A R T I C L E S
Martell et al.
apparently too long to allow sulfur ligation to the heme iron.
According to the available partial density, one conformation
was built with an estimated Fe-S distance of 4.5 Å, as shown
in Figure S4. Judging from the negative residual density around
the sulfur atom, an alternate conformation with a shorter Fe-S
distance was possible, but no consistent model could be refined
because of the limited density.
the hydrophobic pocket, and 1 exhibits no type II conformations
in the crystal structure. Compounds 3, 4, 6, and 8, on the other
hand, all exhibit conformations that hold the thioether sulfur in
close proximity to the heme iron. The best fits are the ethyl
groups in 3 and 4, giving no disorder or alternate conformations
in their crystal structures.
The bulkier three-carbon terminal groups of 6-8 can barely
be accommodated by the hydrophobic pocket, which enables
conformations that hold the sulfur in close proximity to the heme
iron; however, the interactions are not as favorable as those of
3 and 4, leading to high levels of disorder in the entire thioether
tail and a higher population of type I conformation, which can
account for the loss of type II binding toward ferric nNOS.
Although both 6 and 7 have an identical n-propyl tail, the fit is
better with 6. Again, this superior fit results from the shorter
linker in 6, which orients the terminal propyl group differently,
restrained by bond angles along the alkyl chain. The branched
isopropyl group in 8 is also disordered, but it fits better into the
pocket than does the n-propyl group of 7, thereby giving an
Fe-S distance of 2.8 Å compared to >3.0 Å for 7.
Potency and the Fe-S Bond. Unexpectedly, coordination to
the heme iron has no effect on potency. Compound 3 is
essentially equal in potency to 5, even though the latter displays
no type II binding. Furthermore, both 1 and 4 fail to ligate to
ferric heme, with the Fe-S distance for 1 being more than 5
Å, yet both 1 and 4 are more potent than 3. Therefore, the Fe-S
ligand interaction contributes little, if any, to inhibitor binding
affinity. Considering the extensive hydrogen-bonding interac-
tions from the amino acid and amidine groups to the surrounding
protein residues, it is understandable that the thioether tail has
only a small influence on inhibitor binding. Nevertheless, our
results imply that the Fe-S bond formed from an axial thioether
ligand to the heme of a thiolate protein is exceptionally weak,
which is reflected by the rather long distances in the range of
Dithionite-Reduced Crystal Structures. An apparent paradox
in what has been described so far is that type I ligands, such as
4
and 6, showed substantial Fe-S interaction in the structures
using crystals prepared with the nNOS heme domain in its ferric
state, even though the spectral data indicate that these do not
form low-spin complexes when the iron is in the ferric state.
However, 4 and 6 do form low-spin type II complexes with the
heme iron in the ferrous state. It is well-known that metal centers
4
0
can undergo reduction in the X-ray beam, so it could be that
our structures of 4 and 6 complexed to nNOS are really in the
ferrous state owing to X-ray-induced reduction. To test this
hypothesis, dithionite-reduced nNOS-inhibitor crystals were
prepared for 3-5 in the hope that these crystal structures would
strictly reflect the ferrous inhibitor conformation. As it has
previously been reported that the Fe-S bond length for native
2
2
Met ligands can decrease upon heme reduction, we thought
there might be a decrease in the Fe-S distance in our dithionite-
reduced crystal structures. The structure for the 4-nNOS
complex in the reduced state at 2.10 Å resolution revealed the
Fe-S distance to be 2.7 Å (Figure S5, Supporting Information),
which is not significantly different from the Fe-S distance
observed for the structure from the crystal not treated by
dithionite. The data for 3 with the dithionite-reduced crystal at
2
.40 Å were not fully refined because of marginal data quality,
although the electron density and Fe-S distance were very
similar to data obtained from crystals that were not treated with
dithionite. There was also no change found for 5 in the
dithionite-reduced structure. This observation indicates that
the three-carbon amidine-sulfur linker still could not align the
sulfur atom close to the heme iron, even in its ferrous state
2
.5-2.8 Å, as seen in structures reported here. This type of
Fe-S bonding is much weaker than that for the natural
thioether-heme ligation in cytochrome c, for which the Fe-S
distance is ∼2.3 Å. The Fe-S interaction with our NOS
inhibitors is so weak that, without proper hydrophobic contacts
from the terminal alkyl group to the protein, the sulfur atom
cannot ligate to the heme, as seen in the cases of 1 and 2. These
observations underscore the previously documented low intrinsic
(Figure S5). The fact that pretreatment of crystals with dithionite
had very little effect on the structures of these three inhibitors
suggests that reduction may have occurred rapidly in the X-ray
beam.
1
,4-6
Discussion
affinity of thioethers for ferric heme
to what has been widely thought,
and imply that, contrary
coordination of thioethers
9
,27,41
Coordination of Thioether to Heme. All of the thioether
inhibitors reported here exhibit binding to the nNOS active site
similar to that of the substrate L-arginine. The extensive
hydrogen-bonding interactions with the protein from the inhibi-
tor amino acid and amidine groups anchor the inhibitors above
the heme plane. However, there are two factors that determine
whether or not each thioether inhibitor can behave as a heme
ligand: (1) the length of the linker from the amidine to the sulfur
atom and (2) the size of the tail after the sulfur atom. From the
structural data reported here, it is obvious that both one-carbon
and two-carbon linkers can bring the sulfur atom to the vicinity
of the heme iron, while a three-carbon linker, as in 5, is too
long. Whether or not the inhibitor can bind directly to the heme
is also controlled by the size of its terminal alkyl tail; the tail
must fit into the hydrophobic pocket formed by Pro565, Val567,
and Phe584. The terminal methyl groups of 1 and 2, which we
to the heme iron does not generally lead to increased potency,
at least not in heme-thiolate proteins. Furthermore, our results
4
2,43
suggest that while hydrogen-bonding
and electrostatic
44
interactions of native Cys ligands with nearby residues stabilize
heme-thiolate ligation, hydrophobic interactions may play an
important role in facilitating the thioether-iron coordination
of native Met heme ligands in cytochrome c.
There are two possible explanations for why the thioether to
heme interaction seen in our nNOS inhibitor complexes is so
weak, exhibiting a rather long Fe-S distance of 2.5-2.8 Å.
(41) Narayanan, K.; Griffith, O. W. J. Med. Chem. 1994, 37, 885–887.
(
42) (a) Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Uekusa, H.;
Ohashi, Y.; Uchida, T.; Kitagawa, T.; Nagano, T. J. Am. Chem. Soc.
1
999, 121, 11571–11572. (b) Ueyama, N.; Nishikawa, N.; Yamada,
Y.; Okamura, T.; Nakamura, A. J. Am. Chem. Soc. 1996, 118, 12826–
12827.
27
reported earlier, are not large enough to interact favorably with
(
43) Yoshioka, S.; Takahashi, S.; Ishimori, K.; Morishima, I. J. Inorg.
Biochem. 2000, 81, 141–151.
(
40) Beitlich, T.; Kuhnel, K.; Schulze-Briese, C.; Shoeman, R. L.;
(44) (a) Goodin, D. B. J. Biol. Inorg. Chem. 1996, 1, 360–363. (b) Poulos,
T. L. J. Biol. Inorg. Chem. 1996, 1, 356–359.
Schlichting, I. J. Synchrotron Radiat. 2007, 14, 11–23.
8
04 J. AM. CHEM. SOC. 9 VOL. 132, NO. 2, 2010