) of which are supplied as Supporting
Information, contains three octahedral (O6) and a trigonal
bypyramidal (O5) iron(II) sites and displays two quadrupole
doublets in a 3:1 ratio (Figure S9). It is noteworthy that the
iron(II) sites with O6 coordination environments have a larger
isomer shift of 1.30(2) mm/s than the iron(II) site with O5
geometry, 1.17(2) mm/s in this compound.
2
2
δ ) constant × (|ψ (0) | - |ψ (0) | )
(1)
s
S
s
A
with S the sample and A a reference absorber.41 The expectation
2
value |ψs(0)S | describes the s-electron density at the iron
nucleus. The isomer shift of an iron complex is dictated by two
major factors, a direct contribution from 4s-electron density and
indirect contributions from the 3d-electrons that shield the
s-electrons from penetrating the nucleus.42 Since the direct
contribution in the series of ferrous complexes in the table is
fixed, only the indirect contributions are variable. The shielding
can be considered as arising from the nonbonding influence of
Superimposed on the effects of coordination geometry is the
N/O composition of the coordination environment of iron for
the compounds in Table 2. Of particular interest are carboxylate-
bridged diiron(II) species with a single N- and a variable number
of O-atom donors, which includes compounds 1-3 reported
here. These environments represent possibilities for the reduced
forms of bacterial multcomponent monooxygenases and related
6
the 3d electronic configuration of iron(II), attenuated by
3-5
non-heme diiron enzymes. The isomer shift of the diiron(II)
covalency in which ligands donate an effective total of x
electrons from their filled orbitals to the metal 3d-orbitals and
by covalency involving donation of an effective number y of
-1 6
sites in reduced sMMOH is reported to be ∼1.30 mm s ,
which is in the range of six coordinate NO5 iron(II) sites. This
comparison suggests that the metal centers in the enzyme may
be six-coordinate. The X-ray structural work indicates six
ligands in the vicinity of each iron atom, with some of the water
3
d metal electrons into π* ligand acceptor orbitals. Thus, the
effective number of shielding 3d-electrons (Neff) can be de-
scribed by Neff ) 6 + x - y. Because the complexes listed in
Table 2 have ligand frameworks that are only weakly π acidic,
the y contribution can be considered to be negligible. The
covalency contribution, x, is expected to follow the order six-
43
molecules interacting only weakly with the diiron(II) center.
The correlation between coordination number and M o¨ ssbauer
isomer shift for the NOx complexes in Table 2 should assist the
interpretation of results for other carboxylate-bridged diiron
proteins in the literature.
>
five- > four-coordination. Larger isomer shifts will therefore
occur for complexes having higher Neff values, as observed for
Electronic and Resonance Raman Spectroscopy. The
the compounds listed in Table 2. The complex [Fe4(µ-OMe)4-
4-cyanopyridine ligand affords a valuable spectroscopic signa-
Tol
(
O2CAr )4(HOMe)6](HOMe), the synthesis and structure de-
ture for monitoring the chemistry of its carboxylate-bridged
diiron(II) complexes. A similar strategy for investigating the
ferrous heme in a cytochrome P450 appeared recently, while
(
(
(
40) Dabrowiak, J. C.; Merrell, P. H.; Stone, J. A.; Busch, D. H. J. Am. Chem.
Soc. 1973, 95, 6613-6622.
41) Drago, R. S. Physical Methods in Chemistry; W. B. Saunders Co.:
Philadelphia, PA, 1977.
42) Gibb, T. C. In Principles of M o¨ ssbauer Spectroscopy; Chapman & Hall:
New York, 1976; pp 93-101.
44
this report was in preparation. The intense colors of 1 and 2
(43) Whittington, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2001, 123, 827-838.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005 8391

A R T I C L E S
Yoon and Lippard
Figure 3. UV-vis spectra of [Fe2(µ-O2CArTol)4(4-NCC5H4N)2] (1) and
4
F-Ph
[Fe2(µ-O2CAr
)4(4-NCC5H4N)2] (2) in CH2Cl2.
observed in the solid state are also evident in CH2Cl2 solutions
of the compounds, with λmax values occurring at 510 nm (ꢀ )
-1
-1
-1
-1
2
200 cm M ) and 480 nm (ꢀ ) 2300 cm M ), respectively
(Figure 3). Considering that the iron(II) sites of 2 are coordinated
by carboxylates with more electron-withdrawing groups, the
observed difference (blue shift) in the energy of the electronic
transition supports a MLCT assignment. To gain more insight
into the origin of this unusual electronic transition, the resonance
Raman (rR) spectrum of 1 was measured in CH2Cl2 solution at
ambient temperature with excitation at 514 nm. The result is
presented in Figure S10, which displays the spectra of 1 and,
for comparison, 4-NCC5H4N (literature data). Several strong
-1
resonance-enhanced peaks appeared in the 700 cm to 1800
-
1
cm region for 1. These features are also present in the rR
spectrum of the 4-NCC5H4N ligand, indicating that the chro-
mophore involves the N-donor ligand. On the basis of the high
intensities of the electronic transitions, the blue shift of the more
electron-poor iron(II) complex, and the resonance enhanced
peaks corresponding to the 4-NCC5H4N ligand, the visible bands
of 1 and 2 are assigned to an Fe(II) f 4-NCC5H4N charge
transfer (MLCT) transition.
Equilibria in Coordinating Solvents. No marked thermo-
chromism of 1 occurs in CH2Cl2 or toluene solutions of 1. When
coordinating solvents such as THF, acetonitrile, or benzonitrile
are employed, however, the visible band of 1 is absent at
ambient temperature and grows in as the temperature is lowered
Figure 4. (Top) UV-vis spectra of [Fe2(µ-O2CArTol)4(4-NCC5H4N)2] (1)
in THF, measured at variable temperatures. (Bottom) The absorbance change
at 490 nm fit to eq 2; the line represents a least-squares fit to the data
points, and the resulting estimated errors for the each parameter are shown
in parentheses in the text.
a quantitative treatment of the equilibrium. A mathematical
equation describing the model of eq 2 is derived in Appendix
1
(Supporting Information), which assumes no change in the
extinction coefficient (ꢀ) of 1 over the entire temperature range.
The resulting thermodynamic parameters are ∆H ) 19.0 (6) kJ
-
1
-1
-1
mol , ∆S ) -16.2 (16) J mol K , and ꢀ490 ) 4650 (280)
-
1
-1
M cm , with R ) 0.9996, and the fit is shown in Figure 4
bottom). The observed thermochromism arises from the small
(Figure 4, top). The effect is fully reversible. This solvent-
(
dependent thermochromism was further investigated in THF
solution, where the complex is more soluble. Addition of 2 equiv
of 4-NCC5H4N to a THF solution of 1 at ambient temperature
restored its red color, indicating the existence of the equilibrium
depicted in Scheme 2 and described by eq 2,
positive enthalpy and negative entropy for the reaction. The
electron-withdrawing cyano group of the 4-NCC5H4N ligand
weakens its donor strength, contributing to the small positive
enthalpy. The large excess of the coordinating THF solvent
displaces the 4-NCC5H4N ligands from 1. Because of the
tendency for coordinating solvents to react with 1, further studies
were carried out in noncoordinating CH2Cl2.
1
+ 2(THF) h W + 2(4-NCC H N)
(2)
5
4
where W is [Fe2(µ-O2CArTol)2(O2CArTol)2(THF)2]. Fitting the
measured absorbance change over a temperature range afforded
(
44) Ost, T. W. B.; Clark, J. P.; Anderson, J. L. R.; Yellowlees, L. J.; Daff, S.;
Chapman, S. K. J. Biol. Chem. 2004, 279, 48876-48882.
8392 J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005

Dioxygen-Dependent Non-Heme Enzymes
A R T I C L E S
Scheme 2
Water-Dependent Reactions. Recently we reported the
anhydrous CH2Cl2 at room temperature, the UV-vis spectrum
of the resulting red-pink (λmax ) 510 nm) solution exactly
matches that of 1. Dissolving 1 in CH2Cl2 that is saturated with
water produces the electronic transition at 460 nm, characteristic
of 3 in the solid state. These results indicate that 1 and 3 are
related by a water-dependent equilibrium. UV-vis spectra of a
CH2Cl2 solution of 3 were measured over the temperature range
283 K to 193 K (Figure 5). Upon lowering the temperature from
observation of water-dependent interconversions involving
Tol
Tol
the compounds [Fe2(µ-OH2)2(µ-O2CAr )2(O2CAr )2(4-
t
BuC5H4N)2] and [Fe2(OH2)2(µ-O2CAr4F-Ph)2(O2CAr
17
4F-Ph
)2-
17,18
(
THF)2].
A quantitative treatment of the putative equilibrium
processes was not possible, however, due to the absence of
optical spectroscopic bands for following the reactions in
solution. When the yellow-red compound 3 is dissolved in
2
73 to 203 K, there is a gradual shift of the peak maximum
from 510 to 460 nm, with two isosbestic points at 480 and 360
nm. The peak at 510 nm originates from compound 1, and that
at 460 nm is assigned to a water-containing compound 3. The
two isosbestic points suggest that 1 and 3 are the only
spectroscopically active species involved in the thermochromic
behavior over the temperature range from 273 to 203 K. Below
203 K, however, the absorbance at 460 nm continues to increase
and the isosbestic points disappear.
A possible explanation for the observed, reversible thermo-
chromism is the addition of water molecules to 1, as displayed
in Scheme 3. Dissolution of 3 effects the dissociation of water
molecules from the diiron site, generating 1. Binding of water
molecules to 1 decreases the absorbance at 510 nm while
increasing that at 460 nm, indicating the generation of compound
3
. The disappearance of isosbestic points below 203 K can be
explained by both the increased overall molar concentration of
all components in solution as temperature decreases and the
turbidity arising from the generation of the clathrate CH2Cl2‚
3
4H2O at low temperature. The formation of such a clathrate
below -40 °C in 2-4 mM H2O in CH2Cl2 has been noted
4
5
previously. A mathematical equation describing this model
eq 3) is derived in Appendix 2 (Supporting Information).
(
1
+ 2H O h 3
(3)
2
Fitting the temperature dependence of the observed absorb-
ance change at 510 nm resulted in the thermodynamic and
-
1
spectral parameters ∆H ) - 95 (11) kJ mol , ∆S ) -253
-
1
-1
-1
-1
(
1
47) J mol K , ꢀ510 ) 2001 (26) M cm for 1, and ꢀ510 )
-
1
-1
090 (8) M cm for 3 with R ) 0.986 (Figure 5, bottom).
The negative enthalpy results from the energy of bond formation
between water and the diiron(II) site, and the negative entropy
corresponds to a formally termolecular reaction. Although water
binding is not tight at room temperature, it becomes very
favorable at low temperature. Recently reported oxygenation
studies of diiron(II) complexes in a carboxylate-rich coordination
environment described, without explanation, the inhibition of
intermediates generated at low temperature by a trace amount
4
6
of water. We suggest that the binding of water molecules to
(
45) Kryatov, S. V.; Rybak-Akimova, E. V.; MacMurdo, V. L.; Que, L., Jr.
Inorg. Chem. 2001, 40, 2220-2228, and refs. cited therein.
46) Kryatov, S. V.; Chavez, F. A.; Reynolds, A. M.; Rybak-Akimova, E. V.;
Que, L., Jr.; Tolman, W. B. Inorg. Chem. 2004, 43, 2141-2150, and refs.
cited therein.
Figure 5. (Top) UV-vis spectra of [Fe2(µ-O2CArTol)4(4-NCC5H4N)2] (3)
in CH2Cl2, measured at variable temperatures. (Bottom) The absorbance
change at 510 nm fit to eq 3.
(
J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005 8393

A R T I C L E S
Scheme 3
Yoon and Lippard
a
Table 3. Rate Constants (k1obs) for Oxygenation of 1 in CH2Cl2
their carboxylate-rich diiron(II) complexes at low temperature
may be the cause of this behavior.
temperature (K)
k1obs
The properties observed for the present complexes have not
yet been encountered for diiron(II) units having nitrogen-rich
environments, suggesting perhaps that carboxylate-rich coor-
dination spheres might be especially amenable to supporting
diverse geometries depending on the amount of water available.
Our findings thus suggest the possible occurrence of related
water-dependent equilibria in enzymes having several glu-
tamate or aspartate ligands bound to their diiron centers, and
variable numbers of water molecules are indeed encountered at
2
283
93
1.4(3)
0.71(6)
0.40(2)
0.20(1)
0.10(1)
0.045(5)
2
2
2
73
63
53
243
a
k1obs is defined by the following reaction:
k1obs
Fe (II,II) + O2
8final product
2
4
3
47
9
8
the active sites of sMMOH,
RNR-R2,
∆ D, and
ToMOH.10 In reduced sMMOH, for example, there are two
water molecules, compared with no water molecules at all at
derived from time-dependent absorbance changes by using a
nonlinear least-squares fitting procedure based on eq 4, where
9
the diiron(II) core of ∆ D. During catalysis the accessible
A ) A - (A - A )exp(-kobst)
(4)
t
∞
∞
0
amount water at the non-heme diiron(II) sites may differ even
more and function to allow a shared structural motif to achieve
its diverse roles.
A∞ and A0 are the optical absorbances at infinite and zero times,
respectively. The fast oxygenation of 1 in solution contrasts with
its observed inertness to O2 in the solid state, indicating that
the reactive species in solution may not have a quadruply
bridged diiron(II) structure. An equilibrium between doubly and
quadruply bridged isomers of 1 in the solution state is likely to
occur, with concomitant carboxylate shifts. Such an equilibrium
has been proposed and is generally accepted for the analogous
complex, [Fe2(µ-O2CAr )4(4- BuC5H4N)2]. One of these
isomers, probably a doubly bridged complex, would react with
dioxygen.
Competition between dioxygen and water for 1 was inves-
tigated between 193 and 293 K by using stopped-flow kinetics.
To obtain pseudo-first-order conditions, the concentrations of
Effect of Water on the Oxygenation Rate of Compound
1. The influence of water on the oxygenation chemistry of
carboxylate-rich diiron(II) complexes is of interest because the
active sites in reduced sMMOH contain two water molecules
in the first and second coordination spheres. Such water may
modulate the oxygenation reactions of the diiron(II) centers in
the enzyme. The reaction of compound 1 with dioxygen
instantaneously produces a light yellow-green solution. The
kinetics of this process were followed by stopped-flow spec-
troscopy, monitoring the charge-transfer band, a broad feature
centered at 510 nm for 1 (Figure 6). When examined in the
presence of excess dioxygen, the decay of compound 1 follows
a pseudo first-order rate law. Rate constants (Table 3) were
Tol
t
36
1
, water, and dioxygen were maintained at 0.19, 1.00, and 2.9
mM, respectively. The reaction proceeds through the extremely
fast generation of a species that absorbs at 460 nm and
subsequently decays (Figure 7). The first steps were only
detectable at temperatures below 223 K and went to completion
within 1 s. The time-dependent absorbance changes follow a
pseudo first-order rate law and the fit results are listed in Table
4
. The second step proceeds considerably more slowly than the
first. The decay at 460 nm also follows a pseudo first-order
rate law, and rate constants were determined by a linear squares
fit to eq 4. The kinetic behavior can be rationalized by the
following scenario (Scheme 4). The species with an absorption
maximum at 460 nm, the formation of which is correlated with
the decrease of the band at 510 nm, is assigned as the water-
containing compound 3, generated by aquation of 1 (see above),
and the decay of this peak occurs through oxygenation. This
analysis provides two important insights. First, aquation of 1 is
at least 1000 times faster than oxygenation of 1. The rate
-1
constant for aquation (k2obs) of 1 at 223 K is 63.5(8) s , whereas
Figure 6. Spectral changes occurring during oxygenation of 1 (1.74 ×
-
4
1
0
M) without water in CH2Cl2 at 273 K over 20 s.
(47) Nordlund, P.; Eklund, H. J. Mol. Biol. 1993, 232, 123-164.
8394 J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005

Dioxygen-Dependent Non-Heme Enzymes
A R T I C L E S
Scheme 4
Table 5. Selected Interatomic Distances (Å) and Angles (deg) for
a
6
Fe1‚‚‚Fe2
Fe1-O1
Fe1-O2
Fe1-O3
Fe1-O5
Fe1-O7
Fe1-N1
Fe2-O1
Fe2-O2
Fe2-O4
2.8323(10)
1.950(3)
1.978(3)
2.045(3)
2.076(3)
1.955(3)
2.132(4)
1.999(3)
1.945(3)
2.053(3)
Fe2-O6
2.033(3)
2.127(4)
2.888(5)
2.639(5)
2.636(5)
2.814(6)
43.50(10)
43.33(10)
88.19(14)
87.75(14)
Fe2-N3
O8‚‚‚O1
O8‚‚‚O11
O10‚‚‚O13
O10‚‚‚O2
Fe1-O1-Fe2
Fe1-O2-Fe2
O1-Fe1-O2
O1-Fe2-O1
a
Numbers in parentheses are estimated standard deviations of the last
significant figure. Atoms are labeled as indicated in Figure 8.
Figure 7. (Top) Spectral changes that occur during the reaction of 1 (1.60
-4
×
10 M) with dioxygen and water in CH2Cl2 at 213 K over 1 s. (Bottom)
Spectral changes that occur during oxygenation of 1 (1.60 × 10 M) with
Table 6. Selected Interatomic Distances (Å) and Angles (deg) for
-
4
a
7
dioxygen and water in CH2Cl2 at 253 K over 10 s.
Fe1‚‚‚Fe2
Fe2‚‚‚Fe3
Fe1‚‚‚O1
Fe1-O2
Fe1-O3
Fe1-O4
Fe1-O11
Fe2-O3
Fe2-O4
3.064(4)
2.960(3)
1.881(7)
2.029(7)
2.004(7)
2.042(8)
2.103(7)
1.999(7)
2.034(9)
Fe2-O10
2.059(7)
2.085(7)
2.019(8)
2.063(7)
2.181(9)
2.311(3)
99.9(3)
Fe2-O6
Table 4. Rate Constants k2obs and k3obs for Oxygenation of 1 in
CH2Cl2 with Water
Fe2-O12
Fe3-O7
temperature (K)
k2obs (s-1)a
temperature (K)
k3obs (s-1)a
Fe3-N2
Fe3-Cl(1)
Fe1-O3-Fe2
Fe1-O4-Fe2
Fe2-O12-Fe3
2
2
2
2
2
1
23
18
13
08
03
98
62.8(5)
43.0(24)
23.7(17)
11.1(8)
5.1(2)
293
283
273
263
253
243
14.6(7)
9.1(10)
5.5(5)
97.5(4)
95.7(3)
2.7(2)
1.21(17)
0.39(12)
0.12(1)
a
Numbers in parentheses are estimated standard deviations of the last
significant figure. Atoms are labeled as indicated in Figure 9.
2.5(1)
233
a
into the light greenish-yellow CH2Cl2 solution generated upon
oxygenation of 1 affords crystals, an inseparable mixture of the
quadruply bridged diiron(III) complex 6 and the hexairon(III)
species 7. The structure of 6 is depicted in Figure 8, and selected
interatomic distances and angles are listed in Table 5. Two
distorted octahedral iron(III) atoms are bridged by two hydroxide
and two carboxylate ligands. The Fe‚‚‚Fe distance of 2.8323(10)
Å is in the ∼2.85 Å range typical for previously reported
k2obs and k3obs are defined by following the reaction:
k2obs
k3obs
1
+ H O + O
8 3
8 final product
2
2
-
1
that for oxygenation (k1obs) at 243 K is 0.045(5) s . This
dramatic difference in rate constants may originate from the
small but obvious size difference between water and dioxygen
and the hydrogen bond donating ability of the former. Second,
the water-containing compound 3 reacts with dioxygen ca. 10-
fold faster than does 1. We suggest that the lability of the water
ligands in 3 affords an open coordination site for dioxygen
binding (Scheme 4), an event that may facilitate Fe-O2 bond
formation, which is presumably the rate-determining step for
oxidation.
2
+
36
quadruply bridged {Fe2(µ-OH)2(µ-O2CR)2}
cores. Two
intramolecular hydrogen bonding interactions occur between the
terminal, metal-bound carboxylates and the bridging hydroxo
groups, with O-H‚‚‚O distances of 2.818(6) and 2.888(5) Å.
Tol
In addition, two carboxylic acids (HO2CAr ) are located in
proximity to the terminal carboxylates in the crystal lattice,
generating hydrogen-bonding interactions.
The hexairon(III) species 7 was isolated with compound 6.
Its structure is best described by the diagrams in Figure 9, and
a list of selected interatomic distances and angles is included
Isolation and Structural Characterization of Iron(III)
Complexes
4-NCC5H4N)2]‚2(HO2CAr ) (6) and [Fe6(µ-O)2(µ-OH)4(µ-
O2CAr )6(4-NCC5H4N)4(Cl)2] (7). Vapor diffusion of pentane
Tol
Tol
[Fe2(µ-OH)2(µ-O2CAr )2(O2CAr )2-
Tol
(
Tol
J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005 8395

A R T I C L E S
Yoon and Lippard
experiment was performed in the absence of light, the same
mixture of 6 and 7 resulted, indicating that a photochemical
-
reaction is not involved in generating the Cl anion.
Mechanistic Considerations. Based on the spectroscopic,
structural, and kinetic data obtained, a mechanism is proposed
to account for the observed chemical conversions (Scheme 4).
An equilibrium between the doubly and quadruply bridged
isomers of 1 in the solution state can occur through carboxylate
shifts. One of these isomers, most likely the doubly bridged
complex, reacts with dioxygen faster than the other, quadruply
bridged one. This explanation accounts for the marked difference
in the oxygenation of solid versus solution samples of 1. In the
competition between water and dioxygen, aquation occurs first
and the resulting aqua complex then reacts with dioxygen. The
faster reaction rate of the aquated species compared to 1 is
ascribed to the favorable formation of an available coordination
site generated by rapid, reversible dissociation of water. Oxida-
-
tion of the solvent, CH2Cl2, generates Cl anions, which
coordinate to the iron(III) site, leading to reorganization of the
diiron species to hexanuclear 7. The dissociated carboxylic acid
interacts with a diiron(III) unit through hydrogen-bonding in
compound 6.
Summary
High-spin 4-cyanopyridine diiron(II) carboxylate complexes
with distinctive charge transfer transitions in the visible region
have been prepared and used to investigate quantitatively their
water-dependent reaction chemistry. Utilizing optical spectros-
copy, we were able both to determine the thermodynamic
constants for the water-dependent equilibrium and to monitor
kinetically the effects of water on the oxygenation of the
diiron(II) complex 1. We propose that a rapidly exchanging
water molecule in 3 will generate an open coordination site,
accelerating the oxygenation step compared to the reaction under
anhydrous conditions. The final products of oxidation are the
Figure 8. Top: ORTEP diagram of [Fe2(µ-OH)2(µ-O2CArTol)2(O2CArTol)2-
Tol
(
4-NCC5H4N)2(OH2)2]‚2(HO2CAr )2 (6) showing 50% probability thermal
ellipsoids for all non-hydrogen atoms. Bottom: View of the structure with
Tol
-
the aromatic rings of Ar CO2 ligands omitted for clarity.
-
dinuclear complex 6 and a Cl -containing iron(III) oligomer.
Finally, by comparison of the new diiron(II) complexes with
related ones in the literature, we established a correlation
between M o¨ ssbauer effect isomer shifts and coordination
number for high-spin iron(II) complexes in an oxygen-rich
coordination environment. The trend was rationalized on the
basis of the s-electron density at the iron nucleus across the
series.
Acknowledgment. This work was supported by Grant
GM32134 from the National Institute of General Medical
Sciences. NMR spectra were measured by using a Bruker 400
spectrometer purchased with assistance from the NIH under
Grant 1S10RR13886-01. We thank Ms. Elizabeth Nolan for
assistance in measuring Raman spectra, which were obtained
in the Harrison Spectroscopy Laboratory at MIT, funded in part
by NSF and NIH.
Figure 9. Top left: ORTEP diagram of 7 showing 50% probability thermal
ellipsoids for all non-hydrogen atoms. Top Right: The aromatic rings of
Tol
-
Ar CO2 ligands are omitted for clarity. Bottom: View of the structure
Tol
-
with the aromatic rings of Ar CO2 and 4-NCC5H4N omitted for clarity.
Supporting Information Available: Experimental details for
Tol
the synthesis and characterization of [Fe4(µ-OMe)4(O2CAr )4-
in Table 6. The structure of 7, which has an inversion center,
(HOMe) ](HOMe), including X-ray crystallographic information
6
-
combines two (µ3-oxo)triiron(III) units through four OH and
(Tables S1, S4; Figure S8); Table S1, also summarizing X-ray
-
Tol
two O2CAr
bridging groups. Four octahedral and two
crystallographic information for 1-7; Tables S2 and S3
providing geometric parameters for 1-4; Figures S1-S5
showing ORTEP diagrams including atom labels for 1-5;
Figures S6, S7, and S9, illustrating M o¨ ssbauer data and fits for
distorted trigonal bipyramidal iron(III) sites result. It is note-
worthy that the structure of 7 also contains two Cl- ligands
coordinated to each of the octahedral iron(III) sites. When the
8396 J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005

Dioxygen-Dependent Non-Heme Enzymes
A R T I C L E S
1
-3, 5, [Fe4(µ-OMe)4(O2CArTol)4(HOMe)6](HOMe), and
This material is available free of charge via the Internet at
http://pubs.acs.org.
4
F-Ph
[Fe(O2CAr
)2(Hdmpz)2]; and resonance Raman spectra of
1
and 4-NCC5H4N at 23 °C (Figure S10). Derivations of eqs 2
and 3 are also supplied as Appendices 1 and 2, respectively.
JA0512531
J. AM. CHEM. SOC.
9
VOL. 127, NO. 23, 2005 8397