ORIGINAL PAPER

Dke1—structure, dynamics, and function: a theoreticaland experimental study elucidating the role of the binding siteshape and the hydrogen-bonding network in catalysis

Hrvoje Brkic • Daniela Buongiorno •

Michael Ramek • Grit Straganz • Sanja Tomic

Received: 30 September 2011 / Accepted: 29 March 2012 / Published online: 20 April 2012

� SBIC 2012

Abstract This study elucidates the role of the protein

structure in the catalysis of b-diketone cleavage at the three-

histidine metal center of diketone cleaving enzyme (Dke1)

by computational methods in correlation with kinetic and

mutational analyses. Molecular dynamics simulations,

using quantum mechanically deduced parameters for the

nonheme Fe(II) cofactor, were performed and showed a

distinct organization of the hydrophilic triad in the free and

substrate-ligated wild-type enzyme. It is shown that in the

free species, the Fe(II) center is coordinated to three histi-

dines and one glutamate, whereas the substrate-ligated,

catalytically competent enzyme–substrate complex has an

Fe(II) center with three-histidine coordination, with a small

fraction of three-histidine, one-glutamate coordination. The

substrate binding modes and channels for the traffic of water

and ligands (2,4-pentandionyl anion, methylglyoxal, and

acetate) were identified. To characterize the impact of the

hydrophobic protein environment around the metal center

on catalysis, a set of hydrophobic residues close to the

active site were targeted. The variations resulted in an up to

tenfold decrease of the O2 reduction rates for the mutants.

Molecular dynamics studies revealed an impact of the

hydrophobic residues on the substrate stabilization in the

active site as well as on the orientations of Glu98 and

Arg80, which have previously been shown to be crucial for

catalysis. Consequently, the Glu98–His104 interaction in

the variants is weaker than in the wild-type complex. The

role of protein structure in stabilizing the primary O2

reduction step in Dke1 is discussed on the basis of our

results.

Keywords Dke1 � Fe2? parameters in nonheme enzyme �Molecular dynamics � Mutants � Random acceleration

molecular dynamics

Abbreviations

Ace Acetone

DPD 1,1-Difluoropentanedione

MD Molecular dynamics

MI 4-Methylimidazole

PD 2,4-Pentanedione

PDA 2,4-Pentanedionyl anion

PDB Protein Data Bank

RAMD Random-acceleration molecular dynamics

Tris Tris(hydroxymethyl)aminomethane

UHF Unrestricted Hartree–Fock

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-012-0898-8) contains supplementarymaterial, which is available to authorized users.

H. Brkic

Medical Faculty Osijek,

University of Osijek,

J. Huttlera 4, 31000 Osijek, Croatia

D. Buongiorno � G. Straganz (&)

Institute of Biotechnology and Biochemical Engineering,

Graz University of Technology,

Petersgasse 12, 8010 Graz, Austria

e-mail: grit.straganz@tugraz.at

M. Ramek

Institute of Physical and Theoretical Chemistry,

Graz University of Technology,

Brockmanngasse 29,

8010 Graz, Austria

S. Tomic (&)

Ruder Boskovic Institute,

Bijenicka 54, 10000 Zagreb, Croatia

e-mail: sanja.tomic@irb.hr

123

J Biol Inorg Chem (2012) 17:801–815

DOI 10.1007/s00775-012-0898-8

Introduction

Acetylacetone dioxygenase from Acinetobacter johnsoii,

Dke1, is a nonheme Fe2?-dependent enzyme that cata-

lyzes the oxidative degradation of b-dicarbonyl com-

pounds [1, 2]. Dke1 has a homotetrameric structure

[Protein Data Bank (PDB) code 3BAL] [3] with each

subunit organized in a single-domain b-barrel fold, a fold

which is characteristic for the cupin superfamily of

proteins. Although Dke1 displays an atypical three-his-

tidine metal binding site that deviates from the typical

two-histidine, one-carboxylate motif [4], it apparently

follows the rationale of O2 activation that is typically

found for mononuclear nonheme iron(II) dioxygenases

[5, 6]. O2 reduction rates have previously been shown to

be influenced by the hydrogen-bonding network in the

active site, as a mutational and kinetic analysis of

hydrophilic residues in the substrate binding pocket

suggests [7]. Two possible mechanisms have been dis-

cussed to account for this effect: firstly, a well-oriented

hydrogen bond, which is provided by a hydrophilic triad

of amino acid moieties (namely Tyr70, Arg80, Glu98)

was suggested to stabilize the transition state of O2

reduction. Secondly, increased water access to the active

site or greater hydrophilicity of the substrate binding

pocket could promote binding of water to the substrate-

ligated metal cofactor, thus shifting the equilibrium of

the metal center from the catalytically competent five-

coordinate form to an inept, water-protected six-coordi-

nate species.

In this work we elucidate the role of the protein

structure in the catalysis of b-diketone cleavage at the

three-histidine metal center of Dke1 by molecular mod-

eling studies. Structural dynamic studies on free and

substrate-ligated wild-type enzyme were performed,

interactions among residues that are potentially important

for catalysis were determined, and channels for water,

substrate, and product trafficking were identified. To

perturb the active site of Dke1, a set of hydrophobic

residues lining or close to the active site were subjected

to mutational analysis. The impact of these variations on

catalytic O2 reduction rates was characterized, and the

results were correlated with the results of computational

studies on the respective substrate-ligated variants. The

studies were focused on characterizing factors with a

putative impact on O2 reduction rates, namely, (a) the

geometry of the metal center and the organization of the

hydrophilic triad residues Tyr70, Arg80, and Glu98,

(b) residue-based protein flexibility, (c) the ability of the

active site to stabilize water molecules, and (d) the

impact of the selected variations on the affinity of the

active site for O2 molecules.

Materials and methods

Computational methods

System preparation

The crystal structure of the ligand-free Dke1 was extracted

from the PDB (code 3BAL), and the zinc ion present in the

structure was replaced with Fe2?.

A complex between the wild-type enzyme and 2,4-

pentanedionyl anion [PDA; CH(CH3CO)2-] was built, and

the complexes with the mutants were prepared from this by

replacement of the respective amino acid side chain. Sub-

strate binding into the active site of Dke1 was examined by

steered molecular dynamics (MD) simulations [8] as

implemented in the Amber 10 program package (http://

ambermd.org/) [9]. The center of two oxygen atoms of the

substrate was pulled towards Fe2? with a force constant of

50 kcal/(mol A) and an optimal (equilibrium) distance of

2.5 A. The Amber ff03 force field [10] and the general

AMBER force field were used to parameterize the protein

and the substrate, respectively. Parameters for the substrate

were derived using Antechamber in the Amber 10 program

package (http://ambermd.org/antechamber.html). For the

iron cation, Fe2?, the parameters were derived using

quantum mechanical calculations (vide infra). We used the

minimal set of parameters, which ensured the proper Fe2?

coordination geometry, but at the same time did not sup-

press the natural behavior of the system.

Nonpolar hydrogen atoms were added with tleap, which

is part of the Amber 10 program package, and polar

hydrogen atoms were added manually to optimize the

hydrogen-bonding network in the protein.

Iron parameter derivation

Model complexes of high-spin Fe2? with several ligands

were defined and their geometry was optimized by unre-

stricted Hartree–Fock (UHF) calculations. The triple-zeta

valence basis set TZV plus polarization functions on car-

bon, nitrogen, and oxygen as coded in the program

GAMESS [11] was used. For hydrogen, carbon, nitrogen,

and oxygen this basis set is identical to 6-311G*. For iron it

is characterized by the contraction {5111111111/

41111111/411} and yields an energy of -1,262.33880651

a.u. for the neutral atom in the 5D state (see note 1 in the

electronic supplementary material for comparison with

other sources). To cover the simultaneous interaction of

Fe2? in the specific system (namely, with water, a nitrogen

triad, and carbonyl oxygen atoms), the following ligands

were used: PDA, water, acetone (Ace), ammonia, and

4-methylimidazole (MI). The optimized structures were of

802 J Biol Inorg Chem (2012) 17:801–815

123

D2h symmetry for Fe2?�6H2O, Fe2?�2Ace�4H2O, and

Fe�2PDA�2H2O, C2v symmetry for Fe2?�5H2O, Ci symmetry

for Fe2?�2MI�4H2O, and C3 symmetry for Fe2?�3MI�3H2O and

Fe2?�3NH3�3H2O. However, the Fe2?�6H2O, Fe2?�5H2O, and

Fe2?�3NH3�3H2O systems were the key structures.

Fe2?�6H2O is a Th structure that is Jahn–Teller-distorted

to D2h symmetry with three different Fe–O distances,

2.1600, 2.1828, and 2.2336 A, for a chemically identical

substituent. Clearly, such a structure cannot be modeled by

any force field that is based on harmonic-like terms with

single values for equilibrium distances or angles, like the

Amber force fields are [9, 10] (see note 2 in the electronic

supplementary material). Since a Jahn–Teller distortion is a

feature limited to systems of high symmetry and a degen-

erate ground state, it can safely be ignored for iron in a

protein and the various Fe–O distances can be averaged to

a common value.

Fe2?�5H2O was generated from Fe2?�6H2O by removal

of one water molecule. Interestingly, its structure did not

change much during the UHF geometry optimization and

converged to a slightly distorted pyramid with Fe–O

distances of 2.1827 A (axial), 2.1716 A, and 2.1016 A and

O–Fe–O angles of 92.29� and 106.25�. The octahedral

coordination pattern for Fe2? therefore appears to be a quite

stable one, even if one ligand position remains vacant.

For Fe2?�3NH3�3H2O, the initial structure was chosen

with the catalytic triad in mind: the three ammonia mole-

cules form the upper layer of a sandwich, and the three water

molecules form the lower layer. The converged structure is

of C3 symmetry with key distances of 2.2660 A (Fe–O) and

2.2736 A (Fe–N), and key angles of 83.65� (O–Fe–O),

96.45� (N–Fe–N), and 88.01�/170.82� (O–Fe–N). The same

remark as made above for the Fe–O distances in the

Fe2?�6H2O system has to be repeated here for the O–Fe–N

angle: no force field that is based on harmonic-like terms

with single values for equilibrium distances or angles can

model such a multivalued situation correctly. As a conse-

quence, we reduced the O–Fe–N force constant to a small

value.

To determine the Amber force constants Kr, the opti-

mized model structures were used as starting points for

single-point UHF energy calculations at slightly distorted

geometries.

The detailed description of the procedure is given in

note 3 in the electronic supplementary material. The actual

parameters were obtained by averaging the values obtained

for the model complexes listed. The set of parameters used

for the subsequent MD simulations of the large systems is

given in Table S1. In brief, the iron–histidine and iron–

substrate interactions are described using bonding as well

as nonbonding interaction terms, whereas all other inter-

actions with the iron ion are described using solely the

nonbonding terms.

Molecular dynamics simulations: wild-type protein–ligand

complexes

Free Dke1 as well as the complexes with PDA were placed in

a truncated octahedron filled with TIP3P-type water mole-

cules [12]. Besides water molecules, Na? ions were added to

neutralize the system and were placed near charged amino

acids at the protein surface. The resulting systems consisting

of approximately 36,000 atoms were simulated using peri-

odic boundary conditions. The electrostatic interactions

were calculated using the particle-mesh Ewald method [13].

Before MD simulations were performed, the protein

geometry was optimized in four cycles with different

constraints. In the first cycle (10,000 steps), water and

substrate molecules were relaxed, whereas the protein and

the iron ion were constrained using a harmonic potential

with a very high force constant of 500 kcal/(mol A2). In

the second and third cycles (also 10,000 steps each), the

constraint on the iron ion was reduced first to 50 kcal/

(mol A2) and then to 32 kcal/(mol A2), and the constraint

on the protein backbone was 50 and 10 kcal/(mol A2),

respectively. In the final cycle, the constraint on the protein

backbone was 5 kcal/(mol A2) and that on the iron ion was

12 kcal/(mol A2). The energy minimization procedure was

the same in all four cycles: The first 5,000 steps of steepest

descent were followed by conjugate gradient optimization

steps.

After energy minimization, the system was heated from 0

to 300 K in 100 ps using a canonical ensemble with con-

stant volume and temperature (NVT) conditions, followed

by six equilibration stages (NPT conditions), 100 ps each,

during which the water density was adjusted and the initial

constraints on the protein and the metal ion were gradually

reduced. Isotropic scaling was used to ensure a system

density of 1.023 (?0.001) g/cm3. The equilibrated system

was then subjected to productive MD simulations at con-

stant temperature (300 K) and pressure (1 atm, the NPT

ensemble). During the first nanosecond of these simula-

tions, the weak constraints were used, and the subsequent

MD simulations were accomplished without any restraints.

The time step during preequilibration, equilibration, and the

first 2 ns of the productive MD simulations was 1 fs, and

after that 2- and 1-fs steps were used for simulations from 3

to 13 ns and from 14 to 20 ns, respectively. After these,

another set of either 20 or 30 ns of MD simulations with

approximately two times weaker iron bonding parameters

followed (see Table S1). The temperature was held con-

stant, using Langevin dynamics with a collision frequency

of 1 ps-1 and a time step 1 fs. Bonds involving hydrogen

atoms were constrained using the SHAKE algorithm. The

structures were sampled every 1.0 ps and trajectories were

analyzed using the program ptraj within the Amber program

suite. For each product at least two productive trajectories

J Biol Inorg Chem (2012) 17:801–815 803

123

were generated in a way that after 13 ns two to three parallel

simulations were performed with different random number

seeds.

Simulations of the substrates (reactant and products)

expulsion from the Dke1 active site

To explore possible pathways by which the substrates

PDA, methylglyoxal, and acetate can enter and leave the

Dke1 active site, random-acceleration MD (RAMD) sim-

ulations [14] were performed. Simulation of substrate

egress from a protein active site is very demanding because

it might occur on a timescale much longer than the time-

scale currently possible for standard MD simulations of

proteins, especially when the substrate binding site is not

exposed to solvent, but is buried in the protein interior, as is

the case in Dke1. The RAMD method overcomes this

problem by applying a small, artificial, randomly orientated

force to the center of mass of the ligand to enhance the

probability of its spontaneous exit. To obtain statistically

significant results, for each of the simulated complexes we

performed 15 RAMD simulations with an acceleration in

the range from 0.17 to 0.30 kcal/(g A) (atomic mass given

in grams per mole).

The RAMD simulations were run for 250 ps or until the

distance between the centers of mass of Dke1 and the PDA

molecule became greater than 30 A. The time step was the

same as used in MD simulations (1 fs). The direction of the

force was kept for 40 time steps (40 fs). If a ligand did not

move by more than 0.01 A during this period, a new

direction was chosen randomly, otherwise the same force

was applied for the next period of 40 time steps.

For the purpose of RAMD calculations, the force con-

stants for the bonding parameters including iron and sub-

strate oxygen(s) were set to zero, namely, the constants

related to bonds, angles, and dihedral angles (see Table

S1).

GRID calculations: determination of the possible

low-energy positions of O2

The most favorable positions of the O2 molecule within the

active center were determined using the program GRID

[15]. The interaction energies were calculated at the nodes

of the regular three-dimensional grid with a 1-A spacing.

The size of the grid was adjusted to extend at least 10 A in

all directions from the metal ion, ensuring that it was large

enough to consider the effect of mutations in the active site.

Calculations were preformed on optimized structures, after

about 20 ns of unconstrained MD simulation. Only the

positions found within 3.1 A from the metal ion, with an

interaction energy below -5 kcal/mol, were taken into

account.

Experimental methods

Construction of Dke1 variants

Site-directed mutations were introduced via a standard two-

stage PCR protocol [16]. In brief, for each variant, forward

and reverse oligonucleotide primers of 25 nucleotides

bearing the respective intended mutation at their central

position were designed and were used in two separate pri-

mer extension reactions with the plasmid vector pKYB1-

dke1-strep [17] as a template according to standard proce-

dures. The latter contains the full-length dke1 gene, which is

fused to an oligonucleotide that encodes a C-terminal ten

amino acid affinity tag [18]. The affinity tag does not alter

the biochemical and catalytic properties of Dke1, as dem-

onstrated previously [16]. After four cycles of separate

amplification with 3 U of Pfu DNA polymerase (Promega,

Madison, WI, USA) 18 cycles of amplification of the

combined reaction mixtures were applied using an anneal-

ing temperature of 62 �C. The final extension phase was

15 min at 72 �C. Template DNA was digested with DpnI,

and the amplified plasmid vectors were transformed into

electrocompetent cells of Escherichia coli BL21 (DE3)

(Stratagene, La Jolla, CA, USA) via standard procedures.

Plasmid DNA from the resulting clones was isolated via a

miniprep kit (Wizard Plus SV Minipreps, Promega) and was

subjected to dideoxy sequencing of the entire dke1 gene in

order to verify that the desired mutation had been intro-

duced into the otherwise unaltered dke1 gene.

Production and purification of protein

Recombinant Dke1 was produced according to the proce-

dures described previously [17]. Purification was per-

formed according to reported protocols by applying affinity

chromatography with a 5-mL Strep-Tactin column (IBA,

Gottingen, Germany). Eluted protein was subjected to

buffer exchange by three cycles of desalting via NAP-25TM

columns (GE Healthcare, Chalfont St. Giles, UK) using

20 mM tris(hydroxymethyl)aminomethane (Tris)/HCl

buffer, pH 7.5. The resulting protein preparations were

concentrated to 5 mg/mL or greater with Vivaspin centri-

fugation concentrator tubes (Vivascience, Hanover, Ger-

many) and stored at -20 �C. The purity of all the enzyme

preparations was more than 95 %, as estimated by sodium

dodecyl sulfate gel electrophoresis.

Determination of Fe2? and Fe3? contents and kinetics

of Fe2? detachment

The content of protein-bound Fe2? in purified enzyme

preparations was determined spectrophotometrically by

monitoring the formation of a colored solution of the

804 J Biol Inorg Chem (2012) 17:801–815

123

complex between Fe2? and 3-(2-pyridyl)-5,6-di(2-furyl)-

1,2,4-triazine-50,500-disulfonic acid disodium salt

(e592nm = 35.5 mM-1 cm-1) [19, 20] as described previ-

ously [6]. Total iron was determined by adding ascorbic

acid/sodium ascorbate solution (pH 7.5, 10 mM) at a final

concentration of 10 mM to the assay mixture directly prior

to the addition of the protein sample in order to reduce

Fe3? to Fe2?. To determine Fe2? detachment rates, the

increase in absorbance due to Fe2?–3-(2-pyridyl)-5,6-di(2-

furyl)-1,2,4-triazine-50,500-disulfonic acid disodium salt

complex formation was continually measured over a period

of 12 h at 25 �C, pH 7.5 and in the presence of excess 3-(2-

pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-50,500-disulfonic acid

disodium salt (20 mM). These conditions allowed the

determination of pseudo-first-order kinetic constants of

iron detachment (note that at 3-(2-pyridyl)-5,6-di(2-furyl)-

1,2,4-triazine-50,500-disulfonic acid disodium salt concen-

trations of 20 mM or greater, detachment of Fe2? (less than

40 lM) does not significantly depend on the 3-(2-pyridyl)-

5,6-di(2-furyl)-1,2,4-triazine-50,500-disulfonic acid diso-

dium salt concentration). The curves were generally

biphasic and were therefore best fitted to a double-expo-

nential equation of the form

A ¼ a1 exp �k1Fetð Þ þ a2 exp �k2Fetð Þ; ð1Þ

where A is the absorbance, t is time, a1 and a2 are the

respective amplitudes of absorbance, and k1Fe and k2Fe are

the pseudo-first-order kinetic constants of iron detachment.

Protein concentration

Protein concentration was determined by measuring the

absorbance at 280 nm of the appropriately diluted purified

enzyme preparation in Tris buffer (20 mM, pH 7.5) and

applying a theoretically calculated absorption coefficient

from the respective sequence based on the method of

Edelhoch [21] and the parameters of Pace et al. [22] using

the program ProtParam (http://www.expasy.ch).

Gel filtration

The molecular mass of the native enzyme was estimated

via analytical gel filtration using a Superdex 200 gel fil-

tration column (3-mL bed volume, GE Healthcare) by

applying a flow rate of 0.2 mL/min. A low molecular mass

standard for gel filtration (GE Healthcare) and wild-type

Dke1 were used as calibrants. Detection was at 280 nm.

Circular dichrosim spectroscopy

Near-UV circular dichroism spectra of protein preparations

(1 mg/mL) were recorded with a J-715 spectropolarimeter

(JASCO, Easton, MD, USA) at 25 �C in Tris buffer

(20 mM, pH 7.5) using a cylindrical cell with 0.2-mm path

length. The following instrument parameters were applied:

step resolution 0.2 nm, scan speed 50 nm/min, response

1 s, bandwidth 1 nm. For each sample, five spectra were

recorded in the wavelength range from 190 to 260 nm,

averaged, and corrected by a blank spectrum of Tris buffer.

The circular dichroism signal was converted to the mean

residue ellipticity and processed using the program

DICHROWEB [23, 24] to calculate the secondary structure

composition, whereas the program STRIDE [25] was used

to determine the relative content of secondary-structure

elements in the crystal structure of Dke1 (PDB code 3BAL).

Steady-state and pre-steady-state kinetic measurements

Steady-state kinetic studies with 2,4-pentanedione (PD) and

1,1-difluoropentanedione (DPD) were performed spectro-

photometrically with a DU 800 UV–vis spectrophotometer

(Beckmann Coulter, Fullerton, CA, USA) at 25 �C in Tris

buffer (20 mM, pH 7.5), whereby the absorbance trace at

the wavelength of maximum substrate absorbance was

monitored as described previously [26]. Specific activities

were generally related to the concentration of Fe2? in the

protein sample. O2 concentrations were varied by mixing

N2- and O2-purged buffers in different quantities. The

respective O2 concentrations were monitored using a

Microtox TX3-AOT O2 sensor (PreSens, Regensburg,

Germany) as described previously. To determine the

enzymatic activities, a series of classic initial rate mea-

surements at various substrate and O2 concentrations and a

nonlinear fit of the Henri–Michaelis–Menten equation for a

single-substrate enzymatic reaction to the data were per-

formed. This method gave reliable kcatapp values, but the

corresponding KM values were too small for direct assess-

ment. To estimate KM values, therefore, the integrated form

of the Michaelis–Menten equation was applied to expanded

reaction time courses, whereby 50 lM substrate was used

and the rates were corrected for the decrease in O2 con-

centration as described previously [7].

Fast kinetic analysis

Formation and decay of the iron(II) enzyme–substrate

complex was assessed with an SX.18MV stopped-flow UV–

vis spectrophotometer (Applied Photophysics, Leatherhead,

UK) equipped with a modular optical system. Data acqui-

sition and analysis were done using Applied Photophysics

software. Detection was by a photodiode-array detector in

the range from 280 to 700 nm using a logarithmic time

base. The prototypical transition of the substrate-ligated

metal center was monitored at the wavelength of its maxi-

mum absorbance (kmax PD = 420 nm, kmax DPD = 450 nm)

[26]. In the stopped-flow experiment, stock solutions of

J Biol Inorg Chem (2012) 17:801–815 805

123

substrate (380 lM) and iron(II) enzyme (400 lM) were

mixed in equal volumes. The substrate concentration is

fully saturating under steady-state conditions (KMapp

\ 10 lM). The reaction was conducted at 25 �C in air-

saturated 20 mM Tris/HCl buffer (pH 7.5). Substrate

binding and decay occurred in distinct time domains and the

kinetics were fit separately via formulas with a single

exponential as described previously [26]:

AES- buildupð Þ ¼ AESmax 1� exp �k1tð Þ½ � ð2ÞAES- break-downð Þ ¼ AESmax exp �k2tð Þ; ð3Þ

where AES is the absorbance of the enzyme–substrate

complex at 420 nm (PD) and 450 nm (DPD), respectively,

AESmax is the theoretical maximum absorbance under the

conditions used, and k1 and k2 are the respective time

constants for formation and breakdown of the complex.

Results

To gain insights into the structural basis for the Dke1-

catalyzed reaction and the differences in catalytic perfor-

mance between the wild-type protein and its mutants, we

first investigated the structure of free and substrate-ligated

Dke1 and then compared the results with experimental and

in silico results for the respective mutants. Using MD

simulations, we investigated dynamic and structural prop-

erties of the analyzed systems, whereby we focused on the

structure, geometry, and rigidity of the metal center, the

affinity of the substrate binding pocket for water and O2,

and characterization of water-, substrate-, and product-

trafficking paths.

Simulations of the wild-type protein, free

and in complex with 2,4-pentanedione

We performed two independent MD simulations (50 and

19.5 ns long) for the ligand-free Dke1 and three indepen-

dent MD simulations (40, 20, and 19.5 ns long) for its

complex with PDA.

First coordination sphere and hydrophilic triad

In the crystal structure of the ligand-free Dke1 (PDB code

3BAL), the metal ion is coordinated with three histidine

residues, His62, His64, and His104, whereby His62 and

His64 coordinate the metal ion via Ne and His104 coor-

dinates via Nd. During MD simulations this geometry

changed to either distorted octahedral or trigonal bipyra-

midal coordination. First, the Glu98 side chain reoriented

(rotation about the Cb–Cc and Cc–Cd bonds) and one

(occasionally both) of its carboxyl oxygen atoms entered

the Fe2? coordination sphere. Subsequently (almost at the

same time) one or two water molecules entered the metal

binding site (see Fig. S1) and either a five-coordinate or a

six-coordinate geometry was established (Fig. 1). After

several nanoseconds (5.2 ns in the A chain, 17.2 ns in the

B chain, 1.4 ns in the C chain, and 3.8 ns in the D chain) a

Fig. 1 Fe2? coordination in the wild-type (WT) protein. Fe2? is five-

coordinated for about 23 % of the entire simulation time (a). The

octahedral coordination of the iron cation with one of the Glu98

oxygen molecules in the equatorial plane and one in the axial

position, opposite Ne of His62, is present for about 28 % of the entire

time (b), whereas the one with two water molecules and His104 Ndand His64 Ne in the equatorial plane and the Glu98 oxygen and Ne of

His62 in the axial position (c) is present for about 49 % of the entire

simulation time. Amino acid residues coordinating the metal iron are

shown in stick representation, and Fe2? is represented as a pink ball

806 J Biol Inorg Chem (2012) 17:801–815

123

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10

4(N

E2

)3

5G

lu9

8

[OE

1(2

)]

His

10

4(N

E2

)9

Arg

80

–G

lu9

8(%

)

Arg

80

(NE

)G

lu9

8

[OE

1(2

)]

63

Arg

80

(NE

)G

lu9

8[O

E1

(2)]

34

Arg

80

(NE

)G

lu9

8

[OE

1(2

)]

27

Arg

80

(NE

)G

lu9

8

[OE

1(2

)]

39

Arg

80

(NE

)G

lu9

8

[OE

1(2

)]

75

Arg

80

(NH

2)

Glu

98

[OE

1(2

)]

64

Arg

80

(NH

2)

Glu

98

[OE

1(2

)]

20

Arg

80

(NH

2)

Glu

98

[OE

1(2

)]

39

Arg

80

(NH

2)

Glu

98

[OE

1(2

)]

38

Arg

80

(NH

2)

Glu

98

[OE

1(2

)]

72

Arg

80

–T

yr7

0(%

)

Arg

80

(NH

2)

Ty

r70

(OH

)2

5A

rg8

0(N

H2

)T

yr7

0(O

H)

14

Arg

80

(NH

2)

Ty

r70

(OH

)1

2A

rg8

0(N

H2

)T

yr7

0(O

H)

12

Arg

80

(NH

2)

Ty

r70

(OH

)3

Gly

68

–G

lu9

8(%

)

Gly

68

(O)

Glu

98

(N)

10

0G

ly6

8(O

)G

lu9

8(N

)7

4G

ly6

8(O

)G

lu9

8(N

)8

7G

ly6

8(O

)G

lu9

8(N

)6

4G

ly6

8(O

)G

lu9

8(N

)8

7

Gly

68

(O)

Glu

98

(CG

)5

Gly

68

(O)

Glu

98

(CG

)1

3G

ly6

8(O

)G

lu9

8(C

G)

8

Glu

98

–T

yr7

0(%

)

Glu

98

[OE

1(2

)]

Ty

r70

(OH

)2

0G

lu9

8

[OE

1(2

)]

Ty

r70

(OH

)4

Glu

98

[OE

1(2

)]

Ty

r70

(OH

)1

1G

lu9

8

[OE

1(2

)]

Ty

r70

(OH

)2

4G

lu9

8

[OE

1(2

)]

Ty

r70

(OH

)4

1

Glu

11

–A

rg8

0(%

)

Glu

11

[OE

1(2

)]

Arg

80

(NH

1)

1G

lu1

1

[OE

1(2

)]

Arg

80

(NH

1)

12

Glu

11

[OE

1(2

)]

Arg

80

(NH

1)

1G

lu1

1

[OE

1(2

)]

Arg

80

(NH

1)

2G

lu1

1

[OE

1(2

)]

Arg

80

(NH

1)

9

Glu

11

[OE

1(2

)]

Arg

80

(NH

2)

4G

lu1

1

[OE

1(2

)]

Arg

80

(NH

2)

13

Glu

11

[OE

1(2

)]

Arg

80

(NH

2)

47

Glu

11

[OE

1(2

)]

Arg

80

(NH

2)

9G

lu1

1

[OE

1(2

)]

Arg

80

(NH

2)

23

Glu

11

[OE

1(2

)]

Arg

80

(NE

)5

Glu

11

[OE

1(2

)]

Arg

80

(NE

)1

1G

lu1

1

[OE

1(2

)]

Arg

80

(NE

)3

3G

lu1

1

[OE

1(2

)]

Arg

80

(NE

)1

3G

lu1

1

[OE

1(2

)]

Arg

80

(NE

)1

7

Glu

11

–H

is1

04

(%)

Glu

11

[OE

1(2

)]

His

10

4(N

E2

)8

4G

lu1

1

[OE

1(2

)]

His

10

4(N

E2

)3

Glu

11

[OE

1(2

)]

His

10

4(N

E2

)5

7G

lu1

1

[OE

1(2

)]

His

10

4(N

E2

)1

Glu

11

[OE

1(2

)]

His

10

4(N

E2

)3

5

Th

ep

erce

nta

ge

of

the

enti

reti

me

of

the

un

con

stra

ined

mo

lecu

lar

dy

nam

ics

sim

ula

tio

nd

uri

ng

wh

ich

ace

rtai

nin

tera

ctio

nex

iste

dis

giv

en.

An

inte

ract

ion

issp

ecifi

edas

ah

yd

rog

enb

on

dif

the

dis

tan

ceb

etw

een

the

hy

dro

gen

do

no

ran

dac

cep

tor

was

bel

ow

3.7

A,

and

ifth

ed

on

or–

acce

pto

ran

gle

dev

iate

dle

ssb

yth

an6

0�

fro

m1

80�

J Biol Inorg Chem (2012) 17:801–815 807

123

second water molecule approached the Fe2? ion and

replaced one of the Glu98 oxygens in the first coordination

sphere of iron (see Fig. S1b). The respective coordination

of the Fe2? ion, with a subtle interplay/exchange of water

molecules in its coordination sphere, was retained

throughout the remaining period (about 15 ns) of the MD

simulation. The scenario was in principle similar in all four

Dke1 subunits, with a delay (about 2 ns) in subunit D. The

resulting structure of the metal binding site is stabilized by

a hydrogen bond or strong electrostatic network, Tyr70–

Arg80–Glu98–Gly68 (Table 1), as well as by the van der

Waals and electrostatic interactions established between

the amino acid residues from the first coordination sphere

and neighboring amino acid residues. In detail, the car-

boxylate oxygen of Glu98 is hydrogen-bonded to the

Arg80 guanidino group, and via its backbone to Gly68.

With the side chains of His64 and His104, Glu98 interacts

electrostatically (Fig. 2). The His64 amide nitrogen is

hydrogen-bonded to the Ala102 carbonyl group, and the

His104 imidazole is hydrogen-bonded to the carboxylate

oxygen of Glu11 from the neighboring unit. His62 and

His104 backbones are bonded by two strong hydrogen

bonds. For 59 % of the simulation time of 50 ns, His62

interacted with the Ala60 carbonyl group by forming a

weak hydrogen bond (Fig. 2).

The Phe115 and Phe59 residues, which form a hydro-

phobic patch on one side of the substrate binding pocket,

interact by van der Waals forces and weak CH–p (face-to-

edge) interactions. Leu123 and Phe119, stabilized by the

CH–p and van der Waals interactions with Trp47 form the

other hydrophobic patch (see Fig. S2).

Water channel(s)

During the first 30 ns, water molecules exclusively used

channel T1 to enter the active site. However, since the

separation between the two b strands defining the entrance

of channel T2 increased slightly during the simulations,

water molecules often also entered the active site through

this channel in the last 10 ns of the MD simulations (Fig.

S3). Arg80 defines the border between these two channel

entrances on the Dke1surface (Fig. S3). T1 enters the

active site adjacent to the loop between two b strands

containing residues Thr107 and Gly105. T2 passes by

residues Glu11 and Glu12 (from the neighboring subunit),

and near Tyr70 proceeds to the active site.

Substrate-ligated Dke1

The initial orientation of the substrate in the Dke1 binding

site changed slightly during simulations and finally stabi-

lized in one of two overlapping binding modes (see Fig.

S4). In both binding modes, the substrate (PD) coordinates

the iron ion bidentately and forms a distorted octahedral

coordination together with three histidines and one water

molecule. In the following calculations we considered the

more populated mode (80 %).

First coordination sphere and hydrophilic triad

In the complex between the wild-type protein and PDA, the

octahedral coordination of the iron cation is established by

three histidine residues, the substrate oxygen atoms, and

one water molecule (see Fig. 3). During the MD simula-

tions, Fe2? was predominately six-coordinate. According

to spectroscopic findings [6], there is a mixture of active

sites with and without a sixth ligand (water); in this respect

the strong donor effect of the substrate, which is believed

to result in lower affinity for the sixth ligand, water, cannot

be mimicked via molecular mechanical calculations in

these studies. In the Dke1–PDA complex, Glu98 was

expelled from the first metal coordination sphere, and

formed a strong hydrogen bond with His104 (see Fig. 4;

Table 1), which lasted for more than 21 ns during the 40-ns

MD simulation. However, its interaction with Arg80

decreased (see Table 1). The hydrogen bond between

Glu98 and His104 and that between Arg80 and Glu98 were

weakened to electrostatic interactions during the last 10

and 20 ns of the simulations, respectively. Concurrently,

the Tyr70–Arg80 interaction also became weaker, but the

interaction between Arg80 and Glu11 from the neighboring

subunit (see Fig. 4; Table 1) increased. However, there is a

hydrogen-bond interaction between the aforementioned

glutamate and His104 in the native Dke1 during almost the

entire MD simulation (Table 1), whereas in the complex

Fig. 2 Hydrogen-bonding network in the structure of the substrate-

free, WT enzyme extracted from subunit A obtained after 20 ns of

molecular dynamics (MD) simulation. Amino acid residues are shown

in stick representation, and Fe2? is represented as a pink ball.Hydrogen bonds and strong electrostatic interactions are shown as

dashed lines colored dark blue and cyan, respectively. The life times

of the specified hydrogen bonds are given in Table 1. Glu11 is from

the neighboring unit

808 J Biol Inorg Chem (2012) 17:801–815

123

this interaction does not exist for long. Also, during the

complex simulations a substantial electrostatic interaction

was established between Tyr70 and Glu12 from the

neighboring unit (see Fig. 4); the mean distance between

the Tyr70 side chain oxygen and Glu12 amide oxygen is

about 5.5 A.

Phe59, Phe115, Phe119, Phe125, Leu123, Met117, and

Met77 constitute the predominantly hydrophobic binding

pocket, in which the substrate is accommodated (see

Fig. S5). The substrate interacts by CH–p electrostatic

interactions with Phe59 (in both substrate orientations) and

with Met117 via H–S electrostatic interactions during the

entire MD simulations. Phe115 is stabilized by face-to-face

stacking interactions with Phe51 and by face-to-edge

interactions with Phe59. Met77 interacts with the phenyl

ring of Tyr70 via CH–p interactions (see the Fig. S6 for

some of these interactions).

Water channels

Upon substrate ligation, water molecules were displaced

and left the active site. Therefore, the pathways by which

water can enter and leave the protein were identified.

During the MD simulations of the complex, water mole-

cules used channels T1 and T2 to enter the enzyme active

site (see Fig. S3). Comparison of the free protein structure

and the protein structure in the complex with PDA revealed

that during the simulation of the latter, Arg80 reoriented

and its side chain came close to entrance to the T2 channel

(in the ‘‘free’’ protein, Arg80 is close to the entrance to the

T1 channel). The proximity of Arg80 increased the polarity

of the entrance to the T2 channel and consequently water

molecules are, differently from when it is close to the

entrance to the T1 channel, more often guided to enter the

protein through the T2 channel than through the T1 chan-

nel. To roughly estimate the width of the T1 and T2

channels, we calculated the solvent-accessible surface area

for the amino acid residues defining the entrance to the

channels (at the protein surface) and found that they are

roughly 400 and 300 A2, respectively. Their mean values

during the simulations were 395 ± 28 and 286 ± 25 A2,

respectively, for ligand-free protein and 50 ns of MD

simulations and 443 ± 35 and 280 ± 29 A2, respectively,

for the complex 40 ns of MD simulation. Owing to the

presence of the bidentately coordinated substrate, PDA, the

number of water molecules in the first coordination sphere

of iron is reduced (see Fig. S7, Table S2). However, in the

more distant regions of the active site, namely, up to 3.5 A

from the iron cation, the number of water molecules was

similar, i.e., from 16 to 21 ns and from 36 to 40 ns (an

equilibrium population of water molecules in the protein

was established at about 5 ns in both systems), and the

mean number of water molecules per subunit was 1.9 and

Fig. 3 The first coordination sphere of the complex between the WT

protein and 2,4-pentanedionyl anion (PDA) determined via MD simula-

tion (extracted from subunit A obtained after 21 ns of MD simulation of

the WT–PDA complex). The WT–PDA complex shows octahedral

coordination. Histidine residues coordinating the metal ion and substrate

are shown in stick representation, and Fe2? is represented as a pink ball

Fig. 4 Orientation of the hydrophilic triad Tyr70, Arg80, and Glu98

suggested to be important for stabilization of the transition state [7] of

the WT–PDA complex. The dashed blue line represents the strong

hydrogen bond between the His104 imidazole and the carboxyl group

of Glu98, whereas the dashed cyan line represents the electrostatic

interaction that occasionally forms a hydrogen bond during the MD

simulation. The orientation displayed is from subunit A obtained by

optimizing the WT–PDA complex after 21 ns of MD simulation.

His8, Glu11, and Glu12 are from the neighboring subunit

J Biol Inorg Chem (2012) 17:801–815 809

123

1.8 for free and substrate-ligated wild-type protein,

respectively (in the region up to 5 A, the average number

of water molecules is about 3.7 in both cases).

Protein flexibility

The substrate binding significantly rigidified the protein

structure, and significantly reduced fluctuations of the

otherwise very flexible turn-spanning residues 80–87 (see

Fig. 5).

In summary, a comparison of free and substrate-ligated

iron(II) wild-type Dke1 showed that the metal center in

Dke1 has three-histidine, one-carboxylate ligation in the

resting state and becomes a metal center with three-histi-

dine ligation when the substrate binds [with a small frac-

tion of three-histidine, one-gultamate ligation (11 %)—this

type of ligation appeared only in one subunit when the

system was simulated for more than 22 ns]. The hydro-

philic triad reorients in a way to enable hydrogen-bond

formation between Glu98 and His104.

Mutational analysis of Dke1

The impact of the hydrophilic active-site residues on

catalysis has been characterized previously [7]. It has been

suggested that Glu98, Arg80, and Tyr70 are positioned to

provide a hydrogen-bonding network for the stabilization of

the transition state for O2 reduction. Alternatively, it was

suggested that the residues, which form a hydrophilic gate,

limit access of water molecules to the active site and thus

support formation of the catalytically competent five-

coordinate metal center. (A mixture of five- and six-coor-

dinate substrate-ligated metal centers is observed in Dke1

[6]). To investigate the influence of the hydrophobic resi-

dues in the binding site on the O2 reduction rates, we con-

sequently subjected the active site of Dke1 to mutational

analysis. Phenylalanine residues near the metal center were

replaced by alanine with the aim of increasing the active-

site cavity size and of creating additional space for water in

the active site during catalysis. Such variations are also of

relevance when adapting the scaffold of Dke1 to accept

larger substrates. Expression vectors of five variants,

namely, Phe119Ala, Phe59Ala, Phe115Ala, and Phe125Ala

were constructed. However, the gene product of Phe125Ala

could not be expressed in our hands; the other variant

proteins were produced and purified and O2 reduction rates

were assessed.

Structural characterization of Dke1 variants

To verify that the mutations did not significantly alter the

overall fold and structures of the variants, the secondary

structure composition of all variants was determined via

circular dichroism spectroscopy. The data are summarized

in Table S3 along with results from wild-type Dke1 [27]

and previously reported variants [7]. In summary, similar

contents of secondary structure and no major deviations

from the composition of wild-type Dke1 were found, which

implies that the variants are correctly folded. Furthermore,

the quaternary structures of all the variants were assessed

via gel filtration. Elution chromatograms generally showed

one predominant peak at an elution time that corresponded

to a molecular mass of approximately 70 kDa and was the

same as for wild-type Dke1, within experimental error

(Table S3). Whereas most samples showed a monodisperse

composition, Phe59Ala preparations showed a shoulder,

which accounted for an estimated 5 % of the total absor-

bance area at 280 nm and had an estimated size in the

range 100–200 kDa. However, the higher molecular mass

species were not separated well enough to allow efficient

removal of the impurity by gel filtration. Taken together,

these data clearly show that the overall structure of the

Dke1 variants was not significantly altered by the respec-

tive substitutions.

Metal binding and iron detachment kinetics

All preparations of purified protein retained substantial

amounts of Fe(II) throughout purification, with contents

ranging from approximately 30 to 70 %. Typical metal

contents are given in Table S4. To assess the amount of

Fe3? in the enzyme preparations, the spectrophotometric

Amino acid fluctuations Wild Type

00.5

11.5

22.5

33.5

44.5

5

1 9 17 25 33 41 49 57 65 73 81 89 97 105 113 121 129 137 145

Amino acid residue N°R

MS

F [

Å]

WT_cplx

WT_free

Fig. 5 Root mean square

fluctuation (RMSF) calculated

for the free enzyme and its

complex with PDA. RMSF was

calculated over the last 20 ns (of

40 ns) of the MD simulations,

and was averaged over all

subunits

810 J Biol Inorg Chem (2012) 17:801–815

123

assays were repeated in the presence of 10 mM ascorbic

acid in order to reduce putative Fe3? in the sample to Fe2?

and thus determine the amount of total iron. Generally,

Fe3? contents of 5 % or less were found in the enzyme

preparations. The results are summarized in Table S4. Iron

detachment kinetics were measured as described in

‘‘Experimental methods.’’ The kinetic constants for Fe2?

detachment were rather similar to those for the wild type,

with the most significant exception being the Phe59Ala

variant, which shows an approximately tenfold acceleration

of the ‘‘faster’’ rate of the biphasic iron detachment, mir-

rored by k1Fe. Notably this is not mirrored in an overall

decrease of metal content in the respective enzyme prep-

arations, which indicates a similar affinity of the Phe59Ala

variant for Fe2? compared to Dke1 wild type enzyme. The

determination of thermodynamic Fe2? dissociation con-

stants as described previously [27] revealed large errors

and did not allow us to differentiate between the different

variants and we could only confirm estimated general Kd

values below 50 lM (data not shown).

Enzyme kinetic analysis of the Dke1 variants

Steady-state enzyme kinetic measurements of the variants

Phe59Ala, Phe115Ala, and Phe119Ala were performed at air

saturation using the substrates PD and DPD. (The catalytic

rates for the substrate 1,1,1-trifluoropentanedione were too

small to allow steady-state kinetic measurements.) The

results are summarized in Table 2. All three variants,

Phe59Ala, Phe115Ala, and Phe119Ala, showed a significant

decrease in kcatapp, and a strong impact of the electronic sub-

strate structure on kcatapp was generally observed as previously

described for wild-type Dke1 [17]. KM values were generally

estimated to be below 5 lM; however, the sensitivity of the

spectrophotometric method was too low to allow their pre-

cise determination. Catalytic rates were dependent on O2 up

to 0.6 mM O2 (Fig. S8), in analogy to wild-type Dke1.

Furthermore, single-turnover kinetics of substrate conver-

sion was recorded. Therefore, ligation of the substrate to the

metal center and the subsequent decay of the complex were

monitored by measuring the formation of an absorption band

that evolves upon ligation of the diketonate substrate to the

Fe2? cofactor. Similar bands, where a substrate’s keto moi-

ety coordinates to a mononuclear nonheme Fe2? center, have

been assigned to metal-to-ligand charge transfer transitions

[28]. Anaerobic complexes of the Dke1 variants with PD and

DPD, respectively, gave transitions that were similar

regarding kmax and intensities to those for the respective

wild-type Dke1 complex, with a low-energy absorption band

centered at 420 nm (e = 0.25 ± 3 mM-1 cm-1) and

450 nm (e = 0.28 ± 3 mM-1 cm-1), respectively [26].

Single-turnover kinetics at air saturation showed a buildup of

the enzyme substrate complex to approximately 100 % of

the expected value, followed by a breakdown. Signal for-

mation and decay occurred in different time domains, similar

to the principal characteristics of substrate binding in wild-

type Dke1. Also in analogy, the traces of PD binding were

apparently biphasic, with an initial formation of the enzyme–

substrate complex that was too fast to be assessed and that

accounted for about half of the total amplitude of the

enzyme–substrate complex. The rates of DPD binding were

significantly lower and showed monophasic behavior. The

apparent first-order kinetic constants of PD and DPD binding

(k1 PD and k1 DPD) are shown in Table 2. Whereas the rates

of substrate binding were generally up to fivefold slower in

the Phe59Ala and Phe119Ala variants, the rates were actu-

ally significantly accelerated for the Phe115Ala variant,

leading to full binding of PD to the metal center of the variant

within 3 ms, so the binding event could not be monitored.

The impact of the described variations on substrate binding

was, however, less pronounced than that of the substitution

of phenylalanine for Tyr70, which has been described pre-

viously [26].

The traces of the decay of the enzyme–substrate com-

plex were fit to the equation d[ES]/dt = -k2[ES][O2],

which describes the dependence of the cleavage rate of the

enzyme–substrate complex on the concentration of O2, and

consequently, second-order rate constants (k2 PD and

Table 2 Steady-state and single-turnover kinetics of substrate binding and conversion in wild-type Dke1 [17] and variants

Variant k1 PD (s-1) k2 PD (s-1 mM-1) k1 DPD (s-1) k2 DPD (s-1 mM-1) kcat PDapp (s-1) kcat DPD

app (s-1)

Wild type [26] �/180 [26] 28 55/10 0.13 6.6 0.036

Tyr70Phe [7] 7.15 ND 1.9/0.3 ND 1.27 0.0015

Phe59Ala �/36 2.5 9.8 0.03 0.7 0.008

Phe115Ala �200 7 95 0.08 1.4 0.018

Phe119 Ala �/45 15 24 0.09 3.2 0.022

k1 PD and k1 DPD are the apparent first-order rate constants of PD and DPD binding, respectively, obtained from single-turnover kinetics (in cases

of biphasic binding characteristics, both values are given), whereas k2 PD and k2 DPD are the cO2-dependent second-order rate constants of the

subsequent decay of the enzyme–substrate complex. kcat PDapp and kcat DPD

app are the apparent turnover numbers as determined via steady-state

kinetics at air saturation ðcO2¼ 0:26 mM). The values given have an experimental error of 15 % or less

ND not determined

J Biol Inorg Chem (2012) 17:801–815 811

123

k2 DPD) were determined. When transformed to apparent

turnover numbers at cO2¼ 0:26 mM, these corresponded

well to the actually determined kcatapp values at air saturation.

This finding implies that the enzyme–substrate complex

decays at the same cO2-dependent rate as the rate-deter-

mining step of the reaction, in analogy to wild-type Dke1.

Similar to wild-type Dke1, and previously described vari-

ants, a strong correlation of the velocity of the reaction with

the electronic substrate structure is found, whereby a higher

energy of the occupied molecular orbital increases

kcat=KMO2with a coefficient of Dlog (kcat

app)/DeHOMO =

4 eV-1 (see Fig. S8). This indicates that the rate-deter-

mining step involves the transfer of one or more electrons

from the substrate. Taken together, these data imply that,

like in wild-type Dke1, the rate-determining step is the

oxidation of the enzyme–substrate complex by O2.

Computational analysis of the substrate-ligated Dke1

variants

To elucidate the structural basis for the reduced O2

reduction rates in the respective protein variants, MD

simulations of the respective variants, Phe115Ala,

Phe119Ala, and Phe59Ala, ligated with PDA were per-

formed. For each complex, MD simulations of at least

60 ns in total (20 and 40 ns) were performed (for details

see ‘‘Computational methods’’).

First coordination sphere and hydrophilic triad

The octahedral coordination of the iron cation observed in

the wild-type complex is preserved in these complexes as

well. Analysis of the structure of the amino acid residues

important for the stabilization of the metal ion and for the

protein activity revealed that the positions of His62, His64,

and His104 were well conserved during the all simulations

(altogether 60 ns, two independent simulations, 20 and

40 ns long). In contrast, the orientation of Glu98 in the

complexes with mutated proteins changed compared with its

orientation in the complex between the wild-type enzyme

and PDA, resulting in a weakening of its interaction with

His104 (Table 1). The strength of the interaction between

Glu98 and Arg80 was not been significantly affected in the

complexes with Phe59Ala and Phe115Ala variants, whereas

in the complex with Phe119 it increased. Because of the

reorientation of Glu98, hydrogen bonds between the Arg80

side chain, Ne and NH2, and the Glu98 carboxyl (Ne—OE1/2

and NH2—OE1/2) were disrupted during the initial stage of

the MD simulations of the complexes, but the subsequent

reorientation of Arg80 resulted in their reestablishment. In

the new position, the Arg80 side chain interacts more effi-

ciently with the carboxyl groups of Glu11 from the neigh-

boring unit. The most striking change of the Arg80 position

was found in the Phe115Ala mutant, where the complete

turn following Arg80 (80–87 amino acid residue) is dis-

placed with respect to its position in the wild-type protein

(Fig. S10). In this complex, Arg80 is positioned to perfectly

line the entrance of the T1 channel, making it more hydro-

philic than in the other systems; consequently, in the

Phe115Ala–PDA complex, water molecules mostly use this

channel to enter the protein (see Table S5).

The entry of water could be influenced by the protein

flexibility as well [29], and single point mutations might

induce changes in the local protein flexibility. Analysis of

the relative root mean square fluctuations of the protein

backbone in different complexes during the last 20 ns (of

40 ns) of MD simulations revealed that the selected

mutations resulted in an increase of the protein flexibility,

especially in the region between residues 80 and 89 (see

Fig. S11). The exception is the N terminus (the first 15

amino acid residues), which is more rigid in the Phe59Ala

and Phe119Ala mutants. To summarize, the Phe ? Ala

mutations induced a weakening of the Glu98–His104

interaction and increased the flexibility of the protein.

Analysis of water trafficking through channels T1 and T2

revealed that it depends mostly on the orientation of polar

residues such as Arg80 at the entrances to the channels.

Results of GRID calculations

To investigate a putative impact of the mutations on sta-

bilization of the O2 molecule in the active site, we per-

formed GRID calculations. We used a 3.1-A sphere to

locate possible minima of O2 molecules. The results (see

Table S6) imply that the oxygen affinity in the Phe59Ala

and Phe119Ala variants is comparable to that in the wild-

type protein. However, as outlined in Fig. S12, the respec-

tive sites of highest O2 affinity are somewhat displaced in

the mutants. Notably, the replacement of Phe115 by alanine

leads to destruction of the high-affinity O2 binding site.

Ligand expulsion from the Dke1 active site

To see how mutations influence the substrate (PDA) binding,

we performed RAMD simulations and studied the pathways

the substrate uses to enter the active site of the protein. For

each complex we performed 15 RAMD simulations and

found that in the wild-type protein the substrate mostly used

the T1 channel (the same channel we found water molecules

use to enter the enzyme active site), whereas in the com-

plexes with mutants the substrate more often used the other

routes as well (Table 3). It is interesting that in the

Phe115Ala mutant, the substrate often used the predomi-

nantly hydrophobic T3 channel, which was only rarely used

by water molecules (see the above analysis of the MD sim-

ulations; Fig. 6). This channel extends from the binding site

812 J Biol Inorg Chem (2012) 17:801–815

123

where it is lined with Phe51, Phe59, Phe115, and Phe125 to

the Dke1 surface ending close to Ala27 and Thr148. At the

end where it enters the active site, the channel is wider in the

Phe59Ala and Phe115Ala variants than it is in the native

protein; consequently, for the substrate transport in the native

protein, it was used in 40 % of cases, whereas in the

Phe59Ala and Phe115Ala mutants, it was used in 65 and

85 % of cases, respectively. The T3 channel is also the main

route for the products of the catalytic reaction (methylgly-

oxal and acetate) to exit in the wild-type complex (no sim-

ulations of product exit were performed for variants).

Discussion

First coordination sphere of the metal center of wild-

type Dke1

For the purpose of MD simulations of the metalloenzyme,

the parameters for the nonheme iron ion in Dke1 were

derived using Hartree–Fock quantum mechanical calcula-

tions. During the simulations of the free Dke1 as well as its

complex with PDA, the Fe2? ion was mostly six-coordi-

nated (distorted octahedral coordination), with the five-

coordinated form being present for about one tenth of the

simulation time. This is in good agreement with experi-

mental data [6], where a six-coordinate species was found

for free wild-type Dke1, whereas the substrate-ligated

complex was a mixture of six- and five-coordinate species.

Whereas in the work of Diebold et al. [6] it was suggested

that the Glu98 residue in wild-type Dke1 stabilizes a metal-

bound water molecule in the resting enzyme, the results

from MD simulations now suggest that Glu98 itself binds to

the metal center, establishing in this way a more charge-

neutral center within the hydrophobic core of the protein.

The crystal structure of Fe(III)-quercetin dioxygenase1 [31]

and the structure of Fe(II)-acireductone dioxygenase1,

which was solved by NMR and X-ray absorption spec-

troscopy [32] revealed six-coordinated metal centers—

three-histidine, one-carboxylate ligated—in the resting

enzyme—comparable to the results of our MD simulations.

However, upon substrate binding, Glu98 is eliminated from

the first coordination sphere, so the catalytically competent

active site displays a three-histidine-ligated Fe2? center.

Substrate, product, and water trafficking in Dke1

We found that substrate binding pushed the Glu98 carboxyl

away from Fe2? and disrupted the three-histidine, one-

glutamate coordination of the iron ion. Also, it rigidified

the enzyme structure and influenced the distribution of

possible paths for water to enter the active site.

Substrates egress from the enzyme active-site pocket

was simulated using RAMD. These simulations revealed

two main routes that may be used by a substrate molecule

to enter and leave the Dke1 binding site. In the wild-type

Table 3 Results of random-acceleration molecular dynamics simulations for substrate and products in the complexes (with the WT and mutated

enzymes)

Channel WT–PDA Phe59Ala–PDA Phe115Ala–PDA Phe119Ala–PDA WT–MG WT–AC

Channel

T1 40 20 10 20 0 15

T2 15 0 5 15 0 0

T3 40 65 85 60 100 77

Other 5 15 0 5 0 8

In the second to fifth columns, the substrate expulsion paths in the complexes with the WT protein and mutants are given; in the last two columns,

the expulsion paths of the products in the WT complex are given. Each simulation was performed 20 times, and the percentage of usage of each

of the tunnels, either by substrate or by product molecules, is given

PDA 2,4-pentanedionyl anion, MG methylglyoxal, AC acetate

Fig. 6 Water and substrate entering/exiting channels determined

during MD and random-acceleration MD simulations as determined

for WT Dke1 (dark blue T1, magenta T2, and orange T3). The

substrate and water used the same paths to pass through the protein.

The channels are displayed in subunit A of the Dke1–PDA complex.

The tunnel topology was the same for the respective variants. (The

figure was prepared with the help of CAVER [30])

1 Structures of an Fe(III) containing quercentin 2,3-dioxygenase from

Bacillus subtilis (PDB code 1Y3T) and of Fe(II) ligated acireductone

dioxygenase from Klebsiella oxytoca (PDB code 2HJ1).

J Biol Inorg Chem (2012) 17:801–815 813

123

protein, PDA was mostly transported through the T1

channel, extending from Arg80 at the surface to Thr107 in

the binding pocket, whereas in the variants the T3 channel

was used most often, the bottom end of which is sur-

rounded with hydrophobic residues Phe51, Phe59, Phe115,

and Phe125. Since alanine is smaller than phenylalanine, it

is wider in the Phe59Ala and Phe115Ala mutants and more

passable than in the wild-type protein.

Since the product molecules (methylglyoxal and acetate)

used the T3 channel exclusively to exit the protein, their

exchange with the substrate in the variant is expected to be

hindered to some extent in comparison with the exchange

in the native protein.

The role of the protein environment in O2 reduction

catalysis

Mutational analysis, both experimental and theoretical,

showed that the single point mutations of the hydrophobic

residues lining the binding site do not influence significantly

the overall protein fold. However, MD simulations revealed

an increase of the enzyme flexibility for the variants, espe-

cially for the most flexible region, the region between amino

acid residues 80–87, of which Arg80 is a part. Arg80 inter-

acts with Glu98 and Tyr70, amino acid residues from the

enzyme active site, and directs the water molecules to enter

either the T1 or the T2 channel leading to the metal ion. The

kinetic analysis revealed that changes of the hydrophobic

residues (Phe ? Ala mutations) lead to an up to tenfold

decrease of the O2 reduction rate. Furthermore, the analysis

of the protein complexes with PDA showed that whereas the

interactions of the amino acid residues important for the

stabilization of the metal ion were well conserved in all

complexes during the MD simulations, the Glu98–His104

interaction was much stronger in the complex with the wild-

type enzyme than in the Phe ? Ala mutants. Previous

experimental studies have shown that mutation of Glu98 has

a strong, 100-fold, impact on the rate-determining step of O2

reduction. Consequently, structural perturbations that

impede the correct positioning of Glu98 may also lead to a

decrease in O2 reduction rates. We propose that the hydrogen

bond between His104 and Glu98, where His104 donates its

H? from Ne to the Glu98 carboxyl group, renders His104

more electronegative, thus stabilizing an Fe3? species,

which is believed to be a transient species during O2 reduc-

tion [28]. Previously suggested catalysis-promoting struc-

tural features [7], such as increased accessibility of water to

the active site or a putative hydrogen bond positioned by the

hydrophilic triad such that it could stabilize the transition

state of O2 reduction, were not supported by our study.

Acknowledgments This work is the result of the bilateral Croatian–

Austrian collaboration project ‘‘Metal centers in enzymes and

proteins.’’ The authors acknowledge the Ministry of Science, Edu-

cation, and Sport of the Republic of Croatia (project 098-0982913-

2748) and the Austrian Academic Exchange Service (WTZ project

HR 26/2008) for their financial support. G.D.S. acknowledges the

support of the FWF (P18828).

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