Structural and functional bis(dithiolene)-molybdenum/tungsten active site analogues of the dimethylsulfoxide reductase enzyme family [13]

Full text of DOI:10.1021/ja001197y

7410
J. Am. Chem. Soc. 2000, 122, 7410-7411
Structural and Functional
Bis(dithiolene)-Molybdenum/Tungsten Active Site
Analogues of the Dimethylsulfoxide Reductase
Enzyme Family
Booyong S. Lim, Kie-Moon Sung, and R. H. Holm*
Figure 1. Minimal structures of the desoxo MoIV and monooxo MoVIO
centers in Rs DMSOR, together with the pterin dithiolene cofactor ligand
(S2pd).
Department of Chemistry and Chemical Biology
HarVard UniVersity, Cambridge, Massachusetts 02138
ReceiVed April 6, 2000
Determination of structures of molybdenum oxotransferase/
hydroxylase enzymes1 by the combined results of crystallography
and X-ray absorption spectroscopy enables the conception of
synthetic analogues designed to disclose the intrinsic structural,
electronic, and reactivity properties of the catalytic sites. Members
of the dimethylsulfoxide reductase (DMSOR) enzyme family1a
include, inter alia, DMSOR itself and trimethylamine N-oxide
reductase (TMAOR) and contain a cofactor with two pterin
dithiolene (S2pd) ligands bound to molybdenum. The DMSOR
of Rhodobacter sphaeroides (Rs) has been shown to function by
a direct oxo transfer pathway from isotope labeling2 and resonance
Raman spectroscopy;3 TMAOR is likely to behave similarly.
Redetermination of the X-ray structure of oxidized Rs DMSOR
by Schindelin and co-workers4 at higher (1.3 Å) resolution than
the initial structure (2.2 Å)5 reveals two distinct molybdenum
environments, the catalytically competent monooxo site [MoVIO-
(O‚Ser)(S2pd)2] with symmetric dithiolene binding, and five-
coordinate [MoVIO2(O‚Ser)(S2pd)]. Crystallographic5 and EXAFS6
results are consistent with the minimal reduced site formulation
[MoIV(O‚Ser)(S2pd)2]. The DMSOR of Rhodobacter capsulatus
(Rc) is reported to contain seven-coordinate dioxo and six-
coordinate sites in its oxidized (1.82 Å) and reduced (2.8 Å) forms,
respectively.7 A related seven-coordinate site may exist in the
TMAOR of Shewanella massilia (2.5 Å).8 Although the issue
currently remains open, it is possible that the same or a similar
type of site disorder occurs in these enzymes as in Rs DMSOR.
In pursuing oxo transfer reactivity (Figure 1), we adopt the
minimal reaction paradigm MIV + XO T MVIO + X, utilize
complexes whose bis(dimethyl)dithiolene ligand structure closely
resembles that of the pterin dithiolene,9,10 and include systems
with M ) Mo and W in view of the existence of TMAOR
isoenzymes.11 Our first generation of desoxo M(IV) and monooxo
M(VI) structural/reactivity analogues utilized the aromatic dithio-
lene benzene-1,2-dithiolate (bdt) with M ) Mo12 and W.13
Figure 2. Structures of complexes 1 (left) and 4 (right) with 50% thermal
ellipsoids. Selected interatomic distances (Å) and angles (deg): for 1,
Mo-O1 1.895(5), mean Mo-S 2.317(3), mean C-C (ring) 1.33(1),
mean C-S 1.768(6), Mo-O1-C9 135.0(4), mean O1-Mo-S
110.1(2); for 4, W-O1 1.728(3), W-O2 1.994(4), W-S1 2.492(1), mean
W-S(2-4) 2.42(1), mean C-C (ring) 1.34(1), mean C-S 1.73(2),
O1-W-O2 93.3(2), O1-W-S1 146.0(1), O2-W-S4 154.4(1),
S2-W-S3 153.49(5).
Reaction of [M(CO)2(S2C2Me2)2]9,14 with 1 equiv each of
NaOPh and Et4NCl in acetonitrile followed by standard workup
affords the desoxo complexes [MIV(OPh)(S2C2Me2)2]1-, M ) Mo
(1, brown, 47%) and W (2, green-brown, 66%),10 as Et4N+ salts.15
The complexes are isostructural and possess the square-pyramidal
stereochemistry shown for 116 (Figure 2). Related complexes of
both metals with isopropoxide and 2-adamantyloxide have been
prepared by similar reactions and shown to have square-pyramidal
structures; anionic oxygen ligands are intended to simulate serinate
binding. In reactions similar to those of bdt complexes,12,13
treatment of acetonitrile solutions of 1 and 2 with 1-1.5 equiv
of Me3NO yields the monooxo complexes [MVIO(OPh)-
(S2C2Me2)2]1-, M ) Mo (3, green) and W (4, red-violet, 76%).
All attempts to isolate unstable 3 have led to the recovery in high
yield of [MoVO(S2C2Me2)2]1- (5), identified by comparison of
absorption and EPR spectra15 with those of an authentic sample.9
Complex 3 has been securely established by comparative spec-
troscopic properties with 4, including a feature-by-feature red-
(1) (a) Hille, R. Chem. ReV. 1996, 96, 2757-2816. (b) Roma˜o, M. J.;
Kna¨blein, J.; Huber, R.; Moura, J. J. G. Prog. Biophys. Mol. Biol. 1997, 68,
121-144. (c) Kisker, C.; Schindelin, H.; Rees, D. C. Annu. ReV. Biochem.
1997, 66, 233-267.
(12) Donahue, J. P.; Goldsmith, C. R.; Nadiminti, U.; Holm, R. H. J. Am.
Chem. Soc. 1998, 120, 12869-12881.
(13) Lorber, C.; Donahue, J. P.; Goddard, C. A.; Nordlander, E.; Holm, R.
H. J. Am. Chem. Soc. 1998, 120, 8102-8112.
(2) Schultz, B. E.; Holm, R. H.; Hille, R. J. Am. Chem. Soc. 1995, 117,
827-828.
(3) Garton, S. D.; Hilton, J.; Oku, H.; Crouse, B. R.; Rajagopalan, K. V.;
Johnson, M. K. J. Am. Chem. Soc. 1997, 119, 12906-12917.
(4) Li, H.-K.; Temple, C.; Rajagopalan, K. V.; Schindelin, H. J. Am. Chem.
Soc. 2000, 122, in press.
(14) Goddard, C. A.; Holm, R. H. Inorg. Chem. 1999, 38, 5389-5398.
(15) All reactions and measurements were conducted under anaerobic
conditions. Reactions were performed in acetonitrile solutions at ambient
temperature. Absorption spectra (acetonitrile): λmax 3/4 395(sh)/337, 475 (sh)/
408(sh), 610/514, 795/637 nm. Kinetics parameters for the conversions 1 f
5, 2 f 4, and 3 f 5 were obtained from reactions monitored by
(5) Schindelin, H.; Kisker, C.; Hilton, J.; Rajagopalan, K. V.; Rees, D. C.
Science 1996, 272, 1615-1621.
(6) (a) George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Soc.
1996, 118, 1113-1117. (b) George, G. N.; Hilton, J.; Temple, C.; Prince, R.
C.; Rajagopalan, K. V. J. Am. Chem. Soc. 1999, 121, 1256-1266.
(7) (a) McAlpine, A. S.; McEwan, A. G.; Shaw, A. L.; Bailey, S. J. Biol.
Inorg. Chem. 1997, 2, 690-701. (b) McAlpine, A. S.; McEwan, A. G.; Bailey,
S. J. Mol. Biol. 1998, 275, 613-623.
1
spectrophotometry. H NMR (CD3CN): δ 2.54 (1), 2.61 (2), 2.26 (3), 2.19
(4). Complexes 3 and 4 are fluxional at ambient temperature. IR (KBr): νMO
917 (16O), 874 (18O) cm-1 (5); 895 (16O), 848 cm-1 (18O) (4); EPR (acetonitrile,
298 K):
g ) 1.996, aMo ) 28.8 G (5). All (isolated) compounds gave
satisfactory elemental analyses.
(8) Czjzek, M.; Dos Santos, J.-P.; Pommier, J.; Giordano, G.; Me´jean, V.;
Haser, R. J. Mol. Biol. 1998, 284, 435-447.
(16) (a) (Et4N)[1]: monoclinic (P21/c), a ) 17.56(2) Å, b ) 8.263(8) Å,
c ) 18.70(2) Å, â ) 102.01(1)°, Z ) 4, R1(wR2) ) 7.00(16.66)%. (b) (Et4N)-
[4]: monoclinic (P21/c), a ) 10.0883(4) Å, b ) 15.8029(6) Å, c )
16.8607(6) Å, â ) 98.602(1)°, Z ) 4, R1(wR2) ) 3.61(6.61)%. Data were
collected at 213 K with Mo KR radiation, and structures were solved with
direct methods and refined by standard procedures; absorption corrections
(SADABS) were applied.
(9) Lim, B. S.; Donahue, J. P.; Holm, R. H. Inorg. Chem. 2000, 39, 263-
273.
(10) Sung, K.-M.; Holm, R. H. Inorg. Chem. 2000, 39, 1275-1281.
(11) Buc, J.; Santini, C.-L.; Giordani, R.; Czjzek, M.; Wu, L.-F.; Giordano,
G. Mol. Microbiol. 1999, 32, 159-168.
10.1021/ja001197y CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/15/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 30, 2000 7411
isolated product was shown to be 5 (30% 18O enrichment) by IR
spectroscopy15 and mass spectrometry. Formation of 5 from 1 in
an oxo transfer system is accompanied by the formation of ca. 1
equiv of phenol (1H NMR). We consider it probable that 3
undergoes an internal redox reaction with the formation of 5 and
a phenoxyl radical, which abstracts a hydrogen atom from solvent
to form phenol. Under pseudo-first-order conditions at 289-340
K, the slow oxo transfer conversion 1 + R2SO f 3 + R2S
proceeds followed by the rapid decomposition reaction 3 f 5 +
PhOH (k/[1] ) 0.12(1) M-1 s-1 at room temperature). The overall
transformation 1 f 5 is very clean, with a tight isosbestic point
observed at 552 nm; under these conditions, 3 does not ac-
cumulate. Kinetics parameters (Table 1) again include ∆Sq values
consistent with an associative pathway. The relative rates k7/k8
≈ 115 (Mo) and 23 (W) arise because of the sterically less
hindered ring structure of (CH2)4SO in the transition state.
These results demonstrate that bis(dialkyldithiolene)Mo(IV)/
W(IV) complexes with a dithiolene ligand structure and axial
ligand closely related to those present in Rs DMSOR, and possibly
other members of the DMSOR family, sustain direct oxo transfer
with biological substrates. The relative rates kW/kMo ) 6.0 ((CH2)4-
SO) and ∼30 (Me2SO) refer to processes in which the metal is
Table 1. Kinetics Data for Oxo Transfer Reactions of Mo/W
Bis(dithiolene) Complexes with Sulfoxides
M
R2SO
k298 K (M-1 s-1
)
∆Hq (kcal/mol)
∆Sq (eu)
Mo
(CH3)2SO
(CH2)4SO
(CH3)2SO
(CH2)4SO
1.3 × 10-6 a
1.5(2) × 10-4
3.9(4) × 10-5
9.0(3) × 10-4
14.8(5)
10.1(4)
14.4(2)
11.6(4)
-36(1)
-39(1)
-30(1)
-33(1)
W
a Obtained from the Eyring plot.
shifted absorption spectrum.15 In contrast to 3, (Et4N)[4] is readily
isolated as a stable black crystalline solid by addition of ether to
the reaction mixture. Acetonitrile solutions of 4 undergo very slow
decomposition, yielding [WVO(S2C2Me2)2]1- (6).14 Complex 4 has
a distorted octahedral structure16 (Figure 2), similar to those of
[MO(OSiPh2But)(bdt)2]1- (M ) Mo,12 W13), in which the oxo
ligand exerts a trans influence on the W-S1 bond length
(2.492(1) Å) relative to the other W-S distances (mean 2.42(1)
Å). As in the six-coordinate site of Rs DMSOR, the two oxygen
ligands are cis, but the enzyme site is described as distorted
trigonal prismatic with no sulfur atom trans to the oxo ligand.4
Chelate ring C-C and C-S bond distances point to an enedithi-
olate ligand description, as is the case for all five-coordinate bis-
(dithiolene)Mo/W complexes thus far examined.9,10,14
Complexes 1 and 2 support oxo transfer reactions of highly
variable rates with a variety of molecules XO, including the
biological substrates Me3NO and Me2SO. Direct oxo transfer was
demonstrated in rapid reactions with Ph2Se18O (62% enriched),
which after product isolation gave 5 and 4, respectively, detected
by appropriately shifted νMO absorptions in their infrared spectra.15
Reaction of both complexes with 1 equiv of Me3NO is immediate,
thereby allowing identification of 3 in situ15 before any appreciable
decomposition. However, reactions with sulfoxides are relatively
slow, even when neat (CH2)4SO (7) or Me2SO (8) are used as
solvents. The clean tungsten conversion 2 + R2SO f 4 + R2S
was observed in acetonitrile at 290-333 K with both sulfoxides
in systems showing a tight isosbestic point at 337 nm. The final
spectrum is that of isolated 4 measured separately. Spectropho-
tometric analysis of reactions conducted under pseudo-first-order
conditions affords the kinetics parameters in Table 1. Reactions
are second-order; the large negative entropies of activation are
consistent with an associative transition state in which, presum-
ably, the phenolate ligand and sulfoxide occupy cis positions in
a structure not far removed from that of 4.
oxidized; kW > kMo is anticipated from the result that kMo
kW for the second-order reactions [MVIO2(S2C2(CN)2)2]2-
.
+
(RO)3-nR′nP f [MIVO(S2C2(CN)2)2]2- + (RO)3-nR′nPO, in which
the metal is reduced.18 Further, kW/kMo ≈ 25 for the oxidation of
[MIVO(bdt)2]2- by Me3NO.19 Rates of reduction of Me2SO by
Escherichia coli (Ec) DMSOR (kcat/KM ) 1.9 × 106 M-1 s-1 20
)
are much faster than in analogue systems and may arise in part
from a protein-induced substrate-bound transition state closer to
the distorted trigonal prismatic oxidized enzyme site than is 3/4.
The second-order rate constant for reoxidation of Me2S-reduced
Rc DMSOR with Me2SO is 4.3 × 102 M-1 s-1 21
. These analogue
systems retain three features of the Ec TMAOR isoenzymes:
substrate discrimination k7/k8 ) 40 by the tungstoenzyme, kW
>
kMo (ratio 2.3) for reduction of Me3NO, and a much slower
(possibly nil) rate of reduction of (CH2)4SO and Me2SO by the
molybdoenzyme.11 These features suggest active sites similar to
the structures of 1/2 but subject to modulation by protein structure
and environment. Clearly, bis(dithiolene) complexes 1 and 2 are
intrinsically reactive to both N-oxides and S-oxides. Future reports
will describe additional structural and reactivity features of bis-
(dithiolene)Mo/W analogue systems.
Acknowledgment. This research was supported by NSF Grant CHE
98-76457. We thank Prof. H. Schindelin for access to ref 4 prior to
publication.
Supporting Information Available: Crystallographic data for the
compounds (Et4N)[1] and (Et4N)[4] (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
JA001197Y
Systems in acetonitrile based on molybdenum complex 1
behave similarly, but react more slowly and with the difference
that the final product is 5 rather than 3. For example, the system
1/440 equiv of (CH2)4SO at 323 K for 8 h formed 92-93% 5
based on absorption spectra and integrated EPR signal intensities.
Tetrahydrothiophene was detected by GC. To prove direct oxo
transfer to Mo(IV) from a sulfoxide, complex 1 in acetonitrile
was treated with 25 equiv of 2-thiaindane S-oxide17 (82% 18O-
enriched, from 2-thiaindane S-dibromide and H218O) for 2 d. The
(17) Holland, H. L.; Turner, C. D.; Andreana,. P. R.; Nguyen, D. Can. J.
Chem. 1999, 77, 463-471.
(18) Tucci, G. C.; Donahue, J. P.; Holm, R. H. Inorg. Chem. 1998, 37,
1602-1608.
(19) Ueyama, N.; Oku, H.; Nakamura, A. J. Am. Chem. Soc. 1992, 114,
7310-7311.
(20) Simala-Grant, J. L.; Weiner, J. H. Eur. J. Biochem. 1998, 251, 510-
515.
(21) Adams, B.; Smith, A. T.; Bailey, S.; McEwan, A. G.; Bray, R. C.
Biochemistry 1999, 38, 8501-8511.