Bioorganometallic mechanism of action, and inhibition, of IspH

Article abstract of DOI:10.1073/pnas.0911087107

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K _M;V_max), EPR and 1H, 2H, 13C, 31P, and 57Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP parent molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π?σ metallacycle or η²-alkenyl complex. The complex is poised to interact with H⁺ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η¹-allyl complex. After reduction, this forms an η³-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by ¹H, ²H, and ¹³C ENDOR, where hyperfine couplings of approximately 6 MHz for ¹³C and 10 MHz for ¹H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe₄S ₄-containing proteins, such as IspG.

Full text of DOI:10.1073/pnas.0911087107

Bioorganometallic mechanism of action,
and inhibition, of IspH
Weixue Wanga, Ke Wangb, Yi-Liang Liua, Joo-Hwan Noa, Jikun Lia, Mark J. Nilgesc, and Eric Oldfielda,b,1
aCenter for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801; bDepartment of Chemistry, University of Illinois, 600 South
c
Mathews Avenue, Urbana, IL 61801; Illinois EPR Research Center, 506 South Mathews Avenue, Urbana, IL 61801
Edited* by Jack Halpern, The University of Chicago, Chicago, IL, and approved December 29, 2009 (received for review September 25, 2009)
We have investigated the mechanism of action of Aquifex aeolicus
IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) re-
ductase], together with its inhibition, using a combination of
site-directed mutagenesis (KM; Vmax), EPR and 1H, 2H, 13C, 31P,
and 57Fe-electron-nuclear double resonance (ENDOR) spectroscopy.
On addition of HMBPP to an (unreactive) E126A IspH mutant, a re-
action intermediate forms that has a very similar EPR spectrum to
those seen previously with the HMBPP “parent” molecules, ethy-
lene and allyl alcohol, bound to a nitrogenase FeMo cofactor.
The EPR spectrum is broadened on 57Fe labeling and there is no
evidence for the formation of allyl radicals. When combined with
ENDOR spectroscopy, the results indicate formation of an organo-
metallic species with HMBPP, a π∕σ “metallacycle” or η2-alkenyl
complex. The complex is poised to interact with Hþ from E126
(and H124) in reduced wt IspH, resulting in loss of water and for-
mation of an η1-allyl complex. After reduction, this forms an η3-allyl
π-complex (i.e. containing an allyl anion) that on protonation (at C2
or C4) results in product formation. We find that alkyne dipho-
sphates (such as propargyl diphosphate) are potent IspH inhibitors
and likewise form metallacycle complexes, as evidenced by 1H, 2H,
and 13C ENDOR, where hyperfine couplings of approximately
6 MHz for 13C and 10 MHz for 1H, are observed. Overall, the
results are of broad general interest because they provide new in-
sights into IspH catalysis and inhibition, involving organometallic
species, and may be applicable to other Fe4S4-containing proteins,
such as IspG.
article, we focus on the last enzyme in the nonmevalonate path-
way, IspH (LytB), with the goal of obtaining a better under-
standing of its mechanism of action, and inhibition.
The IspH (LytB) enzyme HMBPP (E-4-hydroxy-3-methyl-
but-2-enyl diphosphate) reductase (EC 1.17.1.2) catalyzes the
2Hþ∕2e− reduction of HMBPP (3) to form an approximately
5∶1 mixture of IPP and DMAPP:
The enzyme is essential for survival and is not found in humans,
so is an attractive target for drug development (11). The struc-
tures of IspH from Aquifex aeolicus (12) and Escherichia coli
(13) have recently been reported and indicate trefoil-like protein
structures with a central Fe3S4 cluster (14), whereas EPR (15),
Mössbauer (16, 17), reconstitution and catalytic activity (15,
17) measurements have all been interpreted as indicating that
an Fe4S4 cluster is the catalytically active species. Ligand-free
IspH has an “open” structure (12), whereas IspH cocrystallized
with diphosphate has a “closed” structure (13) in which a serine-
X-asparagine (SXN) loop is involved in hydrogen bonding with a
PPi ligand. The mechanism of action of IspH is controversial
and there have been many different proposals (13, 15, 18–21)
(Fig. S1). However, none of these models has yet been supported
by any spectroscopic evidence, and none have led to the devel-
opment of IspH inhibitors. Here, we report spectroscopic results
that indicate the involvement in catalysis of π∕σ metallacycle
intermediates similar to those found for ethylene and allyl alcohol
when bound to a nitrogenase FeMo cofactor (22–24). Then,
based on these results, we show that alkynes can inhibit IspH,
forming once again, metallacyles or π∕σ complexes.
enzyme inhibition ∣ iron-sulfur protein ∣ isoprenoid biosynthesis ∣
nonmevalonate pathway
nzymes that catalyze the formation of isoprenoids are of in-
E
terest as drug targets. There are two main pathways involved
in the early steps in isoprenoid biosynthesis: The mevalonate
pathway found in animals and in pathogens such as Staphylococ-
cus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative
agents of staph infections, Chagas’ disease and the leishma-
niases), and the nonmevalonate or Rohmer pathway found in
most pathogenic bacteria, as well as in the malaria parasite,
Plasmodium falciparum (1). Both pathways lead to formation
of the C5-isoprenoids isopentenyl diphosphate (IPP, 1) and
dimethylallyl diphosphate (DMAPP, 2). In the later stages of iso-
prenoid biosynthesis, these C5-compounds then form the farnesyl
diphosphate (FPP) and geranylgeranyl diphosphate (GGPP)
used in protein prenylation, sterol, and carotenoid biosynthesis.
Understanding how the enzymes catalyzing these “downstream”
events function has led to a better understanding of e.g. how FPP
synthase (2) and GGPP synthase function, and can be inhibited
(3); the discovery that bisphosphonates have potent antiparasitic
activity (4); the clinical use of amiodarone (a squalene oxidase
and oxidosqualene cyclase inhibitor) against Chagas’ disease
(5; 6) and leishmaniasis (7); anticancer agents that inhibit both
FPPS and GGPPS (8); as well as the discovery that cholesterol
lowering agents (squalene synthase inhibitors) can function as
antivirulence agents, against S. aureus (9). However, there have
been few compounds discovered that block the nonmevalonate
pathway, fosmidomycin being the notable exception (10). In this
Results and Discussion
The Role of Protein Residues. We first investigated the role of
protein residues in the IspH mechanism. In previous work, we
noted that in addition to E126, His42, and His124 were also
totally conserved residues, were located in the active site region,
and were likely essential for catalytic activity, a conclusion now
supported by mutagenesis results (13). However, the exact role
of these residues was unclear. We thus determined the KM and
Vmax values for three mutants: H42A, H124A, and E126A. In
the case of the E126A mutant, activity was so low (Vmax
<
0.025 μmol min−1 mg−1) that KM could not be measured. But
with the H124A mutant, we found that although Vmax was low
(0.05 μmol min−1 mg−1 versus 1.16 μmol min−1 mg−1 for the
wild-type enzyme), KM was essentially unchanged (7 μM versus
Author contributions: W.W., K.W., and E.O. designed research; W.W., K.W., Y.-L.L., J.-H.N.,
J.L., M.J.N., and E.O. performed research; W.W., J.-H.N., and E.O. analyzed data; K.W. and
J.L. contributed new reagents/analytic tools; and E.O. wrote the paper.
The authors declare no conflict of interest.
This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: eo@chad.scs.uiuc.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0911087107/DCSupplemental.
4522–4527 ∣ PNAS ∣ March 9, 2010 ∣ vol. 107 ∣ no. 10
www.pnas.org/cgi/doi/10.1073/pnas.0911087107

5 μM, for the wild-type enzyme). This indicates that H124 is not a
major contributor to substrate binding, but is essential for
catalysis, suggesting that H124 may be involved in delivering Hþ
to E126 and the bound HMBPP. In the case of H42, however,
we find in the H42A mutant that there is an increase in KM (from
7–74 μM), indicating a role in substrate binding, consistent with
the crystallographic observation that H42 hydrogen bonds to a
bound diphosphate ligand (13). There is, nevertheless, also
a 5-fold decrease in Vmax, due perhaps to the possibility that
several more distal residues could also be involved in proton
transfer.
tion) are, interestingly, quite similar to those seen previously with
ethylene and allyl alcohol bound to the α-70Ala mutant of a nitro-
genase FeMo cofactor protein [ethylene: g ¼ 2.123; 1.978; 1.949
(24); allyl alcohol: g ¼ 2.123; 1.998; 1.986 (22)] with, on average,
only a j0.01j difference between the IspH and nitrogenase g-
values. In nitrogenase, ethylene and allyl alcohol have been
shown [via ENDOR and/or density functional theory calculations
(22–24)] to bind as π∕σ “metallacycles,” as shown e.g. in Fig. 1E.
And because HMBPP is simply a substituted alkene, one possible
explanation of the E126A þ HMBPP spectrum is that HMBPP
binds to reduced IspH in a similar manner, that is, as a π or
π∕σ “metallacycle” or “η2-alkenyl” complex, similar to that shown
in Fig. 1F (which is based on the nitrogenase/allyl alcohol struc-
ture and contains Mo and “X”). That such a complex could form
with HMBPP is supported by the results of ligand docking calcu-
lations using Glide (25) (Fig. 1G and H) in which it can be seen
that HMBPP can bind with its diphosphate occupying the “PPi”
site seen crystallographically, at the same time that its double
bond interacts with the unique fourth Fe, added computationally
here as described previously (12). Based on the observation that
only reduced Fe4S4 clusters bind alkynes (26), it seemed likely
that only the reduced IspH would bind HMBPP in this manner,
and to investigate this binding question in more detail, we used
ENDOR to probe the HMBPP- reduced IspH interaction in more
detail.
EPR of IspH with Bound Ligands. As shown by Wolff et. al. (15), as-
isolated, oxidized IspH (from E. coli) has low activity and exhibits
an EPR spectrum characteristic of an S ¼ 1∕2 ½Fe3S4ꢀþ cluster.
On reconstitution (with DTT, Fe3þ and S2−) and under reducing
(excess dithionite) conditions, a broad, S ¼ 1∕2 EPR spectrum
characteristic of an ½Fe4S4ꢀþ cluster is obtained (15), essentially
identical to that we find for reduced A. aeolicus IspH (Fig. 1A).
On addition of HMBPP, the spectrum sharpens and several com-
ponents can be seen (Fig. 1B). This suggests that HMBPP might
bind to the cluster, or that HMBPP reacts and that the IPP/
DMAPP products bind to the cluster, or that both occur. The ob-
servation that the IPP product can bind is supported by the ob-
servation that the spectrum of reduced, wild-type IspH plus IPP
(Fig. 1C) is similar to that of the HMBPP spectrum. Clearly, this
apparent reactivity complicates any determination of the details
of HMBPP binding, so we next investigated the binding of
HMBPP to the E126A mutant of IspH. In our previous work
(12) we proposed that E126 was a key catalytic residue, a sugges-
tion now confirmed by mutagenesis results (13). If E126 is inac-
tivated by mutagenesis (to Ala), this should block catalysis,
and might enable observation of an early reaction intermediate.
As shown in Fig. 1D, the spectrum of the reduced E126A
mutant þ HMBPP is in fact now quite different to that seen
with the wild-type enzyme þ HMBPP, confirming this idea.
The spectrum is broadened in an 57Fe-labeled sample
(Fig. S2C), indicating an ½Fe4S4ꢀþrather than an organic radical,
origin. Moreover, the g-values (g ¼ 2.124; 1.999; 1.958, by simula-
2H, 13C, 31P, and 57Fe ENDOR of IspH þ Alkene Ligands. A test of this
π∕σ metallacycle complex formation hypothesis with reduced
IspH is that there should be significant hyperfine couplings
(A) with the ligand if it indeed bonds to the S ¼ 1∕2, ½Fe4S4ꢀþ
cluster. We thus prepared the following series of 2H and 13C
isotopically labeled HMBPPs (4–6), and investigated their EN-
DOR spectra when bound to IspH.
Fig. 1. 9 GHz EPR of A. aeolicus IspH, and molecular models for ligand interactions. (A) Wild-type IspH. (B) Wild-type IspH þ HMBPP. (C) Wild-type IspH þ IPP.
(D) E126A IspH þ HMBPP. Spectra were all obtained on samples reduced with 20 equivalents sodium dithionite (see SI Text for more details), and are essentially
identical to those obtained in samples prepared via photoreduction (e.g. Fig. S2A and B). Microwave power was 1 mW for A–C, and 0.1 mW for D. (E) Structure
of nitrogenase Fe/Mo cofactor þ allyl alcohol. (F) As (E) but edited to more clearly show the allyl alcohol binding site. Mo (Cyan); “X” (Blue). (G) Glide docking
result for HMBPP docking to IspH active site region. (H) As (G) but after molecular mechanics optimization.
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On binding 4 to E126A IspH, we clearly see (Fig. 2A, Bottom
Trace) a low frequency feature in the Mims ENDOR (27) differ-
ence spectrum attributable to the methyl deuterons, character-
ized by A ∼ 0.4 MHz. At higher frequency, there is a peak
centered at the 31P Larmor frequency, but with a very small
(<0.1 MHz) hyperfine coupling, in the ENDOR spectra of both
3 and 4 with IspH (Fig. 2A, Inset in Black). Essentially the same
results are found on binding of 5 (which contains a 4-2H1 group),
inset in blue above the 5-2H3 spectra, with A again being approxi-
mately 0.4 MHz for 2H and <0.1 MHz for 31P. With [u-13C]
HMBPP, 6, there are two major features (having
A ¼ 1.70; 0.80 MHz, Fig. 2B), which we attribute to the olefinic
carbons, 13C2; 3. Signals from 13C1, 4 and 5 are not observed, due
we believe to very small couplings. These EPR and 2H, 13C EN-
DOR results support the idea that HMBPP bound to reduced
IspH forms a metallacycle (that is, a π or η2-alkenyl σ-like com-
plex), just as found with ethylene and allyl alcohol binding to the
nitrogenase FeMo protein cofactor (22–24).
both EPR and ENDOR spectroscopy indicate π or π∕σ complex
formation with reduced IspH and HMBPP, leading to the cata-
lytic mechanism proposal described in the following.
Catalytic Mechanism of IspH. The models shown in Fig. 1G and H
indicate that when the HMBPP diphosphate docks to the “PPi”
site and the double bond forms a π∕σ alkenyl complex with the
(reduced) Fe4S4 cluster, the CO2H group of E126 is approxi-
mately 2.3 Å from the HMBPP 4-OH group. This then suggests
the catalytic mechanism shown in Fig. 3 in which: (i) After
initially binding to the oxidized cluster via oxygen (Fig. 3A),
on reduction a π (or π∕σ) complex forms (Fig. 3B and C), “acti-
vating” the substrate for the subsequent deoxygenation. The
structure shown in Fig. 3B is the π-complex, but the proposed
reaction can arguably be better followed via the η2-alkenyl/metal-
lacycle, Fig. 3C. (ii) This reduced complex is then protonated via
E126 (and H124A), resulting in formation of water and an η1-allyl
complex, Fig. 3D. (iii) After a second reduction, the η3-allyl com-
plex forms (Fig. 3E and F), with protonation at C2 or C4 then
forming the two products, DMAPP and IPP (Fig. 3G and H).
One potential drawback of this mechanism might be that the
E126 carboxyl is not acidic enough to protonate the 4-OH. How-
ever, H124 is close by (approximately 5 Å from the H124 Nδ to
E126 carboxyl oxygen, PDB file #3F7T), and could be involved in
increasing the acidity of E126, either directly or via an intervening
H2O. The source of Hþ in the second part of reaction, the
reductive cleavage, could be via E126 (in the case of DMAPP
formation), or solvent (e.g. H3Oþ in a “water” site), for IPP
(because there are no other obvious Hþ donors apparent).
57Fe ENDOR spectra of ½57Feꢀ-IspH ꢁ 3 also support the idea
that 3 may bind directly to the Fe4S4 cluster. The 57Fe Davies
ENDOR spectrum (27) (π∕2mw ¼ 12 ns, nonselective pulse) of
IspH in the absence of any added ligands is broad (Fig. 2C)
and there are three major features present. On binding 3, a shar-
per ENDOR spectrum is seen. Using selective (π∕2mw ¼ 48 ns)
pulses in a Davies ENDOR spectrum, features attributable to 1H
as well as 57Fe are observed (Fig. 2D); with nonselective pulses
(π∕2mw ¼ 12 ns) only two major Fe features are apparent
(Fig. 2E) and these features (as expected) are absent in a control
(½56Feꢀ IspH þ 3) spectrum (Fig. 2F). The difference spectrum
(2E-2F) is shown in Fig. 2G. No additional small 57Fe hyperfine
couplings are observed, as confirmed by Mims ENDOR. These
results show that there are major changes in the electronic struc-
ture of the reduced Fe4S4 cluster on HMBPP addition, and
strongly support the idea that HMBPP binds chemically to the
cluster. This in turn supports the idea that the hyperfine couplings
13
seen in the 2H∕ C ENDOR results are not solely due to a dipolar
or through space interactions but rather, arise (at least in part)
from through bond, Fermi contact couplings. So, the results of
Fig. 3. Proposed mechanism of action of IspH. (A) Oxidized Fe4S4 cluster plus
HMBPP (12); (B) the Fe4S4 cluster has been reduced by one electron; the struc-
ture is generally similar to that found for allyl alcohol bound to nitrogenase;
(C) π∕σ complex (shown for simplicity as the η2-alkenyl metallacycle) is pro-
tonated by E126/H124A; (D) After protonation, the 4-OH is removed as H2O,
forming an η1-allyl complex; (E) the Fe4S4 cluster is reduced by second e−, and
subsequently forms a η3-allyl (π) complex, (F); (G) protonation at C4 results in
DMAPP product; (H) IPP is formed by protonation at C2.
Fig. 2. ENDOR spectra of IspH with HMBPP. (A) E126A IspH þ 4 (Black) or 5
(Blue). Spectra are difference spectra (2H-1H); the 31P region is the
unsubtracted ENDOR result. (B) E126A IspH þ 6 minus E126A þ 3, showing
solely 13C ENDOR. (C) Unliganded wild-type ½u-57Feꢀ-IspH, with natural
abundance 57Fe control subtracted. (D) ½u-57Feꢀ-E126A IspH þ HMBPP, 57Fe
ENDOR, Davies selective pulse. (E) As (D) but nonselective pulse. (F) As (D)
but natural abundance 57Fe-IspH. (G) Difference spectrum of (E) and (F).
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Wang et al.

A simplified version of the mechanism, together with the
locations of the reactants/intermediates/products in IspH is illus-
trated in Fig. 4. In Fig. 4A, we show a docking pose for HMBPP
bound to reduced IspH (PDB file #3F7T; chosen because it con-
tains a PPi ligand and adopts the “closed” conformation) in which
the diphosphate hydrogen bonds to S225, N227, and H42 while
the side-chain fits the pocket near the Fe4S4 cluster. Note of
course that, based on the spectroscopic and activity results, we
show an Fe4S4 (not an Fe3S4) cluster. The double bond in
HMBPP forms a π (or π∕σ) complex with the reduced Fe4S4
cluster, based on the ENDOR results. As is well known in organ-
ometallic chemistry, such complexes are typically neither
completely π nor completely σ, as discussed by Pelmenschikov
et al. (23), but for convenience in drawing reaction mechanisms,
is shown as a σ (η2-alkenyl) complex in Figs. 3 and 4. On proton-
ation of the 4-OH by E126 (possibly involving H124 and H2O),
the η1-allyl complex and water form, Fig. 4B. After the second
reduction, the η3-complex forms (Fig. 4C), which is then proto-
nated to form the two products (Fig. 4D). Although radical tran-
sition states or reactive intermediates may also be involved, the
EPR and ENDOR spectra we do observe are unlikely to arise
from allyl radicals because (i) allyl radicals would have 2 or 3 par-
tially resolved peaks around the free electron g-value; (ii) they
would have very large [approximately 50 MHz (28)] 13C hyperfine
couplings; (iii) they would not broaden on 57Fe-labeling; plus (iv),
the E126A mutant is essentially unreactive. The model proposed
is, attractive because it invokes well-established precedent for the
formation of π-complexes between Fe4S4 clusters and unsatu-
rated species (alkynes, cyclopentadienides) (26, 29); such com-
plexes are seen with the “parents” ethylene and allyl alcohol
bound to the nitrogenase FeMo cofactor; we observe hyperfine
couplings with bound substrate; docking puts the double bond
next to the unique Fe, and the 4-OH close to the putative Hþ
donor, E126; and there is no evidence for any allyl radical inter-
mediates.
Inhibition was very low, Ki > 10 mM. On the other hand,
propargyl diphosphate (9) was a more potent IspH inhibitor
(Fig. 5B) with a Ki ¼ 0.97 μM. This indicates that acetylenic
diphosphates bind to IspH, most likely with their diphosphates
occupying the “PPi” site, while their alkyne groups bind to the
fourth Fe, forming a π∕σ metallacycle. A docking pose for 9
bound to IspH is illustrated in Fig. 5C and clearly indicates that
the alkyne is in close apposition with the fourth Fe, forming a
metallacycle.
If such metallacycles form, it should be possible to investigate
them spectroscopically. In particular, we expect to see major
changes in the ½Fe4S4ꢀþ EPR spectra, plus, ENDOR should
indicate sizeable hyperfine couplings. The EPR spectra of IspH
(þ8; 9, and 10) are shown in Fig. 5D–F and indicate large g-value
changes (from Fig. 1A) on ligand addition, suggesting an interac-
tion with the Fe4S4 cluster. The Davies 1H ENDOR spectrum
(Fig. 6A) of IspH þ 8 shows an approximately 10 MHz hyperfine
coupling feature, while in the Davies ENDOR spectrum of
IspH þ ½3-2H1ꢀ-8, the 10 MHz feature is greatly attenuated
(Fig. 6A, Inset) and is replaced at low field by a 1.5 MHz hyperfine
1
coupling feature in the (2H − H) difference Mims ENDOR
spectrum (Fig. 6B), attributable to the 3-deuteron. The large
2
1H and H hyperfine couplings are similar to those seen in the
nitrogenase system (22) and indicate formation of an organo-
metallic species.
Additional evidence for formation of Fe-C bonds per se come
from the results of ENDOR experiments using both ½13C3ꢀ-8 and
½13C3ꢀ-9 (Fig. 6C and D), where we see strong 13C ENDOR peaks
having quite large (6 MHz) hyperfine couplings. This, together
with the docking result (Fig. 5C), suggests that both 8 and 9 bind
to the Fe4S4 cluster at the fourth Fe, forming a π∕σ complex,
as with acetylene binding to model Fe4S4 clusters (26, 30). Also
present in the ENDOR spectrum of 9 bound to IspH is a feature
due to 31P (Fig. 6D Inset, hyperfine coupling ¼0.3 MHz). We
favor a sideways-on π-complex rather than the alternative end-
on binding mode because (i) diphenylacetylene (7), which lacks
a terminal H, binds to reduced Fe4S4 clusters (31) but as with
acetylene (30), is still cis-reduced [by 2H (31)]; (ii) the shift in
the C ≡ C vibrational Raman spectrum (26) of C2H2 on binding
to reduced Fe4S4 clusters is relatively small (approximately
60 cm−1), whereas shifts seen on acetylide formation (or in
mononuclear complexes) are typically 2–3x larger (32); (iii),
compound 10, which cannot bind end-on to IspH, still exhibits
(Fig. 5F) a rhombic EPR spectrum similar to that seen with
the terminal alkynes, Fig. 5D and E; and (iv), the observation
of the 2H ENDOR signal from the terminal 2H in 8 is consistent
with this binding mode: FeH would have a larger coupling, while
SH would exchange away.
The Metallacycle Model Leads to Novel Inhibitors. The binding of
acetylene to simpler Fe4S4 clusters, forming π complexes, Fig. 5A,
2−∕3−
has been observed in model systems such as ½Fe4S4ðSPhÞ4ꢀ
2−
(26, 30), and diphenylacetylene (7) binds to ½Fe4S4ðSPhÞ4ꢀ
where it can be cis-hydrogenated by NaBH4 (31). This leads to
the idea that acetylenes might bind to, and thus inhibit, IspH.
We thus next investigated the inhibition of IspH by propargyl
alcohol, 8:
Fig. 4. Protein-based structural models for IspH catalysis. (A) HMBPP docks to the reduced active site with diphosphate binding to S225, N227, and H42, double
bond forms π∕σ complex with Fe4S4 cluster; (B) HMBPP 4-OH is protonated by E126, loss of H2O results in η1-allyl complex; (C) cluster is rereduced by one
electron, forming an η3-allyl (π) complex; (D) η3-allyl complex is protonated at C4 forming DMAPP and oxidized cluster. Protonating the alternate η3-allyl
complex (or η1-allyl species) at C2 results in IPP product formation (as in Fig. 3). Numbering is for the A. aeolicus protein.
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Fig. 5. Inhibition of IspH by alkynes. (A) Proposed binding mode of acetylene to an Fe4S4 cluster (26, 30). (B) IspH inhibition by 9; IC50 ¼ 6.7 μM; Ki ¼ 0.97 μM.
(C) Glide docking pose for 9 bound to IspH. The E. coli IspH structure (PDB 3F7T) and numbering are shown. (D–F) EPR spectra of three alkynes interacting with
IspH: (D) 8; (E) 9; (F) 10. Microwave power was 0.05 mW. All spectra show major g-value changes from those seen in the inhibitor-free protein, Fig. 1A.
The 57Fe ENDOR spectra of ½u-57Feꢀ-IspH þ 8 (and a 56Fe
control) are shown is Fig. 6E–G. The Davies selective pulse spec-
trum (π∕2mw ¼ 48 ns, Fig. 6E) has both 1H and 57Fe contribu-
tions. The 1H signal is edited out by using nonselective pulses
(π∕2mw ¼ 12 ns), Fig. 6F, and now we see two main features
with hyperfine couplings of 28; 35 MHz. No signal is seen with
the 56Fe control sample (Fig. 2G) under the same conditions.
Likewise, no signals are apparent in the low frequency region
when using a Mims pulse sequence, ruling out the presence of
very small 57Fe hyperfine couplings. Overall, these 57Fe ENDOR
results are quite similar to those seen with HMBPP, Fig. 2, and
are quite different to those seen in the absence of these ligands,
supporting again a direct interaction with the cluster.
Conclusions
In summary, the mechanism we propose is unique in that it in-
volves a novel, bioorganometallic mechanism for IspH catalysis,
that is, the formation of transition state/reactive intermediates
(π∕σ complexes; η3-allyl complexes) containing metal-carbon
bonds, together with the involvement of at least two residues
(H124, E126) that deliver Hþ to the active site. The ability of
olefins (as well as acetylenes and cyclopentadienides) to bind
to ½Fe4S4ꢀþ (or ½Fe6Mo2S8ðSPhÞ9ꢀ clusters) is well known in
3−
model systems, as well as in the nitrogenase system, and provides
precedent for the structures proposed. Of particular importance
is the observation that ethylene and allyl alcohol (HMBPP
“minus” the Me and −CH2OPP substituents), form π∕σ com-
Fig. 6. Alkyne inhibitor ENDOR results. (A) IspH þ 8, Davies selective pulse, showing a large 1H hyperfine coupling that is attenuated (Inset, Blue) in an
80% ½3-2H1ꢀ-8 labeled sample. (B) IspH þ ½3-2H1ꢀ-8, (2H − 1H) difference Mims ENDOR spectrum, showing a 2H ENDOR signal with 1.5 MHz coupling.
(C) IspH þ ½u-13C3ꢀ-8, difference Mims ENDOR spectrum showing solely 13C ENDOR. (D) IspH þ ½u-13C3ꢀ-9 difference Mims ENDOR spectrum showing solely
13C ENDOR; the inset shows the 31P Mims ENDOR signals from IspHþ unlabeled 9. (E) ½u-57Feꢀ-IspH þ 8, Davies selective pulse. (F) The same as (E), but with
Davies nonselective pulse. (G) Natural abundance 57Fe control, Davies nonselective pulse.
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Wang et al.

plexes with Fe (in the nitrogenase FeMo cofactor), and similar
metallacycles are likely to form (as reactive intermediates or tran-
sition states) when HMBPP binds to reduced IspH, with the
4-OH then being ideally poised to be protonated (by E126/
H124). This π∕σ bonding would not be present with an Fe3S4
cluster, which just presents 3S2− to the olefin. When compared
with previous proposals, the results we have described above
differ in several important respects. First, organometallic species
(i.e. containing Fe-C bonds) are involved. Second, we find no
evidence for stable, carbon-based radicals. Third, there are spe-
cific roles for key catalytic residues (in particular, E126). Fourth,
the mechanism requires the presence of a fourth, unique Fe. The
likely involvement of an η3 (π) complex (the allyl anion) is also
attractive, because other anionic π-complexes, cyclopentadie-
nides, are well known to form π-complexes with Fe4S4 clusters.
These results are also of broader general interest because they
have led to the discovery of the first μM inhibitor of IspH,
and based again on EPR, ENDOR, and computational docking
results, we propose that these types of inhibitors bind into the
IspH active site with their diphosphates occupying the “PPi” site,
while their alkyne groups form π∕σ complexes with the unique
fourth Fe. Overall, these results can be expected to lead to
new types of inhibitors of IspH, as well as of other Fe4S4 cluster-
containing proteins containing “unique” fourth Fe atoms, such as
IspG, where again, organometallic species are likely to be in-
volved in catalysis.
Methods
Samples for EPR spectroscopy were reduced either by adding Na2S2O4 or
by photoreduction in the presence of 5-deazaflavin. EPR spectra were col-
lected at X-band using a Varian E-122 spectrometer together with an Air
Products helium cryostat. Data acquisition parameters were typically
field center ¼ 3250 G;
field sweep ¼ 800 G;
modulation ¼ 100 kHz;
modulation amplitude ¼ 5 G; time constant ¼ 32 ms; 60
s
per scan; 8 s
between each scan; and temperature ¼ 15 K. Samples for ENDOR spectro-
scopy were reduced by adding Na2S2O4. Pulsed ENDOR spectra were
obtained on a Bruker ElexSys E-580-10 FT-EPR X-band EPR spectrometer using
an ENI A 300RF amplifier and an Oxford Instruments CF935 cryostat at 15 K
(8 K for unliganded wt ½57Feꢀ-IspH). Davies pulsed ENDOR experiments were
carried out for 57Fe samples and the IspH þ 8 sample using a three pulse
scheme (πmw-T-π∕2mw-τ-πmw-τ-echo, πrf applied during T) (27) with the exci-
tation field set to correspond to g2. 57Fe Davies ENDOR spectra were collected
with either π∕2mw ¼ 12 ns (nonselective) or π∕2mw ¼ 48 ns (selective)
pulse excitation. The selective pulse scheme was also used for acquiring
1H ENDOR spectra of IspH þ 8. For other samples, Mims pulsed ENDOR with
a three pulse scheme (π∕2mw-τ-π∕2mw-T-π∕2mw-τ-echo, π∕2mw ¼ 16 ns, and
πRF applied during T) (27) was used, with the excitation field set to cor-
respond to g2. τ-averaging (32 spectra at 8 ns step) was used to reduce
the blind spots that arise from the τ-dependent oscillations.
Further details of site-directed mutagenesis, protein purification and
reconstitution, enzymatic assays, computational aspects, and compound
synthesis are reported in SI Text, Supplementary Methods.
ACKNOWLEDGMENTS. We thank Pinghua Liu for providing his E. coli IspG plas-
mid, Hassan Jomaa, and Jochen Wiesner for providing their A. aeolicus IspH
plasmid, and Thomas B. Rauchfuss and a reviewer, for helpful suggestions.
This work was supported by the United States Public Health Service (NIH
Grants GM65307 and GM073216).
1. Rohmer M, Grosdemange-Billiard C, Seemann M, Tritsch D (2004) Isoprenoid biosynth-
esis as a novel target for antibacterial and antiparasitic drugs. Curr Opin Invest Dr
5:154–162.
2. Martin MB, Arnold W, Heath HT, III, Urbina JA, Oldfield E (1999) Nitrogen-containing
bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis.
Biochem Biophys Res Commun 263:754–758.
3. Guo RT, et al. (2007) Bisphosphonates target multiple sites in both cis- and trans-
prenyltransferases. Proc Natl Acad Sci USA 104:10022–10027.
4. Rodriguez N, et al. (2002) Radical cure of experimental cutaneous leishmaniasis by the
bisphosphonate pamidronate. J Infect Dis 186:138–140.
5. Benaim G, et al. (2006) Amiodarone has intrinsic anti-Trypanosoma cruzi activity and
acts synergistically with posaconazole. J Med Chem 49:892–899.
6. Paniz-Mondolfi AE, Perez-Alvarez AM, Lanza G, Marquez E, Concepcion JL (2009)
Amiodarone and itraconazole: A rational therapeutic approach for the treatment
of chronic Chagas’ disease. Chemotherapy 55:228–233.
7. Paniz-Mondolfi AE, et al. (2008) Concurrent Chagas’ disease and borderline dissemi-
nated cutaneous leishmaniasis: The role of amiodarone as an antitrypanosomatidae
drug. Ther Clin Risk Manag 4:659–663.
17. Xiao Y, Chu L, Sanakis Y, Liu P (2009) Revisiting the IspH catalytic system in the deox-
yxylulose phosphate pathway: Achieving high activity. J Am Chem Soc 131:9931–9933.
18. Altincicek B, et al. (2002) LytB protein catalyzes the terminal step of the 2- C-methyl-D-
erythritol-4-phosphate pathway of isoprenoid biosynthesis. FEBS Lett 532:437–440.
19. Rohdich F, et al. (2003) The deoxyxylulose phosphate pathway of isoprenoid
biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH
protein. Proc Natl Acad Sci USA 100:1586–1591.
20. Xiao Y, Zhao ZK, Liu
P (2008) Mechanistic studies of IspH in the deoxyxylulose
phosphate pathway: Heterolytic C-O bond cleavage at C4 position. J Am Chem Soc
130:2164–2165.
21. Xiao Y, Liu P (2008) IspH protein of the deoxyxylulose phosphate pathway: Mechanistic
studies with C1-deuterium-labeled substrate and fluorinated analogue. Angew Chem
Int Ed Engl 47:9722–9725.
22. Lee HI, et al. (2004) An organometallic intermediate during alkyne reduction by
nitrogenase. J Am Chem Soc 126:9563–9569.
23. Pelmenschikov V, Case DA, Noodleman L (2008) Ligand-bound S ¼ 1∕2 FeMo-cofactor
of nitrogenase: Hyperfine interaction analysis and implication for the central ligand X
identity. Inorg Chem 47:6162–6172.
8. Zhang Y, et al. (2009) Lipophilic bisphosphonates as dual farnesyl/geranylgeranyl
diphosphate synthase inhibitors: An x-ray and NMR investigation. J Am Chem Soc
131:5153–5162.
24. Lee HI, et al. (2005) Electron inventory, kinetic assignment (En), structure, and bonding
of nitrogenase turnover intermediates with C2H2 and CO.
J Am Chem Soc
9. Liu CI, et al. (2008) A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus
virulence. Science 319:1391–1394.
127:15880–15890.
25. Friesner RA, et al. (2006) Extra precision glide: Docking and scoring incorporating a
10. Borrmann S, et al. (2006) Fosmidomycin plus clindamycin for treatment of pediatric
patients aged 1 to 14 years with Plasmodium falciparum malaria. Antimicrob Agents
Ch 50:2713–2718.
11. Rohmer M (2008) From molecular fossils of nacterial hopanoids to the formation of
isoprene units: Discovery and elucidation of the methylerythritol phosphate pathway.
Lipids 43:1095–1107.
12. Rekittke I, et al. (2008) Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate
reductase, the terminal enzyme of the non-mevalonate pathway. J Am Chem Soc
130:17206–17207.
model of hydrophobic enclosure for protein-ligand complexes.
49:6177–6196.
26. Tanaka K, Nakamoto M, Tsunomori M, Tanaka T (1987) Raman-spectra of the adducts
J Med Chem
2−
3−
of reduced species of ½Fe4S4ðSPhÞ4ꢀ and ½Mo2Fe6S8ðSPhÞ9ꢀ with acetylene. Chem
Lett 613–616.
27. Gemperle C, Schweiger
A (1991) Pulsed electron nuclear double-resonance
methodology. Chem Rev 91:1481–1505.
28. Perera SA, Salemi LM, Bartlett RJ (1996) Hyperfine coupling constants of organic
radicals. J Chem Phys 106:4061.
13. Gräwert T, et al. (2009) Structure of active IspH enzyme from Escherichia coli provides
mechanistic insights into substrate reduction. Angew Chem Int Ed Engl 48:5756–5759.
14. Gräwert T, et al. (2004) IspH protein of Escherichia coli: Studies on iron-sulfur cluster
implementation and catalysis. J Am Chem Soc 126:12847–12855.
15. Wolff M, et al. (2003) Isoprenoid biosynthesis via the methylerythritol phosphate
pathway: The (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH)
from Escherichia coli is a [4Fe-4S] protein. FEBS Lett 541:115–120.
16. Seemann M, et al. (2009) Isoprenoid Biosynthesis via the MEP Pathway: In vivo Möss-
29. Schunn RA, Fritchie CJ, Jr, Prewitt CT (1966) Syntheses of some cyclopentadienyl tran-
sition metal sulfides and the crystal structure of ðC5H5FeSÞ4. Inorg Chem 5:892–899.
30. McMillan RS, Renaud J, Reynolds JG, Holm RH (1979) Biologically related iron-sulfur
clusters as reaction centers. Reduction of acetylene to ethylene in systems based on
½Fe4S4ðSRÞ4ꢀ3−. J Inorg Biochem 11:213–227.
2−
31. Itoh T, Nagano T, Hirobe M (1980) ½Fe4S4ðSRÞ4ꢀ Catalytic reduction of diphenylace-
tylene to cis-stilbene in the presence of NaBH4. Tetrahedron Lett 21:1343–1346.
32. Millen RP, de Faria DLA, Temperini MLA (2001) Vibrational spectra of 2-ethynylpyridine
and its silver salt. Vib Spectrosc 27:89–96.
2þ
bauer spectroscopy identifies a ½4Fe-4Sꢀ center with unusual coordination sphere in
the LytB protein. J Am Chem Soc 131:13184–13185.
Wang et al.
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