Computational methods and crystallography have been coupled to study the structure-function relationships of two heme enzymes: cytochrome c peroxidase (CCP) and nitric oxide synthase (NOS). For NOS, computational approaches were used to resolve ambiguities in the orientation of an inhibitor, 7-nitroindazole, bound in the active site. Calculations of interaction energies between the inhibitor and protein supported one orientation over the other. For CCP, site-directed mutagenesis, crystallography, and computation have helped explain why CCP uniquely stabilizes a tryptophan cation radical during catalysis, unlike other peroxidases.

1. Coupling Crystallography and
Computational Biochemistry in
Understanding Heme Enzyme
Structure and Function
THOMAS L. POULOS, TIFFANY BARROWS, B. BHASKAR,
CHRISTOPHER A. BONAGURA,∗
HUIYING LI
Departments of Biochemistry and Molecular Biology and Physiology and Biophysics,
and the Program in Macromolecular Structure, University of California at Irvine,
Irvine, California 92697-3900
Received 14 September 2001; accepted 16 October 2001
DOI 10.1002/qua.10113
ABSTRACT: Computational methods and crystallography have been coupled to study
structure–function relationships in cytochrome c peroxidase (CCP) and nitric oxide
synthase (NOS). NOS is an important drug target and the structural details of
NOS–inhibitor interactions are essential if structure-based drug design is to be of any
use in the development of therapeutic agents targeted to NOS. The structure of
7-nitroindazole, a potent NOS inhibitor, complexed to one NOS isoform left some
ambiguity in the precise orientation of the inhibitor. Various computational approaches
were used to resolve this ambiguity as well as revealing the energetic basis for tight
binding. With CCP site directed mutagenesis, crystallography and various computational
tools have been used to understand why CCP is unique among the peroxidases in its
ability to stabilize a tryptophan cation radical during catalysis. © 2002 Wiley
Periodicals, Inc. Int J Quantum Chem 88: 211–219, 2002
Key words: peroxidases; nitric oxide synthase; free radicals; computation; energetics;
inhibitor binding; nitroindazoles
Correspondence to: T. L. Poulos; e-mail: poulos@uci.edu.
∗Current address: Plexxikon, Inc., 91 Bolivar St., Berkeley,
CA 94710.
Contract grant sponsor: National Institutes of Health.
International Journal of Quantum Chemistry, Vol. 88, 211–219 (2002)
© 2002 Wiley Periodicals, Inc.

2. POULOS ET AL.
Introduction
Computational approaches for studying struc-
ture–function relationships in proteins and
enzymes have become much easier in recent years
owing primarily to hardware/software advances.
Especially important are user-friendly interfaces
that have opened up the use of computational tools
to the experimentalist. Although our work on heme
enzyme structure and function is based on crystal-
lographic and engineering studies, we have increas-
ingly used computational methods to help guide
experimental design, most of which was inspired by
Gilda Loew. The following article summarizes some
of our work in this area on two enzyme systems: ni-
tric oxide synthase and cytochrome c peroxidase.
NOS catalyzes the five-electron oxidation of
L-Arg to NO and L-citrulline (Scheme 1). The re-
action proceeds in two steps. The first, monooxy-
genation of one L-Arg guanidinium nitrogen to
give hydroxy-L-Arg is a typical P450-type oxida-
tion. The second step, oxidation of hydroxy-L-Arg
to L-citrulline and NO, is not P450-like since only
NADPH-derived electron is required. Both steps
require tetrahydrobiopterin or BH4. As in P450
reducing equivalents are delivered to the heme
domain via a FAD/FMN reductase that transfers
NADPH-derived reducing equivalents to the heme.
However, unlike most P450’s where the P450 and
FAD/FMN reductase are separate polypeptides, in
NOS the flavo-reductase is fused to the C-terminal
end of the heme domain, giving a large protein
ranging in size from 130,000 to 160,000 daltons.
There are three main human NOS isoforms: en-
dothelial NOS (eNOS) regulates vascular tone; neu-
ronal NOS (nNOS) is involved with neuronal trans-
mission; and inducible NOS (iNOS) produces NO as
a cytotoxic agent during the immune response. All
three NOS isoforms have exactly the same architec-
ture. The flavin domain exhibits striking similarities
in both function and sequence to microsomal cy-
tochrome P450 reductase [1] and we can anticipate
that the NOS reductase domain will have a very
similar structure to P450 reductase. This domain is
responsible for funneling reducing equivalents from
NADPH to the site of arginine oxidation. In sharp
contrast, the catalytic heme domain bears no se-
quence homology to P450.
Owing to the role that NO plays in physiological
and pathological states, isoform-selective inhibition
of NOS is important. For example, in septic
shock NOS inhibitors can restore vascular tone
and blood pressure [2]. Blocking NO production
by nNOS limits ischemia-elicited infarct size
in animal models [3]. Moreover, NO has been
found to stimulate breast cancer tumor growth [4]
while NOS inhibitors have been shown to block
tumor growth [5]. The culprit appears to be
iNOS-generated NO. Not surprisingly, a number
of pharmaceutical companies have active research
programs on NOS and NO related pathological
conditions. Currently used inhibitors of NOS are
arginine analogues and hence bind to the heme
domain. It appears that efforts to design isoform
specific NOS inhibitors will be directed at this site
although more recent studies have been directed
toward blocking assembly of the active iNOS
dimer [6]. That the heme domain is the target for
isoform-selective inhibitors is one reason we fo-
cused our initial efforts on the heme domains alone.
CCP is a much simpler enzyme. Like other heme
peroxidases, CCP consists of a single polypeptide of
MW ≈ 30,000 and one noncovalently bound heme.
CCP catalyzes the peroxide-dependent oxidation of
ferrocyt.c in the following multistep reaction:
Step 1:
Fe3+
Trp
Resting State
+ H2O2 → Fe4+
–OTrp·
Compound I
+ H2O
Step 2:
Fe4+
=OTrp·
Compound I
+ cyt. c Fe2+
→ Fe4+
–OTrp
Compound II
+ cyt. c Fe3+
Step 3:
Fe4+
–OTrp
Compound II
+ cyt. c Fe2+
→ Fe3+
Trp
Resting State
+ cyt. c Fe3+
+ H2O
SCHEME 1.
212 VOL. 88, NO. 1

3. HEME ENZYME STRUCTURE AND FUNCTION
FIGURE 1. The CCP active site.
In step 1 CCP is oxidized by H2O2 to give com-
pound I. In compound I one electron has been
removed from the iron to give Fe(IV) and one
from Trp191 whose location in the active site is
shown in Figure 1. In step 2, CCP compound I
forms a complex with ferrocyt c which delivers one
electron to the Trp191 radical. In step 3 a second
ferrocyt c molecule reduces compound II back to
the resting state. This mechanism is characteristic
of most heme peroxidase with two important ex-
ceptions. First, CCP uses ferrocyt c as a reducing
substrate while most other well studied peroxi-
dases utilize small organic molecules. Second, CCP
forms a stable cationic Trp radical [7, 8] while other
peroxidases form a cationic heme radical [9]. Both
differences have been the subject of extensive inves-
tigations.
NOS Inhibition
To date crystallographic studies on NOS have fo-
cused on the heme domain alone [10 – 13] (Fig. 2).
Most well known NOS inhibitors are L-Arg-like
compounds and form H-bonds with Glu363 simi-
lar to the way L-Arg interacts with Glu363 (Fig. 3).
There are, however, other NOS inhibitors that have
no structural homology to L-Arg. Of these we have
investigated 7-nitroindazole or 7NI (Fig. 4) most
thoroughly. 7NI and the 3-bromo derivative, 7NIBr,
FIGURE 2. The dimeric heme domain structure of
eNOS. Each subunit consists of 481 residues. A Zn ion is
loicated at the bottom of the dimer interface where it is
tetrahedrally coordinated by pairs of symmetry related
Cys residues. Note the location of the pterin cofactor
(BH4) near the heme.
bind to the various NOS isoforms with a submicro-
molar dissociation constant [14 – 18]. To understand
why 7NI is such a potent inhibitor, we determined
the crystal structure of 7NI complexed to the heme
domain of eNOS (in press). The electron density
map is very clear, allowing for an unambiguous lo-
cation of the inhibitor, but the precise orientation
proved problematic (Fig. 4). We suspected that the
orientation shown designated as orientation A in
FIGURE 3. The active site region of nNOS.
The substrate, L-Arg, is held in place by a series of
H-bonds which are conserved in all NOS isoforms. Note
that the pterin cofactor and L-Arg H-bond with the same
heme propionate. These interactions explain the
interdependence of pterin and L-Arg binding.
INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY 213

4. POULOS ET AL.
FIGURE 4. The 2.1 Å 2Fo-Fc omit electron density map contoured at 1σ of 7NI bound to the eNOS active site.
Although the electron density is very clear, at this resolution it is not possible to unambiguously choose between the
two orientations.
Figure 4 should be favored owing to H-bonds that
can form between the nitro group and nearby pro-
tein atoms (Fig. 5). To choose between the two pos-
sibilities, we turned to some relatively straightfor-
ward computational approaches. The first of these
involved simple energetic calculations. Which ori-
entation of 7NI gives the lowest energy? The eNOS
model was prepared by first adding H-atoms us-
ing the MSI INSIGHT II software. We used the cvff
forcefield provided by MSI but for the heme and
thilolate ligand, we used those parameters devel-
oped by Danni Harris and Gilda Loew. Charges
on 7NI were obtained from a HF 6-31G calculation
using Gaussian 98. The models were energy min-
imized followed by an analysis of the interaction
energies between the 7NI inhibitor and surrounding
protein and heme groups. Since the heme domain
is a dimer, the energies can be analyzed in both
active sites and the values reported in Table I are
the average of both subunits. There is very little
difference in van der Waals energy most of which
derives from stacking between the indazole and
heme rings which is very much the same in both
orientations. However, there is a substantial differ-
FIGURE 5. 7NIBr bound to the eNOS active site. The heme and side chains of E63 from the eNOS-substrate complex
(dark bonds) are superimposed on the 7NIBr structure. Note that E363 must adopt a new conformation in order to make
form for the inhibitor. Motion of E363 requires that the heme propionate also move away from the pterin pocket.
As a result pterin–heme interactions are weakened which allows 7NIBr to bind in the pterin pocket.
214 VOL. 88, NO. 1

5. HEME ENZYME STRUCTURE AND FUNCTION
TABLE I
Comparison of protein-inhibitor energetics in the two
possible orientations of 7NI.
Energies in kcal/mol
orientation of 7-Nl Van der Waals Coloumbic
A −47.12 −12.02
B −47.30 −8.25
ence in Coloumbic interactions. We attribute this to
H-bonds that can form between the nitro group and
nearby protein atoms in orientation A (Fig. 5).
Biochemists are more interested in free energies
and equilibrium constants than absolute binding
energies. Since the inhibitor is transferring from sol-
vent to the enzyme active site, a critical energetic
component is the relative solvation energy of the
inhibitor. To analyze this problem we turned to
the MOLARIS software using the protein dipoles
Langevin dipoles (PDLD) method developed by
Arieh Warshel and colleagues [19]. In this method
the protein is modeled in three distinct regions.
Region 1 contains the atoms whose solvation en-
ergy we wish to know which in this case is all
the 7NI atoms. Region 2 contains those protein
atoms close enough to the inhibitor to significantly
influence the solvation energy. Region 3 defines
a sphere of Langevin dipoles that surround re-
gions 1 and 2. Region 4 surrounds region 3 and
is assigned the dielectric of the bulk solvent. The
main idea behind the PDLD method is that the es-
sential physics of the surrounding solvent can be
modeled as dipoles on a spherical grid rather than
explicit solvent molecules. This saves considerable
computational time and allows an equilibrium state
to be reached in a relatively short computational
time frame. The Gsolvation is computed in both bulk
solvent and in the protein active site and G =
Gprotein − Gsolvent is computed. We carried out
this calculation every ps over a 25 ps molecular dy-
namics simulation, giving 25 G values. This is
important since charge–charge interactions are quite
sensitive to the exact positioning of the charged
groups and will vary. Therefore, allowing the sys-
tem to move in a molecular dynamics simulation
provides a clearer picture on the variability of G
as a function of molecular configuration and also
allows the mean G and standard deviation to
be computed. For orientations A and B the G
values are 0.02 ± 0.19 and 0.81 ± 0.37 kcal/mol, re-
spectively, indicating that orientation A (Fig. 4) is
slightly favored which agrees with the straight en-
ergetic analysis. Since orientation A is favored most
likely due to the H-bonding possibilities of the nitro
group, we next repeated the calculation in orien-
tation A but without the nitro group. In this case
G = 1.4 ± 0.2 kcal/mol which illustrates that the
nitro is important.
We next solved the structure of 7NI with a
bromine atom attached to the 3 position. Since Br is
electron dense relative to C, N, and O it should
be possible to unambiguously determine the orien-
tation of the inhibitor. As shown in Figure 6 the
Br atom is situated in a large lobe of electron density
enabling an unambiguous determination that 7NIBr
adopts orientation A. Another feature of the 7NIBr
complex is that the inhibitor binds in the pterin
pocket as well as the active site pocket. The 7NI
inhibitors also are the only NOS inhibitors that we
have studied so far that lead to any substantial con-
formational change. In Figure 5 note that Glu363
adopts different conformations. In the L-Arg com-
plex, Glu363 is oriented “in” toward L-Arg where it
forms H-bonds with the L-Arg guanidinium group.
However, when 7NI or 7NIBr binds, the Glu363 side
chain swings out toward the heme propionate as
it must in order to make room for the inhibitor.
This leads to a steric/electrostatic clash between
Glu363 and the heme propionate so the propionate
group also must move. This same heme propionate
H-bonds with the pterin cofactor (Fig. 3). Therefore,
we suspect one reason 7NIBr can bind in the pterin
pocket is due in part to a weakening of the pterin–
heme interactions owing to the structural changes
that must take place when the inhibitor binds in the
active site.
CCP
An unusual feature of the CCP reaction is that
in compound I Trp191 forms a stable cationic rad-
ical while other heme peroxidases form a por-
phyrin cation radical. Initially the reason for this dif-
ference looked straightforward. Those peroxidases
that form a heme radical in compound I have a Phe
in place of Trp191 (Fig. 1) and since Trp is easier to
oxidize than Phe, it appeared that simple differences
in the redox potential of the benzene and indole
rings could explain why CCP forms a Trp radical.
However, another peroxidase, ascrobate peroxidase
or APX, has a Trp located exactly in the same po-
sition as Trp191 in CCP; yet APX forms a heme
INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY 215

6. POULOS ET AL.
FIGURE 6. The 1.65 Å 2Fo-Fc electron density map of 7NIBr bound in the eNOS active site contoured at 1σ
(thin lines) and 5σ (thick lines).
radical as in other peroxidases [20]. This was puz-
zling until it was noted that APX and CCP have
an important structural difference that could con-
tribute to the stability of a Trp radical. APX has
a K+
ion located ≈8 Å away from the Trp (Fig. 7)
while CCP has a water at this position [21]. We pos-
tulated that the K+
ion destabilizes the + charge on
a cationic Trp radical in APX to the extent that the
heme is preferrentially oxidized. Therefore, the elec-
trostatic environment surrounding Trp191 in CCP
favors formation of a cationic Trp radical. This view
is consistent with “cavity” mutants where Trp191
is replaced by a smaller amino acid which favors
the binding of positively charged molecules into the
engineered cavity [22, 23]. To further test this hy-
pothesis, the APX K+
site has been engineered into
CCP and the resulting mutants analyzed by deter-
mining crystal structures and measuring activities
and EPR properties [24 – 26]. The structures show
that the engineered K+
site is basically the same as
the authentic site in APX (Fig. 8). The EPR signal
of the Trp191 cation radical is a distinctive feature
in CCP Compound I and has proven to be a valu-
able tool for gauging the stability of the Trp radical.
The EPR signal of the K+
CCP mutants is substan-
tially weakened and short lived, showing that the
Trp191 radical is much less stable in the H+
mutant.
However, the Trp radical still forms which means
that other structural features help to stabilize the
Trp191 radical. We therefore have begun to mutate
other residues close to Trp191 to the correspond-
ing residues in APX. The two most recent targets
are Met230 and Met231 which are Leu and Gln
in APX (Fig. 1). The EPR signal in this mutant
FIGURE 7. The CCP molecule showing the location of
the engineered K+ site. The K+ ion and Trp191 are
≈8 Å apart.
216 VOL. 88, NO. 1

7. HEME ENZYME STRUCTURE AND FUNCTION
FIGURE 8. A comparison of the K+ site in APX with the engineered K+ site in CCP.
is even weaker, suggesting that the electronegative
sulfur atoms of Met230 and Met231 contribute to
stabilization of the Trp191 cationic radical.
It would be extremely useful to have a computa-
tional method to predict which mutants will either
stabilize or destabilize the radical especially if elec-
trostatic stabilization is the primary factor involved.
Various theroretical approaches have been taken to
understand the Trp radical in CCP. While most ex-
perimental approaches favor a cationic Trp191 rad-
ical, an ab initio minimal basis set molecular orbital
coupled with electrostatic calculations favors a neu-
tral Trp191 radical where the indole ring proton is
transferred to Asp235 (Fig. 1) [27]. However, a den-
sity functional (DFT-B3LYP) calculation indicates
a cationic Trp191 radical [28] while another ab ini-
tio set of calculations on 3-methylindole also favors
a cationic Trp191 radical [29]. To more fully under-
stand how the protein contributes to stabilization of
the Trp191 cationic radical, the PDLD method has
been employed [30]. These calculations indicate that
the K+
contributes only partially to destabilization
of the Trp cationic radical. We, too, have used the
PDLD method as implemented in MOLARIS to cal-
culate the relative solvation energy of the Trp191
radical in the protein compared to water just as we
did with the NOS inhibitor. These calculations were
carried out on wild type and mutant crystal struc-
tures. Since the K+
ion site is net neutral owing
to interactions with the engineered Asp199 (Fig. 8),
these two groups were made net neutral when test-
ing the effects of the K+
site. The results of the
MOLARIS calculations shown in Table II are very
similar to an earlier study using very similar meth-
TABLE II
Solvation free energies and relative EPR signal of the Trp191 radical in wild-type CCP, APX, and two
CCP mutants.
Relative
Protein Charge on K+ Charge on Asp239 G (kcal/mol) EPR signal
Wild type — — −5.18 + 1.13 1.0
APX +1 −1 −2.93 ± 0.8
APX 0 0 −3.39 ± 0.62
Mutant 1 +1 −1 −5.03 ± 0.89 0.24
Mutant 1 0 0 −6.29 ± 0.88
Mutant 2 +1 −1 −3.71 ± 0.92 0.05
In both mutants 1 and 2, the APX cation site has been engineered into CCP. Mutant 2 is the same as mutant 1 except Met230
and Met231 have been changed to Leu and Gln, respectively.
INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY 217

8. POULOS ET AL.
ods [30]. Note that in APX a cationic Trp radical is
much less stable than in wild-type CCP and that the
K+
site does contribute to destabilization of the Trp
radical in APX. CCP mutant 1 has the APX K+
site
engineered in and here, too, the K+
site contributes
about the same to destabilization of the Trp191 radi-
cal as it does in APX. Mutant 2 is mutant 1 with both
Met230 and 231 changed to corresponding residues
in APX, Leu, and Gln. Here stabilization of the
Trp191 radical is substantially reduced, indicating
that the Met sulfur atoms are important in contribut-
ing to stabilization of the Trp191 radical.
Summary
Overall the computational methods used in both
the NOS and CCP projects have proven extremely
useful guides in relating energetics to what we see
in the crystal structures. In addition, these com-
putational tools are useful guides in developing
hypotheses that can be tested by suitably designed
experiments. NOS is a good example of where the
crystallography alone did not allow for an unam-
biguous orientation of the inhibitor to be deter-
mined. Analyzing the problem with suitable com-
putational tools, however, provided a clear choice.
With CCP it does appear that the PDLD method [19]
has some predictive value on the stability of the
Trp191 cation radical. It should be noted, however,
that the MOLARIS calculations were carried out on
real crystal structures and not modeled mutants. Ul-
timately we would like to be able to predict the
outcome of the experiment with the computer be-
fore making the mutants. One limitation here is
correctly modeling the mutant. This may be possible
with CCP since CCP has proven to be remarkably
resilient to mutagenesis. CCP can absorb a number
of amino acid substitutions without any significant
change in structure outside of the immediate vicin-
ity of the mutation which are much easier to predict
in silico. The real problem arises when there are
substantial mutant-induced changes that must be
correctly modeled if the predictive computational
methods are to be of any use.
ACKNOWLEDGMENTS
This work was supported in part by grants from
the National Institutes of Health.
BRIEF PERSONAL NOTE BY TLP
1984 was an important year for me. It was
in that year that I first met Gilda Loew at my
first P450 meeting where I first publicly presented
the P450cam X-ray structure. Without question the
longest lasting effect of that eventful summer was
meeting Gilda. She was nervous about her up-
coming presentation and wanted to practice her
talk. Apparently I seemed liked a sympathetic au-
dience so she dragged me into a side room, sat
me down, and presented her lecture. Ever since
we were friends and collaborators, and we man-
aged to team up at various meetings throughout
the world. This misplaced New Yorker had an in-
satiable appetite for culture and science. On one
trip we flew together from San Francisco to Lon-
don and upon our arrival, she shoved me into a taxi
at the airport and off we went to the British Mu-
seum. I, of course, was jet lagged and wanted some
rest but Gilda would have none of this. Her energy
and enthusiasm were boundless and infectious. Pro-
fessionally I will always be grateful that Gilda and
her team helped to introduce computational meth-
ods into my lab. All the assistance I needed was
a phone call or e-mail away although I don’t think
she ever warmed to the rather impersonal nature
of e-mail. She was enormously generous in sharing
her resources and knowledge. As the years went by
and I began to learn more about Gilda, her accom-
plishments, and the many obstacles she faced along
the way, my respect and admiration for Gilda grew.
Even more than her excellent science, I will always
remember Gilda as an outstanding example of how
to succeed against the odds yet retain a youthful en-
thusiasm and delight in science and in living life to
the fullest. She is missed as both a friend and a col-
league.
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