Selectivity of thiolate ligand and preference of substrate in model reactions of dissimilatory nitrate reductase

Article abstract of DOI:10.1021/ic7024268

Complexes analogous to the active site of dissimilatory nitrate reductase from Desulfovibrio desulfuricans are synthesized. The hexacoordinated complexes [PPh₄][Mo^IV(PPh₃)(SR)(mnt)₂] (R = -CH₂CH₃ (1), -CH₂Ph (2)) released PPh ₃ in solution to generate the active model cofactor, {Mo ^IV(SR)(mnt)2}^1-, ready with a site for nitrate binding. Kinetics for nitrate reduction by the complexes 1 and 2 followed Michaelis-Menten saturation kinetics with a faster rate in the case of 1 (V _Max = 3.2 × 10^-2 s^-1, K_M = 2.3 × 10^-4 M) than that reported earlier (V_Max = 4.2 × 10^-3 s^-1, K_M = 4.3 × 10 ^-4 M) (Majumdar, A.; Pal, K.; Sarkar, S. J. Am. Chem. Soc. 2006, 128, 4196-4197). The oxidized molybdenum species may be reduced back by PPh ₃ to the starting complex, and a catalytic cycle involving [Bu ₄N][NO₃] and PPh₃ as the oxidizing and reducing substrates, respectively, is established with the complexes 1 and 2. Isostructural complexes, [Et₄N][Mo^IV(PPh ₃)(X)(mnt)₂] (X = -Br (3), -I (4)) did not show any reductive activity toward nitrate. The selectivity of the thiolate ligand for the functional activity and the cessation of such activity in isostructural halo complexes demonstrate the necessity of thiolate coordination. Electrochemical data of all these complexes correlate the ability of the thiolated species for such oxotransfer activity. Compounds 1 and 2 are capable of reducing substrates like TMANO or DMSO, but after the initial 15-20% conversion, the product trimethylamine or dimethylsulfide formed interacts with the active parent complexes 1 and 2 thereby slowing down further oxo-transfer reaction similar to feedback type reactions. From the functional nitrate reduction, the molybdenum species finally reacts with the nitrite formed leading to nitrosylation similar to the NO evolution reaction by periplasmic nitrate reductase from Pseudomonas dentrificans. All these complexes (1-4) are characterized structurally by X-ray, elemental analysis, electrochemistry, electronic, FT-IR, mass and ³¹P NMR spectroscopic measurements.

Full text of DOI:10.1021/ic7024268

Inorg. Chem. 2008, 47, 3393-3401
Selectivity of Thiolate Ligand and Preference of Substrate in Model
Reactions of Dissimilatory Nitrate Reductase†
Amit Majumdar, Kuntal Pal, and Sabyasachi Sarkar*
Department of Chemistry, IIT Kanpur, Kanpur 208016, India
Received December 17, 2007
Complexes analogous to the active site of dissimilatory nitrate reductase from Desulfovibrio desulfuricans are
synthesized. The hexacoordinated complexes [PPh4][MoIV(PPh3)(SR)(mnt)2] (R ) -CH2CH3 (1), -CH2Ph (2))
released PPh3 in solution to generate the active model cofactor, {MoIV(SR)(mnt)2}1-, ready with a site for nitrate
binding. Kinetics for nitrate reduction by the complexes 1 and 2 followed Michaelis-Menten saturation kinetics
with a faster rate in the case of 1 (VMax ) 3.2 × 10-2 s-1, KM ) 2.3 × 10-4 M) than that reported earlier (VMax
) 4.2 × 10-3 s-1, KM ) 4.3 × 10-4 M) (Majumdar, A.; Pal, K.; Sarkar, S. J. Am. Chem. Soc. 2006, 128,
4196–4197). The oxidized molybdenum species may be reduced back by PPh3 to the starting complex, and a
catalytic cycle involving [Bu4N][NO3] and PPh3 as the oxidizing and reducing substrates, respectively, is established
with the complexes 1 and 2. Isostructural complexes, [Et4N][MoIV(PPh3)(X)(mnt)2] (X ) -Br (3), -I (4)) did not
show any reductive activity toward nitrate. The selectivity of the thiolate ligand for the functional activity and the
cessation of such activity in isostructural halo complexes demonstrate the necessity of thiolate coordination.
Electrochemical data of all these complexes correlate the ability of the thiolated species for such oxotransfer activity.
Compounds 1 and 2 are capable of reducing substrates like TMANO or DMSO, but after the initial 15–20% conversion,
the product trimethylamine or dimethylsulfide formed interacts with the active parent complexes 1 and 2 thereby
slowing down further oxo-transfer reaction similar to feedback type reactions. From the functional nitrate reduction,
the molybdenum species finally reacts with the nitrite formed leading to nitrosylation similar to the NO evolution
reaction by periplasmic nitrate reductase from Pseudomonas dentrificans. All these complexes (1– 4) are characterized
structurally by X-ray, elemental analysis, electrochemistry, electronic, FT-IR, mass and 31P NMR spectroscopic
measurements.
Introduction
DesulfoVibrio desulfuricans, the oxidized active site has been
structurally characterized as [MoVI(OHx)(S.Cys)(S2Pd)2] (x
) 1 or 2) by X-ray crystallography,3 and the reduced desoxo
site [MoIV(S.Cys)(S2Pd)2] has been proposed to mediate the
important oxotransfer reaction (eq 1).
Under Hille classification1 of molybdenum oxotransferase
enzymes, the DMSOR (dimethylsulfoxide reductase) family
conserves two pterin dithiolene (S2Pd)2 moieties and one
protein-derived ligand which normally varies with the
Ser.O-, Cys.S-, or Cys.Se- residue in their cofactor. In
addition to DMSOR and TMANOR (trimethylamine N-oxide
reductase), the DMSOR family includes dissimilatory nitrate
reductase.1 This nitrate reductase catalyzes the reduction of
nitrate to nitrite (Figure 1) and is found in certain bacteria
having the ability to grow on nitrate and use it for their
respiration. In dissmilatory nitrate reductase (NiR)2 from
NO3- + 2H+ + 2e- h NO2- + H2O
(1)
It has been proposed3 that nitrate is first bound to the
molybdenum center of the reduced cofactor with the forma-
tion of an enzyme-substrate complex, followed by the
essential oxotransfer and the elimination of the nitrite ion
with the formation of oxidized molybdenum cofactor,
[MoVI(O)(S.Cys)(S2Pd)2]. This oxo form is believed to be
very protophilic, resulting in the formation of protonated
†
Dedicated in the memory of Prof. Edward I. Stiefel.
* To whom correspondence should be addressed. E-mail: abya@
iitk.ac.in.
(3) 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.; Romao, M. J. Structure 1999, 7, 65–79.
(1) Hille, R. Chem. ReV. 1996, 96, 2757–2816.
(2) Abbreviations are given in Chart 1.
10.1021/ic7024268 CCC: $40.75  2008 American Chemical Society
Inorganic Chemistry, Vol. 47, No. 8, 2008 3393
Published on Web 03/13/2008

Majumdar et al.
Chart 1. Designation of Complexes and Abbreviations
Figure 1. Description of the reduced and oxidized sites of dissimilatory
nitrate reductase (DesulfoVibrio desulfuricans) and the proposed reaction
pathway. Also shown is the pyranopterindithiolate cofactor (R is absent or
a nucleotide).
A preliminary study on this functional analogue reaction has
been reported.11 To mimic cysteinyl thiolate coordination
of the native protein,3 we now refined our study by
synthesizing molybdenum(IV) bis(dithiolene) complexes
(Chart 1) containing simple aliphatic thiolates. The com-
plexes, [PPh4][MoIV(PPh3)(SCH2CH3)(mnt)2].CH2Cl2 (1) and
[PPh4][MoIV(PPh3)(SCH2Ph)(mnt)2] (2) reduce nitrate to
nitrite. Kinetics investigation of the reaction using
[Bu4N][NO3] as the source of nitrate revealed enzymatic
saturation kinetics behavior by following Michaelis-Menten
kinetics. Compounds 1, 2, and 5 (Chart 1) are capable of
reducing TMANO or DMSO, but in the case of DMSO, the
initial oxo transfer reaction was inhibited by the accumulated
reaction product, dimethylsulfide, by blocking the substrate
binding site for further reaction, which is similar to feedback
inhibition. In the case of the TMANO reduction, the product
trimethylamine, being basic, combines with the trace amount
of moisture present even in dry solvent and thereby
decomposes the active species responsible for TMANO
reduction. The formation of DMS-coordinated complexes of
1, 2, or 5 can be monitored spectroscopically, but these could
not be isolated because they are unstable; this led to the
formation of stable tris-dithiolene complexes. Complexes
[Et4N][MoIV(PPh3)(Br)(mnt)2] (3) and [Et4N][MoIV-
(PPh3)(I)(mnt)2] (4), isostructural to 1 and 2, have been
synthesized to compare their ability to participate in similar
oxo transfer reactions. However, these complexes do not
show any reductive activity toward nitrate, TMANO, or
DMSO. The inability to isolate the oxidized complex in the
nitrate reduction is caused by the nitrosylation of the
molybdenum species11 by the reaction product, nitrite. Such
NO evolution or nitrosylation to the Fe-S cluster is known12
from perplasmic nitrate reductase isolated from Pseudomonas
oxidized species [MoVI(OHx)(S.Cys)(S2Pd)2] (x ) 1 or 2).3
To understand the role of the varied protein derived ligands
precisely, molybdoenzymes of the DMSOR family are
studied by site-directed mutagenesis.4 It is known that this
protein–ligand attached to molybdenum in an active site
mainly controls the specificity of the active site toward a
substrate because the replacement of protein–ligand serinate
(in DMSOR2) by cysteinate or by selenocysteinate changes
the properties of the enzymes along with their activity. The
structure of a second dissimilatory nitrate reductase from
Escherichia coli has been crystallographically established5
which contains a [MoIV(O2C.Asp)(S2Pd)2] site with an
unsymmetrically coordinated carboxylate group.
Nitrate reduction by Mo(IV) complexes devoid of any
dithiolene coordination is well-known.6 Pentacoordinated
molybdenum(IV) bis(dithiolene) monothiolate complexes
with resemblance to the active site of NiR have been
reported,7 which with nitrate7a underwent decomposition8
to [MoV(S2C2Me2)3]1-. However, the corresponding tungsten
complex was shown to reduce nitrate with a second-order
reaction kinetics.8 In an attempt to show [MoIV-
(SPh)(S2C2Me2)2]1- as the active model cofactor, the irradia-
tion on the hexacoordinated complex, [MoIV(CO)-
(SPh)(S2C2Me2)2]1- to remove CO resulted in decomposi-
tion7a forming [MoV(S2C2Me2)3]1-. Following the success
of the 1,2-dicyanoethylenedithiolate (mnt2-)9 ligand in the
synthesis of the Mo(VI) complex to perform the functional
analogue reaction of sulfite oxidase,10 we used this ligand
for the synthesis of desoxo complexes in relevance to NiR.
(4) Hilton, J. C.; Temple, C. A.; Rajagopalan, K. V. J. Biol. Chem. 1999,
274, 8428.
(5) Bertero, M. G.; Rothery, R. A.; Palak, M.; Hou, C.; Lim, D.; Blasco,
F.; Weiner, J. H.; Strynadka, N. C. J. Nat. Struct. Biol. 2003, 10, 681–
687.
(9) (a) Bahr, G; Schleitzer, B. Chem. Ber 1955, 88, 1771. (b) Bahr, G.
Angew. Chem. 1956, 68, 525. (c) Stiefel, E. I.; Bennet, L. E.; Dori,
Z.; Crawford, T. H.; Simo, C.; Gray, H. B. Inorg. Chem. 1970, 9,
281–286. (d) Davidson, A; Holm, R. H. Inorg. Synth 1967, 10, 8.
(10) Das, S. K.; Chaudhury, P. K.; Biswas, D.; Sarkar, S. J. Am. Chem.
Soc. 1994, 116, 9061–9070.
(6) (a) Berg, J. M.; Holm, R. H. J. Am. Chem. Soc. 1985, 107, 925–932.
(b) Craig, J. A.; Holm, R. H. J. Am. Chem. Soc. 1989, 111, 2111–
2115.
(7) (a) Lim, B. S.; Donahue, J. P.; Holm, R. H. Inorg. Chem. 2000, 39,
263–273. (b) Musgrave, K. B.; Lim, B. S.; Sung, K.-M.; Holm, R. H.;
Hedman, B.; Hodgson, K. O. Inorg. Chem. 2000, 39, 5238–5247. (c)
Lim, B. S.; Holm, R. H. J. Am. Chem. Soc. 2001, 123, 1920–1930.
(8) Jiang, J.; Holm, R. H. Inorg. Chem. 2005, 44, 1068–1072.
(11) Majumdar, A.; Pal, K.; Sarkar, S. J. Am. Chem. Soc. 2006, 128, 4196–
4197.
(12) Butler, C. S.; Charnock, J. M.; Bennett, B.; Sears, H. J.; Reilly, A. J.;
Ferguson, S. J.; Garner, C. D.; Lowe, D. J.; Thomson, A. J.; Berks,
B. C.; Richardson, D. J. Biochemistry 1999, 38, 9000–9012.
3394 Inorganic Chemistry, Vol. 47, No. 8, 2008

Model Reactions of Dissimilatory Nitrate Reductase
IR (CsI pellet): ν 352, 345 (MoIV-Sdithiolene), 326 (MoIV-SEt) cm-1
.
dentrificans. Such NO production is now known as a
common feature because there are reports13 that nitrate
reductase from plants uses nitrite generated from nitrate to
produce NO.
31P NMR: δ 49.88, s. MS: m/z ) 700.908 ([MoIV(PPh3)-
(SCH2CH3)(mnt)2]1-), 438.880 ([MoIV (SCH2CH3)(mnt)2]1-).
[PPh4][MoIV(PPh3)(SCH2Ph)(mnt)2] (2). To a solution of 4.28 g
(4 mmol) of [PPh4]2[MoIVO(mnt)2] and 10.49 g (40 mmol) of PPh3
in 100 mL of dichloromethane,1 mL of methanesulphonic acid was
slowly added at 0 °C with constant stirring. The initial green color
of the solution changed to bright red. After 10 min, 2.85 mL of
PhCH2SH (24 mmol) was added into this solution. Stirring was
continued for another 15 min at 0 °C when the solution became
dark violet. Petroleum ether (60-80 °C) was added to initiate
cloudiness, and the mixture was allowed to stand overnight at 4
°C to yield dark violet block shaped diffraction quality crystals.
These were filtered and washed with petroleum ether and dried
under inert atmosphere. Yield: 3.96 g (90%). Required for
C57H42MoN4P2S5: C, 62.17; H, 3.84; N, 5.09; S, 14.56%. Found:
C, 62.25; H, 3.92; N, 5.16; S 14.62%. Absorption spectrum
(dichloromethane) λmax (ꢀM): 387 (5510), 512 (4375), 555 (3690)
nm. IR (KBr pellet): ν 2200 (CN), 3055 (aromatic CH stretching),
724 (aromatic CH bending) cm-1. IR (CsI pellet): ν 350, 343
Experimental Section
Materials and Methods. All reactions and manipulations were
performed under argon atmosphere using modified Schlenk tech-
nique. CH3CH2SH and PhCH2SH were obtained from Lancaster
and [Et4N][I] was obtained from Spectrochem, India. PPh4Br and
[Bu4N][NO3] were obtained from Alfa-Essar and Aldrich, respec-
tively. [Et4N][Br], Et3N, and CH3SO3H were obtained from S. D.
Fine Chemicals, Ltd., India. Solvents were dried and distilled by
standard procedure. [Et4N]2[MoIVO(mnt)2] and [PPh4]2[MoIVO-
(mnt)2] were prepared following the process reported earlier.10,14,15
Infrared spectra were recorded on a Bruker Vertex 70 FT-IR
spectrophotometer as pressed KBr and CsI disks. Elemental analyses
for carbon, hydrogen, nitrogen, and sulfur were recorded with
Perkin-Elmer 2400 microanalyser. Mass spectra (ESI, negative ion)
were recorded on a Waters Micromass Q-TOF Premier mass
spectrometer. Absorption spectroscopic measurements and kinetics
measurements for nitrate reduction by complexes 1 and 2 were
performed in a USB 2000 (Ocean Optics Inc.) UV–visible spec-
trophotometer equipped with fiber optics. Cyclic voltammetric
measurements were made with a BASi Epsilon-EC Bioanalytical
Systems, Inc. instrument. Cyclic voltammograms and differential
pulse polarographs of 10-3 M solution of the compounds were
recorded (see Supporting Information) with a glassy carbon
electrode as working electrode, 0.2 M Bu4NClO4 as supporting
electrolyte, Ag /AgCl electrode as reference electrode, and a
platinum auxiliary electrode. Sample solutions were prepared in
presence of PPh3 in dichloromethane. PPh3 was used externally to
stabilize the complexes in solution. All electrochemical experiments
were done under argon atmosphere at 298 K. Potentials are
referenced against internal ferrocene (Fc) and are reported relative
to the Ag /AgCl electrode (E1/2(Fc+/Fc) ) 0.459 V vs Ag/AgCl
electrode).
(MoIV-Sdithiolene), 324 (MoIV-SCH Ph) cm-1.31P NMR: δ 49.58, s.
2
MS: m/z ) 763.068 ([MoIV(PPh3)(SCH2Ph)(mnt)2]1-), 500.833
([MoIV (SCH2Ph)(mnt)2]1-).
[Et4N][MoIV(PPh3)(Br)(mnt)2] (3). To a solution of 2.62 g (4
mmol) of [Et4N]2[MoIVO(mnt)2], 10.49 g (40 mmol) of PPh3 in
100 mL of dichloromethane was added 1 mL methanesulphonic
acid dropwise with constant stirring at 0 °C. After the bright red
solution was stirred for 10 min., 5.04 g (24 mmol) of Et4NBrwas
added. Stirring was continued for another 15 min at 0 °C to get a
clear red solution. The addition of petroleum ether (60-80 °C) and
standing overnight at 4 °C produced reddish orange crystals that
were contaminated with an oily mass. The whole mass was filtered,
washed with isopropyl alcohol, followed by diethyl ether, to remove
the oily part and dried in vacuum to get needle-shaped dark reddish
orange diffraction quality crystals. Yield: 3.05 g (90%). Required
for C34H35BrMoN5PS4: C, 48.11; H, 4.16; N, 8.25; S, 15.11%.
Found: C, 48.22; H, 4.21; N, 8.31; S 15.21%. Absorption spectrum
(dichloromethane) λmax (ꢀM): 364 (5150), 400 (5500) 489 (3275),
519 (3000) nm. IR (KBr pellet): ν 2200 (CN), 3050 (aromatic CH
stretching), 730 (aromatic CH bending) cm-1.31P NMR: δ 49.76,
s. MS: m/z ) 718.826 ([MoIV(PPh3)(Br)(mnt)2]1-), 456.724
([MoIV(Br)(mnt)2]1-).
Synthesis.
[PPh4][MoIV(PPh3)(SCH2CH3)(mnt)2].CH2Cl2
(1). To a solution of 4.28 g (4 mmol) of [PPh4]2[MoIVO(mnt)2]
and 10.49 g (40 mmol) of PPh3 in 100 mL of dichloromethane at
0 °C was slowly added 1 mL of methanesulphonic acid with
constant stirring. Initial green color of the solution turned bright
red after 10 min, and then 1.77 mL of CH3CH2SH (24 mmol) was
added to this solution. Stirring was continued for another 15 min
at 0 °C when the solution acquired a dark violet color. Addition of
petroleum ether (60-80 °C) resulted in incipient cloudiness and
on standing overnight at 4 °C dark violet block shaped diffraction
quality crystals separated out. These were filtered, washed with
petroleum ether, and dried under inert atmosphere. Yield: 4.046 g
(90%). Analysis required for C53H42Cl2MoN4P2S5: C, 53.65; H,
3.67; N, 4.63; S, 13.26%. Found: C, 53.75; H, 3.76; N, 4.75; S,
13.32%. Absorption spectrum (dichloromethane) λmax (ꢀM): 387
(6350), 512 (4920), 555 (3985) nm. IR (KBr pellet): ν 2200 (CN),
[Et4N][MoIV(PPh3)(I)(mnt)2].CH2Cl2 (4). To a solution of
2.62 g (4 mmol) of [Et4N]2[MoIVO(mnt)2], 10.49 g (40 mmol) of
PPh3 in 100 mL of dichloromethane at 0 °C, was added 1 mL of
methanesulphonic acid dropwise with constant stirring. The solution
became bright red after 10 min, and 6.17 g (24 mmol) of Et4NI
was added to this solution. Stirring was continued for another 15
min 0 °C, and the solution became dark violet in color. Petroleum
ether (60 °C-80 °C) was added to the filtrate to initiate incipient
cloudiness, and the mixture was allowed to stand overnight at 4
°C to form dark violet crystals that were contaminated with an oily
mass. The whole mass was filtered, washed with water, followed
by isopropyl alcohol and diethyl ether, to remove the oily part and
dried in vacuum to get the block-shaped violet diffraction quality
crystals. Yield: 3.00 g (85%). Required for C35H37Cl2IMoN5PS4:
C, 42.86; H, 3.80; N, 7.14; S, 13.08%. Found: C, 42.92; H, 3.89;
N, 7.21; S 13.17%. Absorption spectrum (dichloromethane) λmax
(ꢀM): 378 (7190), 511 (2800), 568 (2710) nm. IR (KBr pellet): ν
2200 (CN), 3050 (aromatic CH stretching), 740 (aromatic CH
3055 (aromatic CH stretching), 724 (aromatic CH bending) cm-1
.
(13) (a) Desikan, R.; Griffiths, R.; Hancock, J.; Neill, S. Proc. Natl. Acad.
Sci. 2002, 99, 16314–16318. (b) Rockel, P.; Strube, F.; Rockel, A.;
Wildt, J.; Kaiser, W. M. J. Expt. Bot. 2002, 53, 103–110. (c) Lamotte,
O.; Courtois, C.; Barnavon, L.; Pugin, A.; Wendehenne, D. Planta
2005, 221, 1–4.
(14) Maity, R.; Nagarajan, K.; Sarkar, S. J. Mol. Struct. 2003, 656, 169–
176.
bending) cm-1 31P NMR: δ 49.16, s. MS: m/z ) 766.463
.
(15) Nagrajan, K.; Joshi, H. K.; Chaudhury, P. K.; Pal, K.; Cooney, J. A.;
Enemark, J. H.; Sarkar, S. Inorg. Chem. 2004, 43, 4532–4533.
([MoIV(PPh3)(I)(mnt)2]1-), 504.710 ([MoIV(I)(mnt)2]1-).
Inorganic Chemistry, Vol. 47, No. 8, 2008 3395

Majumdar et al.
Kinetics Measurements. These reactions were performed in
dichloromethane solution. Electronic spectroscopic and kinetics
measurements for nitrate reduction by complexes 1 and 2 were
performed using a USB 2000 (Ocean Optics Inc.) UV–visible
spectrophotometer equipped with fiber optics. Nitrate reduction was
followed by observing the appearance of the 770 (in case of 1)
and 790 nm bands (in case of 2). Sharp isosbestic points
demonstrated that these two oxo transfer reactions proceeded cleanly
in the first phase of completion of the reaction
program. The structure was solved using SIR9718 and refined using
SHELXL-97.19 Crystal structures were viewed using ORTEP.20 The
space group of the compounds was determined based on the lack
of systematic absence and intensity statistics. Full-matrix least-
squares/difference Fourier cycles were performed which located
the remaining non-hydrogen atoms. All non-hydrogen atoms were
refined with anisotropic displacement parameters. Complex 1
contains one cation, one anion, and one dichloromethane in the
asymmetric unit. The structure suffered from disorder of unidentified
solvent (CH2Cl2/petroleum ether) in the lattice, which was not
included in the refinement but was taken care of by the SQUEEZE-
procedure (from PLATON). The volume occupied by the solvent
was 235.3 Å3, and the number of electrons per unit cell deduced
by SQUEEZE was 31.8. Complexes 2 and 3 possess only one anion
and one cation in their respective asymmetric units. Complex 4
contains half of a cation, half of an anion, and half of a
dichloromethane molecule in its asymmetric unit. One phenyl ring
of the triphenylphosphine moiety in the anion of complex 4 is
disordered over two positions which were refined with free part
instruction using SHELXL-97.19
[MoIV(PPh2)(SCH2CH3)(mnt)2]1-
f
1-
[MoVI(O) SCH CH mnt 2]
652 nm
680 nm
(
)
3
)
(
)
(
2
[MoIV(PPh3)(SCH2Ph)(mnt)2]1-
f
VI
1-
(
)
(
)
[Mo (O) SCH Ph mnt 2]
(
)
2
Reaction systems contained concentrations of 1 × 10-4 M for
complexes 1 and 2 with an excess substrate [(Bu4N)(NO3)]
concentration in the range of 1.25-3 × 10-4 M. For each complex,
reactions were run at least three times at each substrate concentration
at 25 °C under first-order conditions. Plots of (-ln Ct) [Ct ) (ꢀ2C0
- OD)/(ꢀ2 - ꢀ1), where ꢀ1 and ꢀ2 are the molar absorption
coefficients of the thiolate complexes (1 or 2) and their oxidized
species at 770 and 790 nm, respectively, versus time were linear,
from which kobs values were calculated at each substrate concentra-
tion. Plot of 1/kobs versus 1/[(Bu4N)(NO3)] (Lineweaver-Burk plot)
yielded the VMax and KM values. Errors (see Supporting Information)
were estimated using the EO4FDF NAG FORTRAN Library routine
document.16 Because the oxidized species corresponding to com-
plexes 1 and 2 could not be isolated because of their subsequent
reaction with product nitrite, ꢀ2 values for the complexes 1 and 2
were calculated from the maximum absorbance observed at 770
and 790 nm during the reaction course with [Bu4N][NO3] assuming
full conversion.
Determination of Nitrite. Nitrite generated in the system
containing [Bu4N][NO3] and complexes 1 or 2 was measured
quantitatively following a colorimetric method.17 Immediately after
completion of the oxo transfer reaction, the solvent in the reaction
mixture was removed under vacuum, and the residue was extracted
with water. Griess reagent (1:1 mixture of 0.1% N/1-napthyleth-
ylenediamine dihydrochloride in water and 1% sulfanilamide in
5% phosphoric acid) was added to the extract to obtain a purple
solution. Absorbance at 550 nm was measured, and the concentra-
tion was calculated with the help of a standard curve made with
preknown concentration of NaNO2. Nitrite generated in these
systems corresponded to about 90% of the theoretical conversion
for a complete reaction.
Results and Discussion
Synthesis and Reactivity of Compounds. In the complex,
[MoIVO(mnt)2]2-,10,14 the molybdenyl group, {MoIVdO},
behaves like the carbonyl group as the {ModO} double bond
can easily be protonated with the addition of an acid (HX)
to yield {MoIV(OH)(X)} group, and when the counteranion,
X, is a non-coordinating anion like methanesulfonate, the
species responds to hydrolysis finally to yield the stable tris21
species, [MoIV(mnt)3]2- in 67% yield based on the molyb-
denum present. Its formation can be near quantitative on
addition of 1 equiv of free mnt anion into the reaction
medium.22 The direct formation of the bluish green tris
product may be prevented once the acidification is carried
out in the presence of triphenylphosphine where the initial
brownish green color of the solution rapidly changed to red
presumably because of the formation of the probable reaction
intermediate, {MoIV(PPh3)(OH)(mnt)2}(Figure 2). Addition
of CH3CH2SH, PhCH2SH, Et4NBr, and Et4NI at this stage
into this red solution yielded the complexes 1–4 (Chart 1).
The presence of excess PPh3 at this stage is crucial in
isolating these complexes in excellent yield because excess
PPh3 prevents the back reaction in forming the hypothetical
lone hydroxo species which rapidly changed to the tris21
complex. Complexes 1 and 2 dissociate readily in solution
generating the active pentacoordinated species, [MoIV(S-
R)(mnt)2]1- (R ) -CH2CH3, -CH2Ph), which are competent
X-ray Structure Determinations. Suitable diffraction quality
single crystals were obtained from the crystallization procedures
described in each synthesis. The crystals used in the analyses were
glued to glass fibers and mounted on BRUKER SMART APEX
diffractometer. The instrument was equipped with CCD area
detector, and data were collected using graphite-monochromated
Mo KR radiation (λ ) 0.71069 Å) at low temperature (100 K).
Cell constants were obtained from the least-squares refinement of
three-dimensional centroids through the use of CCD recording of
narrow ω-rotation frames, completing almost all-reciprocal space
in the stated θ range. All data were collected with SMART 5.628
(BRUKER, 2003), and were integrated with the BRUKER SAINT
(18) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giaco-
vazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna,
R. J. Appl. Crystallogr. 1999, 32, 115–119.
(19) Sheldrick, G. M. SHELX97, Programs for Crystal Structure Analysis,
(release 97–2); University of Göttingen: Göttingen, Germany, 1997.
(20) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
(21) (a) Brown, G. F.; Stiefel, E. I. Inorg. Chem. 1973, 12, 2140–2147.
(b) Xu, H.-Wu.; Chen, Z.-N; Wu, J.-G Acta Crystallogr. E 2002, 58,
m631. (c) Formitchev, D. V.; Lim, B. S.; Holm, R. H. Inorg. Chem.
2001, 40, 645–654. (d) Wang, K.; McConnachie, J. M.; Stiefel, E. I.
Inorg. Chem. 1999, 38, 4334–4341. (e) Matsubayashi, G.; Douki, K.;
Tamura, H.; Nakano, M.; Mori, W. Inorg. Chem. 1993, 32, 5990–
5996. (f) Draganjac, M.; Coucouvanis, D. J. Am. Chem. Soc. 1983,
105, 139–140.
(16) Box, G. E. P.; Hunter, W. G.; Hunter, J. S. In Statistics for Experiments;
John Wiley & Sons: New York, 1978.
(17) Basset, J.; Denney, R. C.; Jeffery, G. H.; Mendham, J. In Vogel’s
Textbook of QuantitatiVe Chemical Analysis; Longman Scientific and
Technical: Harlow, Essex, U.K., 1989; pp 702 ff.
(22) Das, S. K.; Biswas, D.; Maiti, R.; Sarkar, S. J. Am. Chem. Soc. 1996,
118, 1387–1397.
3396 Inorganic Chemistry, Vol. 47, No. 8, 2008

Model Reactions of Dissimilatory Nitrate Reductase
Figure 3. Structure (ORTEP view) of anions of 1 (a) and 4 (b) showing
50% probability thermal ellipsoids with atom labeling scheme. Hydrogen
atoms are omitted for clarity.
as observed in other reported bis(dithiolene) molybdenum
complexes.7,10,11 There is very little variation in the Mo-P
bond distance within the series (1–4) and the structurally
similar complexes (5 and 6) (Chart 1). The Mo-P bond
distance is longest (2.590 Å) in the case of complex 2 and
shortest (2.538 Å) in the case of complex 5. Complex 1
exhibits slightly longer Mo-S(thiolate) bond distance (2.367
Å) than in the case of complex 2 (2.338 Å) or complex 5
(2.336 Å). The Mo-S(thiolate) bond distance in complexes
1, 2, and 5 are quite longer than that in the related
molybdenum bis(dithiolene) complexes (2.3127b,c and 2.320
Å)7a but appreciably shorter than that reported (2.469 Å) in
the case of another related molybdenum bis(dithiolene)
complex, [Et4N][MoIV(CO)(SPh)(S2C2Me2)2].7a In complexes
1, 2, and 5, the distance between thiolate sulfur atom and
phosphorus atom of triphenylphosphine moiety ranges from
3.066 (complex 1) to 3.098 Å (complex 5), while the
Mo-S(thiolate)-P angle ranges from 76.29° (complex 1)
to 77.00° (complex 2). Mo-X bond distances are 2.626 (X
) Br) and 2.834 Å (X ) I) in complexes 3 and 4,
respectively. Displacement of Mo atom from the S4 (dithi-
olene sulfur) mean plane (see Table 2) varies from 0.815
(in complex 3) to 0.857 Å (in complex 2).
Oxo Transfer Reaction with Nitrate. The reaction of
nitrate with ([Bu4N][NO3]) as the oxo-donor substrate was
found to be successful only with two complexes (1 and 2)
within the series (1–4). Complexes 1 and 2 individually react
with [Bu4N][NO3] in dichloromethane medium at varying
rates. When such reactions with complexes 1 and 2 are
monitored by 31P NMR spectroscopy with [Bu4N][NO3] as
substrate in 1:2 molar ratio, only one signal appeared11 (other
than the signal at 20.50 ppm corresponding to PPh4+) at 24.97
ppm corresponding to triphenylphosphine oxide (Ph3PO) as
shown in the eqs 2 and 3.
Figure 2. Schematic representation for the synthesis of complexes 1–6.
to reduce nitrate to nitrite. The formation of nitrite form
nitrate in such reaction can be confirmed by Griess’s reagent
with blank tests as controls in the absence of 1 or 2.
Reversible dissociation of triphenylphosphine (PPh3) from
the complexes 1 and 2 has been monitored by 31P NMR
spectroscopy (see Supporting Information). With time, the
peak intensity (δ 49.89, s) due to PPh3 bound to molybde-
num(IV) center (1) decreases with the concomitant increase
of the peak intensity (δ -8.16, s) because of dissociated free
PPh3. Reversibility of the triphenylphosphine dissociation
was confirmed by the addition of free PPh3 in the solution,
while monitoring the 31P NMR spectroscopy which restored
the maximum intensity of the δ 49.89 peak responsible for
the PPh3 bound to molybdenum(IV) (1). Compounds 3, 4,
and 6 (Chart 1) also dissociate in solution, but the dissocia-
tion was found to be very slow: no appreciable dissociation
up to 2 h was noticed. With time, these are slowly converted
to the thermodynamically stable tris21 [MoIV(mnt)3]2- com-
plex in dichloromethane solution (see Supporting Informa-
tion). Complex 5 is rapidly converted to 3, 4, and 6 with
Et4NBr, Et4NI, and Et4NCl, respectively. Even the presence
of PhCH2Br is sufficient, albeit slowly, to trigger the
conversion of 5 to 3 (see Supporting Information), whereas
the thiolate groups in complexes 1 and 2 cannot be
substituted even in the presence of a large excess of
respective halides. These observations showed the stability
of the aliphatic thiolato coordination in the reactive species
to interact with the nitrate ion.
X-Ray Structure Description. All four complexes (1–4)
are characterized by X-ray structure determination. All the
four complexes are isostructural, and structures of 1 and 4
are shown in Figure 3 (see Supporting Information for the
structures of 2 and 3), and the leading structural parameters
are collected in Table 1. The selected bond distances are
presented in Table 2. For the most part, these parameters
are in good agreement with the other related molybdenum
bis(dithiolene) complexes.10,11,14,15 The ranges of mean C-C
and S-C bond distances are 1.33–1.34 and 1.73–1.79 Å,
respectively, which are sufficiently close to the typical bond
lengths (sp2)CdC(sp2) ) 1.331(9) Å and S-C(sp2) ) 1.75(2)
Å to establish the ligand mnt9 as a classical ene-1,2-dithiolate,
MoIV PPh SR mnt 2 - + NO3- f
(
)(
)
[
]
)( )(
(
)
3
Mo O SR mnt 2 - + NO2- + PPh3
VI
[
]
(
)
R ) -CH2CH3 (1), - CH2Ph (2), - Ph (6) (2)
VI
Mo O SR mnt 2 - + PPh3 f
[
]
(
)( )(
)
IV
Mo SR mnt 2 - + Ph3PO
[
]
(
)(
)
R ) -CH2CH3 (1), - CH2Ph (2), - Ph (6) (3)
The oxidized species, {[MoVI(O)(SR)(mnt)2]1-}, produced
through the reduction of nitrate (eq 2) is reduced back (eq
Inorganic Chemistry, Vol. 47, No. 8, 2008 3397

Majumdar et al.
Table 1. Crystallographic Dataa for Complexes 1–4
complexes
1
2
3
4
formula
fw
cryst syst
space group
T (K)
C53H42Cl2MoN4P2S5
1124.04
triclinic
P1
100
C57H42MoN4P2S5
1101.18
monoclinic
P21/n
C34H35BrMoN5PS4
848.76
orthorhombic
Pna21
C35H37Cl2IMoN5PS4
980.69
orthorhombic
Pnma
100
100
100
Z
2
4
4
4
a (Å)
b (Å)
c (Å)
12.645(5)
14.499(5)
16.709(5)
113.494(5)
93.162(5)
91.779(5)
2800.5(17)
1.333
13.976(5)
20.679(5)
19.302(5)
90
109.979(5)
90
5243(3)
1.395
0.552
13.275(5)
18.470(5)
15.227(5)
90
90
90
3734(2)
1.510
1.719
2.04–28.29
1.116
0.0639 (0.1121)
13.616(5)
15.386(5)
18.900(5)
90
90
90
3960(2)
1.645
1.530
2.15–28.31
1.043
0.0506 (0.1139)
R (deg)
ꢀ (deg)
γ (deg)
V (Å3)
dcalcd (g/cm3)
µ (mm-1
)
0.611
2.16–28.77
0.909
θ range (deg)
2.20–28.39
1.070
0.0869 (0.1517)
GOF (F2)
R1b (wR2)c (%)
0.0736 (0.1628)
a Mo KR radiation. b R1 ) ∑|F0 - Fc|/∑F0. c wR2 ) {∑[w(F0 - Fc )2]/∑[w(F0 )2]}1/2
.
2
2
2
Table 2. Selected Bond Distances (Å) for Complexes 1–4
distances
Mo(1)-P(1)PPh3
Mo(1)-S(5)thiolate
nm (Complex 2, see Supporting Information) indicated clean
reduction of nitrate in both the cases. The oxidized species
{[MoVI(O)(SR)(mnt)2]1-}, despite numerous attempts, could
not be isolated so far. This is because of its participation in
the successive reactions with the nitrite produced in the oxo
transfer reaction. The oxidized species once formed slowly
started to change into another species using the available
nitrite present in the medium. It was found that the
concentration of the nitrite produced steadily increased during
the first 1.5 and 3 min (in 1 and 2, respectively) in these
oxo transfer reactions, but on standing for more than 10 min,
the concentration of the nitrite started decreasing in the
solution and after an hour the available concentration of
nitrite from the solution decreased appreciably. Routine
workup procedure of the resultant solution on standing
yielded known orange-brown species23 [MoIV(NO)2(mnt)2]2-.
Characterization of this species with the aid of IR spectros-
copy (νNO, KBr disk 1730 and 1625 cm-1) present as the
final molybdenum product11 helped in explaining our in-
ability in isolating the oxidized molybdenum species. Sul-
famic acid being insoluble in dichloromethane could not be
used to scavenge6 the nitrite ion produced in the oxo-transfer
reaction to protect the oxidized species formed to participate
further reaction with nitrite ion. Interestingly, periplasmic
nitrate reductase from P. dentrificans, while reacting with
nitrate as substrate was shown to produce NO and part of
the Fe-S proteins was shown to be nitrosylated with the
formation of {FeNO} moiety.12 Further, groups of plant
nitrate reductase have been shown to use nitrite to produce
NO.13 The nitrosylation of molybdenum in the model
systems may occur because of the absence of protein which
may control the gate of substrate and product for proper
channelization to control local concentration around the
active metal center for the necessary enzymatic reaction. The
addition of nitrate to the solution of 3, 4, and 6 led to
formation of the tris21 complex almost instantaneously,
whereas this conversion in the absence of nitrate or any other
1
2
3
4
2.59(2) 2.59(2) 2.54(2) 2.55(2)
2.37(2) 2.34(2)
displacement of Mo(1) from S4 0.841
(dithiolene sulfur) mean plane
0.857
0.815
0.817
3) to {[MoIV(SR)(mnt)2]1-} (R ) -SCH2CH3, -SCH2Ph)
with concomitant formation of triphenylphosphine oxide
(Ph3PO). Appearance of only one signal corresponding to
Ph3PO indicated the reaction of nitrate with 1 and 2 to be
almost quantitative. Nitrate reduction by complexes 1 and 2
in dichloromethane solution were monitored by UV-vis
spectroscopy (Figure 4 for 1, see Supporting Information for
2). During the progress of the reaction, the intensity of the
parent absorption band at 512 nm along with the shoulder
at 555 nm started to decrease with time. Complex 1 showed
full development of a new band at 770 nm within 1.5 min
(Figure 4), whereas in the case of complex 2, complete
development of a new band at 790 nm occurred within 3
min (see Supporting Information). Appearance of tight
isosbestic points at 652 nm (Complex 1, Figure 4) and 680
Figure 4. Spectrophotometric monitoring of the reaction of
[PPh4][MoIV(PPh3)(SCH2CH3)(mnt)2].CH2Cl2 (1) (2 × 10-4 M) with
[Bu4N][NO3] (10 × 10-4 M) in dichloromethane. Total time ) 90 s. Scan
rate ) 5 s/scan.
(23) (a) McCleverty, J. A. Prog. Inor. Chem. 1968, 10, 49. (b) Connelly,
N. G.; Locke, J; McCleverty, J. A.; Phipps, D. A.; Ratcliff, B Inorg.
Chem. 1970, 9, 278–280.
3398 Inorganic Chemistry, Vol. 47, No. 8, 2008

Model Reactions of Dissimilatory Nitrate Reductase
reagent is relatively slow (see Supporting Information for 3
and 4). Monitoring these reactions by 31P NMR clearly
showed that the presence of nitrate or other ligands com-
pletely dissociated the bound PPh3 from the molybdenum,
whereas in the absence of any external ion or ligand there
remained an equilibrium between the bound and unbound
PPh3, and in the presence of excess of PPh3, the hexacoor-
dinated complexes became stable for a longer time. The
addition of nitrate influenced the release of bound PPh3 to
produce free pentacoordinated species like [MoIV(X)(mnt)2]1-
(X ) -Br (3), -I (4), and -Cl (6)) which even on binding
of nitrate at the released site are not capable to reduce nitrate
and hence are rapidly converted to the thermodynamically
stable tris21 complex. These results clearly indicate the
indispensable role of thiolate ligation in the synthetic model
complexes (1, 2, and 5) in their response to show oxo-transfer
reaction similar to native dissimilatory nitrate reductase.
UV-vis spectroscopy with 1: 2.5 molar ratios of complexes
(see Supporting Information). During the progress of the
reaction with DMSO, complexes 1 and 2 showed gradual
decrease in the intensity of the absorption band at 512 nm
along with the shoulder at 555 nm with concomitant
development of a new band at 770 and 790 nm, respectively
(see Supporting Information). Complex 5 exhibited gradual
decrease of the intense absorption band at 515 nm, along
with the shoulder at 570 nm with concomitant development
of the 770 nm band (see Supporting Information). The
progress of reaction with TMANO as substrate showed
similar behavior for these complexes. These reactions are
not clean and are associated with uncharacterized side
reactions leading ultimately to the formation of “tris” species.
Interestingly with DMSO (or TMANO) reaction a residual
purple color quite similar to the parent complex (1, 2, and
5) is retained even at the end of the maximum development
of the new band at 770 and 790 nm. On the basis of the 31
P
Oxo Transfer Reaction with DMSO and TMANO.
Complexes 1, 2, and 5 are found to reduce DMSO, as well
as TMANO as shown in eqs 4 and 5. The oxidized species,
{[MoVI(O)(SR)(mnt)2]1}, produced through the reactions (eq
4 and 5) are reduced back to
NMR data for Ph3PO in comparison with the nitrate reducing
systems, an estimated 15–20% conversion was found in case
of oxo transfer reaction with TMANO or DMSO. DMSO
reduction was also confirmed by passing a slow stream of
dinitrogen through the reaction mixture to drive out the
formed dimethylsulfide first through a trap and then through
an aqueous solution of HgCl2, leading to the white precipita-
tion of (Me2S)2(HgCl2)3. Quantification of this solid when
washed and dried indicated roughly 15% conversion. When
1 or 2 (also 5) in solution was treated with dimethysulfide
(DMS), the color of the solution changed immediately. 31P
NMR spectroscopic measurements of 1 and 2 (also 5) in
dichloromethane with excess DMS showed complete dis-
placement of PPh3 (δ -8.16 ppm, s) by dimethylsulfide. The
coordination of DMS in native DMSOR has been reported.25
In the present case, the binding of DMS at Mo(IV) site can
be observed by monitoring the changes in the electronic
spectrum of the starting complex upon addition of DMS (see
Supporting Information). However, such coordinated species
though relatively stable but cannot be isolated in the solid
state as it slowly changes to tris21 complex (see Supporting
Information). Reaction of DMSO with complexes 1 and 2
in presence of DMS shows partial development of the
absorbance bands characteristic of tris species (see Support-
ing Information). This chemistry is simple as excess DMS
replaced PPh3 and formed a new passive complex where the
DMS coordination competes with nitrate coordination.
Kinetics of Nitrate Reduction. Kinetics measurements
for the reduction of nitrate by the complexes 1 and 2 revealed
that the reactions follow Michaelis-Menten kinetics. The
Michaelis plots for the reduction of nitrate by the complex
1 is shown along with the Lineweaver-Burk plots as inset
in Figure 5 (see Supporting Information for 2). Kinetics
results are presented in Table 3. Nitrate reduction by the
complex 1 is much faster than that with complex 2 or
complex 5 reported earlier.11 Ratio of VMax calculated for
MoIV PPh SR mnt 2 - + Me3SO f
(
)(
)
[
]
(
)
3
Mo (O) SR mnt 2 - + Me2S + PPh3
VI
[
]
(
)(
)
R ) -CH2CH3 (1), - CH2Ph (2), - Ph (5) (4)
MoIV PPh SR mnt 2 - + Me3NO f
(
)(
)
[
]
(
)
3
Mo (O) SR mnt 2 - + Me3N + PPh3
VI
[
]
(
)(
)
R ) -CH2CH3 (1), - CH2Ph (2), - Ph (5) (5)
{[MoIV(SR)(mnt)2]1-} (R ) -SCH2CH3, -SCH2Ph, -SPh)
according to eq 3, with concomitant formation of triph-
enylphosphine oxide (Ph3PO). This was confirmed by the
observation that when the reaction with complexes 1, 2, or
5 were monitored by 31P NMR spectroscopy with DMSO
or TMANO as substrate in 1: 2.5 molar ratio, a signal
appeared at 24.97 ppm corresponding to triphenylphosphine
oxide (Ph3PO). It is known that in the absence of any catalyst,
DMSO and Ph3P do not react for at least 1 h even at 189
°C.24 Also it is checked that TMANO and Ph3P together
(1:1 mole ratio) in dichloromethane at ambient condition (25
°C) did not produce any identifiable Ph3PO up to a period
of 1 h. Therefore the presence of Ph3PO in the reaction
mixture strongly supports that the starting species participated
in oxo transfer reaction with DMSO (or TMANO) resulting
its oxidation which in turn was reduced by the partially
released Ph3P. Along with the presence of 24.97 ppm signal
for Ph3PO and 20.50 ppm signal for PPh4 , two 31P signals
+
characteristic of the MoIV bound PPh3 and free PPh3 were
observed in the case of complexes 1, 2, and 5 when allowed
to react with DMSO (or TMANO). The presence of MoIV-
bound PPh3 and free PPh3 suggested that reactions 4 and 5
are not complete with DMSO and TMANO as substrates
respectively. DMSO (or TMANO) reduction by these
complexes in dichloromethane solution was monitored by
(25) (a) Bray, R. C.; Adams, B.; Smith, A. T.; Richards, R. L.; Lowe, D. J.;
Bailey, S. Biochemistry 2001, 40, 9810–9820. (b) Adams, B.; Smith,
A. T.; Bailey, S.; McEwan, A. G.; Bray, R. C. Biochemistry 1999,
38, 8501–8511.
(24) Szmant, H. E.; Cox, O. J. Org. Chem. 1966, 31, 1595.
Inorganic Chemistry, Vol. 47, No. 8, 2008 3399

Majumdar et al.
1 and 2, respectively. The low turnover number for 1
compared to that for 2 or that obtained earlier with 5 (TON
) 50 mmol-1 s-1)11 is the result of the formation of the
oxidized species, {[MoVI(O)(SR)(mnt)2]1-}, at a much faster
rate with 1 compared with that with 2 or 5, and therefore, a
large extent of the oxidized product is consumed by
competing nitrosylating reactions by the generated product
nitrite prior to its reaction with PPh3 to form Ph3PO.
Structural Comparison of the Synthesized Com-
plexes with the Native Active Site. Structural environment
around the active site normally tunes the activity of the
central atom to interact with the substrate and govern the
follow-up reactions. An attempt has been made to compare
the structures of the synthesized complexes (1–6) with that
of the oxidized state of the active site for native NiR3 (see
Supporting Information for superposed structures and cor-
responding dihedral angles between the dithiolene planes).
It was found that the dithiolene planes of synthesized thiolate
(1, 2, and 5) complexes and native active site come closer
when traveling from complex 5 to 2 to 1, and the distorted
octahedral geometry of the complexes became closer to the
trigonal prismatic geometry of the native active site. More-
over the planes became more parallel when traveling from
complex 5 to 2 to 1, and the best match within the thiolate
series was found with complex 1. In spite of the “dithiolene
fold angle”26 effect, which does not allow matching of the
oxidized (VI) and reduced (IV) bis(dithiolene) Mo species
exactly, the structural fitting of the two dithiolene planes with
the native active site can be correlated with the reactivity.
A close analysis would require the availability of the
structural data from the identical oxidation state of the model
and native systems.
One can sum up that the active pentacoordinated model
complex allowed the substrate nitrate to bind to the site
vacated by PPh3, whereby the all important oxo transfer
reaction occurred. The reactivity of the synthesized com-
plexes followed a set pattern comparing their redox potential
data (see Table 4). The reactive thiolato species showed a
gradual decrease in the Epa potential (CV and DPP) from
complex 5 to 2 to 1, indicating that the molybdenum center
became more and more prone to oxidation (see Supporting
Information for cyclic voltametric traces and differential
pulse polarographs). We observed a decade ago22 that the
redox potential value measured for the active complex or
the active enzyme (E) can not reflect in any way its capability
to respond to a redox reaction when such reaction required
the formation of an enzyme–substrate complex (ES). For any
atom-transfer reaction obeying Michaelis–Menten complex-
ation, it is the thermodynamics of this complex (ES) which
should be counted for its transformation to E′P (enzyme-
–product) reaction. However, the Epa data for 1 showed that
it is the easiest one to oxidize among the thiolate series,
which in reality also exhibited best in its desired reaction
for the reduction of nitrate. It should be noted that the
Figure 5. Dependence of the rate of reaction of [PPh4][MoIV(-
PPh3)(SCH2CH3)(mnt)2] · CH2Cl2 (1) with 1–3 equiv of (Bu4N)(NO3) in
dichloromethane at 25 °C on [(Bu4N)(NO3)]. Inset: the corresponding double
reciprocal plot.
Figure 6. Catalytic cycle for nitrate reduction using {[(MoIV(SR)(mnt)2]1-
}
as the active catalyst and PPh3 as the reductant of its putative oxidized
form {[(MoVIO(SR)(mnt)2]1-}.
complex 1 to that calculated for complexes 2 and 5 are 4.48
and 7.68, respectively.
Catalysis of Oxo Transfer. It has been possible to couple
reaction 2 and 3, with the result that the nitrate reduction
(eq 6) can be made catalytic with the use of PPh3 to
regenerate the reactive pentacoordinated Mo(IV) complex.
The catalytic cycle involving [Bu4N][NO3] and PPh3 as the
NO3- + PPh3 f NO2- + Ph3PO
(6)
oxidizing and reducing substrate, respectively, is established
as shown in Figure 6. The initiation of the reaction starts on
the dissociation of PPh3 from the thiolato complexes. PPh3
recovered and Ph3PO produced within a minute of reaction
were confirmed by isolating, purifying, and checking melting
points (80 and 152 °C, respectively) and by 31P NMR
spectroscopy (-8.16 and 24.97 ppm compared with com-
mercial PPh3 and Ph3PO, respectively).11 Suitable controls
demonstrated no reaction between nitrate and PPh3 (1:1 mol
ratio, 25 °C, 1 h) in the absence of these thiolato complexes
as catalyst. On the basis of nitrite produced (measured by
standard colorimetric method), PPh3 recovered, and Ph3PO
produced, the turnover numbers (TON) for such reactions
were found out to be 10 and 40 mmol-1 s-1 for complexes
(26) (a) Joshi, H. K.; Cooney, J. A.; Inscore, F. E.; Gruhn, N. E.;
Lichtenberger, D. L.; Enemark, J. H. Proc. Natl. Acad. Sci. 2003, 100,
3719–3724. (b) Lauher, J. W.; Hoffmann, R. J. Am. Chem. Soc. 1976,
98, 1729–1742.
3400 Inorganic Chemistry, Vol. 47, No. 8, 2008

Model Reactions of Dissimilatory Nitrate Reductase
Table 3. Kinetics Results for Nitrate Reduction by Complexes 1, 2, and 5
a
b
complexes
KM
VMax
[PPh4][MoIV(PPh3)(SCH2CH3)(mnt)2]·CH2Cl2 (1)
[PPh4][MoIV(PPh3)(SCH2Ph)(mnt)2] (2)
[Et4N][MoIV(PPh3)(SPh)(mnt)2]·CH2Cl2 (5)11
b
2.3 × 10-4
1.4 × 10-4
4.3 × 10-4
M
M
M
3.2 × 10-2 s-1
7.1 × 10-3 s-1
4.2 × 10-3 s-1
a KM, kinetically determined Michaelis constant. VMax, maximum velocity that can be achieved.
Table 4. Redox Potentials (Oxidation Region Only) for Complexes 1–6
obtained from these model complexes. The fastest reduction
of nitrate in the synthetic series occurred with 1 containing
ethanethiolate coordination to molybdenum (IV). Ethanethiol
(CH3CH2SH) being a simple aliphatic thiol without any kind
of imposed steric bulk7,8 resembles cysteinate coordination
and therefore established complex 1 to be the closest model
system analogous to the active site of dissimilatory nitrate
reductase from DesulfoVibrio desulfuricans. Complexes 1,
2, and 5 are found to be reluctant to reduce DMSO because
of product inhibition, and such inhibitory reactions are well-
known under the feedback (regulatory) enzymes. Interest-
ingly nitrate reductases from different sources are shown to
reduce nitrate beyond nitrite with the production of NO.13
The inability of isolating the oxidized molybdenum product
was shown to be caused by a similar reaction of the produced
nitrite with the oxidized species to nitrosylate the molybde-
num complex to known [Mo(NO)2(mnt)2]2-. It is to be noted
that the starting reduced complexes are also capable of
consuming nitrite slowly as found separately and in the
presence of excess of PPh3. The competitive reactivity of
reduction of oxidized complex by PPh3, nitrosylations of the
oxidized and starting complexes by nitrite, and also the
reaction between PPh3 and any formed NO are the possible
participating reactions. At the initial stage of the reaction
the reduction of nitrate to nitrite is dominant and unique
enough to respond to the reaction similar to NiR. The
corresponding phenolate- and selenothiolate-substituted com-
plexes are yet to be fully characterized which would have
been the best systems to testify selectivity of the thiolate
ligand in relevance to other molybdoenzymes.
complexes
Eipa (V)a
E (V) from DPP
1
0.698
0.715
0.750
0.900
0.869
0.920
0.689
0.702
0.769
0.887
0.853
0.876
2
511
3
4
611
a i, irreversible.
electrochemical measurements were carried out by addition
of excess PPh3 into the solution to prevent decomposition
of these complexes, and therefore, the observed electro-
chemistry is related to the PPh3-bound inactive forms of the
hexacoordinated species, and the electrochemical data are
by no means related to those of the active pentacoordinated
species. However, from the structural geometry point of view,
the PPh3 attached complexes may mimic NO3- bound state
of the precursor complex (ES type) ready for oxo transfer
reaction, and in that sense, the electrochemical data may be
meaningful. A detailed energy level calculation of these
systems is under progress to understand the electronic
populations.
Relation to Enzyme. The present work provides a detailed
study of some analogue systems of nitrate reductase for the
reduction of biologically relevant substrate, nitrate. These
complexes possess an axial thiolate ligand which very closely
simulates cysteinate coordination in dissimilatory nitrate
reductase from DesulfoVibrio desulfuricans. The analogue
systems (1, 2, and 5) reduce nitrate, and the reaction follows
Michaelis-Menten kinetics, similar to the native enzyme.
As with other members of this family, there has been little
mechanistic work on dissimilatory nitrate reductase,1 al-
though the steady-state kinetics behavior has been examined
in case of the dissimilatory nitrate reductase from Escherichia
coli. Using ubiquinol as electron donor, the intact enzyme
is found to operate via a ping-pong mechanism, exhibiting
Acknowledgment. A.M. and K.P. gratefully acknowledge
predoctoral fellowships from the CSIR, New Delhi, and S.S.
thanks DST, New Delhi, for funding the project. A.M. thanks
Dr. P. K.Chaudhuri, Pune University, India, for useful
discussions regarding kinetics experiments.
the following kinetics parameters: kcat ) 7.2 s-1, KM
ubiquinol
) 78 µM, KMnitrate ) 2 µM, and kcat/KMnitrate ) 5 × 106 M-1
Supporting Information Available: ORTEP figures for the
structures of 2 and 3, kinetic plot of nitrate reduction reaction by
2, 31P NMR spectroscopic monitoring of PPh3 dissociation for
complex 1 and 2, UV–vis spectroscopic monitoring of reactivity
(reaction of 2 with nitrate, TMANO and DMSO reduction by
complexes 1, 2, and 5, reaction of 1 with DMSO in presence of
DMS, conversion of in situ generated [MoIV(DMS)(SR)(mnt)2]1-
complexes (2, R ) -CH2Ph; 5, R ) -Ph) to tris complex, tris
formation reaction of complexes 3 and 4, conversion of complex 5
to complex 3 with PhCH2Br), cyclic voltametric traces and
differential pulse polarographs, errors in kinetics experiments, and
crystallographic data (in CIF format) for the complexes 1–4 and
structural superposition for the complexes 1–6. This material is
available free of charge via the Internet at http://pubs.acs.org.
s-1.27
When these values are compared with that of the analogue
systems (Table 3) discussed here (1 and 2) and earlier (5),11
the conclusion is inescapable that in terms of first-order
kinetics parameters, the native reduced enzyme systems are
orders of magnitude more reactive than the model com-
pounds 1, 2, and 5. However, when reduced viologen dyes
are used as the reducing substrate, the KM value of 200 µM28
was observed for soluble NarGH catalytic dimer of nitrate
reductase, which is surprisingly close to the KM values
(27) Morpeth, F. F.; Boxer, D. H. Biochemistry. 1985, 24, 40.
(28) Buc, J.; Santini, C.-L.; Blasco, F.; Giordani, R.; Cardena, M. L.;
Chippaux, M.; Cornish-Bowden, A.; Giordano, G. Eur. J. Biochem.
1995, 234, 766.
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Inorganic Chemistry, Vol. 47, No. 8, 2008 3401