Analogue reaction systems of selenate reductase

Article abstract of DOI:10.1021/ic0521630

Analogue reaction systems of selenate reductase, which reduces substrate in the overall enzymatic reaction SeO₄^2- + 2H⁺ + 2e^- → SeO₃^2- + H₂O, have been developed using bis(dithiolene) complexes of Mo^IV and W^IV. On the basis of the results of EXAFS analysis of the oxidized and reduced enzyme, the minimal reaction Mo^IVOH + SeO₄^2- → Mo^VIO(OH) + SeO₃^2- is probable. The square pyramidal complexes [M(OMe)(S₂C₂Me₂)₂]^1- (M = Mo, W) were prepared as structural analogues of the reduced enzyme site. The systems, [ML(S₂C₂Me₂)₂]^1-/SeO ₄^2- (L = OMe, OPh, SC₆H₂-2,4,6-Prⁱ₃) in acetonitrile, cleanly reduce selenate to selenite in second-order reactions whose negative entropies of activation implicate associative transition states. Rate constants at 298 K are in the 10^-2-10^-4 M^-1 s^-1 range with ΔS^? = -12 to -34 eu. When rate constants are compared with previous data for the reduction of (CH₂)₄SO, Ph₃AsO, and nitrate by oxygen atom transfer, reactivity trends dependent on the metal, axial ligand L, and substrate are identified. As in all other cases of substrate reduction by oxo transfer, the kinetic metal effect k₂^W > k₂^Mo holds. A proposal from primary sequence alignments suggesting that a conserved Asp residue is a likely ligand in the type II enzymes in the DMSO reductase family has been pursued by synthesis of the [Mo^IV(O₂CR)(S₂C₂Me₂) ₂]^1- (R = Ph, Bu^t) complexes. The species display symmetrical η²-carboxylate binding and distorted trigonal prismatic stereochemistry. They serve as possible structural analogues of the reduced sites of nitrate, selenate, and perchlorate reductases under the proposed aspartate coordination. Carboxylate binding has been crystallographically demonstrated for one nitrate reductase, but not for the other two enzymes.

Full text of DOI:10.1021/ic0521630

Inorg. Chem. 2006, 45, 2979−2988
Analogue Reaction Systems of Selenate Reductase
Jun-Jieh Wang, Christian Tessier, and R. H. Holm*
Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
Received December 20, 2005
2
-
Analogue reaction systems of selenate reductase, which reduces substrate in the overall enzymatic reaction SeO4
2-
+
2H+
+
2e-
f
SeO3
+
H2O, have been developed using bis(dithiolene) complexes of MoIV and WIV. On the
basis of the results of EXAFS analysis of the oxidized and reduced enzyme, the minimal reaction MoIVOH
+
2-
2
-
-
SeO4
f
MoVIO(OH)
+
SeO3 is probable. The square pyramidal complexes [M(OMe)(S2C2Me2)2]1 (M
)
Mo,
W) were prepared as structural analogues of the reduced enzyme site. The systems, [ML(S2C2Me2)2]1-/SeO4 (L
2
-
)
OMe, OPh, SC6H2-2,4,6-Pri3) in acetonitrile, cleanly reduce selenate to selenite in second-order reactions whose
2
negative entropies of activation implicate associative transition states. Rate constants at 298 K are in the 10-
−
4
1
1
10- M- s- range with
∆
Sq ) −12 to
−
34 eu. When rate constants are compared with previous data for the
reduction of (CH2)4SO, Ph3AsO, and nitrate by oxygen atom transfer, reactivity trends dependent on the metal,
axial ligand L, and substrate are identified. As in all other cases of substrate reduction by oxo transfer, the kinetic
W
Mo
metal effect k2 > k2 holds. A proposal from primary sequence alignments suggesting that a conserved Asp
residue is a likely ligand in the type II enzymes in the DMSO reductase family has been pursued by synthesis of
-
the [MoIV(O2CR)(S2C2Me2)2]1 (R
) η
Ph, But) complexes. The species display symmetrical 2-carboxylate binding
and distorted trigonal prismatic stereochemistry. They serve as possible structural analogues of the reduced sites
of nitrate, selenate, and perchlorate reductases under the proposed aspartate coordination. Carboxylate binding
has been crystallographically demonstrated for one nitrate reductase, but not for the other two enzymes.
-
Introduction
) W; L ) RO-, RS-, RCO2 ).8-11 Functional reaction
systems capable of reducing biological S-oxide and N-oxide
substrates5,9,12 and nitrate13 have been demonstrated using
these molecules as stoichiometric reactants. These systems
proceed by primary oxygen atom (oxo) transfer reactions14
and display second-order kinetics that implicate associative
transition states.
Research in this laboratory on biomimetic molybdenum
and tungsten chemistry1 has resulted in the synthesis and
characterization of active site structural analogues and the
development of analogue reaction systems of members of
the sulfite oxidase and DMSO reductase (DMSOR) enzyme
families.2 Square-pyramidal [MIVL(S2C2Me2)2]1- and dis-
torted octahedral [MVIOL(S2C2Me2)2]1- complexes (M )
Mo; L ) RO-, RS-)3-5 are accurate representations of the
reduced and oxidized native enzyme sites, respectively, of
the DMSOR family.6 They also apply to at least one tungsten
isoenzyme7 and, potentially, to other tungstoenzyme sites (M
With results from the foregoing reaction systems in hand,
we have turned our attention to other biological substrates
to learn which of them can be transformed to the biological
product by primary oxo transfer reactions. Among the
enzymes of interest in this context are arsenite oxidase and
selenate reductase.15,16 We have recently reported oxo transfer
reactions of the type MIV + AsVO f MVIO + AsIII with M
* To whom correspondence should be addressed. E-mail: holm@
chemistry.harvard.edu.
(1) Enemark, J. H.; Cooney, J. J. A.; Wang, J.-J.; Holm, R. H. Chem.
ReV. 2004, 104, 1175-1200.
(7) Garner, C. D.; Stewart, L. J. Met. Ions Biol. Syst. 2002, 39, 699-
726.
(2) Hille, R. Chem. ReV. 1996, 96, 2757-2816.
(3) Donahue, J. P.; Goldsmith, C. R.; Nadiminti, U.; Holm, R. H. J. Am.
Chem. Soc. 1998, 120, 12869-12881.
(8) Sung, K.-M.; Holm, R. H. Inorg. Chem. 2000, 39, 1275-1281.
(9) Sung, K.-M.; Holm, R. H. J. Am. Chem. Soc. 2001, 123, 1931-1943.
(10) Sung, K.-M.; Holm, R. H. Inorg. Chem. 2001, 40, 4518-4525.
(11) Jiang, J.; Holm, R. H. Inorg. Chem. 2004, 43, 1302-1310.
(12) Sung, K.-M.; Holm, R. H. J. Am. Chem. Soc. 2002, 124, 4312-4320.
(13) Jiang, J.; Holm, R. H. Inorg. Chem. 2005, 44, 1068-1072.
(14) Holm, R. H. Chem. ReV. 1987, 87, 1401-1449.
(4) Lim, B. S.; Donahue, J. P.; Holm, R. H. Inorg. Chem. 2000, 39, 263-
273.
(5) Lim, B. S.; Holm, R. H. J. Am. Chem. Soc. 2001, 123, 1920-1930.
(6) Kisker, C.; Schindelin, H.; Rees, D. C. Annu. ReV. Biochem. 1997,
66, 233-267.
10.1021/ic0521630 CCC: $33.50
Published on Web 03/10/2006
© 2006 American Chemical Society
Inorganic Chemistry, Vol. 45, No. 7, 2006 2979

Wang et al.
Figure 1. Depiction of the selenate reductase reaction based on the {MoIV(OH)(S2pd)2} and {MoVIO(OH)(S2pd)} active site structures postulated from
X-ray absorption spectroscopy, and that of analoguous reaction systems based on complexes of the general type [MIV(QR)(S2C2Me2)2]1- (M ) Mo, W; Q
) O, S). Also included are likely formulations of the associative transition states, 77Se chemical shifts, and the structure of the pyranopterindithiolene ligand
dianion (S2pd; R absent or contains a nucleotide, usually guanine).
) Mo and W.17 Selenate reductase (SeR)18 is a periplasmic
molybdoenzyme that catalyzes the reaction in Figure 1.
Selenate reduction to selenite has been associated with the
respiratory electron-transfer chains of certain bacteria, in-
cluding T. selenatis, whose SeR is the most thoroughly
characterized. The enzyme has been crystallized,19 but its
full structure is not currently available. EXAFS analysis has
led to the postulated active site structures in Figure 1.20 The
oxidized form reveals ∼4 Mo-S interactions at 2.33 Å, one
ModO bond at 1.68 Å, and one Mo-O bond at 1.81 Å.
The reduced form shows ∼4 Mo-S interactions at 2.32 Å
and one or two Mo-O bonds at 2.22 Å. With recognition
of the qualifications applied to these site formulations,20 we
have proceeded with them as guides for the correct structures
of the two oxidation states and, thereafter, as reactant and
product in potential oxo transfer reactions. Under the Hille
classification,2 the probable presence of two pyranopterin-
dithiolene ligands places SeR, like arsenite oxidase with
similar site structures,21 in the DMSOR family. Within this
family, the sites of these two enzymes differ from other
members by apparently not involving protein ligands. We
demonstrate in this investigation that selenate is reducible
to selenite by molybdenum- and tungsten-mediated oxo
transfer reactions.
Experimental Section
Preparation of Compounds. All reactions and manipulations
were performed under a pure dinitrogen atmosphere using either
an inert atmosphere box or standard Schlenk techniques.
Methanol was distilled from magnesium; acetonitrile, ether,
dichloromethane, and THF were freshly purified using an Innovative
Technology solvent purification system and stored over 4-Å
molecular sieves. The compounds Na2SeO3, Na2SeO4, Ph3AsO, and
(CH2)4SO (distilled over molecular sieves) were commercial
samples (Aldrich). The compounds (Et4N)(RCO2) (R ) But, Ph)
were prepared by equimolar reaction of RCO2H and 25% Et4NOH
in methanol and obtained as colorless crystalline solids after
recrystallization from acetonitrile/ether. The Et4N+ salts of
[M(OPh)(S2C2Me2)2]1- (M ) Mo,5 W8) and [M(SC6H2-2,4,6-
Pri3)(S2C2Me2)2]1- (M ) Mo,4 W11) were prepared as described.
In the following preparations, all volume reduction and evaporation
steps were performed in vacuo.
(Et4N)2[SeO4]. Reactions were performed in subdued light. To
a solution of 3.74 g (20.0 mmol) of Na2SeO4 in 30 mL of water
was added a solution of 6.90 g (40.6 mmol) of AgNO3 in 40 mL
of water. The reaction mixture was stirred for 1 h, and the white
solid was collected, washed with water, methanol, and ether to yield
6.60 g (92%) of Ag2SeO4. A mixture of 2.50 g (7.00 mmol) of
Ag2SeO4 and 2.09 g (10.0 mmol) of Et4NBr was treated with 150
mL of methanol. The reaction mixture was stirred for 24 h and
filtered, and the filtrate taken to dryness.
(15) Schro¨der, I.; Rech, S.; Krafft, T.; Macy, J. M. J. Biol. Chem. 1997,
272, 23765-23768.
(16) Watts, C. A.; Ridley, H.; Dridge, E. J.; Leaver, J. T.; Reilly, A. J.;
Richardson, D. J.; Butler, C. S. Biochem. Soc. Trans. 2005, 33, 173-
175.
(17) Wang, J.-J.; Kryatova, O.; Rybak-Akimova, E. V.; Holm, R. H. Inorg.
Chem. 2004, 43, 8092-8101.
(18) Abbreviations are given in the chart.
(19) Maher, M. J.; Macy, J. M. Acta Crystallogr. 2002, D58, 706-708.
(20) Maher, M. J.; Santini, J.; Pickering, I. J.; Prince, R. C.; Macy, J. M.;
George, G. N. Inorg. Chem. 2004, 43, 402-404.
(21) Conrads, T.; Hemann, C.; George, G. N.; Pickering, I. J.; Prince, R.
C.; Hille, R. J. Am. Chem. Soc. 2002, 124, 11276-11277.
The solid was washed with water, methanol, and ether to give
the product as 1.76 g (87%) of a white solid, which upon
recrystallization from acetonitrile/hexane was obtained as colorless
rectangular crystals. 77Se NMR (MeCN): δ 1038.
(Et4N)[HSeO3]. The previous preparation was followed using
Na2SeO3 instead of Na2SeO4. The product was isolated as colorless
blocklike crystals (85%) after recrystallization from acetonitrile/
hexane and was identified as the hydrogenselenite salt by an X-ray
structure determination. 77Se NMR (MeCN): δ 1360.
2980 Inorganic Chemistry, Vol. 45, No. 7, 2006

Selenate Reductase
Chart 1
(Et4N)[W(OMe)(S2C2Me2)2]. Method A. To a frozen solution
of 120 mg (0.25 mmol) of [W(CO)2(S2C2Me2)2]22,23 in 2 mL of
THF in a dry ice-acetone bath was added a suspension of 13.5 mg
(0.25 mmol) of NaOMe in 0.25 mL of acetonitrile. The frozen
mixture was allowed to warm to -20 °C, resulting in a color change
from reddish-violet to bluish-violet. The reaction solution was
treated with a solution of 42 mg (0.25 mmol) of Et4NCl in 1 mL
of acetonitrile at -20 °C and stirred for 5 min. The dark violet
solid obtained after solvent removal was washed with ether (3 × 5
mL, -20 °C) and dissolved in 1.5 mL of acetonitrile, and the
solution was filtered. The filtrate was allowed to stand at -20 °C
for 2 d, during which a mixture of large brown blocklike crystals
and a much smaller amount of violet needle-shaped crystals formed.
The brown crystals were mechanically separated, washed with ether
(3 × 5 mL, -20 °C), and dissolved in 1 mL of acetonitrile. Ether
(20 mL) was layered on the solution, which was allowed to stand
for 2 d. The product was obtained as 73 mg (50%) of large brown
crystals. 1H NMR (CD3CN, anion): δ 2.57 (s, 4), 3.47 (s, 1).
Absorption spectrum (acetonitrile): λmax (ꢀM) 287 (11300), 312
(9800), 385 (sh, 1600), 426 (900), 551 (800) nm. Anal. Calcd for
C17H35NOS4W: C, 35.11; H, 6.07; N, 2.41; S, 22.01. Found: C,
34.98; H, 5.96; N, 2.34; S, 22.16.
DMSO, DMSO reductase; Nar, membrane-bound nitrate reductase; R*,
C6H2-2,4,6-Pri3; S2pd, pyranopterindithiolene(2-) cofactor ligand; SeR,
selenate reductase; XAS, x-ray absorption spectroscopy.
immediately, was stirred for 30 min, and was filtered. The filtrate
was layered with 25 mL of ether. The solid that separated was
washed with ether (3 × 3 mL) and dried to produce 77 mg (68%)
of a red-brown microcrystalline product. 1H NMR (CD3CN,
anion): δ 0.84 (s, 4), 2.65 (s, 3). IR (KBr): νCOO 1438 cm-1
.
Absorption spectrum (acetonitrile): λmax (ꢀM) 270 (4800), 328
(1850), 379 (sh, 1700), 409 (2300), 491 (1850), 734 (200) nm. Anal.
Calcd for C21H41NMoO2S4: C, 44.75; H, 7.34; N, 2.49; S, 22.71.
Found: C, 44.85; H, 7.28; N, 2.43; S, 22.68.
Method B. All steps were carried out at -20 °C. A solid mixture
of 120 mg (0.25 mmol) of [W(CO)2(S2C2Me2)2], 13.5 mg (0.25
mmol) of NaOMe, and 42 mg (0.25 mmol) of Et4NCl was dissolved
in a minimal volume (ca. 2 mL) of acetonitrile/THF (1:1 v/v). The
bluish-violet solution that formed within 1 min was stirred for 20
min. Ether (40 mL) was layered on the solution. The product was
obtained upon standing for 2 d, followed by the separation of
crystals as in method A, as 78 mg (54%) of brown crystals. Spectral
properties are the same as those of the product obtained by Method
A.
(Et4N)[Mo(O2CPh)(S2C2Me2)2]. To a mixture of 77.7 mg (0.20
mmol) of [Mo(CO)2(S2C2Me2)2] and 50.2 mg (0.20 mmol) of
(Et4N)(O2CPh) was added 4 mL of THF and 1 mL of acetonitrile.
The orange-red solution that formed immediately was stirred for 1
h, resulting in a precipitate, which was collected and washed with
ether/THF (2:1 v/v, 3 × 3 mL) and ether (3 × 3 mL) to give the
1
product as 80 mg (69%) of an orange-red solid. H NMR (CD3-
CN, anion): δ 2.69 (s, 12), 7.34 (t, 2), 7.51 (t, 1), 7.69 (d, 2). IR
(KBr): νCOO 1442 cm-1. Absorption spectrum (acetonitrile): λmax
(ꢀM) 272 (6200), 331 (27000), 370 (2800), 416 (3350), 497 (2700)
nm. Anal. Calcd. for C23H37MoNO2S4: C, 47.33; H, 6.40; N, 2.40;
S, 21.93. Found: C, 47.19; H, 6.40; N, 2.40; S, 21.91.
(Et4N)[Mo(OMe)(S2C2Me2)2]. This compound was prepared by
procedures analogous to those for (Et4N)[W(OMe)(S2C2Me2)2] with
[Mo(CO)2(S2C2Me2)2]4,22 and was isolated as orange-brown block-
like crystals (40%). 1H NMR (CD3CN, anion): δ 2.50 (s, 4), 3.42
(s, 1). Absorption spectrum (acetonitrile): λmax (ꢀM) 325 (9700),
388 (sh, 4300), 458 (sh, 1800), 550 (sh, 960), 728 (400) nm. This
compound was identified by an X-ray structure determination.
In the following sections, complexes 1-14 are designated as in
Chart 1.
X-ray Structure Determinations. The structures of the eight
compounds in Table 1 were determined. Suitable crystals were
obtained by layering several volume equivalents of ether on
acetonitrile solutions which were maintained at 273 K for at least
12 h. Crystals were coated in paratone-N oil and mounted on either
a Bruker SMART 1K or APEX CCD-based diffractometer equipped
with an LT-2 low-temperature apparatus operating at 213 K. Data
were collected with ω scans of 0.3°/frame for 30 s (60 s for 7 and
8), so that 971-1647 frames were collected for a hemisphere of
data. The first 50 frames were recollected at the end of the data
collection to monitor for decay; no significant decay was detected
for any compound. Cell parameters were retrieved using SMART
software and refined using SAINT software on all reflections. Data
integration was performed with SAINT, which corrects for Lorentz
polarization and decay. Absorption corrections were applied using
SADABS. Space groups were assigned unambiguously by analysis
of symmetry and systematic absences determined by XPREP. The
compound (Et4N)[1] occurred as a twinned crystal, as determined
by the TwinRotMax routine in PLATON and confirmed by ROTAX
software. A 2-fold axis along the -1 0 1 reciprocal lattice direction
was observed. The data were detwinned by incorporation of TWIN
instruction in SHELXTL with the matrix 0 0 -1, 0 -1 0, -1 0 0.
The compound (Et4N)[7] was at first unsuccessfully refined using
a unit cell 4× smaller (a ) 9.449 Å, b ) 16.399 Å, c ) 17.678 Å;
(Et4N)[WO(OMe)(S2C2Me2)2]. The procedure is similar to that
for compounds of the type (Et4N)[WO(OR)(S2C2Me2)2].8,9 To a
frozen orange-brown solution of 116 mg (0.20 mmol) of (Et4N)-
[W(OMe)(S2C2Me2)2] in 2.5 mL of acetonitrile in a dry ice-acetone
bath was injected a solution of 18.0 mg (0.24 mmol) of Me3NO in
0.2 mL of acetonitrile. The frozen mixture was warmed to room
temperature over 20 min, resulting in a color change to greenish-
brown. The solution was layered with 40 mL of cold ether and
maintained at -20 °C over 2 d. The solid obtained by decantation
of the pale greenish-brown supernatant was washed with cold ether
(-20 °C, 3 × 5 mL) and dried to yield the product as 92.0 mg
(77%) of a brown-black crystalline solid. 1H NMR (CD3CN,
anion): δ 2.17 (s, 4), 4.08 (s, 1). IR (KBr): νWO 891 cm-1. Absorp-
tion spectrum (acetonitrile): λmax (ꢀM) 330 (6500), 477 (4500), 625
(3000) nm. Anal. Calcd for C17H35NO2S4W: C, 34.17; H, 5.91; N,
2.35; S, 21.42. Found: C, 34.30; H, 6.03; N, 2.40; S, 21.58.
(Et4N)[Mo(O2CBut)(S2C2Me2)2]. To a violet solution of 77.7
mg (0.20 mmol) of [Mo(CO)2(S2C2Me2)2] in 2 mL of THF was
added a solution of 46.2 mg (0.20 mmol) of (Et4N)(O2CBut) in 2
mL of acetonitrile. The reaction mixture became orange-red
(22) Fomitchev, D. V.; Lim, B. S.; Holm, R. H. Inorg. Chem. 2001, 40,
645-654.
(23) Goddard, C. A.; Holm, R. H. Inorg. Chem. 1999, 38, 5389-5398.
Inorganic Chemistry, Vol. 45, No. 7, 2006 2981

Wang et al.
Table 1. Crystallographic Data
(Et4N)[1]
(Et4N)[12]
‚MeCN
[Et4N]2[SeO4]
(Et4N)[2]
(Et4N)[7]
(Et4N)[8]
(Et4N)[9]
‚MeCN
[Et4N][HSeO3]
C8H21NO3Se
formula
C17H35Mo
NOS4
C17H35N
OS4W
581.55
P21/c
C21H41Mo
NO2S4
563.73
Pca21
16
C23H37Mo
NO2S4
583.72
P21/n
C17H35N
O2S4W
597.55
P21/n
C26H47Mo2
N2OS8
852.02
P21/c
4
C18H43N3O4Se
fw
493.64
P21/n
8
444.51
P21/n
4
258.22
P1h
2
space group
Z
4
4
4
a (Å)
b (Å)
c (Å)
22.978(8)
9.645(3)
22.968(8)
10.118(2)
11.058(2)
21.161(4)
37.741(11)
16.381(5)
17.663(5)
8.919(2)
23.807(4)
12.949(3)
8.979(3)
16.925(5)
15.476(5)
18.158(2)
11.582(2)
18.029(2)
13.182(7)
14.176(7)
13.370(7)
7.926(6)
8.909(7)
9.146(7)
80.64(1)
65.15(1)
77.19(2)
569.7(8)
1.505
3.276
2.35-28.05
1.060
3.76
9.45
R (deg)
â (deg)
γ (deg)
V (Å3)
109.56(1)
97.029(3)
105.303(4)
91.944(5)
104.997(2)
114.35(2)
4797(3)
1.367
0.900
0.94-25.00
1.052
2349.8(8)
1.644
5.277
1.94-25.00
1.050
10920(6)
1.372
0.803
1.08-25.00
1.209
2652.0(10)
1.462
0.830
1.71-26.00
1.095
2350.5(13)
1.689
5.281
1.78-27.86
1.255
3662.4(8)
1.545
1.164
1.16-26.00
1.032
2276(2)
1.297
1.675
1.83-27.88
1.052
d
calcd (g/cm3)
µa (mm-1
)
θ range (deg)
GOF (F2)
R1,b %
6.86
14.50
4.04
10.09
7.34
14.25
3.96
9.21
3.60
8.39
2.96
6.89
3.33
8.34
wR2,c %
a
2
2
2
Mo KR radiation. b R1 ) ∑||Fo| - |Fc||/∑|Fo|. c wR2 ) {∑[w(Fo - Fc )2]/∑[w(Fo )2]}1/2
Pna21 or Pnma) that was found by the matrix routine in SMART.
Very large thermal parameters, as well as strong residual electron
density, were observed. Upon careful examination of the collected
frames, weak reflections not accounted for in the unit cell
determination were observed and included in the cell index routine
in SMART to generate the unit cell parameters in Table 1. Missing
symmetry in all crystals was checked with PLATON; none was
found. Structures were solved by direct methods and refined against
all data by full-matrix least-squares techniques on F2 using the
SHELXL-97 package. All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms were placed at idealized positions
on carbon atoms. Crystal parameters and agreement factors are
reported in Table 1.24
Kinetics Measurements. All reactions were performed under
strictly anaerobic conditions in acetonitrile (Burdick & Jackson)
which was stored over 4 Å molecular sieves prior to use. Oxo
transfer reactions were monitored with a Varian Cary 3 spectro-
photometer equipped with a cell compartment thermostated to (0.5
°C and a multicell changer. Thermal equilibrium was reached by
placing a 1 mm quartz cell containing 0.24-0.32 mL of the solution
of the complex in the cell compartment at least 5 min prior to the
start of a reaction. Reactions were initiated upon injection of the
substrate solution containing oxo donor XO by means of a gastight
syringe through the rubber septum cap of the cell followed by rapid
shaking of the reaction mixture. Reaction systems contained the
initial concentrations [1-6]0 ) 1.37-2.14 mM, [SeO42-]0 ) 11.1-
29.4 mM, [(CH2)4SO]0 ) 1.59-3.27 M, and [Ph3AsO]0 ) 3.8-
11.3 mM. Sharp isosbestic points were observed for each reaction
system, indicating the lack of detectable intermediates. For each
substrate, reactions were conducted at five temperatures in the 278-
333 K range. At each temperature, at least five independent runs
with different initial concentrations of XO were performed under
pseudo-first-order conditions. Plots of kobs versus [XO]0 were linear
and yielded overall second-order rate constants, k2, at each
temperature. Final values of k2 were averages from multiple
independent runs. Activation parameters were determined from
Eyring plots of k2 versus (kBT/h)[exp(∆Sq/R - ∆Hq/RT)]. Standard
deviations were estimated using linear least-squares error analysis
with uniform weighting of the data points.
Mercury 400 and 300 spectrometers. Chemical shifts of 77Se{1H}
spectra are referenced to Me2Se. Infrared spectra were determined
with a Nicolet Nexus 470 FT-IR instrument. Absorption spectra
were obtained with a Varian Cary 50 Bio spectrophotometer.
Results and Discussion
Reaction Systems. From the reduced des-oxo SeR site
structure in Figure 1, a desirable molybdenum reactant in
substrate reduction by oxo transfer is a bis(dithiolene)MoIV-
(OH) complex. Hydrolysis of the complexes [M(OPh)-
(S2C2Me2)2]1- (M ) Mo, W) in aqueous acetonitrile media
yields [MO(S2C2Me2)2]2-.5,9 While a hydroxo species is a
likely intermediate in these reactions, no MIV-OH dithiolene
complexes have been isolated or otherwise identified in these
or other systems. In the absence of hydroxo species, we have
utilized the methoxide complexes 1 and 2 whose preparation
is outlined in Figure 2. Here methoxide is the closest
available simulator of hydroxide provided that SeR is an
oxotransferase. Note that the enzyme is a reductase, not a
hydroxylase which utilizes bound hydroxide as a nucleophile
in oxidase reactions.25 We have also utilized phenolate
complexes 3 and 4 as additional reactants with axial anionic
oxygen ligands and for comparison with previous kinetics
results. Thiolate complexes 5 and 6 are structural analogues
of the reduced sites of nitrate reductase with axial cysteinate
ligation26 and relate the present work to our recent study of
nitrate reduction.13 Although SeR is not known to be a
tungstoenzyme, we have included WIV reactants to provide
additional examples of the kinetic metal effect and to
otherwise follow our past practice of elaborating in parallel
the chemistry of analogous molybdenum and tungsten
systems. The stable WVIO complex 9 when detected in an
oxo transfer reaction demonstrates the fate of the oxygen
atom transferred from substrate. This compound has been
isolated and structurally characterized. Corresponding com-
Other Physical Measurements. All measurements were made
under anaerobic conditions. NMR spectra were recorded on Varian
(25) Hille, R. Arch. Biochem. Biophys. 2005, 433, 107-116.
(26) Dias, J. M.; Than, M. E.; Humm, A.; Huber, R.; Bourenkov, G. P.;
Bartunik, H. D.; Bursakov, S.; Calvete, J.; Caldeira, J.; Carneiro, C.;
Moura, J. J. G.; Moura, I.; Roma˜o, M. J. Structure 1999, 7, 65-79.
(24) See paragraph at the end of this article for Supporting Information.
2982 Inorganic Chemistry, Vol. 45, No. 7, 2006

Selenate Reductase
Figure 2. Scheme for the synthesis of the complexes [M(OMe)(S2C2Me2)2]1-, [Mo(O2R)(S2C2Me2)2]1-, and [WO(OMe)(S2C2Me2)2]1-
.
plexes of the type [MoO(OR)(S2C2Me2)2]1-, while sometimes
detectable in solution, are unstable because of autoreduction
to [MoVO(S2C2Me2)2]1-.5
Selenate was utilized as anhydrous (Et4N)2[SeO4], which
is freely soluble in acetonitrile. This compound was prepared
in high yield by simple metathesis reactions. The Me4N+
salt has been obtained by an ion-exchange procedure.27 When
Na2SeO3 was used in the metathesis reactions, the only
product isolated was the hydrogenselenite compound (Et4N)-
[HSeO3], a probable consequence of the greater basicity of
-
selenite versus selenate (pKa ) 6.58 for HSeO3 , 2.05 for
-
HSeO4 ).
Structural Features. While crystals containing complexes
1 and 2 with the same cation would be expected to be
isomorphous, as is the case with, for example, the Et4N+
salts of 8 and [W(O2CPh)(S2C2Me2)2]1-,10 different packing
patterns are found. This behavior has been observed previ-
ously with the Et4N+ salts of 35 and 4.8 The structure of 2,
provided in Figure 3, reveals these essential features: square
pyramidal geometry with the metal atom displaced 0.75 Å
above the S4 mean plane toward the axial methoxide ligand,
a trans S-W-S angle of 142.6(7)° and a dihedral angle of
129.6° between the chelate ring planes. For isostructural
MoIV complex 1 (not shown), the mean Mo-S distance of
2.33(1) Å agrees very well with the 2.32 Å value from the
EXAFS analysis of the reduced enzyme. The mean Mo-O
distance of 1.862(3) Å differs largely from the value of 2.22
Å from the EXAFS investigation, where the identification
of oxygen ligands is described as tentative because of the
correlation with intense Mo-S scattering.20 The values for
1 are averaged over two crystallographically independent
anions; this complex is practically isometric with 2.24 For 2,
the W-O bond length is 1.824(5) Å. Recent geometry-
optimized DFT calculations of [M(OMe)(S2C2H2)2]1- place
the M-OMe bond length at 1.887 Å (Mo) and 1.874 Å
(W).28 Consequently, an authentic M-OH distance in the
enzyme site is expected to lie in the 1.8-1.9 Å range and is
found without exception for MIV-OR bonds in these and
other bis(dithiolene) complexes by crystallography4,5,8,9 and
Figure 3. Structures of tungsten complexes showing 50% probability
ellipsoids and atom labeling schemes. Selected (mean) bond distances (Å)
and angles (deg) for [W(OMe)(S2C2Me2)2]1- (upper): W-O ) 1.824(5),
W-S ) 2.33(1), S-C ) 1.778(5), C-C ) 1.323(5), S1-W-S2 ) 81.91-
(6), S3-W-S4 ) 82.51(7), O-W-S ) 107.9(3)-109.4(2), δ ) 0.746-
(1), θ ) 129.6(1). For [WO(OMe)(S2C2Me2)2]1- (lower): W-O1 )
1.724(4), W-O2 ) 1.926(4), W-S1,2,3 ) 2.40(1), W-S4 ) 2.523(2),
O1-W-O2 ) 93.8(2), S1-W-S2 ) 79.53(5), S3-W-S4 ) 78.17(5),
S2-W-S4 ) 89.61(5), S1-W-S3 ) 157.05(5), θ ) 91.3(1). All but the
last two S-W-S angles are chelate ring bite angles.
XAS.29,30 The long M-O interaction in the enzyme is
presumably the basis for the des-oxo designation.20
Structures of the substrate selenate ion and of the hydro-
genselenite ion are shown in Figure 4. These structures were
(29) Musgrave, K. B.; Donahue, J. P.; Lorber, C.; Holm, R. H.; Hedman,
B.; Hodgson, K. O. J. Am. Chem. Soc. 1999, 121, 10297-10307.
(30) Musgrave, K. B.; Lim, B. S.; Sung, K.-M.; Holm, R. H.; Hedman,
B.; Hodgson, K. O. Inorg. Chem. 2000, 39, 5238-5247.
(27) Malchus, M.; Jansen, M. Z. Naturforsch. 1998, 53b, 704-710.
(28) McNamara, J. P.; Hillier, I. H.; Bhachu, T. S.; Garner, C. D. Dalton
Trans. 2005, 3572-3579.
Inorganic Chemistry, Vol. 45, No. 7, 2006 2983

Wang et al.
Figure 4. Structures of (Et4N)2[SeO4] and (Et4N)[HSeO3]. In the latter,
the hydrogen bonding pattern to a symmetry equivalent molecule A is
shown. Selected (mean) bond distances and angles: for [SeO4]2- Se-O )
1.641(3), O-SeO ) 109.5(2); for [HSO3]- Se-O1 ) 1.678(2), Se-O2 )
1.639(2), Se-O3 ) 1.758(2), O1‚‚‚O3A ) 2.691(3), O1-Se-O2 )
107.1(1), O1-Se-O3 ) 101.3(1), O2-Se-O3 ) 99.9(1).
used to identify the compounds and demonstrate the absence
of water or other protic solvates in the crystals. The
tetrahedral structure of SeO42- in the monoclinic Et4N+ salt
acetonitrile monosolvate displays angles and distances
indistinguishable from those of cubic (Me4N)2[SeO4]27 and
Li2SeO4‚H2O.31 The close O3‚‚‚O1 contact (2.691(3) Å) in
the Et4N+ salt of HSeO3- indicates the presence of hydrogen
bonding. A hydrogen atom was placed on atom O3 with Se-
O3 ) 1.758(2) Å, and its position was constrained in the
O3‚‚‚O1 direction, resulting in an O1A‚‚‚H3 bond distance
of 1.88 Å. Prior structures of hydrogenselenite compounds
with organic counterions reveal hydrogen bonding between
the anion and cation and a pattern of Se-O distances similar
to that found here.32-35 None of these structures contain
mutually hydrogen-bonded hydrogenselenite anions.
The product complex derived from 2 by oxo transfer is
WVIO complex 9, whose structure is found in Figure 3. Its
principal features are a cis disposition of the oxo and
methoxide ligands, a dihedral angle, θ, of 91.3° between
chelate rings, a pronounced trans effect of the oxo ligand
causing the W-S4 bond to be 0.11-0.13 Å longer than
the other three W-S distances, and a trans ligand angle,
S1-W-S3, of 157.1°. This angle describes a structure
intermediate between the octahedral (180°) and trigonal
prismatic (136°) limits.36 The structure of 9 is closely
Figure 5. Structures of [Mo(O2CPh)(S2C2Me2)2]1- and [Mo2O(S2C2Me2)4]1-
showing 50% probability ellipsoids and atom labeling schemes.
comparable to those of 109 and 11,11 which are also oxo
transfer products.
In the course of preparing methoxide complexes 1 and 2,
bluish-violet reaction mixtures were always observed, and a
small amount of violet byproduct was encountered when
isolating the desired brown product compounds. Because of
its persistent appearance, the purple crystals obtained in the
preparation of 1 were crystallographically examined. This
material was shown to be (Et4N)[12], containing a mixed-
valence binuclear complex whose structure is included in
Figure 5. It is formally (but not uniquely) an addition product
of [Mo(S2C2Me2)3]1-, a known compound4 which we have
often encountered as an impurity in the synthesis of other
molybdenum dithiolenes, and the neutral fragment [MoO-
(S2C2Me2)]. The origin of the oxo ligand has not been
established. Metric features are available elsewhere.24
Reduction of Selenate by Oxo Transfer. The reaction
systems utilizing MIV complexes 1-6 are depicted in Figure
1. The overall process is reaction 1, resulting in the formation
of MVIO products and selenite. Of the former, only WVIO
(31) Johnston, M. G.; Harrison, W. T. A. Acta Crystallogr. 2003, E59,
i25-i27.
(32) Ritchie, L. K.; Harrison, W. T. A. Acta Crystallogr. 2003, E59, o1296-
o1298.
(33) Nemec, I.; Chudoba, V.; Havlicek, D.; Cisarova´, I.; Micka, Z. J. Solid
State Chem. 2001, 161, 312-318.
(34) de Matos Gomes, E.; Nogueria, E.; Fernandes, I.; Belsley, M.; Paixaˆo,
J. A.; Matos Beja, A.; Ramos Silva, M.; Mart´ın-Gil, J.; Mart´ın-Gil,
F.; Mano, J. F. Acta Crystallogr. 2001, B57, 828-832.
(35) Neˆmec, I.; Cisarova´, I.; Micka, Z. J. Solid State Chem. 1998, 140,
71-82.
(36) Beswick, C. L.; Schulman, J. M.; Stiefel, E. I. Prog. Inorg. Chem.
2004, 52, 55-110.
2984 Inorganic Chemistry, Vol. 45, No. 7, 2006

Selenate Reductase
Figure 7. 77Se NMR spectrum for the system [W(SC6H2-2,4,6-Pri3)(S2C2-
2-
2-
Me2)2]1-/SeO4 starting with 1 equiv of 6 and 4 equiv of SeO4 after 1
h in acetonitrile solution at ambient temperature.
Table 2. Kinetics Parameters for Oxygen Atom Transfer Reactions in
Selected Systems [MIV(QR)(S2C2Me2)2]1-/XO in Acetonitrile at 298 K
k2
j
M
QR
XO
(M -1 s-1
)
∆Hq (kcal/mol) ∆Sq (eu)
Mod OPh
SeO4
7.2(4) × 10-4
3.2(2) × 10-3
1.4(2) × 10-3
3.3(3) × 10-3
3.6 × 10-2
12(1)
-34(3)
-20(2)
-30(1)
-28(2)
-12(2)
-20(3)
-39(1)
-16(2)
-37(2)
-33(1)
-33(1)
-26(1)
-16(3)
2-
Moe SC6H2Pri3 SeO4
14.8(6)
12.2(4)
12.8(6)
15.8(7)
12.6(9)
10.1(4)
16.8(6)
12.0(2)
11.6(4)
7.1(3)
2-
2-
2-
2-
Wg
OMe
SeO4
SeO4
Wd,g OPh
We
Wa
Mob,f OPh
SC6H2Pri3 SeO4
SC6H2Pri3 NO3
d
-
1.7(1) × 10-1
(CH2)4SO 1.5(2) × 10-4
Moa SC6H2Pri3 (CH2)4SO 1.4(1) × 10-3
Wh
OMe
(CH2)4SO 8.5(1) × 10-5
Wc,f,h OPh
Wi
OMe
Wc,i OPh
(CH2)4SO 9.0(3) × 10-4
Ph3AsO
Ph3AsO
2.0(1)
3.1(1)
9.3(3)
14.6(9)
Wa
SC6H2Pri3 (CH2)4SO 3.5(1) × 10-2
Mo
a Ref 13. b Ref 5. c Ref 9. d k2W/k2Mo ) 4.6. e k2W/k2Mo ) 11. f k2W/k2
) 6.0. g k2OPh/k2
perimentally determined values.
) 2.4. h k2OPh/k2
) 11. i k2OPh/k2
) 1.6. j Ex-
OMe
OMe
OMe
on 1 whose spectra did not show clean isosbestic points; the
kinetics of this system were not pursued. For the systems in
Figure 6, the final spectra are identical to the spectra of
authentic complexes 9 and 11. The formation of these
products can also be established by the characteristic methyl
chemical shifts of WVIO complexes (δ 2.1-2.3 in acetoni-
trile). The formation of selenite is readily demonstrated by
77Se NMR spectra. For example, in the reaction based on
tungsten complex 6, the spectrum in Figure 7 reveals the
signal of substrate SeO42- at δ 1038 and product SeO32- at
Figure 6. Spectrophotometric monitoring of reactions: (upper) the system
2-
[W(OMe)(S2C2Me2)2]1-/SeO4 with [2]0 ) 1.48 mM, [SeO42-]0 ) 14.3
mM, and spectra recorded every 5 min; (lower) the system [W(SC6H2-
2-
2,4,6-Pri3)(S2C2Me2)2]1-/SeO4 with [6]0 ) 1.51 mM, [SeO42-]0 ) 14.3
mM, and spectra recorded every 3 min. Reactions were conducted in
acetonitrile solutions at 298 K. Arrows indicate changes in absorbance with
time; band maxima and isosbestic points are indicated.
-
δ 1268. The product is not HSeO3 , which occurs at δ 1360.
complexes 9-11 are stable to isolation. Reaction 1 is an
example of the generalized oxo transfer paradigm 2. The
corresponding MoVIO complexes are formed in reaction 1
but, as noted above, are not stable. Reactions were monitored
spectrophotometrically, and data were analyzed as in previous
work.5,9,13,17 Illustrative spectra are shown for the systems
based on complexes 2 and 6 in Figure 6. These and other
reaction systems develop tight isosbestic points and proceed
The values for selenate and selenite are very close to reported
values in aqueous solution.37
Rate constants and activation parameters for five reactions
(eq 1) and for other oxo transfer reactions previously
determined and included for comparison purposes are
contained in Table 2. All reactions were found to follow the
second-order rate law (eq 3) with rate constants k2 in the
10-2-10-4 M-1 s-1 range. As for all other oxo transfer
reactions mediated by bis(dithiolene)MIV complexes, activa-
tion entropies are large and negative, indicative of an
associative transition state such as that represented in Figure
1. If this state has the features of that determined from
the experimental results for the reduction of (CH2)4SO
by WIV complexes, it is occurs early in the reaction
[MIVL(S2C2Me2)2]1- + SeO42- f
[MVIOL(S2C2Me2)2]1- + SeO3 (1)
2-
M
IV + XO f MVI + X
-d[MIV]/dt ) k2[MIV][XO]
(2)
(3)
cleanly to completion. The exception is the reaction based
(37) Duddeck, H. Annu. Rep. NMR Spectrosc. 2004, 52, 105-166.
Inorganic Chemistry, Vol. 45, No. 7, 2006 2985

Wang et al.
2-
coordinate and is mainly W‚‚‚OSeO3 bond-making in
58% of ∆Gq. The occurrence of Me3NO (61 kcal/mol) and
(CH2)4SO (86 kcal/mol) and the ends of series 6 may be
related to the indicated bond dissociation energies, but the
position of Ph3AsO (103 kcal/mol) resists explanation on
character.12,38
Complex Structure and Rates. The data of Table 2 reveal
that the nature of the axial ligand and the identity of the
metal measurably influence reaction rates when other factors
are constant. In regard to the indicated axial ligands, the rate
order 4 for tungsten complexes and 5 for molybdenum
complexes holds for selenate reduction and also applies to
2-
this basis. The consistent position, SeO4 J (CH2)4SO, is
difficult to analyze because of the charge difference and the
unknown bond-dissociation energy of selenate. We have
previously demonstrated the reduction of Ph2SeO by complex
3 in acetonitrile.5 Given the usual periodic trend in bond
energies, exemplified by R3P-S/Se bond energy data,40,41
the order D(S-O) > D(Se-O) is expected. However, we
can find no experimental data on Se-O bond energies in
organic or inorganic compounds.
S
O
the reduction of (CH2)4SO.13 Further, k2 /k2 ) 28 for the
reduction of nitrate to nitrite by 6 and [W(OC6H2-2,4,6-
Pri3)(S2C2Me2)2]1-.13 The effect is attributed to a more
6 (SR*) > 4 (OPh) > 2 (OMe)
5 (SR*) > 3 (OPh)
(4)
(5)
Carboxylate Binding. Members of the DMSOR enzyme
family assigned under the Hille scheme2 have been further
classified into types I, II, and III on the basis of protein
sequence alignments of metal-binding regions.42 Type II
includes, among others, T. selenatis SeR, a perchlorate
reductase, and membrane-bound respiratory nitrate re-
ductase, which in its entirety contains three subunits and
is designated NarGHI. The defining feature of type II is a
highly conserved Asp residue which is postulated to function
as a ligand at the molybdenum site. Binding of the Asp
carboxylate group in two molybdenum site structures in the
catalytic subunit NarG has been confirmed by crystal-
lography. The structure of E. coli NarGH42 contains the
oxidized site {MoVIO(O2C‚Asp)(S2pd)} with bond distances
of Mo-S ) 2.45 Å (mean) and ModO ) 1.8 Å and η1-
syn-carboxylate coordination. The structure of E. coli
NarGHI,43 determined before the NarGH result, is reported
to contain a {Mo(O2C‚Asp)(S2pd)2} site with distorted
trigonal prismatic geometry, Mo-S ≈ 2.4 Å, and unsym-
metrical η2-carboxylate binding reflected by Mo-O ≈ 1.9
and 2.4 Å. The unit is presumed to contain MoVI, but the
apparent absence of an oxo ligand renders this oxidation state
suspect: MoIV or MoV is more appropriate. Both types of
carboxylate binding are precedented;44,45 however, nitrate
reductase is the first example of carboxylate coordination
to molybdenum in proteins. Although the sequence align-
ment comparison raises the possibility of an Asp ligand in
SeR, none was found in the structure of T. selenatis SeR,
deduced from XAS.20 Further, the enzyme is specific for
selenate; nitrate reductase activity was not detected,15 and
nitrate reductases are described as ineffective in selenate
reduction.16
accessible metal site, despite the larger axial ligand sub-
stituent R*, which is made possible by the W-S bond length
of 611 being 0.46-0.50 Å longer than the W-O bonds of 2
and 4.8 The rate constant ratios k2OPh/k2OMe ) 1.6 (Ph3AsO)
and 2.4 (SeO42-) for tungsten complexes with the indicated
substrates are consistent with the cyclohexane A value
measure of steric size, which indicates that methoxyl (0.55-
0.75 kcal/mol) is comparable with phenoxyl (0.65 kcal/
mol).39 The largest departure of this ratio from unity thus
OPh
far observed is with (CH2)4SO as substrate, for which k2
k2
/
OMe
) 11. Binding through oxygen (or sulfur) is more
difficult on a steric basis than for the other two substrates.
Last, the ratios k2W/k2Mo ) 4.6-11 are a further demonstra-
tion of the kinetic metal effect by which tungsten com-
plexes at constant ligation and substrate react faster than
molybdenum complexes in substrate reduction by oxo
transfer.9,13,17
Substrate Reactivity Trends. Sufficient data are now
available to allow recognition of certain trends in oxo transfer
reactivity of substrate measured by k2 in acetonitrile with
constant metal reactant. Rate order 6 applies wholly or in
part to 2-6, with qualification necessary only because not
all substrates have been tested with each complex. The less
extensive rate order 7 established with tungsten complex 6
affords placement of nitrate before selenate and probably
after Ph3AsO, but this has not been experimentally estab-
lished. As previous considerations of reaction rates in
reductive oxo transfer reveal,5,9,17 identifying a substrate
reactivity trend is much easier than explaining it. The
R3NO > Ph3AsO > SeO42- > (CH2)4SO
(6)
(7)
Sequence alignment considerations do not appear to apply
in the case of T. selenatis SeR, but this is the only SeR on
2-
NO3- > SeO4 ≈ (CH2)4SO
(39) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic Chemistry;
University Science Books: Sausalito, CA, 2004; p 104.
(40) Holm, R. H.; Donahue, J. P. Polyhedron 1993, 12, 571-589.
(41) Capps, K. B.; Wixmerten, B.; Bauer, A.; Hoff, C. D. Inorg. Chem.
1998, 37, 2861-2864.
problem lies in the inherent differences in substrate stereo-
chemistry, basicities, and other properties and the fact that
the free energies of activation contain substantial enthalpic
and entropic contributions which are difficult to evaluate
separately. For all but two systems in Table 2, |T∆Sq| is 29-
(42) Jormakka, M.; Richardson, D.; Byrne, B.; Iwata, S. Structure 2004,
12, 95-104.
(43) Bertero, M. G.; Rothery, R. A.; Palak, M.; Hou, C.; Lim, D.; Blasco,
F.; Weiner, J. H.; Strynadka, N. C. J. Nature Struct. Biol. 2003, 10,
681-687.
(38) Note that DFT calculations on the systems [MIV(OMe)(S2C2H2)2]1-
/
(44) Carrell, C. J.; Carrell, H. L.; Erlebacher, J.; Glusker, J. P. J. Am. Chem.
Soc. 1988, 110, 8651-8656.
Me2SO (M ) Mo, W) predict a later transition state with appreciable
M‚‚‚O bond making and S‚‚‚O bond weakening and a geometry
approaching that of WVIO complex 9 or 10.28
(45) Holm, R. H.; Kennepohl, P.; Solomon, E. I. Chem. ReV. 1996, 96,
2239-2314.
2986 Inorganic Chemistry, Vol. 45, No. 7, 2006

Selenate Reductase
trophotometry and 1H NMR for periods up to 20 h at room
temperature. Under these conditions, oxo transfer reactions
of complexes such as 1-6 with N-oxides, S-oxides, nitrate,
and selenate readily proceed. Perchlorate was included to
search for perchlorate reductase activity.46,47 Observations
Table 3. Bond Distances (Å) and Angles (deg) of
[Mo(O2CR)(S2C2Me2)2]1- Complexes
R ) Ph
R ) But f
Mo-O1
Mo-O2
Mo-Sa
S-Ca
2.189(2)
2.205(2)
2.332(6)
1.755(4)
1.34(1)
2.19(1)
2.17(1)
2.32(2)
1.78(2)
1.32(2)
-
are briefly summarized as follows: 4/ClO4 , no appreciable
-
reaction; 8/ClO4 , reaction but 13 could not be identified;
C-Cb
14/SeO42-, unidentified reaction, trace of WVIO product (in
S1-Mo-S2
S3-Mo-S4
S1-Mo-S4
S2-Mo-S3
S1-Mo-S3
S2-Mo-S4
O1-Mo-O2
O1-C9-O2
O1-Mo-S1
O1-Mo-S2
O1-Mo-S3
O1-Mo-S4
O2-Mo-S1
O2-Mo-S2
O2-Mo-S3
O2-Mo-S4
81.19(3)
81.60(3)
84.41(3)
84.74(3)
137.20(3)
140.92(3)
60.64(9)
119.4(3)
132.51(7)
88.32(7)
86.89(7)
127.01(7)
90.87(6)
128.48(7)
128.49(6)
87.68(6)
82.4(2)
81.4(2)
83.0(1)
85.4(2)
138.2(2)
140.4(2)
60.2(4)
119(2)
130.1(3)
86.6(3)
88.6(3)
129.9(3)
89.3(3)
127.1(3)
128.9(3)
89.2(3)
aqueous solution with Na2SeO4 a green-blue precipitate
-
formed); 14/ClO4 , nil reaction. The WIV complex 1423 was
examined because it represents an unprotonated version of
the active site of reduced SeR (Figure 1), which could be
reactive in selenate reduction. Carboxylate complexes can
sustain oxo transfer reactions, as shown by the synthesis of
(Et4N)[13] (41%) from 8 and Ph3AsO.11 However, when the
system 8/Ph3AsO was examined at the dilute concentrations
appropriate for kinetics determination, spectral complexity
indicated more than one reaction.
Summary. The following are the principal results and
conclusions of this investigation.
(1) The square pyramidal complexes [MIV(OMe)-
(S2C2Me2)2]1- (M ) Mo, W) have been prepared as
analogues of the reduced site in T. selenatis selenate
reductase, which, from Mo EXAFS analysis, contains two
pyranopterindithiolene ligands and an axial hydroxide. The
Mo-OMe distance of 1.862 Å provides a measure of the
expected Mo-OH bond length in the enzyme, whose
reported value may be artifactually long.
MoS1S2/MoO1O2c
MoS3S4/MoO1O2c
119.3(1)
115.6(1)
116.8(3)
117.9(3)
δd
θe
0.816(1)
125.0(1)
0.807(3)
125.2(2)
a Mean of four. b Mean of two. c Dihedral angle. d Displacement Mo
atom from MoS4 mean plane. e Dihedral angle of MoS2 planes. f Data
for one of four independent anions, all of which are dimensionally very
similar.
(2) Bis(dithiolene) complexes, [MIVL(S2C2Me2)2]1- (M )
Mo, W; L ) RO-, R*S-, cleanly reduce selenate to selenate
in second-order oxygen atom transfer reactions that pro-
ceed through associative transitions states such as that
illustrated in Figure 1. We are unaware of any previous
nonenzymatic reactions resulting in reduction of selenate by
oxo transfer.
which there is any active site structural information. To
establish possible structural analogues of the reduced sites
of selenate, nitrate, and perchlorate reductases with carboxy-
late ligands and to investigate any accompanying reactivity
in substrate reduction, we have prepared the MoIV carboxylate
complexes 7 and 8 in ca. 70% yield by the procedure in
Figure 2. The two complexes are isostructural; metric features
of both are collected in Table 3, and the structure of 8 is
provided in Figure 5. The complexes are six-coordinate with
trans ligand angles of 137.0-141.9°, indicating a close
approach to idealized trigonal prismatic geometry. The
molybdenum atoms are displaced 0.81-0.82 Å toward the
carboxylates, which bind as symmetrical η2-ligands with
Mo-O distances of 2.17-2.21 Å. Chelate rings are canted
at a dihedral angle of 125°; mean Mo-S distances are 2.32-
2.33 Å. These complexes are isostructural with the corre-
sponding species [W(O2CR)(S2C2Me2)2]1-.10,11 The structures
of 7 and 8 are taken to represent unconstrained versions of
the (apparently reduced) site of NarGHI. Further, complex
13, for which W-S ) 2.41-2.45 Å, WdO ) 1.72 Å, W-O
) 2.07 Å, and trans S-W-S ) 143.5°,11 should fulfill the
same role for the oxidized NarG site.
(3) Second-order rate constants for the reactions (1) cover
the range of 3.6 × 10-2 to 7.2 × 10-4 M-1 s-1, are dependent
upon axial ligand (series 4 and 5), and manifest the kinetic
metal effect in substrate reduction (k2W > k2Mo). Comparison
with previous results leads to recognition of substrate
reactivity trends (series 6 and 7) which, however, are not
easily explained in their entirety.
(4) The [MoIV(O2CR)(S2C2Me2)2]1- complexes are readily
prepared, have approximate trigonal prismatic stereochem-
istry with symmetrical η2-carboxylate binding, and are
unconstrained structural analogues of the reduced site in the
Nar nitrate reductase and, perhaps, also of sites in selenate
and perchlorate reductases if sequence alignment arguments
apply.
(5) The complexes in (2) are capable of reducing at least
four types of biological substrates (N-oxides, S-oxides,
nitrate, selenate) in oxo transfer reactions, all of which have
the characteristics in (2). Analoguous reaction systems cannot
To investigate the effect of carboxylate and other axial
ligands on reactivity, additional systems were investigated
using WIV complexes (1-6 mM), because of the greater ease
of oxidation and stability of possible WVIO products, and
selenate and perchlorate (3-20 equiv) as potential substrates
in acetonitrile. Systems were monitored by UV-vis spec-
(46) Kengen, S. W. M.; Rikken, G. B.; Hagen, W. R.; van Ginkel, C. G.;
Stams, A. J. M. J. Bacteriol. 1999, 181, 6706-6711.
(47) Okeke, B. C.; Frankenberger, W. T., Jr. Microbiol. Res. 2003, 158,
337-344.
Inorganic Chemistry, Vol. 45, No. 7, 2006 2987

Wang et al.
prove an enzyme pathway or mechanism but can disclose
what is possible. Given the close structural relationship of
analogue complexes and the immediate coordination envi-
ronment of enzyme sites in the same oxidation state,
enzymatic transformation of the substrates by atom transfer
is highly probable. With one DMSOR, this reaction has been
proven by isotope labeling.48
Acknowledgment. This research was supported by NSF
Grant CHE 0237419. C.T. was supported by a fellowship
from the Quebec Nature and Technology Research Fund.
Supporting Information Available: X-ray crystallographic files
in CIF format for the structure determinations of the compounds
in Table 1. This material is available free of charge via the Internet
at http://pubs.acs.org.
(48) Schultz, B. E.; Holm, R. H.; Hille, R. J. Am. Chem. Soc. 1995, 117,
827-828.
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