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Chemical Physics Letters 403 (2005) 35–41

Substrate binding in the active site of cytochrome P450cam

Marcel Swart *, Andre R. Groenhof, Andreas W. Ehlers, Koop Lammertsma *

Organic and Inorganic Chemistry, Vrije Universiteit Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Received 20 October 2004; in final form 21 December 2004

Available online 11 January 2005

Abstract

We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calcu-

lations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of

the reaction taking place. The DFT interaction energy is well reproduced by QM/MM calculations, which allows for application of

QM/MM to the catalytic cycle of cytochrome P450s. The substrate is distorted considerably due to the presence of the protein envi-

ronment, which however does not have a large impact on the strong hydrogen bonding interactions.

� 2004 Elsevier B.V. All rights reserved.

1. Introduction

The family of P450 enzymes is involved in the catal-

ysis of oxygenase reactions on organic compounds, suchas the hydroxylation of camphor by the P450cam en-

zyme, the single-most studied of all P450 enzymes

[1,2]. In this enzyme, the substrate is hydroxylated regio-

and stereo-specifically (98–100%) at the 5-position, lead-

ing to 5-exo camphorol (see Scheme 1).

A tyrosine residue (Tyr96) has been linked to this

specificity by keeping the substrate bound in a specific

orientation to the active species (a heme group) therebypositioning it in an optimal orientation for hydroxyl-

ation at the 5 position [1]. A hydrogen bond between

the hydroxyl group of tyrosine and the carbonyl group

of the substrate (see Scheme 2) is thought to be respon-

sible for this tight positioning and the connected high

degree of regio- and stereo-selectivity; when tyrosine is

mutated for phenylalanine [1], the specificity and reac-

tion rate is reduced, and the amount of side-productsincreases.

0009-2614/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cplett.2004.12.092

* Corresponding authors. Fax: +31 20 4447488.

E-mail addresses: swart@chem.vu.nl, m.swart@few.vu.nl (M.

Swart), lammert@chem.vu.nl (K. Lammertsma).

It is evident that a hydrogen bond may help in fixat-

ing the position of the substrate relative to the active

species to maximize the propensity of the reaction taking

place. However, it is not yet clear (a) whether this partic-ular hydrogen bond is strong enough for keeping the

substrate position fixed, or that other (steric) factors

play a role; (b) if hydrogen bonding is prominent,

whether it is reproduced in hybrid QM/MM calcula-

tions; (c) if the substrate is optimally placed for maxi-

mum binding to occur; (d) what the influence of the

protein environment on the hydrogen bond and its con-

stituents is; (e) whether the substrate deforms when it ispresent in the active site. In this contribution, we address

these questions by using density functional theory

(DFT) [3–5] calculations.

2. Computational details

All calculations have been performed with the

amsterdam density functional (ADF) program [6,7]

using the Becke [8]–Perdew [9] exchange-correlation

functional, which is well suited for geometry optimiza-

tions [10]. A triple-f basis set of Slater type orbitals(STOs; TZP) [11] was used with the frozen-core approx-

imation [7].

O

H

H3C

O

Tyr96camphor

HOCC dihedral angle

Scheme 2. Hydrogen bond between Tyr96 and camphor.

P450cam

54

3

2

87

9

10

1 6O O

OH

H

Scheme 1. Hydroxylation catalyzed by P450cam enzyme.

36 M. Swart et al. / Chemical Physics Letters 403 (2005) 35–41

We use the ADF energy decomposition scheme, as

described in detail elsewhere [12]. In this scheme, the to-

tal energy of the system (the H-bond energy) consists of

the interaction energy between the two fragments and

the preparation energy:

DEtot ¼ DEintxn þ DEprep;DEintxn ¼ DEPauli þ DEelstat þ DEoi:

The interaction energy (DEintxn) can be further decom-

posed into meaningful components like Pauli repulsion

(DEPauli), electrostatic interactions (DEelstat) and orbital

interactions/polarization energy (DEoi). The preparation

(or deformation) energy (DEprep) is the sum of the ener-gies needed for bringing a fragment from its equilibrium

structure to the geometry of the total system.

In this study, we focus mainly on the side-chain of the

tyrosine residue, cutting it off at the Cbeta position, and

replacing the connection to the backbone by a

hydrogen.

The QM/MM calculations [13] were performed using

a recent implementation, with the AMBER95 [14] forcefield for describing the interactions within the protein

environment, as well as the interactions of the QM

atoms with the MM atoms. For the MM interactions

of camphor, we used regular (C, O, CT, H1) atomtypes

and employed the MDC-q [15] charges as obtained at

the equilibrium structure of camphor.

3. X-ray and DFT structures of Tyr96-camphor adduct

The coordinates for the two fragments (tyrosine,

camphor) were taken initially from the crystal structure

of substrate bound ferric P450cam (entry 1DZ4 in the

PDB-database) [16]; as there were two chains present

in the crystal structure, the fragments were superim-

posed and the resulting coordinates averaged. Hydrogen

atoms were added and their positions optimized while

keeping the (non-hydrogen) atoms from the crystalstructure fixed. Subsequently, a full geometry optimiza-

tion was performed without freezing any atom. The lat-

ter geometry will be referred to as the theoretical

geometry, while the former (with the non-hydrogen crys-

tal coordinates fixed) will be referred to as the experi-

mental geometry. We also optimized the geometry with

QM/MM calculations on the complete enzyme (see be-

low), which will be referred to as the QM/MM geome-try. Finally, two full geometry optimizations were

carried out for the tyrosine and camphor fragments sep-

arately, the outcome of which will be referred to as the

fragment geometry of either tyrosine or camphor. The

latter are the equilibrium structures for the separate

fragments.

For either one of these structures, we will report the

energy profile as a function of the hydroxyl groupHOCC dihedral (see Scheme 2), when the hydrogen is

rotated from the hydrogen-bonding position (around

180�) to non-bonding (ca. 0�) and back.

4. Interaction between the fragments

First, we investigate the strength of the H-bond forthe theoretical structure. In Fig. 1, we report the interac-

tion energy and its components, when the hydroxyl

group is rotated from a non-bonding (u = 0�) to a

hydrogen-bonding (ca. 130–230�) orientation, and back

to non-bonding (360�). The interaction between the two

fragments is favorable only in a narrow region, when the

strong Pauli repulsion is compensated by the favorable

orbital and electrostatic interactions.The strength of the H-bond corresponds to the sum

of the interaction and the preparation energy. In Fig.

2 we report the change of the H-bond energy (total)

upon rotation together with the interaction energy

(intxn) and the preparation energy of the tyrosine

(tyr). The preparation energy of camphor has a con-

stant, small value (see below) and is therefore not

shown. The hydrogen-bonding interactions are foundunchanged in the energy profile. However, a barrier of

3.2 kcal/mol is found, for the H-bond (total) energy, be-

tween the non-bonding (u=0�) and hydrogen-bonding

(u = 180�) orientation, which results solely from the

rotational energy profile of the tyrosine fragment.

In order to investigate the influence of the protein we

now turn to the experimental structure. The striking dif-

ference between the experimental and theoretical geome-try is the rotranslational shift of one of the fragments as

a whole (see Fig. 3).

-20

-10

0

10

20

0 60 120 180 240 300 360

HOCC dihedral angle (˚)

Interaction Energy(kcal/mol)

intxn

electrostatics

Pauli repulsion

orbital interactions

Fig. 1. Interaction energy as function of Tyr–HOCC dihedral angle u in theoretical geometry.

-10

0

10

0 60 120 180 240 300 360

HOCC dihedral angle (˚)

Energy(kcal/mol)

intxn

total

tyr

intxn-qmmm

Fig. 2. H-bond strength as function of Tyr–HOCC dihedral angle u in theoretical geometry.

Fig. 3. Superposition of fragments in experimental (ball-and-stick,

grey), QM/MM (cylinders, grey) and theoretical (ball-and-stick, black)

structure.

M. Swart et al. / Chemical Physics Letters 403 (2005) 35–41 37

Strong hydrogen bonding interactions (6–8 kcal/mol)

are observed, however the interaction energy between

the two fragments is different between the experimental

and theoretical geometry. In the theoretical geometry

the two fragments have a stabilizing hydrogen-bonding

interaction of 7.8 kcal/mol (7.4 for the total H-bond

strength), while it is only 6.3 in the experimental geom-

etry (see Table 1). The difference between the two iscaused mainly by Pauli repulsion, which is 1.6 kcal/

mol lower for the theoretical geometry; both electro-

static and orbital interactions/polarization effects differ

by only 0.1–0.2 kcal/mol between the experimental and

theoretical geometry. Due to the rotranslational shift

the position of the minimum is moved from u = 180�in the theoretical geometry towards u = 210� for the

experimental structure (see Fig. 4).Also given in Table 1 are the energies for the non-

bonded complex, i.e., the situation where the hydroxyl

proton of tyrosine is pointing away from the carbonyl

oxygen of camphor (HOCC dihedral angle, see Scheme

2, of 0�). The interaction is found to be unfavorable,

resulting from a large contribution of Pauli repulsion

Table 1

Interaction energy (kcal/mol) and its components for tyrosine–camphor complex

H-bond INTXN Paulia El.St.b OI/Pc

Hydrogen-bonded complex

Exp. geom.d �6.31 +16.65 �13.57 �9.39

Theor. geom.e �7.35 �7.83 +15.04 �13.70 �9.17

Non-bonded complex

Exp. geom.f +5.27 +6.06 +0.21 �1.01

Theor. geom.f +4.61 +5.51 +0.15 �1.05

a Pauli repulsion.b Electrostatic interactions.c Orbital interactions and polarization effects.d Tyrosine HOCC dihedral angle u 210�.e Tyrosine HOCC dihedral angle u 180�.f Tyrosine HOCC dihedral angle u 0�.

-10

0

10

0 60 120 180 240 300 360

HOCC dihedral angle (˚)

Energy(kcal/mol)

prot-i

prot-t

intxn-exp

intxn-qmmm

Fig. 4. Interaction as function of Tyr–HOCC dihedral angle in experimental (exp) [total energy not given for experimental geometry due to large

preparation energy of camphor (vide infra)] and QM/MM (prot; see Fig. 5) [DFT interaction (prot-i) and H-bond (prot-t) energy for camphor–

tyrosine adduct at QM/MM (protein environment taken into account, vide infra) optimized geometry] geometry.

38 M. Swart et al. / Chemical Physics Letters 403 (2005) 35–41

in combination with weak electrostatic and orbital inter-

actions (see Fig. 5).

4.1. QM/MM investigation

In subsequent studies, we would like to investigate

the catalytic cycle of cytochrome P450cam with the en-

zyme environment present explicitly in hybrid QM/

MM calculations. As the heme group (the active species)

is present also in other enzymes, with a broad range offunctions (e.g., oxygen/electron transfer) [17], a substan-

tial influence on the enzyme function must result from

the protein environment. Although many theoretical

studies have already been performed on the active site

of this enzyme, only few have included the enzyme envi-

ronment explicitly in the calculations. For the QM/MM

approach, the division of the substrate containing active

site as QM part and the protein as MM part seems themost logical choice. A good description of the interac-

tions between both parts is essential, where the H-bond

between camphor and the tyrosine provides an ideal test

case. Therefore, we calculated the QM/MM interactionenergy between tyrosine and camphor (i.e., similar as

above), with camphor in the QM and tyrosine in the

MM system. For the MM system, an appropriate force

field (such as AMBER95 [14]) should be able to give a

reliable description of the interactions.

The QM/MM interaction energies for the theoretical

and experimental structure are also given in Figs. 2

and 4. Apart from a downward shift of approximatelyonly 2 kcal/mol, it is very similar to the DFT interaction

energy. The difference in energy between the non-bond-

ing and hydrogen-bonding orientation is reproduced

very well (ca. 11 kcal/mol QM/MM vs. 12 kcal/mol

DFT), and the preferred orientation (u around 210�for the experimental structure, 180� for the theoretical

structure) is equal for both the QM/MM and DFT cal-

culations. Therefore, we conclude that the use of QM/MM calculations is valid for this system. This will not

only reduce significantly the size of the QM part in these

Fig. 5. QM/MM setup: MM (left) and QM system (right; Tyr96 is shown (in grey) for clarity).

M. Swart et al. / Chemical Physics Letters 403 (2005) 35–41 39

calculations, with corresponding reduction of CPU-

time, but also gives confidence that the important

hydrogen-bonding interactions are still adequately

described.

4.2. Deformation of the fragments upon binding

The deformation energy is similar in both cases(0.2 kcal/mol), but the distortion is much larger due to

the influence of the protein environment. The tyrosine

part of the experimental geometry is only slightly de-

formed from its equilibrium (fragment) structure; taking

the tyrosine fragment geometry as reference, the coordi-

nates of the tyrosine part of the experimental geometry

show a root-mean-square-deviation (RMSD) of only

0.02 A (non-hydrogens only) or 0.03 A (hydrogens in-cluded). This also shows up in the preparation energy,

which is only 1.3 kcal/mol for the tyrosine part of the

experimental geometry (see Table 2). Naturally, the tyro-

sine part of the theoretical geometry resembles more clo-

sely the fragment geometry of tyrosine, with a RMSD of

0.004 A (non-hydrogens only), and a preparation energy

of only 0.3 kcal/mol.

Table 2

RMSDa (A) and preparation energy (kcal/mol) with respect to

fragment data

RMSD Eprep

Experimental geometry

Tyrosine part 0.020 1.28

Camphor part 0.086 20.01

Theoretical geometry

Tyrosine part 0.004 0.28

Camphor part 0.012 0.20

QM/MM geometry

Camphor part 0.058 0.42

a Root-mean-square-deviation of coordinates of non-hydrogen

atoms, with respect to coordinates from fragment geometry.

The camphor part of the theoretical structure is also

very similar to the fragment structure, which shows up

in the RMSD (0.01 A) and the preparation energy

(0.2 kcal/mol). In fact, only the C@O double bond is

slightly elongated (0.002 A) by the H-bond. On the

other hand, the experimental geometry of camphor

shows a large distortion in the protein structure

(RMSD = 0.09 A,DEprep = 20 kcal/mol). Several causesmay lie at the origin of this distortion; e.g. it may result

from steric hindrance (or other strain) imposed by the

protein environment, a less than adequate force field

description for the substrate as used in the elucidation

of the crystal structure, or even simply be an artifact

of the X-ray data analysis. After all, the RMSD of

0.09 A is still smaller than the estimated experimental

accuracy (0.1–0.3 A).The main difference is a severely shortened bond

between C5 and C6 of only 1.511 A (Table 3). Interest-

ingly, the same bond is theoretically predicted to be

1.559 A and in a recent crystallographic study of

Table 3

Selected coordinates (A, �) of camphor in the experimental, theoretical

and QM/MM geometry

Bonda Experimentalb Theoreticalc QM/MMc

C1–C2 1.529 1.537 1.543

C2–C3 1.522 1.539 1.543

C3–C4 1.524 1.542 1.544

C4–C5 1.528 1.547 1.548

C5–C6 1.511 1.559 1.559

C6–C1 1.570 1.568 1.571

C1–C7 1.561 1.576 1.575

C4–C7 1.558 1.567 1.563

C1–C10 1.529 1.517 1.522

C2@O 1.208 1.220 1.222

C1–C2–C3@O 180.1 181.2 184.6

a See Scheme 1 for atom numbering.b Ref. [18].c This work.

40 M. Swart et al. / Chemical Physics Letters 403 (2005) 35–41

uncomplexed camphor found to be even longer

(1.576 A) [18].

4.3. Taking the protein environment into account

In order to find the origin of the distortion of thesubstrate when it is present in the active site, we

decided to go one step further and optimize the geom-

etry of the substrate within the enzyme by means of

QM/MM calculations. The active site was treated with

DFT (QM) while the interactions with the protein envi-

ronment were described by a classical (MM) force field

(AMBER95 [14]). The active site consisted of the iron–

porphyrin moiety, the cysteine residue that acts as axialligand to iron, and the substrate; the MM system con-

sisted of the remaining amino acid residues and crystal

waters, in total 7262 atoms. Only the atoms of the sub-

strate were allowed to relax in the geometry optimiza-

tion, apart from a preoptimization of the coordinates

of the protons (which are absent in the crystal

structure).

The QM/MM optimization of the substrate leads toonly a marginal reduction of the RMSD (relative to

the fragment structure), from 0.09 A for the experi-

mental geometry to 0.06 A for the QM/MM geometry,

but is accompanied by a significant reduction of the

preparation energy, from 20 to only 0.4 kcal/mol.

The distortion between the fragment and theoretical

structure can be attributed to the hydrogen bonding

interactions between camphor and tyrosine, while thatbetween the theoretical and the QM/MM structure

may be attributed to the influence of the protein

environment.

In Table 3, we report the bond length for the exper-

imental, theoretical and QM/MM structures. The QM/

MM distances differ only slightly from the theoretical

ones (only up to 0.006 A). In general, the QM/MM dis-

tances are elongated, which can be expected from theinfluence of the surroundings. The shortened C5–C6

bond observed in the experimental structure could

not be reproduced by the QM/MM calculations,

though it is worth noting that this is one of the few

bonds that is not elongated by this approach. How-

ever, the most striking feature of the QM/MM struc-

ture results from changes of the angles around the

carbonyl group of camphor, which is accompanied bythe shift of camphor as a whole (see Fig. 3). The latter

results in a RMSD between the QM/MM and theoret-

ical structure of camphor–tyrosine of 0.25 A, to which

the former contributes a still significant amount of

0.05 A.

Even though the crystal structure may have inaccu-

racies in the camphor structure, our QM/MM versus

QM calculations provide evidence to suggest that thesubstrate is indeed distorted in the active site of the

P450cam enzyme. The influence of this distortion on

the catalytic activity will be addressed in future

studies.

5. Concluding remarks

We have investigated the hydrogen bond between a

tyrosine residue (Tyr96) and substrate of the P450cam

enzyme, which is linked to the high regio- and stereo-

specificity observed in the enzymatic hydroxylation of

camphor. The interactions were computed at a few dif-

ferent structures, either coming from X-ray data, DFT

calculations or QM/MM calculations.

Indeed, we find the existence of strong hydrogenbonding interactions between the two fragments, that

are strong enough (>6 kcal/mol) to account for the high

specificity as observed experimentally. With the ADF

energy decomposition analysis, all three components

are shown to contribute significantly to the strong

hydrogen bonding interactions. The considerable Pauli

repulsion energy of +16.7 kcal/mol is overcome by

favorable electrostatic and orbital interactions of�13.6 and �9.4 kcal/mol, respectively. The hydrogen-

bond interactions are well reproduced by QM/MM cal-

culations, which allows for the application of QM/MM

to the complete enzyme.

When we compare the structures with the equilib-

rium structure of the separate tyrosine or substrate

fragment, we find only small deviations for tyrosine.

For camphor however, we find a relatively small devi-ation for the DFT structure, but severe distortions

both in the X-ray and QM/MM structure. The protein

environment therefore has a large influence on the con-

formation of the substrate. However, despite the lar-

gely distorted camphor, the deformation energy

needed is small and the hydrogen bonding interactions

are hardly affected.

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