Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase

Article abstract of DOI:10.1021/ja046971m

Ru(II)- and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L) ₂L']^2t, L' is a perfluorinated biphenyl bridge connecting 4,4′-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Δ65 and Δ114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Δ65 and Δ114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Rudiimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO)₃L₁L₁']⁺, where L ₁ is 4,7-dimethylphenanthroline and L₁' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F₈bp-im) (4) or F (F₉bp) (5), also form complexes with Δ114. Binding of 4 shifts the Δ114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Δ114 dissociation constant is 100 ± 80 nM. Rewire 5 binds Δ114 with a K_d of 5 ± 2 μM, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe.

Full text of DOI:10.1021/ja046971m

Published on Web 03/17/2005
Luminescent Ruthenium(II)- and Rhenium(I)-Diimine Wires
Bind Nitric Oxide Synthase
Alexander R. Dunn,† Wendy Belliston-Bittner,† Jay R. Winkler,†
Elizabeth D. Getzoff,‡ Dennis J. Stuehr,§ and Harry B. Gray*,†
Contribution from the Beckman Institute, California Institute of Technology,
Pasadena, California 91125, Department of Molecular Biology and Skaggs Institute for
Chemical Biology, The Scripps Research Institute, MS MB4, 10550 North Torrey Pines Road,
La Jolla, California 92037, and Department of Immunology, Lerner Research Institute,
The CleVeland Clinic, CleVeland, Ohio 44195
Received May 22, 2004; Revised Manuscript Received January 27, 2005; E-mail: hbgray@caltech.edu
Abstract: Ru(II)- and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase
(iNOSoxy). In the ruthenium wires, [Ru(L)2L′]2+, L′ is a perfluorinated biphenyl bridge connecting 4,4′-
dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3).
2 binds in the active site of the murine iNOSoxy truncation mutants ∆65 and ∆114, as demonstrated by a
shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind ∆65 and ∆114, as evidenced by biphasic
luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1
or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data
suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch
thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru-
diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the
reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO)3L1L1′]+, where L1 is 4,7-
dimethylphenanthroline and L1′ is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole
to a distal imidazole (F8bp-im) (4) or F (F9bp) (5), also form complexes with ∆114. Binding of 4 shifts the
∆114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-
state luminescence measurements establish that the 4:∆114 dissociation constant is 100 ( 80 nM. Re-
wire 5 binds ∆114 with a Kd of 5 ( 2 µM, causing partial displacement of water from the heme iron. Our
finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly,
we have demonstrated the power of time-resolved FET measurements in the characterization of small
molecule:protein interactions that otherwise would be difficult to observe.
Introduction
Full-length NOS consists of oxygenase, reductase, and
calmodulin binding domains. The NOS oxygenase domain
The enzyme nitric oxide synthase (NOS) is the major
biological source of nitric oxide (NO), a secondary messenger
acting in a myriad of circumstances that include neuronal
development, regulation of blood pressure, apoptosis, neuro-
transmission, and immunological response.1-7 Because of the
central importance of NO, NOS has been implicated in septic
shock, inflammation, a variety of neurodegenerative disorders,
and heart disease.8-10
(NOSoxy), which contains cysteine-ligated heme and tetrahy-
drobiopterin (H4B) cofactors, catalyzes the conversion of
arginine and molecular oxygen to NO and citrulline.11 The
electrons necessary for this reaction are provided by the
reductase module, which is attached to NOSoxy by a calmodu-
lin-binding linker.12,13 NOS functions as a homodimer; the
reductase module from one half of the dimer reduces the
oxygenase domain of the other.14,15 Calmodulin binding is
† California Institute of Technology.
(8) Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. ReV. Pharmacol. Toxicol. 1999,
39, 191-220.
(9) Heales, S. J. R.; Bolan˜os, J. P.; Stewart, V. C.; Brookes, P. S.; Land, J.
M.; Clark, J. B. Biochim. Biophys. Acta 1999, 1410, 215-228.
(10) Hingorani, A. D.; Liang, C. F.; Fatibene, J.; Lyon, A.; Monteith, S.; Parsons,
A.; Haydock, S.; Hopper, R. V.; Stephens, N. G.; O’Shaughnessy, K. M.;
Brown, M. J. Circulation 1999, 100, 1515-1520.
(11) Stuehr, D. J. Biochim. Biophys. Acta 1999, 1411, 217-230.
(12) Cho, H. J.; Xie, Q. W.; Calaycay, J.; Mumford, R. A.; Swiderek, K. M.;
Lee, T. D.; Nathan, C. J. Exp. Med. 1992, 176, 599-604.
(13) Bredt, D. S.; Hwang, P. M.; Glatt, C. E.; Lowenstein, C.; Reed, R. R.;
Snyder, S. H. Nature 1991, 351, 714-718.
(14) Crane, B. R.; Rosenfeld, R. J.; Arvai, A. S.; Ghosh, D. K.; Ghosh, S.;
Tainer, J. A.; Stuehr, D. J.; Getzoff, E. D. EMBO J. 1999, 18, 6271-
6281.
‡ The Scripps Research Institute.
§ The Cleveland Clinic.
(1) Kendrick, K. M.; Guevara-Guzman, R.; Zorrilla, J.; Hinton, M. R.; Broad,
K. D.; Mimmack, M.; Ohkura, S. Nature 1997, 388, 670-674.
(2) Huang, P. L.; Huang, Z. H.; Mashimo, H.; Bloch, K. D.; Moskowitz, M.
A.; Bevan, J. A.; Fishman, M. C. Nature 1995, 377, 239-242.
(3) Ko, G. Y.; Kelly, P. T. J. Neurosci. 1999, 19, 6784-6794.
(4) Luth, H. J.; Holzer, M.; Gertz, H. J.; Arendt, T. Brain Res. 2000, 852,
45-55.
(5) Mize, R. R.; Dawson, T. M.; Dawson, V. L.; Friedlander, M. J. Nitric Oxide
in Brain DeVelopment, Plasticity and Disease; Elesevier: Amsterdam, 1998.
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A R T I C L E S
Dunn et al.
known to modulate electron transfer and, hence, catalysis.16-18
Several crystal structures of the NOS oxygenase domain have
been determined,19-21 but the structure of the full-length enzyme
remains elusive.
High-spin, dimeric ∆65 iNOS was generated by incubating ∆65 with
1 mM tetrahydrobiopterin (H4B) and 1 mM arginine (Arg) for 2 h at
4 °C before diluting the sample to final concentrations of 0.1 mM H4B
and 1 mM Arg. Satisfactory Arg and H4B binding was signaled by a
shift of the Soret to 396 nm. NOS extinction coefficients were
determined using the hemochromogen assay; 1 mL of NOS solution
was diluted with 0.125 mL of 0.5 M NaOH and 0.125 mL of pyridine,
and then reduced with several grains of sodium dithionite. The resulting
ferrohemochromogen concentration was calculated using an extinction
coefficient of 31 mM-1 cm-1 at 556 nm. The assays were calibrated
using cytochrome P450cam (ꢀ416 ) 115 mM-1 cm-1).32 The NOS
extinction coefficients calculated using this method are substrate-free
We have a long-standing interest in the high-valent interme-
diates thought to play key roles in heme-mediated oxidations.22-29
To observe these intermediates, we have designed Ru-diimine
photosensitizers (Ru-wires) that bind to the mechanistically
related enzyme cytochrome P450 and inject an electron into
the active site upon excitation with 470-nm light.27 Energy
transfer between the excited state of the Ru-wire and the heme
also serves as a sensitive structural probe.22,29 Like NOSoxy,
cytochrome P450 enzymes possess a cysteine-ligated heme in
the active site and catalyze the oxidation of substrates using
molecular oxygen and two electrons supplied by a reductase
(in the case of P450, a separate protein).30
Given the postulated mechanistic similarities between NOS
and cytochrome P450, we have endeavored to develop similar
photosensitizer-wires for NOS. Our initial investigation showed
that complexes 1-3 bind the oxygenase domain of murine
inducible NOS (iNOSoxy) with micromolar dissociation con-
stants. Intriguingly, a combination of fluorescence energy
transfer (FET) measurements and structural modeling suggests
that 1 and 3 bind to the surface patch thought to interact with
the reductase module. Second generation compounds 4 and 5,
which are structurally analogous to 2 and 3, bind in the iNOSoxy
active site with micro- and nanomolar dissociation constants.
∆65 (-H4B, -Arg) ꢀ422 ) 75 mM-1 cm-1, ∆65 (+H4B, +Arg) ꢀ396
75 mM-1 cm-1, and substrate-free ∆114 ꢀ422 ) 85 mM-1 cm-1
=
.
Ru-wires (1-3) were prepared by the literature procedure.27 The
fluorinated biphenyl bridging moieties for Re-wires 4 and 5 were
synthesized by reacting imidazole and perfluorobiphenyl in dimethyl
sulfoxide. The resulting mono- and disubstituted perfluorobiphenyl-
imidazole ligands were separated by flash silica chromatography. Re-
(dimethylphenanthroline)(CO)3Cl was treated with silver triflate, and
then reacted with either the mono- or disubstituted perfluorobiphenyl-
imidazole ligand to form 4 and 5 as triflate salts.33
Both time-resolved and steady-state spectroscopic measurements
were performed as previously described.27 Luminescence decay profiles
were fit to a biexponential function (eq 1):
I(t) ) c1 e-k t + c2 e-k t
(1)
1
2
using a nonlinear least-squares algorithm. The ratio of enzyme-bound
to free ruthenium complex is c1:c2, where k1 and k2 are the luminescence
decay rate constants for the enzyme-bound and free ruthenium
complexes. Dissociation constants were derived from c1:c2 as previously
described.27
Characteristic FET distances (R0) for the Ru- and Re-diimine wires
with iNOSoxy were calculated from the probe emission and NOS
absorption spectra.27 These distances are 24.3 Å for 1 and 2 with ∆114,
19.6 Å for 3 with ∆114, 32 Å for 4 and 5 with ∆114, 24.3 Å for 1 and
2 with substrate-free ∆65, 19.5 Å for 3 with substrate-free ∆65, 23.9
Å for 1 and 2 with Arg- and H4B-bound ∆65, and 19.3 Å for 3 with
Arg- and H4B-bound ∆65.
Experimental Section
Murine inducible NOS oxygenase domain constructs with N-terminal
truncations at residues 65 (∆65) and 114 (∆114) were prepared as
previously described.31 Small aliquots of the protein solutions were
exchanged into phosphate buffer (50 mM potassium phosphate, 100
mM potassium chloride, pH 7.2) using a desalting column immediately
before use. The measurement of the heme Soret peak at 422 nm verified
successful removal of the dithiothreitol (DTT) present in the storage
buffer.
(15) Siddhanta, U.; Wu, C.; Abu-Soud, H. M.; Zhang, J.; Ghosh, D. K.; Stuehr,
D. J. J. Biol. Chem. 1996, 271, 7309-7312.
Results and Discussion
(16) Kobayashi, K.; Tagawa, S.; Daff, S.; Sagami, I.; Shimizu, T. J. Biol. Chem.
2001, 276, 39864-39871.
Ru-Wires. Two murine iNOSoxy truncation mutants, ∆114
and ∆65, were investigated. Importantly, ∆114 is predominantly
monomeric, while ∆65 exists in a monomer-dimer equilibrium,
forming a strong dimer in the presence of H4B.31 For clarity,
both ∆65 without bound H4B and ∆114 are referred to below
as “monomeric”. Monomeric iNOSoxy has an exposed active
site (see below), while dimeric iNOSoxy has a much more
constricted substrate access channel.
No change in the iNOSoxy heme absorption spectrum was
observed upon stoichiometric addition of 1 or 3 to either ∆114
or ∆65. In contrast, the addition of excess 2 to ∆114 or
monomeric ∆65 (-H4B, -Arg) resulted in a heme Soret shift
from 420 or 422 to 426 nm, consistent with imidazole ligation
of the heme (Figure 1). The absorption spectrum of dimeric
∆65 (+H4B, +Arg) was not altered in the presence of 1-3,
indicating that none of the Ru-wires displaces Arg from the
dimeric iNOSoxy active site.
(17) Panda, K.; Ghosh, S.; Stuehr, D. J. J. Biol. Chem. 2001, 276, 23349-
23356.
(18) Abu-Soud, H. M.; Yoho, L. L.; Stuehr, D. J. J. Biol. Chem. 1994, 269,
32047-32050.
(19) Crane, B. R.; Arvai, A. S.; Gachhui, R.; Wu, C.; Ghosh, D. K.; Getzoff, E.
D.; Stuehr, D. J.; Tainer, J. A. Science 1997, 278, 425-431.
(20) Raman, C. S.; Li, H.; Martasek, P.; Kral, V.; Masters, B. S. S.; Poulos, T.
L. Cell 1998, 95, 939-950.
(21) Poulos, T. L.; Li, H.; Raman, C. S.; Schuller, D. J. AdV. Inorg. Chem.
2001, 51, 243-293.
(22) Dmochowski, I. J.; Crane, B. R.; Wilker, J. J.; Winkler, J. R.; Gray, H. B.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12987-12990.
(23) Wilker, J. J.; Dmochowski, I. J.; Dawson, J. H.; Winkler, J. R.; Gray, H.
B. Angew. Chem., Int. Ed. 1999, 38, 90-92.
(24) Dmochowski, I. J.; Dunn, A. R.; Wilker, J. J.; Crane, B. R.; Green, M. T.;
Dawson, J. H.; Sligar, S. G.; Winkler, J. R.; Gray, H. B. Methods Enzymol.
2002, 357, 120-133.
(25) Low, D. W.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 1996, 118,
117-120.
(26) Berglund, J.; Pascher, T.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc.
1997, 119, 2464-2469.
(27) Dunn, A. R.; Dmochowski, I. J.; Winkler, J. R.; Gray, H. B. J. Am. Chem.
Soc. 2003, 125, 12450-12456.
(28) Dunn, A. R.; Dmochowski, I. J.; Bilwes, A. M.; Gray, H. B.; Crane, B. R.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12420-12425.
(29) (a) Dunn, A. R.; Hays, A. M. A.; Goodin, D. B.; Stout, C. D.; Chiu, R.;
Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2002, 124, 10254-10255.
(b) Hays, A. M. A.; Dunn, A. R.; Chiu, R.; Gray, H. B.; Stout, C. D.;
Goodin, D. B. J. Mol. Biol. 2004, 344, 455-469.
In all cases, biexponential Ru-wire luminescence decays
were observed in the presence of stoichiometric ∆114 or ∆65,
(30) Ortiz de Montellano, P. R. Cytochrome P450: Structure, Mechanism, and
Biochemistry; Plenum Press: New York, 1995.
(31) Ghosh, D. K.; Wu, C.; Pitters, E.; Moloney, M.; Werner, E. R.; Mayer, B.;
Stuehr, D. J. Biochemistry 1997, 36, 10609-10619.
(32) Sligar, S. G. Biochemistry 1976, 15, 5399-5406.
(33) Belliston-Bittner, W.; Dunn, A. R.; Winkler, J. R.; Getzoff, E. D.; Stuehr,
D. J.; Gray, H. B. Manuscript in preparation.
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A R T I C L E S
Table 1. Ru-Wire Dissociation Constants and Ru-Fe Distances from FET Kineticsa
∆114
∆65
∆65 +Arg, +H4B
compound
Kd
(µM)
Ru
−Fe (Å)
Kd
(µM)
Ru
−Fe (Å)
Kd
(
µM)
Ru−Fe (Å)
1
2
3
0.88 ( 0.15
7.1 ( 0.4
0.71 ( 0.09
18.9 ( 0.1
17.8 ( 0.5
20.1 ( 0.1
0.54 ( 0.04
6.5 ( 2.4
0.58 ( 0.16
19.6 ( 0.2
19.3 ( 0.6
20.2 ( 0.4
1.7 ( 0.4
7.2 ( 3.4
0.89 ( 0.15
19.6 ( 0.4
20.9 ( 0.8
21.0 ( 0.3
a Uncertainties are the root-mean-square deviations calculated from independent measurements (3 for ∆114, 2 for ∆65, 3 for ∆65 +H4B, +Arg).
Figure 1. UV-visible absorption spectrum of ∆114 alone (5.7 µM; green)
and bound to 2, corrected for the absorption attributable to the Ru-wire
(+20.5 µM 2; blue).
Figure 3. (A) Model of 2 bound to the exposed heme of monomeric ∆114.
The Ru-Fe distance in this model is 16.9 Å. (B) The NOS dimer, shown
with Ru(bpy)3 (van der Waals surface) docked at the proposed reductase
binding site. The Ru-Fe distance is ∼19 Å, consistent with the Ru-Fe
distances measured for the ∆65:1 and ∆65:3 conjugates.
Figure 2. Sample transient luminescence decay data for 1 (blue) and a
1:1 mixture of 1 and ∆65 (1.8 µM; green). The fast component of the
luminescence decay corresponds to 1 bound to ∆65.
The spectroscopic evidence suggests that 1 and 3 do not bind
in the active site; the heme absorption spectrum is not altered
in the presence of 1 or 3, and the Kd values and Ru-Fe distances
determined for these Ru-wires are not substantially altered by
the addition of H4B and Arg. Addition of stoichiometric 4, which
binds in the iNOS active site (see below), does not alter the
binding of 3 to monomeric ∆65, again demonstrating that 3
does not bind in the active site (Supporting Information).
Consistent with these data, modeling suggests that 1 and 3
cannot fit into the substrate access channel of dimeric ∆65
(+H4B, +Arg), owing to the bulk of the ruthenium tris-bipyridyl
moiety. Instead, the calculated Ru-Fe distances indicate that 1
and 3 may bind on the distal side of the enzyme, at the proposed
binding site of the reductase module (Figure 3b).14 The proposed
binding site is concave and hydrophobic. The Ru-wires present
few opportunities for specific interactions with the protein
surface. Instead, extensive wire:protein hydrophobic contacts
likely mediate binding. Of interest in this regard is that other
RuII-diimines bind cytochrome c oxidase at the physiologically
relevant cytochrome c binding site.34,35
indicating that the Ru-wires bind to the enzyme (Figure 2).
Quenching of the Ru-wire excited state could, in principle,
occur by either energy or electron transfer to the iNOSoxy heme.
Since no electron-transfer products were observed by transient
UV-visible absorption spectroscopy, and because the rate of
luminescence quenching is consistent with FET, energy transfer
is the probable mechanism of Ru-wire luminescence quenching.
The weightings of the fast and slow luminescence decay phases
were used to calculate dissociation constants, while the rates
of energy transfer were used to calculate Ru to heme-Fe
distances. Ru-Fe distances so obtained for Ru-wire:P450cam
conjugates matched those observed in the corresponding crystal
structures to within 0.4 Å.22
The Ru-wires bind with micromolar dissociation constants
and with Ru-Fe distances of 18-21 Å (Table 1). Interestingly,
1 and 3 bind ∆114, ∆65, and dimeric ∆65 (+H4B, +Arg) with
dissociation constants that are virtually identical. The Ru-Fe
distances calculated for 1 and 3 are similar for ∆114 and ∆65
and are unaffected by the presence of H4B and Arg (Table 1).
(34) Zaslavsky, D.; Sadoski, R. C.; Wang, K. F.; Durham, B.; Gennis, R. B.;
Millett, F. Biochemistry 1998, 37, 14910-14916.
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Chart 1. Ru-Wires (1-3) and Re-Wires (4 and 5) Bind iNOSoxy
and also Cytochrome P450cam
Figure 4. (A) 2.2 µM ∆114 alone (green) and with stoichiometric 5 (blue).
Both the slight shift in peak wavelength and development of a pronounced
shoulder indicate partial conversion to high-spin, five-coordinate heme. (B)
6.0 µM ∆114 alone (green) and with stoichiometric 4 (blue). The red shift
in peak wavelength is consistent with imidazole ligation of the heme.
In contrast, the shifts in the absorption spectra of monomeric
∆65 and ∆114 in the presence of excess 2 clearly indicate that
the imidazole of 2 ligates the heme (Figures 1 and 3a).19
Structural modeling suggests that the less bulky 2, which lacks
the adamantyl group of 1 and the tetramethylbipyridine ligands
of 3, can bind in the more exposed active site of monomeric
iNOSoxy (Figure 3a). However, 2 may also bind iNOSoxy at
the site occupied by 1 and 3; 1.5 equivalents of 2 bind to
monomeric ∆65 (-H4B, -Arg) when the Ru-wire is present
in 6-fold excess, and 2 binds to dimeric ∆65 (+H4B, +Arg),
in which the active site is occupied. In contrast to those in 1
and 3, the Ru-Fe distances determined for the 2:∆114 and
2:∆65 +H4B, +Arg conjugates increase markedly from 17.8
to 20.9 Å, suggesting a different mode of binding to monomeric
(∆114) and dimeric (∆65 +H4B, +Arg) iNOSoxy. These data
together suggest that 2 binds to both the active site of monomeric
iNOSoxy and another portion of the protein with similar Kd
values.
Re-Wires. Luminescent rhenium diimines, [Re(CO)3(L2)-
(L2′)]+, where L2 is a 2,2′-bipyridyl or phenanthryl derivative,
and L2′ is a nitrogen donor, such as imidazole or pyridine,
typically have microsecond excited-state lifetimes. Like Ru-
(bpy)32+, the excited states are both good oxidants (∼1.2 V vs
NHE) and reductants (-0.7 V vs NHE).36 In addition, photo-
chemically generated [Re(CO)3(L2)(L2′)]2+ complexes are ex-
tremely strong oxidants (∼1.8 V vs SCE).37
To take advantage of rhenium photochemistry, we synthesized
Re-wires 4 and 5 (Chart 1). The complexes are structurally
similar to 1 and 3, but the Re-photosensitizer is smaller. The
surface areas of [Ru(bpy)3]2+ and [Re(CO)3(dimethylphen-
anthroline)(imidazole)]+ are 650 and 550 Å2, respectively.38
Additionally, the fac-carbonyls of 4 and 5 result in a more
compact profile on one side of the complex, allowing a closer
approach to the protein. The virtually identical absorption spectra
of 4 and 5 are typical of Re-diimines. Both complexes are
luminescent, with emission spectra centered at 560 nm (quantum
yields are 0.055 in phosphate buffer).
Upon addition of 4 to ∆114 murine iNOSoxy, the Soret shifts
from 422 to 426 nm, signaling imidazole ligation to the heme
iron (Figure 4b). Time-resolved luminescence measurements
indicate that 4 is almost completely bound to NOS in 1:1
micromolar solutions (data not shown). Indeed, a Kd could not
be determined from the luminescence decay data due to the
rapidity of the decay39 and the almost complete absence of a
slow decay rate corresponding to free 4 in solution. Instead, a
dissociation constant of 100 ( 80 nM was calculated from a
comparison of the steady-state luminescence spectra of 4 alone
and bound to ∆114 iNOSoxy (Figure 5).
Re-wire 5 causes a blue shift in the ∆114 Soret, indicating
partial conversion to a high-spin, five-coordinate heme (Figure
4a). In addition to a small shift in peak wavelength, a noticeable
shoulder appears in the Soret (Figure 4a), consistent with partial
(35) Zaslavsky, D.; Kaulen, A. D.; Smirnova, I. A.; Vygodina, T.; Konstantinov,
A. A. FEBS Lett. 1993, 336, 389-393.
(36) Connick, W. B.; DiBilio, A. J.; Hill, M. G.; Winkler, J. R.; Gray, H. B.
Inorg. Chim. Acta 1995, 240, 169-173.
(38) Gerstein, M. Acta Crystallogr. A 1992, 48, 271-276.
(39) Initial data suggest that the luminescence quenching is attributable to rapid
electron transfer (ref 33).
(37) Sacksteder, L.; Zipp, A. P.; Brown, E. A.; Streich, J.; Demas, J. N.; Degraff,
B. A. Inorg. Chem. 1990, 29, 4335-4340.
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Luminescent Ru
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A R T I C L E S
electron transfer between the reductase module and oxygenase
domain. Inhibitors that disrupt protein:protein interactions could
be of great value, owing to the biological ubiquity of transient
protein complexes.
The interactions of 2 and 4 with ∆114 are in many ways
analogous to those observed with small molecules that prevent
NOS holoenzyme dimerization.42 Because only iNOS exhibits
an appreciable monomer-dimer equilibrium in vivo, these
inhibitors are highly isoform selective.42 The low dissociation
constant of 4 makes it a useful lead compound for further
development. The ∼70-fold difference in dissociation constants
2+
between 2 and 4 illustrates the steric influence of the Ru(bpy)3
moiety.
The ability of 5 to bind in or near the iNOSoxy active site is
remarkable given its dissimilarity to Arg or known inhibitors.
As with the Ru-wires, it seems likely that binding is driven
principally by hydrophobic interactions. Although 3 binds more
tightly than 5 to NOS, it does not produce a similar shift in the
absorption spectrum, again demonstrating the importance of
steric bulk in modulating wire-NOS interactions.
Figure 5. Steady-state luminescence spectra of 2.6 µM samples of ∆114
(green), 4 (black), and a 1:1 mixture of ∆114 and 4 (blue). The luminescence
of 4 is strongly quenched in the presence of ∆114, making it a sensitive
indicator of the presence of the enzyme. The sharp spike at 710 nm is an
artifact arising from scattered 355-nm excitation light.
Most notably, we have shown the usefulness of FET kinetics
in characterizing small molecule:protein interactions. Conven-
tional UV-visible absorption measurements or competition
binding assays would have overlooked the ability of 1 and 3 to
bind iNOSoxy. What is more, FET measurements yield distance
constraints, which provide additional information about the
interaction between the wire and target enzyme. As demon-
strated by these results, FET data greatly facilitate the formula-
tion of structural models for wire:enzyme conjugates.
displacement of the iron-ligating water from the active site. The
time-resolved luminescence decay spectra (Supporting Informa-
tion) suggest that 5 binds with a dissociation constant of 5 ( 2
µM and a Re-heme distance of ∼18 Å. Both the changes in
the Soret absorption and the calculated Re-Fe distance are
consistent with 5 binding in the active site. Structural modeling
indicates that 5 can fit in the active site of monomeric iNOSoxy,
unlike the sterically bulkier 3. The structural dissimilarities of
5 and Arg make it surprising that 5 binds at all. However, the
relatively exposed active site of the monomeric ∆114 iNOSoxy
provides good surface complementarity with the fluorinated
biphenyl moiety.
Acknowledgment. We thank John Magyar for helpful dis-
cussions. This work was supported by the Fannie and John Hertz
Foundation (A.R.D.), the National Institutes of Health (W.B.,
E.D.G.), and the National Science Foundation (H.B.G., J.R.W.).
Concluding Remarks
Supporting Information Available: Further transient lumi-
nescence decay kinetics. This material is available free of charge
via the Internet at http://pubs.acs.org.
Small molecules that interfere with NOS function are being
investigated as potential treatments for several diseases.40,41 All
currently known inhibitors in this class bind in the active site
of the enzyme. In contrast, Ru-wires or similar compounds
may provide an effective means of NOS inhibition by preventing
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