Eric Oldfield

Jikun Li

Ke Wang

Tatyana I Smirnova

Weixue Wang

CiteSeerX

Pyridine Inhibitor Binding to the 4Fe-4S Protein A. aeolicus IspH 
(LytB):  A HYSCORE Investigation 
Weixue Wang,† Jikun Li,† Ke Wang,‡ Tatyana I. Smirnova,§ and Eric Oldfield*†‡ 
Center for Biophysics and Computational Biology, 607 South Mathews Avenue, and Department of Chemistry, 600 South Mathews 
Avenue, University of Illinois at Urbana–Champaign, Urbana, IL 61801, and Department of Chemistry, North Carolina State Uni-
versity, 2620 Yarbrough Drive, Raleigh, NC 27625 
Fax:  (+1) 217-244-0997 Email:  eo@chad.scs.uiuc.edu 
KEYWORDS Isoprenoid/non-mevalonate/IspH (LytB)/inhibitors/HYSCORE 
IspH is a 4Fe-4S protein that carries out an essential reduc-
tion step in isoprenoid biosynthesis. Using hyperfine sub-
level correlation (HYSCORE) spectroscopy, we show that 
pyridine inhibitors of IspH directly bind to the unique 4th 
Fe in the 4Fe-4S cluster, opening up new routes to inhibi-
tor design, of interest in the context of both anti-bacterial as 
well as anti-malarial drug discovery. 
Isoprenoid biosynthesis is an important target for drug dis-
covery.1  In most pathogenic bacteria as well as in malaria 
parasites, the early stages in isoprenoid biosynthesis are car-
ried out by the methylerythritol phosphate pathway.2  This 
pathway is essential for producing the isoprenoids used in e.g. 
cell wall biosynthesis in bacteria, and in quinone formation 
and, since it is not present in humans, the enzymes involved 
are important targets for the development of new antibiotics.3  
The last two enzymes, IspG and IspH, are unusual 4Fe-4S-
containing proteins that carry out 2H+/2e- reductions of the 
substrates MEcPP (2-C-methyl-D-erythritol-2,4-cyclo 
diphosphate, 1)4-6 and HMBPP (E-1-hydroxy-2-methyl-but-2-
enyl-4-diphosphate, 2) to form the C5 isoprenoids IPP 
(isopentenyl diphosphate, 3) and DMAPP (dimethylally l 
diphosphate, 4) in an ~1:5 ratio,7, 8 as shown in Scheme 1: 
Scheme 1. Reactions catalyzed by the proteins IspG (GcpE) and 
IspH (LytB). 
O
O P O
P
O
OH
OHOH
Me
OH
O
1, MEcPP 2, HMBPP
HO
OPP
IspG (GcpE)
2H+/2e
IspH (LytB)
2H+/2e OPP OPP
+
3, IPP 4, DMAPP
 
In recent work we proposed that both the IspG (EC 
1.17.7.1, HMBPP synthase, also known as GcpE)9 as well as 
the IspH (EC 1.17.1.2, HMBPP reductase, also known as LytB) 
catalyzed reactions involve formation of organometallic spe-
cies (i.e. containing Fe-C bonds).10  Support for the interme-
diacy of organometallic species in catalysis comes indirectly 
from electron paramagnetic resonance (EPR) and electron nu-
clear double resonance (ENDOR) spectroscopy as well as 
mechanistic considerations,10 and more directly, from the ob-
servation that the Fe-C distances (2.6–2.7 Å) between the api-
cal iron atom in the 4Fe-4S cluster and the allylic species seen 
crystallographically in IspH are even shorter than the ones 
observed for bound HMBPP,11 and are far shorter than the 3.6-
3.7 Å sum of the Fe, C van der Waals radii.12  We also found 
that alkynes could be quite potent inhibitors of both IspG and 
IspH, and that EPR and ENDOR spectra indicated that these 
alkynes bound at or very close to the unique 4th Fe in the re-
duced 4Fe-4S cluster.. The ability to inhibit IspG or IspH is of 
interest in the context of the development of anti-infectives, 
and the ability of a given compound to inhibit both enzymes is 
of even more interest since, in principle, it will lead to a de-
crease in drug resistance since both enzymes would be re-
quired to mutate.   
In addition to alkyne inhibitors, we discovered a second 
class of IspH inhibitors, pyridine diphosphates,13 but how 
these bound to the protein was not clear.  Here, we report the 
results of an X-band hyperfine sublevel correlation 
(HYSCORE) spectroscopic as well as a quantum chemical 
investigation, which helps clarify how these inhibitors func-
tion. 
Scheme 2. Aromatic species investigated. 
N
OPP
N
OPP
N
OPP
N
OPP
NCl
OPP
7, BPH-293 8, BPH-1030
9, BPH-1027 10, BPH-1026 11, BPH-1029
N N NH2
5, pyridine 6, 2-aminopyridine
 
 
We first investigated a series of pyridine ligands, 5-11, 
Scheme 2, binding to wild-type IspH from Aquifex aeolicus.  
The continuous-wave EPR spectrum of IspH + pyridine (5) is 
the same as that of the unliganded protein (i.e. in the absence of 
pyridine, Figure S1a), and there is no evidence for any sizeable 
pyridine-14N hyperfine interaction in the HYSCORE spectrum 
(Figure S1b), indicating only very weak binding affinity to 
IspH.  The same results are obtained with the more basic (pKa = 
6.8 vs. 5.2) species 2-aminopyridine (6, Figure S1c).  However, 
on addition of the inhibitor BPH-293 (7, IC50 = 38 µM), the 
EPR spectrum changes13 (Figure S1a) and new signals attrib-
utable to 14N single and double quantum transitions appear in 
the (+,-) quadrant of the HYSCORE spectrum (Figure 1a).  The 
14N hyperfine interaction is quite large, with the hyperfine 
coupling constant being ~8 MHz.  Reconstituted IspH (Figure 
1a) and anaerobically purified IspH (Figure S2) both give the 
same results.  The ortho and para-pyridyl analogs of 7 (com-
pounds 8, IC50 = 1.2 mM and 9, IC50 = 149 µM) show no evi-
dence of any sizeable pyridine-14N hyperfine interaction in 
their HYSCORE spectra (Figure S1d, e), due presumably to 
their inability to bind to the 4th Fe, for “steric” reasons.  
Moreover, chlorine substitution of 7 (compound 10) results in 
loss of all activity (IC50 > 3 mM), due presumably to loss in 
donor-ability of the pyridine nitrogen (the computed pKa val-
ues of the pyridine fragments in 7 and 10 are 4.7 and 0.7, re-
spectively), consistent with the absence of a pyridine-14N 
HYSCORE signal (Figure S1f).  Addition of one CH2 group to 
the side-chain of 7 results in a better inhibitor (11, IC50 = 9.1 
µM), although there is no significant difference between the 
HYSCORE spectra of 7 (Figure 1a) and 11 (Figure S1g), indi-
 
cating that differences in enzyme inhibition are due to differ-
ences in the alkyl diphosphate fragment binding in the active 
site, rather than differences in Fe-pyridine interactions.   
 
 
Figure 1. HYSCORE spectra of A. aeolicus IspH + pyridine inhibitor 
7.  (a) HYSCORE spectra of unlabeled A. aeolicus IspH + 7.  The 
inset shows the CW-EPR spectrum, and the arrow indicates the 
magnetic field position for collecting the HYSCORE data.  (b) 
HYSCORE spectra of 15N-labeled A. aeolicus IspH + 7.  Micro-
wave frequency = 9.66 GHz (a), 9.68 GHz (b); magnetic field was 
set at g2 = 1.921; τ = 136ns. 
These results do not, however, prove that the 14N 
HYSCORE signals in the (+,-) quadrant (Figure 1a and Figure 
S1g) arise directly from the inhibitors 7 and 11 since, in prin-
ciple, inhibitor binding might result in a protein conforma-
tional change and binding of a protein ligand to Fe, e.g. the 
nearby His 42 or 124, which form part of the active site.14 To 
investigate this possibility, we prepared a sample using uni-
formly 15N-labeled IspH and inhibitor 7.  As can be seen in 
Figure 1b, the 14N signals centered at ~3.6 MHz seen in Figure 
1 are no longer present, and are replaced by a signal centered at 
1.5 MHz, the 15N Larmor frequency. Moreover, the 14N signals 
in the (+,-) quadrant are essentially identical to those seen in 
samples prepared using unlabeled IspH (Figure 1a). This 
strongly suggests that the signals centered at ~3.6 MHz arise 
from protein nitrogens near the 4Fe-4S cluster, while the 14N 
signals in the (+,-) quadrant arise from the bound inhibitor 7, 
rather than from any protein residues.  
To begin to better understand the interaction between the 
pyridine inhibitor 7 and IspH, we next simulated the 
HYSCORE spectra of IspH + 7 taken at three different magnetic 
field strengths (Supporting Information, Figures S3a, b and c) 
using the EasySpin program15 (Figures S3d, e and f), finding 
aiso(14N) = 7.4 MHz, Aii(14N) = [6.2 7.6 8.4] MHz for the hyper-
fine interaction, and e2qQ/h = 3.0 MHz for the nuclear quad-
rupole coupling constant.  
This large aiso(14N) is similar to, or even larger than, that of a 
number of systems in which nitrogens directly bind to Fe cen-
ters.  For example, in met-myoglobin the porphyrin nitrogens 
have aiso = 8.11 MHz and 7.8 MHz, and the histidine Nε has 
aiso = 9.28 MHz.16  In a model heme complex, FeTPP(4-MeIm)2 
(TPP, tetraphenylporphyrin; 4-MeIm, 4-methyl imidazole), the 
aiso of  the porphyrin nitrogens is 5.1 MHz, while that of the 
coordinated 4-MeIm is 5.7 MHz.17  In Rieske-type 2Fe-2S pro-
teins, aiso(14N) of the coordinated His nitrogens are approxi-
mately 5 MHz,18 and in the case of the 4Fe-4S enzyme MoaA 
(which also has an unique 4th iron), N1 of the substrate 
guanosine 5’-triphosphate binds to the 4th iron and has aiso 
(14N) =  3.6 MHz.19 On average, these results give an aiso(14N) ~ 
6 MHz for systems containing Fe-N bonds, suggesting that the 
IspH + 7 complex also contains an Fe-N bond. 
The large 14N hyperfine interaction seen in the IspH + 7 
complex might also, at least in principle, indicate that the 
pyridine fragment is just close-by to the reduced 4Fe-4S clus-
ter, without directly bonding to the 4th iron. For example, the 
pyridine group might be protonated and interact with e.g. the 
E126 CO2- group that is close to the cluster; or it could be 
close-by, but deprotonated. Fortunately, determination of the 
14N nuclear quadrupole coupling constant (e2qQ/h) enables an 
answer to this question, since protonated, neutral and metal-
coordinated pyridine ligands have very different e2qQ/h val-
ues.20  
For pyridine itself, the e2qQ/h is 4.6 MHz, but in species in 
which there is a formal +1 charge on N, such as the pyridinium 
ion (12), pyridine-N-oxide (13) and N-methyl pyridinium (14), 
e2qQ/h values of approximately 1 MHz are observed experi-
mentally.20 In the case of pyridine bonded to Fe in 
Fe(CO)4(pyr), 15, e2qQ/h is in between these extreme values 
(e2qQ/h ~ 2.4 MHz), and for Mo(pyr)2(CO)4 as well as 
Cr(CO)4(2,2’-bipyridyl), e2qQ/h ~3.1 MHz.  So, when pyr is 
bonded to Cr, Mo or Fe, the e2qQ/h decreases from the 4.6 MHz 
seen in free pyridine to ~ 2.4 – 3.1 MHz, due to metal-ligand 
bonding, close to the 3.0 MHz value we find from the 14N 
HYSCORE results.   
Scheme 3. Structures discussed in the text.  
Fe
S
S
Fe
S
S
Cys
Cys
N
N
HN
HN
His
His
Fe
S
S
Fe
S
S
Cys
Cys
NHN
His
S
Cys
18 19
N
16
N
H
N
125
Fe
N
O
N
Me
N
14
Fe
13 15
OC CO
OC CO
HN
N3
17
 
To see to what extent these e2qQ/h values might be repro-
duced computationally, we used the Gaussian-09 (Revision 
A.01) program.21  Results are given in Table S1 and are shown 
graphically in Figure 2 and Figure S4.  Clearly, there is a good 
correlation (Figure 2a) between theory and experiment (R2 = 
0.965; slope = 0.963) for a series of model systems, and when 
using [Fe4S4(SMe)3(pyr)]2- (16, Figure 2b) as a model, we find 
e2qQ/h = 2.3 MHz for the pyridine 14N, in quite good accord 
with experiment. 
This large decrease in e2qQ/h, from the 4.6 MHz value found 
for free pyridine to the 2.4 – 3.1 MHz values observed in model 
systems and the IspH + 7 complex, is also seen in proteins in 
which imidazole (histidine) ligands bind to Fe.  For example, 
for imidazole (17) the N3 (deprotonated) e2qQ/h is 4.032 
MHz,22 in good accord with the 3.894 MHz computed using 
DFT.  The e2qQ/h values for solid imidazole and solid his-
tidine are both smaller and essentially identical (3.27 MHz, 
Im; 3.36 MHz, His)23 – due presumably to very strong hydro-
 
gen bonding in the solid state.  But when bound to Fe in met-
alloproteins, e2qQ/h decreases considerably from the 4 MHz 
gas phase value (for imidazole).   
For example, in myoglobins, e2qQ/h ranges from 2.2 – 2.5 
MHz for the directly bonded imidazole nitrogens;24-26 in the 
(Cys)3 (His)1 – coordinated [2Fe-2S] cluster in the human 
mito-NEET protein (18), e2qQ/h = (-) 2.47 MHz;27 and in sev-
eral (Cys)2(His)2-coordinated [2Fe-2S] Rieske-type protein 
(19), e2qQ/h values have been reported to be in the range ~2.2-
2.9 MHz.28-30 Clearly then, the 14N nuclear quadrupole cou-
pling constant decreases from ~4 MHz for the free (gas phase) 
imidazole to ~2.5 MHz when bound to Fe, similar to the de-
crease in e2qQ/h we find with pyr bound to Fe in the 4Fe-4S 
cluster of IspH. 
 
Figure 2. (a) Graph showing correlation between experimental and 
computed e2qQ/h values for a series of model systems; (b) Model 
used in quantum mechanical calculation of the 14N e2qQ/h value for 
IspH+7 complex. 
These results all support the idea that the IspH pyridine in-
hibitors bind to IspH via a Lewis acid/base (Fe4S4 clus-
ter/ligand) mechanism with the donor orbital occupancy (σ) 
decreasing from 2 (pyridine) to ~1.73,20 and that the hyperfine 
coupling seen experimentally is due to this η1-bonding, rather 
than being due to a neutral pyr, or pyr-H+ ligand just being 
close to the 4Fe-4S cluster. This, in turn, suggests that 
stronger Lewis bases (such as imidazole-containing ligands) 
may be more potent IspH inhibitors.  These results also sup-
port the idea that other inhibitors, such as alkynes,10, 13 as well 
as possible reaction intermediates (η3-allyls),10, 11 also act as 
Lewis bases, when interacting with the 4Fe-4S cluster in 
IspH. 
Overall, these results are of interest for several reasons.  
First, we find evidence for an 14N HYSCORE signal when the 
pyridine inhibitor 7 binds to IspH. Based on isotopic label-
ing, this signal is assigned to the pyridine 14N.  Second, the 
experimental e2qQ/h (from simulations of field-dependent 
HYSCORE) is 3 MHz.  This is between the e2qQ/h = 4.6 MHz 
found for pyridine itself and e2qQ/h values of ~1 MHz found in 
pyridinium salts and pyridine-N-oxide,20 and is in fact within 
the 2.4 – 3.1 MHz range of values found for pyridines bound to 
Cr, Mo and Fe carbonyls.23 So, while the ligand may initially 
bind as the cationic species (to e.g. E126), the η1-complex is 
the more stable species.  Third, we report the results of DFT 
calculations of the 14N nuclear quadrupole coupling constant 
(e2qQ/h) in pyridine-containing metal systems finding a good 
correlation between theory and experiment (R2 = 0.965, slope = 
0.963, Figure 2) , in addition to predicting a 2.3 MHz e2qQ/h 
value for a [Fe4S4(SMe)3(pyr)]2- model cluster, in quite good 
accord with experiment (given that the protein was excluded 
from the calculation, and the crystallographic structure of the 
4Fe-4S/pyridine protein-containing complex is not yet 
known). When all published experimental results on pyridine 
and imidazole-containing systems are considered, there is a 
~35 – 40% decrease in the 14N e2qQ/h on metal binding, the 
same as that found in the IspH + 7 system. This again supports 
formation of an η1-complex between IspH and 7, an observation 
of interest in the context of the design of other inhibitors, of 
interest as anti-infective drug leads.  
ACKNOWLEDGMENT We thank Hassan Jomaa, and Jo-
chen Wiesner for providing their IspH plasmid.  This work 
was supported by the United States Public Health Service 
(NIH grants AI074233 and GM065307).  W.W. was sup-
ported by a Predoctoral Fellowship from the American Heart 
Association, Midwest Affiliate (Award 10PRE4430022). EPR 
instrumentation used in this work was supported by NIH 
grants S10RR023614 and S10RR025438, NSF CHE-
0840501, and NCBC 2009-IDG-1015.  Computational re-
search was supported by the National Science Foundation 
through Teragrid resources provided by NCSA under grant 
TG-CHE100060. 
SUPPORTING INFORMATION Details on protein pro-
duction and purification, HYSCORE sample preparation, Sup-
porting Information figures, and full citation for reference 21 
are reported in the Supporting Information available free of 
charge via the Internet at http://pubs.acs.org. 
 
† Center for Biophysics and Computational Biology, Univer-
sity of Illinois at Urbana–Champaign, 607 South Mathews 
Avenue, Urbana, IL 61801 (USA) 
‡ Department of Chemistry, University of Illinois at Urbana–
Champaign, 600 South Mathews Avenue, Urbana, IL 61801 
(USA) 
§ Department of Chemistry, North Carolina State University, 
2620 Yarbrough Drive, Raleigh, NC 27625 (USA) 
 
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Pyridine Inhibitor Binding to the 4Fe-4S Protein A. aeolicus IspH (LytB): A HYSCORE Investigation

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Pyridine Inhibitor Binding to the 4Fe-4S Protein A. aeolicus IspH (LytB): A HYSCORE Investigation

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