Application of DFT methods to the study of the coordination environment of the VO2+ ion in...

ORIGINAL PAPER

Application of DFT methods to the study of the coordinationenvironment of the VO2+ ion in V proteins

Daniele Sanna • Vincent L. Pecoraro •

Giovanni Micera • Eugenio Garribba

Received: 21 December 2011 / Accepted: 18 March 2012 / Published online: 15 April 2012

� SBIC 2012

Abstract Density functional theory (DFT) methods were

used to simulate the environment of vanadium in several

V proteins, such as vanadyl-substituted carboxypeptidase

(sites A and B), vanadyl-substituted chloroplast F1-ATPase

(CF1; site 3), the reduced inactive form of vanadium

bromoperoxidase (VBrPO; low- and high-pH sites), and

vanadyl-substituted imidazole glycerol phosphate dehy-

dratase (IGPD; sites a, b, and c). Structural, electron

paramagnetic resonance, and electron spin echo envelope

modulation parameters were calculated and compared with

the experimental values. All the simulations were per-

formed in water within the framework of the polarizable

continuum model. The angular dependence of ANiso

��

�� and

ANz

�� on the dihedral angle h between the V=O and N–C

bonds and on the angle u between the V=O and V–N

bonds, where N is the coordinated aromatic nitrogen atom,

was also found. From the results it emerges that it is

possible to model the active site of a vanadium protein

through DFT methods and determine its structure through

the comparison between the calculated and experimental

spectroscopic parameters. The calculations confirm that the

donor sets of sites B and A of vanadyl-substituted car-

boxypeptidase are [COO�Glu, H2O, H2O, H2O] and [NHis(||),

NHis(\), COO�Glu, H2O], and that the donor set of site 3 of

CF1-ATPase is [COO�Asp, OHThr, H2O, H2O, NHax2Lys]. For

VBrPO, the coordination modes [NHis(||), NHis(\), OHSer,

H2O, H2Oax] for the low-pH site and [NHis(||), NHis(\),

OHSer, OH–, H2Oax] or [NHis(||), NHis(\), O�Ser, H2O] for the

high-pH site, with an imidazole ring of histidine strongly

displaced from the equatorial plane, can be proposed.

Finally, for sites a, b, and c of IGPD, the subsequent de-

protonation of one, two, and three imidazole rings of his-

tidine and the participation of a carboxylate group of a

glutamate residue ([NHis(||), COO�Glu, H2O, H2O], [NHis(||),

NHis(||), COO�Glu, H2O], and [NHis(||), NHis(||), COO�Glu,

OH-, NaxHis], respectively) seems to be the most plausible

hypothesis.

Keywords Vanadium � Proteins � Density functional

theory methods � Electron paramagnetic resonance

spectroscopy � Electron spin echo envelope modulation

spectroscopy

Introduction

Vanadium plays a number of roles in biological systems

and has been found in many naturally occurring com-

pounds [1, 2]. Vanadium is comparatively rare among

biometals but is an environmentally ubiquitous element,

and is relatively abundant in seawater, where its

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-012-0895-y) contains supplementarymaterial, which is available to authorized users.

D. Sanna

Istituto CNR di Chimica Biomolecolare, Trav. La Crucca 3,

07040 Sassari, Italy

V. L. Pecoraro

Department of Chemistry, University of Michigan,

930 N. University Ave, Ann Arbor, MI 48109-1055, USA

G. Micera � E. Garribba (&)

Dipartimento di Chimica e Farmacia,

Centro Interdisciplinare per lo Sviluppo della Ricerca

Biotecnologica e per lo Studio della Biodiversita della Sardegna,

Universita di Sassari, Via Vienna 2, 07100 Sassari, Italy

e-mail: [email protected]

123

J Biol Inorg Chem (2012) 17:773–790

DOI 10.1007/s00775-012-0895-y

http://dx.doi.org/10.1007/s00775-012-0895-y

concentration is approximately 30 nM, classifying it as the

second most (next to molybdenum) abundant transition

metal in the oceans [2]. In the sea, vanadium is accumu-

lated by the fan worm Pseudopotamilla occelata belonging

to the polychetes (bristle worms) [3], and by ascidians, a

class of the subphylum Tunicata [4]. In ascidians, vana-

dium is found in special blood cells, the vanadocytes, and

is bound by proteins named vanabins [5]. Systematic

investigations on Shewanella oneidensis have shown that

bacteria can use external vanadate as a substrate for elec-

tron delivery [6]. High amounts of vanadium were also

discovered in Amanita muscaria, where vanadium is

present in a compound called amavadin, a low molecular

mass, anionic nonoxidovanadium(IV) complex derived

from N-hydroxyimino-2,20-diisopropionic acid—(S,S)-

H3hidpa—in its triply deprotonated form, hidpa(3-) [7, 8].

Two enzymes contain vanadium, vanadium-dependent

haloperoxidases (VHPOs) [9] and nitrogenase [10]. The

structures of vanadium chloroperoxidase (VClPO) from

Curvularia inaequalis [11] and vanadium bromoperoxidase

(VBrPO) from Ascophyllum nodosum [12] were reported,

in which a vanadate anion H2VO4- is bonded to the protein

via a histidine nitrogen; vanadium is in the center of a

slightly distorted trigonal bipyramid, with the oxygen

donors occupying the three equatorial positions and one

axial position.

In humans, vanadium compounds exhibit a wide variety

of pharmacological properties, and many complexes have

been tested as antiparasitic, spermicidal, antiviral, anti-

HIV, antituberculosis, and antitumor agents [13]. Vana-

dium has been also proved to be one of the most efficient

metal ions with insulin-like effects [14–17]. Moreover,

vanadate inhibits several groups of phosphate-metabolizing

enzymes, such as phosphatases, ribonucleases, and ATP-

ases [1, 18].

Vanadium shows a very rich chemistry and forms an

enormous variety of compounds, in which the most

important oxidation states are ?III, ?IV, and ?V [1, 2].

The ?IV state is dominated by the complexes formed by

the oxidovanadium or vanadyl ion, VO2?, whose impor-

tance is related to their insulin-enhancing activity and to

the stable form of vanadium in biofluids and cellular

environments. In vivo blood studies on rats showed that,

almost independently of the initial oxidation state of the

vanadium compound, the metal ion seems to be transported

in the blood in oxidation state ?IV [19], which can be

further stabilized by interaction with the bioligands present

in the bloodstream.

One of the most important and interesting uses of the

VO2? ion is as a physicochemical marker of the metal

binding sites in proteins: electron paramagnetic resonance

(EPR) patterns of the oxidovanadium ion have been

employed since the early 1970s by Chasteen and coworkers

[20] to extract specific information on the metal binding

sites of peptides and proteins.

The geometry and its modification can be involved in

the activity of naturally occurring compounds. For exam-

ple, the inactivation of VHPOs, owing to the reduction of

VVO to VIVO species, seems to be connected to an irre-

versible structural change [9, 11]. Unfortunately, in the

case of complex systems, such as vanadium proteins and

other natural vanadium species, the resolution of the

structure through X-ray diffraction analysis is often not

possible and, thus, other physicochemical tools must be

used to characterize the compounds and study the proper-

ties and interaction of vanadium with the biomolecules.

One resource is, of course, represented by the usual spec-

troscopic techniques [NMR, EPR, electron spin echo

envelope modulation (ESEEM), electron–nuclear double

resonance, UV–vis, and IR spectroscopies] [2, 21]. How-

ever, over the last few years, density functional theory

(DFT) methods have been demonstrated to give excellent

results in the optimization of the geometries of transition

metal complexes and in the prediction of molecular and

spectroscopic properties [22]. Zampella et al. [23, 24] have

applied DFT methods to investigate structural properties of

complexes related to the resting and peroxo forms of the

active site of VHPO. Recently, we used these methods to

infer the coordination modes of the VO2? ion in the

binding sites of albumin [25] and immunoglobulin G [26]

and to propose that the form with which a cis-octahedral

insulin-enhancing agent is transported in the blood serum is

probably cis-VO(carrier)2(protein), where ’’carrier’’ is a

bidentate monoanionic ligand and the protein can be

albumin, immunoglobulin G or, if the carrier is not a

synergistic anion, also transferrin [26–29].

On these bases, in this work we used DFT methods to

study the coordination environment of VO2? in vanadium

proteins, such as vanadyl-substituted carboxypeptidase

(sites A and B), vanadyl-substituted chloroplast F1-ATPase

(CF1; site 3), the reduced inactive form of VBrPO (low-

and high-pH sites), and vanadyl-substituted imidazole

glycerol phosphate dehydratase (IGPD; sites a, b, and c).

The methods used were first validated on simple VO2?

coordination compounds and, subsequently, applied to the

model compounds of the metal site in vanadium proteins.

DFT calculations

All the calculations presented in this article were per-

formed with Gaussian 03 (revision C.02) [30] and DFT

methods [31].

The structures were optimized with the hybrid

exchange–correlation functionals B3LYP [32, 33],

B3PW91 [32, 34, 35], and B3P86 [32, 36, 37], and the

774 J Biol Inorg Chem (2012) 17:773–790

123

basis set 6-311g or 6-311?g(d) [38]. For all the structures,

minima were verified through frequency calculations. The

solvent (water, methanol, ethanol, or dimethyl sulfoxide)

effect was simulated within the framework of the polariz-

able continuum model (PCM) [39–41].

The metal sites of the vanadium proteins were simulated

using 4-methylimidazole for histidine nitrogen (NHis),

acetate for Glu-COO- or Asp-COO- (COO�Glu, and

COO�Asp), 4-methylphenolate for Tyr-O- (O�Tyr), methoxide

for Ser-O- or Thr-O- (O�Ser and O�Thr), methanol for

Ser-OH or Thr-OH (OHSer and OHThr), methylthiolate for

Cys-S- (S�Cys), and butylamine for Lys-NH2 (NH2Lys)

coordination. The symbols NHis(||), NHis(\), and NHis(\)

indicate an imidazole aromatic ring of a histidine residue

arranged parallel and perpendicular to the V=O bond and

significantly displaced from the equatorial plane of the

VO2? ion, respectively.

The optimized structures were used to calculate the 51V

(Aiso, Ax, Ay, and Az) and 14N (ANiso, AN

x , ANy , and AN

z )

hyperfine coupling constants. The 51V hyperfine coupling

constants were calculated at the BHandHLYP/6-311g(d,p)

level of theory, with the half-and-half functional

BHandHLYP used as incorporated in Gaussian 03. In the

first-order approximation, the 51V hyperfine coupling ten-

sor A has one isotropic contribution deriving from the

Fermi contact (Aiso) and another from the dipolar hyperfine

interaction (tensor T): A = Aiso1 ? T [42, 43]. As dem-

onstrated in the literature, DFT simulations are a valid tool

for predicting EPR parameters of VO2? complexes [44–

46]. Using the half-and-half hybrid BHandHLYP func-

tional and the 6-311g(d,p) basis set, one can calculate the51V hyperfine coupling constant along the z-axis (Az) with a

mean deviation from the experimental value lower than

3 % [47, 48]. It must be remembered that for a VO2?

species the Az value is usually negative, even if in the

literature its absolute value is commonly reported. The 14N

hyperfine coupling constants were calculated at the

BHandH/6-311g(d,p) level of theory, with the functional

BHandH incorporated in the Gaussian 03 package.

BHandHLYP and BHandH include a mixture of exact

Hartree–Fock and DFT methods to calculate the exchange–

correlation (EXC) and this seems to be necessary to simu-

late correctly Aiso and ANiso, which depend on the indirect

core level spin polarization arising from the unpaired spin

density in the metal d orbitals. In particular, BHandHLYP

is defined as 0:5� EHFX þ 0:5� ELSDA

X þ 0:5� EBX þ ELYP

C

and BHandH as 0:5� EHFX þ 0:5� ELSDA

X þ ELYPC , where

EHFX , ELSDA

X , EBX, and ELYP

C are the energies due to the

Hartree–Fock exchange, the local spin density approxi-

mation exchange functional, the gradient-corrected Becke

88 exchange functional, and the gradient-corrected Lee–

Yang–Parr correlation functional, respectively.

Results and discussion

Validation of the DFT methods

The accuracy of the levels of theory adopted in this study in

the prediction of the geometry and spectroscopic parame-

ters (in particular, EPR and ESEEM data) must be first

validated on simple coordination compounds formed by the

VO2? ion. Buhl et al. [49, 50] recently ranked some pop-

ular functionals for the first-row transition metal complexes

in their capability of predicting metal–donor distances.

They observed that, among the functional studied, B3P86

and B3PW91, composed of one exchange and one corre-

lation part, afford the lowest deviations from the experi-

mental values [50]. For this reason, in this work we tested

these two functionals and added B3LYP, which is the most

popular functional and is considered the benchmark for

evaluating the performances of the other functionals.

Bond distances were calculated for nine VO2? com-

plexes with V–O, V–N, and V–S bonds: [VO(H2O)5]2? (1)

[51], [VO(acac)2] (2), where acac is acetylacetonato(-)

[52], [VO(ma)2] (3), where ma is maltolato(-) [53],

[VO(salen)] (4), where salen is N,N0-ethylenebis(salicy-

lideneiminato)(2-) [54], [VO(PAIS)] (5), where PAIS is

N-(2-salicylideneamino)phenyl)pyridine-2-carboxamidato

(2-) [55], trans-[VO(bpb)(H2O)] (6), where bpb is 1,2-

bis(2-carboxamidopyridyl)benzenato(2-) [56], [VO(tma)2]

(7), where tma is 2-methyl-3-oxy-4H-pyran-4-thionato(-)

[57], [VO(mnt)2]2- (8), where mnt is 1,2-dicianoethylene-

1,2-dithiolato(-) [58], and [VO(edt)2]2- (9), where edt is

ethane-1,2-dithiolato(2-) [59]. The functionals B3LYP,

B3PW91, and B3P86, coupled with the basis set 6-311g,

were used; for the structures containing V–S bonds, diffuse

and polarization functions were also added on the sulfur

atom. Finally, the effect of the solvent (water for 1, 3, and

7, methanol for 2, 4, 5, and 8, ethanol for 9, and dimethyl

sulfoxide for 6) was simulated within the framework of the

PCM. The results are shown in Figs. 1, S1, and S2, where

the calculated V–O, V–N, and V–S distances are compared

with the experimental ones. Firstly, there is a general ten-

dency for the all-electron calculations to overestimate the

length of the metal–donor bonds [50]. It is also possible to

observe that the popular B3LYP functional is somewhat

worse than B3PW91 and B3P86, and that B3P86 yields the

best agreement with the experimental values. From the data

obtained, the order of accuracy of the functionals tested in

the prediction of the bond distances is B3P86 [B3PW91 [ B3LYP. To improve further the prediction of

the V–S bonds it is, however, necessary to use a general

basis set (the basis set is indicated as B3P86/gen in the

caption for Fig. S2) with 6-311?g(d) on the sulfur atom

and 6-311g on the other atoms. Finally, all the vanadium–

donor distances closely approach the experimental ones if

J Biol Inorg Chem (2012) 17:773–790 775

123

the solvent effect within the framework of the PCM is also

considered. We will indicate these calculations as B3P86

(PCM) for V–O and V–N bonds (Figs. 1, S1) and B3P86/

gen (PCM) for V–S bonds (Fig. S2). For example, the V–O

distance in [VO(acac)2] goes from 1.994 to 1.984, 1.978,

and 1.974 A when the B3LYP, B3PW91, B3P86, and

B3P86 (PCM) levels of theory are used (experimental

value of 1.969 A); for [VO(PAIS)], the calculated mean

distance of the V–N bond is 2.071, 2.057, 2.052, and

2.046 A, respectively (2.040 A experimental). For

[VO(edt)2]2- mean V–S distances of 2.483, 2.457, 2.449,

2.415, and 2.384 A (2.378 A experimental) are obtained

with the B3LYP, B3PW91, B3P86, B3P86/gen, and

B3P86/gen (PCM) levels of theory.

Therefore, we simulated the vanadium site in vanadium

proteins using the B3P86 functional, the 6-311g basis set

(or B3P86/gen if there are sulfur donors bound to vana-

dium) and the PCM.

The subsequent step is the validation of the DFT sim-

ulations used for the prediction of the EPR (51V hyperfine

coupling constant tensor A) and ESEEM (14N superhy-

perfine coupling constant tensor AN) parameters. In par-

ticular, the 51V hyperfine coupling constant along the z-axis

(Az) allows the identification of the equatorial donors of a

VO2? species through the application of the ‘‘additivity’’

rule, which was proposed by Wuthrich [60], developed by

Chasteen [61], and further improved by Smith et al. [62];

furthermore, it must be noted that vanadium proteins usu-

ally give an anisotropic signal also at room temperature for

the slow rotational motion of a VO2? species in aqueous

solution, with the consequence that only Az can be mea-

sured [63]. With DFT methods it is possible to calculate

EPR parameters of transition metal complexes [22, 64],

and more particularly of a VO2? species [44–48]. In par-

ticular, it has recently been demonstrated that it is possible

to predict Az for a VO2? complex [through a simulation at

the BHandHLYP/6-311g(d,p) level of theory] with a mean

deviation lower than 3 % with respect to the experimental

value [47, 48]. The value of Az was calculated for six VO2?

species with VO(O5), VO(O4), VO(N2O2), VO(N3O),

VO(O2S2), VO(S4), and VO(N4) coordination (the experi-

mental Az were taken from the references indicated):

[VO(H2O)5]2? (1) [61], [VO(acac)2] (2) [65], [VO(salen)]

(4) [66], [VO(PAIS)] (5) [55], [VO(tma)2] (7) [57],

[VO(mnt)2]2- (8) [58], [VO(en)2]2? (10), where en is

ethylenediamine [61], and [VO(imid)4]2? (11), where imid

is imidazole [67]. The hybrid functionals B3LYP,

B3PW91, and B3P86 and the half-and-half hybrid func-

tional BHandHLYP, coupled with the 6-311g(d,p) basis

set, were tested, and the results are presented in Fig. 2. The

atomic coordinates obtained after a simulation at the

B3P86 (PCM) level of theory were used for the compounds

containing V–O and/or V–N bonds, and at the level B3P86/

gen (PCM) for the compounds containing V–S bonds.

Whereas all three hybrid functionals significantly

underestimate Az, the prediction of the half-and-half

hybrid functional BHandHLYP is much more accurate.

The mean of the percent deviation from the experimental

values, calculated as 100 9 (|Az|calcd - |Az|

exptl)/|Az|exptl, is

-16.0 % for B3LYP, -12.1 % for B3PW91, -13.8 % for

B3P86, and -0.1 % for BHandHLYP. The order of

Fig. 1 Comparison of the V–O distances calculated at different

levels of theory with the experimental ones. PCM is polarizable

continuum model

Fig. 2 Comparison of the 51V

Az values calculated at the

different levels of theory with

the experimental ones

776 J Biol Inorg Chem (2012) 17:773–790

123

accuracy in the prediction of Az is BHandH-

LYP � B3PW91 [ B3P86 [ B3LYP. On the basis of our

data, the use of the BHandHLYP functional and the 6-

311g(d,p) basis set after an optimization at the B3P86/6-

311g level of theory (or B3P86/gen for the compounds

with V–S bonds) within the framework of the PCM ensures

a very good prediction of Az. Therefore, it can be supposed

that an analogous agreement between |Az|calcd and |Az|

exptl is

obtained when DFT methods are applied to the model site

of a vanadium protein. As recently pointed out, the better

performance of BHandHLYP than the other functionals is

related to the prediction of the Fermi contact term, which

depends on the indirect core level spin polarization arising

from the unpaired spin density in the metal d orbitals. The

spin polarization is difficult to simulate with high accuracy,

and most of the functionals underestimate it significantly

[45, 68]. The higher fraction of Hartree–Fock exchange in

half-and-half functionals such as BHandHLYP, which is

available in Gaussian 03, improves considerably the pre-

diction of the Fermi contact and, thus, of Az (see ‘‘DFT

calculations’’).

The short relaxation times in EPR spectroscopy produce

lines that are very broad (typically, around 10 G) and this

precludes, in most of the cases, the resolution of the cou-

pling of the unpaired electron on vanadium with the nuclei

in the ligand sphere, quantified by the superhyperfine

coupling constant AL (where L indicates a ligand). Infor-

mation on this superhyperfine coupling can be provided by

ESEEM spectroscopy, a variant of EPR spectroscopy [62,

69–71]. The superhyperfine coupling constants reported for14N are in the range 1–8 MHz and vary with the nature,

position, and orientation of the ligands [72–74]. In studies

on the identification of nitrogen donors bound to vanadium

in VO2? species, both ANiso and AN

z are used. Few compu-

tational studies have been published in which ligand

superhyperfine coupling constants for transition metal

complexes were calculated [75–77] and, to the best of our

knowledge, only one concerns the VO2? ion [77].

Neese [75] showed that for Cu2? complexes, hybrid

functionals such as B3LYP and PWP1 give better predic-

tions than functionals based on the generalized gradient

approximation such as BP and BLYP. The following VO2?

species, for which ESEEM data are available in the liter-

ature, were simulated: [VO(salen)] (4) [72], cis-[VO(ed-

da)(H2O)] (12), where edda is ethylene-N,N0-diacetate(2-)

[72], [VO(hybeb)]2- (13), where hybeb is 1,2-bis(2-hy-

droxybenzamido)benzenate(4-) [78], [VO(meox)2] (14),

where meox is 2-methyl-8-hydroxyquinolinate(-) [79],

and cis-[VO(Hhida)(H2O)] (15), where Hhida is N-(2-

hydroxyethyl)iminodiacetate(2-) [74]. We tested several

functionals: besides B3LYP, B3PW91, B3P86, and

BHandHLYP, also another half-and-half functional

BHandH, which is available in Gaussian 03, was used. The

difference between BHandHLYP and BHandH is the term

EBX due to the gradient-corrected Becke 88 (B) exchange

functional, which is absent in BHandH (see ‘‘DFT calcu-

lations’’). In this case too, we used for the compounds

containing V–O and/or V–N bonds the atomic coordinates

obtained after a simulation at the B3P86 (PCM) level of

theory and for the compounds with V–S bonds those

obtained after a simulation at the B3P86/gen (PCM) level

of theory. The values of ANiso and AN

z calculated are shown

in Fig. S3.

As can be seen, BHandH and B3PW91 perform better

than the other functionals. The order of accuracy is

BHandH * B3PW91 � B3P86[BHandHLYP[B3LYP.

In agreement with the experiments, all the functionals

predict a small value of ANiso and AN

z for an axially coordi-

nated nitrogen, like the amino nitrogen in cis-[VO(Hhida)

(H2O)] [74], but BHandH gives the best results. For this

reason, the latter functional is preferred to the others in the

application to the models of vanadium proteins.

Variation of the 14N superhyperfine coupling constant

with respect to the V=O bond and histidine residue

The characterization of VO2? biomolecules relies strongly

on EPR and ESEEM spectroscopy, particularly through the

application of the ‘‘additivity’’ rule for the prediction of Az.

It has been shown experimentally that the Az values of

VO2? imidazole species have a critical angular dependence

[80] and this has been reconfirmed through DFT methods

[81, 82]. A dependence of the 14N superhyperfine coupling

constant tensor AN on the dihedral angle h between the

V=O and N–C bonds, where C is the carbon that bridges

the two nitrogen atoms in the imidazole ring, has also been

proposed [81]. The data obtained with a scalar relativistic

spin-unrestricted open-shell Kohn–Sham approach can be

fitted by the equation ANiso

��

�� = 7.47 ? 0.59 9 sin(2h -

90). However, as noticed by the authors, ANiso

��

�� is system-

atically larger than the experimental value by approxi-

mately 15 %. In our opinion, this is due to the use of the

BP86 functional, which does not reproduce satisfactorily

the core level spin polarization.

In this work, we reexamined the dependence of ANiso

��

�� on

the dihedral angle h and tested for the first time that of

ANz

��; the calculations were performed on the complex

[VO(imid)(H2O)3]2? by varying the dihedral angle hbetween the V=O and N–C bonds from 0� to 180� (i.e.,

from a parallel to a perpendicular orientation of the aro-

matic ring with respect to the V=O bond). Furthermore,

since it has been observed that ANiso

��

�� for the axial coordi-

nation of an imidazole nitrogen is significantly reduced

J Biol Inorg Chem (2012) 17:773–790 777

123

with respect to that of an equatorial nitrogen [74], we used

DFT calculations to elucidate the dependence of ANiso

��

�� and

ANz

�� on the angle between the V=O and V–N bonds, where

N is the coordinated aromatic nitrogen atom (indicated

with u); in this case u is varied from 90� to 180� (i.e., from

a perfectly equatorial to a perfectly axial imidazole

nitrogen).

The dependence of ANiso

��

�� and AN

z

�� on the dihedral angle

h is displayed in Fig. 3. The values range from 6.3 to

7.0 MHz ( ANiso

��

��) and from 6.6 to 7.7 MHz ( AN

z

��). As

demonstrated in ‘‘Validation of the DFT methods,’’ the use

of the BHandH functional allows us to obtain good

agreement between calculated and experimental values of

the superhyperfine coupling constants. Least-squares fits

of the calculated values of ANiso

��

�� and AN

z

�� yield the curves

of Eqs. 1 and 2.

ANiso

��

�� ¼ 6:65þ 0:34 � sinð2h� 90Þ; ð1Þ

ANz

�� ¼ 7:19þ 0:55� sinð2h� 90Þ: ð2Þ

The variation of ANiso

��

�� and AN

z

�� follows the functional

form of sin(2h - 90) described by Smith et al. [80] for the

imidazole contribution to the vanadium hyperfine coupling

constant. Interestingly, the value calculated in this work is

approximately 11 % smaller than that reported by Saladino

and Larsen [81].

The dependence of ANiso

��

�� and AN

z

�� on the angle u

between the V=O and V–N bonds is shown in Fig. 4. It is

noteworthy that on increasing the value of u, i.e., moving

the imidazole ring from an equatorial toward an axial

position, both ANiso

��

�� and AN

z

�� values strongly decrease; in

particular, ANiso

��

�� goes from 7.3 MHz (u = 90�) to

0.2 MHz (u = 180�). In this case too, a sine function

allows interpolation of the data, with the dependence on the

angle u expressed by Eqs. 3 and 4:

ANiso

��

�� ¼ 3:89þ 3:28� sinð2u� 90Þ; ð3Þ

ANz

�� ¼ 4:33þ 3:09� sinð2u� 90Þ: ð4Þ

Of course, it must be pointed out that the physical

meaning of these equations (which, for the moment, have

to be considered as simple interpolation formulas) must be

clarified.

Application of DFT methods to vanadium proteins:

carboxypeptidase

DeKoch et al. substituted VO2? ion into the active site of

carboxypeptidase A and observed two sets of resonances

with |Az|exptl of 175.8 9 10-4 cm-1 (B site) and

165.9 9 10-4 cm-1 (A site, active site), the first domi-

nating below pH 5 and the second above 5 [83]. They

assigned the first site to a carboxylate coordination and the

second to a coordination environment of two imidazoles

and two water molecules.

We performed DFT simulations in water within the

framework of the PCM (using the B3P86 functional and

the 6-311g basis set) on model complexes of the metal sites

with the following donor sets: [COO�Glu, H2O, H2O, H2O],

[COO�Glu, H2O, H2O, H2O, H2Oax], [NHis(||), NHis(\),

COO�Glu, H2O], [NHis(||), NHis(||), COO�Glu, H2O], and

[NHis(||), NHis(\), COO�Glu, OH-] (Scheme 1). The values

of |Az|calcd (|Az|

calcd denotes the value obtained by DFT

simulations) are reported in Table 1. From the results

discussed in ‘‘Validation of the DFT methods,’’ the struc-

tures that give a deviation of |Az|calcd higher than 2 % with

respect to the experimental values can be rejected; thus, it

can be concluded that the most probable coordination for

sites B and A, respectively, are [COO�Glu, H2O, H2O, H2O],

and [NHis(||), NHis(\), COO�Glu, H2O]. On the basis of the

Fig. 3 Angular dependence of ANiso

��

�� and AN

z

�� on the dihedral angle h

between the V=O and N–C bonds. Values calculated at the BHandH/

6-311g(d,p) level of theory

Fig. 4 Angular dependence of ANiso

��

�� and AN

z

�� on the angle u between

V=O and V–N bonds, where N is the coordinated aromatic nitrogen

atom. Values calculated at the BHandH/6-311g(d,p) level of theory

778 J Biol Inorg Chem (2012) 17:773–790

123

X-ray structure of bovine carboxypeptidase [84], the resi-

dues involved in the metal coordination should be Glu-72,

His-69, and His-196, one water molecule completing the

equatorial plane of VO2? ion. The coordination environ-

ment of vanadium in the two sites of vanadyl-substituted

carboxypeptidase is represented in Fig. 5. The possible

structures of site B with an axially bound water can be

ruled out: it has recently been demonstrated that such an

axial interaction results in a lowering of |Az|exptl [85].

Concerning site A, the coordination of a water molecule

rather than of an OH- ion has to be preferred. The signals

of site A dominate the spectrum at pH 5 and, even if

hydrogen bonds between water and neighboring groups

may facilitate its deprotonation, a pK around 5 seems to be

too low for a VO2? species; as a comparison, the values for

a water molecule coordinated to pyrone and pyridinone

derivatives are in the ranges 8.47–8.78 and 10.47–10.75,

respectively [86, 87]. Furthermore, the arrangement with

one imidazole ring parallel to the V=O bond and the other

perpendicular to it, as predicted by the DFT simulations,

seems to be in agreement with X-ray diffractometric

analysis, which shows that the two aromatic ring planes

form an almost right angle (experimental 69.0� [84]).

Therefore, the coordination modes [COO�Glu, H2O, H2O,

H2O] and [NHis(||), NHis(\), COO�Glu, H2O] proposed in this

work for site B and active site A must be considered as an

additional possibility that must be taken into account with

respect to those [NHis(||), NHis(\), COO-, H2O] and

[NHis(||), NHis(\), COO-, OH-], in which the change in

|Az|exptl is attributed to the deprotonation of the water

molecule to give an OH- ion [80].

The values of Acalcdz for site B are compared in Table 2,

using the atomic coordinates obtained after an optimization

at a progressively more accurate level of theory. It is

noteworthy that the improvement in the optimization

step—B3P86 (PCM)—results in a better prediction of the

spectroscopic data, such as Az. Therefore, the use of

the B3P86 functional and water as the solvent in the

(Glu)CH2COO–

H2O

OH2

V

OOH2

(Glu)CH2COO–

(HO–) H2O

N

V

ON

HN

His

NH

His

(Glu)CH2COO–

H2O

OH2

V

OOH2

OH2

(Glu)CH2COO–

H2O

N

V

O

NH

His

N NH

His(c)

(a) (b)

(d)

Scheme 1 Model complexes

for the metal sites of vanadyl-

substituted carboxypeptidase:

a, b site B and c, d site A. The

models that reproduce well the

experimental results are those in

a and c

Table 1 Calculated and experimental 51V A tensor for the model sites of vanadyl-substituted carboxypeptidase

Vanadium protein Donora,bAcalcd

x Acalcdy Acalcd

z Aexptlx Aexptl

y Aexptlz

Deviation (%)c

Carboxypeptidase (site B) COO�Glu, H2O, H2O, H2O -74.6 -74.3 -176.1 -67.3 -67.3 -175.8 ?0.2

COO�Glu, H2O, H2O, H2O; H2Oax -71.8 -70.3 -171.4 -67.3 -67.3 -175.8 -2.5

Carboxypeptidase (site A) NHis(||), NHis(\), COO�Glu, H2O -61.3 -60.9 -164.9 -61.1 -61.1 -165.9 -0.6

NHis(||), NHis(||), COO�Glu, H2O -60.2 -57.4 -162.0 -61.1 -61.1 -165.9 -2.4

NHis(||), NHis(\), COO�Glu, OH- -54.6 -50.7 -154.9 -61.1 -61.1 -165.9 -6.6

51 V A coupling constants measured in units of 10-4 cm-1

a The donor sets that reproduce well the electron paramagnetic resonance (EPR) parameters are in boldb NHis(||) and NHis(\) indicate an imidazole ring of a histidine residue donor parallel to the V=O bond (dihedral angle of 10� in the calculations)

and perpendicular to the V=O bond (dihedral angle of 70� in the calculations)c Percent deviation of |Az|

calcd from |Az|exptl, expressed as 100 9 (|Az|

calcd - |Az|exptl)/|Az|

exptl

J Biol Inorg Chem (2012) 17:773–790 779

123

framework of the PCM must be recommended if an

accurate prediction of the structure or some spectroscopic

parameter of a metal-containing biocompound is desired.


CF1-ATPase

CF1-ATPase is an extrinsic membrane protein complex

that, in association with the intrinsic membrane complex

F0, couples the proton motive force generated by the light

reactions of photosynthesis to synthesize ATP. In the

crystal structure of the F1-ATPase from bovine mitochon-

dria [88], there are six sites for the binding of Mg2?

nucleotides, of which three are catalytic. Information about

the types of groups that serve as metal ligands to the pro-

tein and how the ligands change upon activation of the F1-

ATPase has been obtained by examining EPR and ESEEM

spectra from vanadyl-substituted spinach chloroplast F1,

where the VO2? ion is bound at specific Mg2? binding sites

[89–91]: this suggests the presence of a coupled nitrogen

nucleus with anomalous properties, including ESEEM

frequencies that lie entirely below 5.0 MHz [91]. The value

of ANiso, around 1.0 MHz, which is comparable with that

displayed by model complexes with an amine nitrogen

trans to the V=O group such as [VO(Hhida)(H2O)] [92,

93], is related to the presence of a lysine residue in the axial

position [74], which was confirmed by the X-ray structure

of the bovine heart enzyme [88].

For site 3, LoBrutto et al. [74] proposed the equatorial

coordination of one carboxylate (aspartate or glutamate),

one hydroxyl side chain belonging to a residue of serine or

threonine, and two water molecules, and the axial binding

of an amino nitrogen of a lysine residue. We tested by DFT

simulations the model complexes of this site and those

involving the deprotonation of one of the two water ligands

and of the hydroxyl group of serine or threonine; the

analysis of the X-ray structure within 5.5 A from the metal

ion suggests that the equatorial plane of the VO2? ion may

be formed by Thr-176, Asp-269, and two water molecules,

and that Lys-175 may occupy the axial position

(Scheme 2). Both Az (from the EPR spectrum) and ANiso

(from the ESEEM spectrum) values were calculated and

compared with the experimental data (Table 3). All three

donor sets tested, i.e., [COO�Asp, OHThr, H2O, H2O,

NHax2Lys], [COO�Asp, OHThr, OH-, H2O, NHax

2Lys], and

[COO�Asp, O�Thr, H2O, H2O, NHax2Lys], with the axial

Fig. 5 Most probable

vanadium environment in

vanadyl-substituted

carboxypeptidase: a site B and

b site A

Table 2 Calculated 51V Az coupling constants (at the BHandHLYP/6-311g(d,p) level of theory) for three types of structure optimization for

vanadyl-substituted carboxypeptidase and vanadyl-substituted chloroplast F1-ATPase (CF1-ATPase)

Vanadium protein Donors Optimization Acalcdz Aexptl

zDeviation (%)a

Carboxypeptidase (site B) COO�Glu, H2O, H2O, H2O B3LYP/6-311g -186.5 -175.8 ?6.1

COO�Glu, H2O, H2O, H2O B3P86/6-311g -180.1 -175.8 ?2.4

COO�Glu, H2O, H2O, H2O B3P86/6-311g (H2O) -176.1 -175.8 ?0.2

CF1-ATPase (site 3) COO�Asp, OHThr, H2O, H2O, NHax2Lys B3LYP/6-311g -177.9 -169.1 ?5.2

COO�Asp, OHThr, H2O, H2O, NHax2Lys B3P86/6-311g -175.2 -169.1 ?3.6

COO�Asp, OHThr, H2O, H2O, NHax2Lys B3P86/6-311g (H2O) -170.9 -169.1 ?1.1

51 V Az coupling constants measured in units of 10-4 cm-1

a Percent deviation of |Az|calcd from |Az|

exptl, expressed as 100 9 (|Az|calcd - |Az|

exptl)/|Az|exptl

780 J Biol Inorg Chem (2012) 17:773–790

123

coordination of amine nitrogen, are compatible with the

low ANiso

��

�� (approximately 1 MHz). Thus, the DFT method

confirms that an axially bound nitrogen donor should give a

small value of ANiso

��

��, in agreement with the experimental

data [74]. However, among the three models tested, only

the first one (Scheme 2a) reproduces satisfactorily Az

(Table 3); thus, the deprotonation of the water molecule or

of the alcoholic group of the Thr-176 residue, which

according to the ‘‘additivity’’ rule should lower signifi-

cantly the value of Az, can be ruled out by DFT calcula-

tions. The plausible structure of the metal site is shown in

Fig. 6.

Furthermore, as observed for site B of carboxypeptidase,

the prediction of Acalcdz becomes better when the optimi-

zation of the metal site is performed in water with the PCM

rather than in the gas phase (Table 2).


VBrPO

VHPOs are metalloenzymes containing a mononuclear

vanadium cofactor that catalyze the oxidation of halides in

the presence of hydrogen peroxide [1, 2, 9]. The first

example of this class was a VBrPO isolated from the

marine alga A. nodosum [94, 95]. Single-crystal X-ray

diffraction analysis and X-ray absorption spectroscopy

(XAS) revealed that the active sites of bromoperoxidase

from A. nodosum [12, 96] and chloroperoxidase from

C. inaequalis [11, 97] are very similar, with vanadium in

the center of a slightly distorted trigonal bipyramid and a

histidine imidazole as the sole protein bound to the metal

ion. Upon reduction to vanadium(IV) by dithionite, the

enzyme is irreversibly inactivated but can be studied by

EPR and ESEEM spectroscopy. The 51V Az values are

167.5 9 10-4 cm-1 at pH 4.2 and 160.1 9 10-4 cm-1 at

pH 8.4 [98]. Modulation frequencies of 3.1, 4.2, 5.3, and

8.1 MHz were observed in the ESEEM spectrum [99]; it

has been assumed that the highest and either the second-

highest or the third-highest frequencies arise from the

DmI = ±2 transition of a histidine nitrogen coordinated in

the equatorial plane of the VO2? ion. In contrast, the fre-

quency centered around 3.1 MHz cannot be attributed to an

equatorial imidazole nitrogen and remains unassigned: an

axial or a nitrogen donor displaced from the equatorial

plane is the most probable candidate.

According to XAS [100] and EPR [98] studies, the vana-

dium center in the reduced form is in a square pyramidal or

octahedral coordination environment and the coordination

sphere is formed by an oxido ligand, two nitrogens (almost

certainly from histidines), and two or three oxygen functions.

Candidates for the oxygen-functional ligands are serine,

aspartic acid, and water. Smith et al. [80] have proposed, as the

set of ligands, two residues of imidazole coordinating vana-

dium with the aromatic nitrogens, one parallel and another

perpendicularly bonded, one residue of serine binding with the

deprotonated –OH group, and one water molecule at low pH

or one OH- ion at high pH completing the equatorial coor-

dination sphere of the metal ion [80]. From an examination of

the crystallographic structure of VBrPO [12], the residues

involved in the vanadium binding should be His-418, His-486,

and Ser-416; for VClPO [11, 97] they may be His-404, His-

496, and Ser-402.

We tested several models of the metal site, pentacoor-

dinated and hexacoordinated, with the coordination of the

serine residue in protonated or deprotonated form, and with

two imidazole rings in parallel (||) or perpendicular (\)

fashion with respect to the V=O bond and in the equatorial

position, the axial position, or in a position strongly dis-

placed from the equatorial plane (\) (Scheme 3; Table 4).

Of course, a good model should reproduce both EPR and

ESEEM results. The relative position of the two histidine

residues, cis or trans, cannot be determined unequivocally

by the examination of the X-ray structure; indeed, the

N–V–N angle is in the range 132–133� for VBrPO [12] and

123–133� for VClPO [11, 97]. In the calculations we chose

(Thr)CH2O

H2O

–OOCCH2(Asp)

V

OOH2

H NH2(CH2)4(Lys)

(Thr)CH2O

HO–

–OOCCH2(Asp)

V

OOH2

H NH2(CH2)4(Lys)

(Thr)CH2O–

H2O

–OOCCH2(Asp)

V

OOH2

NH2(CH2)4(Lys)

(a) (b)

(c)

Scheme 2 Model complexes

for the metal site of vanadyl-

substituted chloroplast

F1-ATPase (CF1-ATPase) (site

3). The model that reproduces

well the experimental results is

that in a

J Biol Inorg Chem (2012) 17:773–790 781

123

the ‘‘cis’’ option; however, the comparison of the data for

the most plausible coordination environments with the cis

or trans arrangement of the histidines suggests that similar

results are obtained (Table 4).

A possible explanation for the change in Az with pH is

the reorientation of one of the imidazole moieties (from

parallel to perpendicular with respect to the V=O direction)

on acidification or, alternatively, the protonation of one of

the ligands at lower pH; from our data, the partial reori-

entation of the two imidazole rings should give a differ-

ence of only 2–3 9 10-4 cm-1 in Az, whereas the

protonation at low pH of the imidazole nitrogens, replaced

by water molecules, should result in a significant increase

of |Az| (donor sets [OHSer, H2O, H2O, H2O] and [OHSer,

H2O, H2O, H2O; H2Oax], see Table 4).

Among the several metal sites that give good agreement

with Az (in bold in Table 4), the possibility that a serine

residue (Ser-416) is in the protonated form, analogously to

what is observed for CF1-ATPase, must be taken into

account. However, among the nine donor sets that repro-

duce the EPR parameters, only those for which the imid-

azole ring of one histidine is strongly displaced from the

equatorial plane [O=V–NHis(\) angle of 130� in the cal-

culations] agree well with the ESEEM data. In fact, if the

two imidazole rings occupy equatorial positions, even with

different orientations with respect to the V=O direction, the

two values of ANiso

��

�� would be very close to each other,

slightly above 6 MHz; in contrast, if one of them occupies

the axial site, one ANiso

��

�� value would be much smaller, as

described in ‘‘Variation of the 14N superhyperfine coupling

constant with respect to the V=O bond and histidine

residue’’ and observed for [VO(Hhida)(H2O)] [74] and

CF1-ATPase (see above). Therefore, as mentioned, one

imidazole ring (the one belonging to His-418) may be

strongly displaced from the equatorial plane, owing to the

steric hindrance of the polypeptide chain, with the otherTa

ble

3C

alcu

late

dan

dex

per

imen

tal

51V

Ate

nso

ran

dA

N iso

� ��

cou

pli

ng

con

stan

tsfo

rth

em

od

elsi

teo

fv

anad

yl-

sub

stit

ute

dC

F1-A

TP

ase

Van

adiu

mp

rote

inD

on

ora

EP

Rp

aram

eter

sbE

SE

EM

par

amet

ersc

Aca

lcd

xA

calc

dy

Aca

lcd

zA

exptl

xA

exptl

yA

exptl

zD

evia

tio

n(%

)dA

N iso

� �� calc

dA

N iso

� �� exptl

mexptl

CF

1-A

TP

ase

(sit

e3

)C

OO� A

sp;O

HT

hr;H

2O;H

2O;N

Hax

2L

ys

-7

1.7

-7

1.1

-1

70

.9-

60

.4-

60

.4-

16

9.1

?1

.10

.9*

1.0

1.0

,3

.2,

3.8

CO

O� A

sp;

OH

Thr;

OH�;

H2O

,NH

ax 2L

ys

-6

1.3

-5

3.1

-1

58

.1-

60

.4-

60

.4-

16

9.1

-6

.50

.9*

1.0

1.0

,3

.2,

3.8

CO

O� A

sp;

O� T

hr;

H2O

,H2O

,NH

ax 2L

ys

-6

5.7

-6

0.6

-1

62

.7-

60

.4-

60

.4-

16

9.1

-3

.80

.9*

1.0

1.0

,3

.2,

3.8

ES

EE

Mel

ectr

on

spin

ech

oen

vel

op

em

od

ula

tio

na

Th

ed

on

or

sets

that

rep

rod

uce

wel

lth

eE

PR

par

amet

ers

are

inb

old

,an

dth

ed

on

or

sets

that

rep

rod

uce

wel

lth

eE

SE

EM

par

amet

ers

are

un

der

lin

edb

51V

Aco

up

lin

gco

nst

ants

mea

sure

din

un

its

of

10

-4

cm-

1

c14N

Ais

oco

up

lin

gco

nst

ants

mea

sure

din

meg

aher

tzd

Per

cen

td

evia

tio

no

f|A

z|calc

dfr

om

|Az|

exptl,

exp

ress

edas

10

09

(|A

z|calc

d-

|Az|

exptl)/

|Az|

exptl

Fig. 6 Most probable vanadium environment in vanadyl-substituted

CF1-ATPase (site 3)

782 J Biol Inorg Chem (2012) 17:773–790

123

occupying an equatorial position; this hypothesis was

advanced previously but was not fully demonstrated, by

LoBrutto et al. [74]. The donor set that best reproduces the

EPR and ESEEM results at pH 4.2 is [NHis(||), NHis(\),

OHSer, H2O, H2Oax]; this is displayed in Scheme 3 and

Fig. 7a. Comparable results are obtained if the two histi-

dines bound to vanadium are in the trans position.

With regard to the structural changes of the donor set

[NHis(||), NHis(\), OHSer, H2O, H2Oax], when the pH is

increased, two possibilities can be advanced on the basis of

our calculations: (1) the equatorial water molecules could

undergo deprotonation to yield an OH- ion and donor set

[NHis(||), NHis(\), OHSer, OH-, H2Oax] or (2) the proton-

ated -OH group of the Ser-416 residue could become Ser-

O- to give the set [NHis(||), NHis(\), O�Ser, H2O] with the

loss of the water from the axial position. The simulations

are in agreement with the X-ray structure of VBrPO [12],

where the O=V–NHis-418 angle is rather different from the

O=V–NHis-486 angle (112.6� vs. 91.2�). The first hypothesis

has the advantage that the structure is hexacoordinated and

the observed change in the Az value is that expected on the

basis of the ‘‘additivity’’ rule (6.9 9 10-4 cm-1); the

second one gives a better agreement with the bond lengths

determined from XAS studies (V=O and mean equatorial

V–O and V–N distances of 1.61, 1.94, and 2.08 A vs. 1.63,

1.91 and 2.11 A determined experimentally [100]). From

an examination of the structure of VBrPO, Lys-341 may be

involved in the deprotonation of the water molecule (the

distance between H2O and NLys-341 is 2.9 A [12]); this is

agreement with the important catalytic role assigned to

Lys-353 in VClPO from C. inaequalis [101]. Therefore,

both the models appear consistent, within the errors of the

extended X-ray absorption fine structure determination,

and one cannot definitively distinguish which model is

correct with the available experimental data; for the

moment, it is not possible to demonstrate which ligand (the

Ser-416 residue or H2O) undergoes deprotonation. The two

structures of the site are reported in Fig. 7b and c. The

coordination environment determined by DFT calculations

is very similar to that proposed by some of us [80]; thus,

the results of this work allow us to suggest additional

structures that must be taken into account in future studies

(HO–) H2O

(Ser)CH2OV

O

OH2

(H)

N NH

His

NNH

His

(HO–) H2O

(Ser)CH2O–

V

O

N NH

His

N NH

His

(HO–) H2O

V

O

N NH

His

N

HN

His(Ser)CH2O

(H)

(HO–) H2O

V

OOH2

N NH

His

(Ser)CH2O

(H)

N

NH

His

H2O

(Ser)CH2O

OH2

V

O

(OH2)

H

OH2

H2O

(Ser)CH2O–

V

ON NH

His

NNH

His

(a)

(e)

(c) (d)

(b)

(f)

Scheme 3 Models complexes

for the metal sites of vanadium

bromoperoxidase (VBrPO). The

models that reproduce well the

experimental results are those in

a, e, and f

J Biol Inorg Chem (2012) 17:773–790 783

123

Table 4 Calculated and experimental 51V A tensor and ANiso

��

�� coupling constants for the model sites of vanadium bromoperoxidase (VBrPO)

Vanadium

protein

Donora,b,c EPR parametersd ESEEM parameterse

Acalcdx Acalcd

y Acalcdz Aexptl

xf Aexptl

yf Aexptl

zf Deviation

(%)gAN

iso

��

��calcd mexptl

VBrPO

(low pH)

NHis(||), NHis(\), OHSer, H2O, H2Oax h -69.0 -68.5 -169.4 -50.2 -50.2 -167.5 ?1.1 5.6, 7.5 3.1, 4.2,

5.3, 8.1

NHis(||), (NHis(\), OHSer, H2O -67.4 -64.8 -167.9 -50.2 -50.2 -167.5 ?0.2 7.3, 7.4 3.1, 4.2,

5.3, 8.1

NHis(||), OHSer, H2O, H2O, NaxHis -70.4 -68.2 -166.8 -50.2 -50.2 -167.5 -0.4 0.8, 7.6 3.1, 4.2,

5.3, 8.1

OHSer, H2O, H2O, H2O -79.4 -76.9 -180.0 -50.2 -50.2 -167.5 ?7.5 – 3.1, 4.2,

5.3, 8.1

OHSer, H2O, H2O, H2O, H2Oax -81.2 -80.4 -177.4 -50.2 -50.2 -167.5 ?5.9 – 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(||), O�Ser, H2O -59.2 -53.6 -158.2 -50.2 -50.2 -167.5 -5.6 6.0, 6.6 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(\), O�Ser, H2O -61.6 -56.1 -160.5 -50.2 -50.2 -167.5 -4.2 6.0, 6.6 3.1, 4.2,

5.3, 8.1

NHis(||), O�Ser, H2O, H2O, NaxHis -60.8 -55.0 -156.5 -50.2 -50.2 -167.5 -6.6 0.7, 6.3 3.1, 4.2,

5.3, 8.1

VBrPO

(high pH)

NHis(||), NHisð\Þ;O�Ser;H2Oi -63.1 -57.2 -161.1 -55.1 -55.1 -160.1 ?0.6 4.8, 6.5 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(\), OHSer, OH–, H2Oax j -62.3 -58.8 -162.0 -55.1 -55.1 -160.1 ?1.2 4.3, 5.0 3.1, 4.2,

5.3, 8.1

NHis(||), NHisð\Þ; O�Ser; H2O, H2Oax -52.4 -46.7 -148.1 -55.1 -55.1 -160.1 -7.5 3.9, 6.1 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(\), OHSer, OH– -57.8 -56.3 -159.5 -55.1 -55.1 -160.1 -0.4 6.3, 6.5 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(||), O�Ser, H2O -59.2 -53.6 -158.2 -55.1 -55.1 -160.1 -1.2 6.0, 6.6 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(\), O�Ser, H2O -61.6 -56.1 -160.5 -55.1 -55.1 -160.1 ?0.3 6.0, 6.6 3.1, 4.2,

5.3, 8.1

NHis(||), NHis(||), O�Ser; OH� -49.0 -44.6 -149.6 -55.1 -55.1 -160.1 -6.6 4.9, 4.9 3.1, 4.2,

5.3, 8.1

NHis(||), NHisð\Þ; O�Ser; OH� -49.3 -45.3 -150.1 -55.1 -55.1 -160.1 -6.2 4.9,5.4 3.1, 4.2,

5.3, 8.1

NHis(||), OHSer, H2O, OH–, NaxHis -64.8 -58.8 -161.0 -55.1 -55.1 -160.1 ?0.6 0.9, 6.0 3.1, 4.2,

5.3, 8.1

NHis(||), O�Ser, H2O, H2O, NaxHis -60.8 -55.0 -156.5 -55.1 -55.1 -160.1 -2.2 0.7, 6.3 3.1, 4.2,

5.3, 8.1

NHis(||), O�Ser, H2O, OH–, NaxHis -48.2 -48.0 -149.6 -55.1 -55.1 -160.1 -6.6 0.7, 4.7 3.1, 4.2,

5.3, 8.1

a The two histidines are in the cis positionb The donor sets that reproduce well the EPR parameters are in bold, and the donor sets that reproduce well the ESEEM parameters are underlinedc NHis(||), NHis(\), and NHis(\) indicate an imidazole ring of a histidine residue donor parallel to the V=O bond (dihedral angle of 10� in the calculations),

perpendicular to the V=O bond (dihedral angle of 70� in the calculations), and significantly displaced from the equatorial plane [O=V–Nim(\) angle of

130� in the calculations]d 51V A coupling constants measured in units of 10-4 cm-1

e 14N Aiso coupling constants measured in megahertzf Parameters measured in 0.1 M sodium citrateg Percent deviation of |Az|



exptl

h For the species with the two histidines in the trans position Acalcdz is -168.7 9 10-4 cm-1 (?0.7 % from |Az|

exptl) and ANiso

��

��calcd are 5.4 and 7.8 MHz

i For the species with the two histidines in the trans position Acalcdz is -160.4 9 10-4 cm-1 (?0.2 % from |Az|

exptl) and ANiso

��


j For the species with the two histidines in the trans position Acalcdz is -160.4 9 10-4 cm-1 (?0.2 % from |Az|

exptl) and ANiso

��


784 J Biol Inorg Chem (2012) 17:773–790

123

on the reduced VBrPO. In this case too, EPR and ESEEM

data for the model species with the two histidine nitrogens

in the trans position are very similar to those obtained

when they are arranged in the cis position (see Table 4);

this result should not be surprising if one considers that the

empirical ‘‘additivity’’ rule does not allow one to dis-

criminate between the two situations and is based only on

the contribution of the equatorial donor functions inde-

pendently of their relative position [61, 62].

A final comment concerns the interpretation of the

ESEEM data. As pointed out above, considering that only

two nitrogens are bound to vanadium, LoBrutto et al. [74]

suggested that two of the four observed components arise

from DmI = ±2 transitions. On the basis of our results, it is

also possible that under the experimental conditions used

for recording the spectrum, both forms of VBrPO (low- and

high-pH forms) may be present in aqueous solution, and so

four—belonging to four slightly different nitrogen

donors—instead of two absorptions may be revealed.


IGPD

IGPD is an enzyme involved in the biosynthesis of histi-

dine catalyzing the dehydration of imidazole glycerol

phosphate to imidazole acetol phosphate [102].

IGPD from Saccharomyces cerevisiae was initially

isolated as an inactive apoprotein [103], which combines

stoichiometrically and specifically with certain divalent

metal cations (Mn2?, Co2?, Cd2?, Fe2?, Ni2?, and Zn2?)

to form a catalytically active metalloenzyme of high

molecular mass (approximately 570 kDa) comprising 24

subunits. The structure of Arabidopsis thaliana IGPD,

which contains a dinucler manganese site, has been

determined to 3.0 A resolution [104]. The different

metalloenzymes have different catalytic activities, with the

Mn2?-containing enzyme being the most active and the

enzyme with Zn2? the least active [105]. Metal ions which

did not assemble the enzyme (Cu2?, Ca2?, and Mg2?)

yielded no catalytic activity.

It has been demonstrated that VO2? is unique in that it is

the only metal ion that assembles the enzyme correctly but

still leaves it enzymatically inactive [105]. For this reason,

VO2? can be used as a spin probe to investigate the metal

center coordination environment by EPR and ESEEM

spectroscopy. The X-band EPR spectrum of polycrystalline

vanadyl-substituted IGPD from S. cerevisiae (pH 7.5)

shows the resonances of three different species (denoted a,

b, and c), indicating multiple, inequivalent coordination

environments, countersigned by |Az| of 169.1 9 10-4,

161.6 9 10-4, and 140.6 9 10-4 cm-1 [105]. The number

of ESEEM resonances and the value of ANiso

��

�� around 7 MHz

suggest histidine rather than lysine ligation [106]; further-

more, the resonances observed below 2.5 MHz may be

associated with an axially coordinated histidine nitrogen. On

the basis of the experimental data, the authors concluded that

the b site has four equatorially coordinated histidines, whereas

the a site has three equatorial histidines and one axial histi-

dine, postulating that the total number of histidine residues

coordinating the vanadium center remains the same and that

one histidine shifts from an equatorial to an axial position

[107]. Moreover, characterization of the c site was not

reported and its very low |Az| value is not compatible with the

binding of only histidine nitrogens. In our opinion, the pre-

vious assignment of the a and b sites must be reexamined

because the model does not explain the spectroscopic

behavior and an evaluation of the c site must be attempted. We

have now done this through a computational approach.

In particular, from pH 6 to 8 the resonances of the b site

and, mainly, of the c site become more intense with respect

to those of the a site [105]. This could indicate the coor-

dination of basic groups that, with increasing pH, replace

the water molecules. Several model complexes have been

examined and two types of complexation scheme tested. In

Fig. 7 Most probable vanadium environment in VBrPO: a low-pH site; b, c high-pH site

J Biol Inorg Chem (2012) 17:773–790 785

123

both approaches the coordination of an axial histidine

donor in the apical site—which may explain the significant

reduction of |Az| for the c site and the resonances below

2.5 MHz in the ESEEM spectrum—was considered. The

first scheme is based on the coordination of one, two, and

three histidine nitrogens bound to VO2? in the a, b, and csites, and the second is based on binding of two, three, and

four histidine nitrogens (Scheme 4 and Table 5).

Scheme 4 Models complexes for the metal sites of vanadyl-substituted-substituted imidazole glycerol phosphate dehydratase (IGPD). The

models that reproduce well the experimental results are those in a–e and j

786 J Biol Inorg Chem (2012) 17:773–790

123

Table 5 Calculated and experimental 51V A tensor and ANiso

��

�� coupling constants for the model sites of vanadyl-substituted imidazole glycerol

phosphate dehydratase (IGPD)

Vanadium

protein

Donora,b His EPR parametersc ESEEM parametersd

Acalcdx Acalcd

y Acalcdz Aexptl

zDeviation

(%)eAN

iso

��

��calcd AN

iso

��

��exptl mexptl

IGPD

(a site)

COO�Glu, H2O, H2O, H2O, H2O 0 -74.6 -74.3 -176.1 -169.1 4.1 – 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), COO�Glu;H2O;H2O 1 -68.0 -64.1 -167.8 -169.1 -0.8 6.8 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHisð\Þ;COO�Glu;H2O;H2O 1 -68.4 -65.6 -169.0 -169.1 -0.1 6.7 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(\), H2O, H2O 2 -67.1 -66.7 -169.0 -169.1 -0.1 7.1, 7.6 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), H2O, H2O 2 -64.5 -62.9 -165.8 -169.1 -2.0 7.3, 7.3 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(\), NHis(\), H2O, H2O 2 -71.1 -70.1 -172.5 -169.1 2.0 7.4, 8.0 7.0 0.8, 1.2, 2.2,

5.0, 9.1

IGPD

(b site)

NHis(||), NHis(||), COO�Glu;H2O 2 -60.2 -57.4 -162.0 -161.6 0.2 7.3, 7.3 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHisð?Þ; COO�Glu; H2O 2 -61.3 -60.9 -164.9 -161.6 2.0 7.1, 7.6 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), COO�Glu; OH� 2 -50.1 -48.2 -150.8 -161.6 -6.7 6.0, 6.0 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHisð?Þ; COO�Glu; OH� 2 -54.6 -50.7 -154.9 -161.6 -4.1 6.0, 6.1 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHis(\), H2O 3 -60.6 -59.4 -162.7 -161.6 0.7 6.8, 6.6, 7.5 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), H2O;H2O;NaxHis 3 -64.3 -62.2 -163.3 -161.6 1.1 7.4, 7.5, 0.9 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHisð?Þ; H2O, H2O; NaxHis 3 -67.1 -65.0 -165.0 -161.6 2.1 7.7, 7.4, 1.0 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(\), NHis(\), H2O 3 -63.0 -61.4 -164.9 -161.6 2.0 7.6, 7.0, 7.1 7.0 0.8, 1.2, 2.2,

5.0, 9.1

IGPD (c site) NHis(||), NHis(||), CCO�Glu;OH�;NaxHis 3 -44.3 -43.7 -145.4 -140.6 3.4 6.9, 6.5, 0.9 7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis

(||), NHis

(||), NHisð?Þ; S�Cys; NaxHis 4 -46.8 -44.1 -143.9 -140.6 2.3 6.9, 6.7,

5.1, 0.8

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHis(\), NHis(\) 4 -58.9 -58.0 -161.5 -140.6 14.9 6.9, 6.9,

7.0, 7.0

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHis(||), NHis(||) 4 -51.4 -51.4 -155.9 -140.6 10.9 7.0, 7.0,

7.0, 7.0

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(\), NHis(\), NHis(\), NHis(\) 4 -67.7 -67.7 -168.7 -140.6 20.0 6.8, 6.8,

6.8, 6.7

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHisð?Þ; H2O; NaxHis 4 -59.4 -58.2 -156.5 -140.6 11.3 6.8, 6.6,

7.6, 0.9

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHisð?Þ; NHisð?Þ; H2O; NaxHis 4 -60.3 -59.6 -158.2 -140.6 12.5 7.1, 7.6,

7.2, 1.2

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHis(\), NHis(\), O�Ser=Thr 4 -60.0 -59.0 -156.1 -140.6 11.0 5.6, 5.6,

5.8, 5.9

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHis(\), NHis(\), O�Tyr 4 -60.9 -56.4 -157.4 -140.6 11.9 5.8, 5.8,

6.3. 5.5

7.0 0.8, 1.2, 2.2,

5.0, 9.1

NHis(||), NHis(||), NHis(\), NHis(\), H2O 4 -54.5 -53.9 -153.4 -140.6 9.1 6.9, 6.9,

7.1, 7.1

7.0 0.8, 1.2, 2.2,

5.0, 9.1

a The donor sets that reproduce well the EPR parameters are in bold, and the donor sets that reproduce well the ESEEM parameters are underlinedb NHis(||), NHis(\), and NHis(\) indicate an imidazole ring of a histidine residue donor parallel to the V=O bond (dihedral angle of 10� in the calculations),

perpendicular to the V=O bond (dihedral angle of 70� in the calculations), and significantly displaced from the equatorial plane [O=V–Nim(\) angle of 130� in the

calculations]c 51V A coupling constants measured in units of 10-4 cm-1

d 14N Aiso coupling constants measured in megahertze Percent deviation of |Az|



exptl

J Biol Inorg Chem (2012) 17:773–790 787

123

The first model starts from the X-ray diffractometric

analysis of Mn2?-containing IGPD [104]: in such a model,

His-169, Glu-173, and His-74 of another chain (His-145,

Glu-77, and His-170 of another chain for the second

manganous ion) plus a water molecule should occupy the

equatorial coordination positions and His-47 should occupy

the axial site. Therefore, it is plausible that, with increasing

pH, the metal coordination changes from [NHis(||), COO�Glu,

H2O, H2O] (a site, with Glu-173 and His-169 or His-74

binding), to [NHis(||), NHis(||), COO�Glu, H2O] (b site, with

Glu-173, His-169, and His-74 coordination), to [NHis(||),

NHis(||), COO�Glu, OH-, NaxHis] (c site, with Glu-173, His-

169, His-74, and axial His-47 binding). The deprotonation

of the water molecule and the axial coordination of His-47

accounts for the decrease of |Az| for the c site which is

observed at pH higher than 7: |Az|calcd is

145.4 9 10-4 cm-1, 3.4 % higher than |Az|exptl. Other

geometric details derived from DFT simulations are in

agreement with the experimental structure: indeed, the two

imidazole rings of His-74 and His-169 are almost parallel

(experimental dihedral angles of 4.0�) and the angles NHis-

74–V–NHis-47 and NHis-169–V–NHis-47 are close to 90�(91.1� and 104.1� experimental).

The second model is based on the subsequent coordi-

nation of two, three, and four histidine nitrogens in sites a,

b, and c. To explain the low value of |Az| for the c site, we

tested the coordination of an axial donor, such as H2O, Tyr-

O-, Ser-O-, or Thr-O-, but this coordination is not suffi-

cient to lower the value of |Az| below 156 9 10-4 cm-1.

Therefore, the equatorial binding of a ligand that gives a

low contribution to |Az|, such as a Cys-S-, plus an axial

histidine nitrogen must be postulated: in this case |Az|calcd is

143.9 9 10-4 cm-1, 2.3 % higher than |Az|exptl. Cysteine

residues have been found in the structure of IGPD of

S. cerevisiae [103], but not in that of A. thaliana [104].

Even if both models can explain the spectroscopic

measurements, the strong point of the first approach is that

the findings are consistent with the X-ray structure and the

metal site found in Mn2?-containing IGPD [104].

ESEEM spectra, containing complicated features, can be

explained as a result of the overlap of the signals of the

different histidine nitrogens present in the three sites: in

particular, the parallel arranged imidazole rings in the aand b sites may account for the resonances above 5.0 MHz,

whereas the axially bound histidine for those below

2.5 MHz.

Therefore, the donor sets [NHis(||), COO�Glu, H2O, H2O],

[NHis(||), NHis(||), COO�Glu, H2O], and [NHis(||), NHis(||),

COO�Glu, OH-, NaxHis] seem to be the most probable for the

a, b, and c sites, respectively. These are shown in Fig. 8.

Conclusions

The combination of DFT and experimental methods pre-

sented in this work has demonstrated that a higher level of

clarity for the vanadyl-substituted active sites of proteins

can be achieved when both theory and spectroscopy are

used in conjunction. Our reanalysis of four very different

vanadium proteins has provided greater certainty of the

true coordination environments in these systems. Thus, we are

close to having a generalizable method not only for predicting

the structure of a simple VO2? complex, but also for simu-

lating in water the chemical environment of vanadium con-

tained in more complicated species, such as biomolecules,

proteins, and enzymes. It will be interesting to verify if these

conclusions are applicable to other vanadium proteins. This

possibility is currently being examined.

References

1. Crans DC, Smee JJ, Gaidamauskas E, Yang L (2004) Chem Rev

104:849–902

2. Rehder D (2008) Bioinorganic vanadium chemistry. Wiley,

Chichester

3. Ishii T, Nakai I, Okoshi K (1995) In: Sigel A, Sigel H (eds)

Metal ions in biological systems, vol 31. Dekker, New York,

pp 491–509

Fig. 8 Most probable vanadium environment in vanadyl-substituted IGPD: a a site, b b site, and c c site

788 J Biol Inorg Chem (2012) 17:773–790

123

4. Michibata H, Uyama T, Kanamori K (1998) In: Tracey AS,

Crans DC (eds) Vanadium compounds: chemistry, biochemistry,

and therapeutic applications. ACS symposium series 711.

American Chemical Society, Washington, pp 248–258

5. Michibata H, Yoshinaga M, Yoshihara M, Kawakami N,

Yamaguchi N, Ueki T (2007) In: Kustin K, Costa Pessoa J,

Crans DC (eds) Vanadium: the versatile metal. ACS symposium

series 974, American Chemical Society, Washington,

pp 264–280

6. Carpentier W, De Smet L, Van Beeumen J, Brige A (2005) J

Bacteriol 187:3293–3301

7. Bayer E (1995) In: Sigel A, Sigel H (eds) Metal ions in bio-

logical systems, vol 31. Dekker, New York, pp 407–421

8. Berry RE, Armstrong EM, Beddoes RL, Collison D, Ertok SN,

Helliwell M, Garner CD (1995) Angew Chem Int Ed Engl

38:795–798

9. Pecoraro VL, Slebodnick CA, Hamstra BJ (1998) In: Tracey AS,

Crans DC (eds) Vanadium compounds: chemistry, biochemistry,

and therapeutic applications. ACS symposium series 711.

American Chemical Society, Washington, pp 157–167

10. Eady RR (1998) In: Tracey AS, Crans DC (eds) Vanadium

compounds: chemistry, biochemistry, and therapeutic applica-

tions. ACS symposium series 711. American Chemical Society,

Washington, pp 363–405

11. Messerschmidt A, Wever R (1996) Proc Natl Acad Sci USA

93:392–396

12. Weyand M, Hecht HJ, Kiesz M, Liaud MF, Vilter H, Schomburg

D (1999) J Mol Biol 293:595–611

13. Costa Pessoa J, Tomaz I (2010) Curr Med Chem 17:3701-3738

14. Thompson KH, Orvig C (2001) Coord Chem Rev

219–221:1033–1053 and references therein

15. Thompson KH, Orvig C (2006) J Inorg Biochem

100:1925–1935

16. Shechter Y, Goldwaser I, Mironchik M, Fridkin M, Gefel D

(2003) Coord Chem Rev 237:3–11

17. Sakurai H, Yoshikawa Y, Yasui H (2008) Chem Soc Rev

37:2383–2392

18. Stankiewicz PJ, Tracey AS, Crans DC (1995) In: Sigel A, Sigel

H (eds) Metal ions in biological systems, vol 31. Dekker, New

York, pp 287–324

19. Yasui H, Takechi K, Sakurai H (2000) J Inorg Biochem

78:185–196

20. Chasteen ND (1998) In: Sigel A, Sigel H (eds) Metal ions in

biological systems, vol 31. Dekker, New York, pp 231–247

21. Garner CD, Collison D, Mabbs FE, In: Sigel A, Sigel H (eds)

Metal ions in biological systems, vol 31. Dekker, New York,

pp 617–670

22. Neese F (2009) Coord Chem Rev 253:526–563

23. Zampella G, Kravitz JY, Webster CE, Fantucci P, Hall MB,

Carlson HA, Pecoraro VL, De Gioia L (2004) Inorg Chem

43:4127–4136

24. Zampella G, Fantucci P, Pecoraro VL, De Gioia L (2005) J Am

Chem Soc 127:953–960

25. Sanna D, Garribba E, Micera G (2009) J Inorg Biochem

103:648–655

26. Sanna D, Micera G, Garribba E (2011) Inorg Chem

50:3717–3728

27. Sanna D, Micera G, Garribba E (2010) Inorg Chem 49:174–187

28. Sanna D, Buglyo P, Micera G, Garribba E (2010) J Biol Inorg

Chem 15:825–839

29. Sanna D, Buglyo P, Bıro L, Micera G, Garribba E (2012)

Metallomics 4:33–36

30. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,

Cheeseman JR, Montgomery JA Jr, Vreven T, Kudin KN,

Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Men-

nucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji

H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida

M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X,

Knox JE, Hratchian HP, Cross JB, Adamo C, Jaramillo J,

Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R,

Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA,

Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Dan-

iels AD, Strain MC, Farkas O, Malick DK, Rabuck AD,

Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG,

Clifford S, Cioslowski J, Stefanov BB, Liu G, Liashenko A,

Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham

MA, Peng CY, Nanayakkara A, Challacombe M, Gill PMW,

Johnson B, Chen W, Wong MW, Gonzalez C, Pople, JA (2004)

Gaussian 03, revision C.02. Gaussian, Wallingford

31. Parr RG, Yang W (1989) Density-functional theory of atoms

and molecules. Oxford University Press, Oxford

32. Becke AD (1993) J Chem Phys 98:5648–5652

33. Lee C, Yang W, Parr RG (1988) Phys Rev B 37:785–789

34. Perdew JP (1991) In: Ziesche P, Eischrig H (eds) Electronic

structure of solids. Akademie, Berlin, pp 11–20

35. Perdew JP, Wang Y (1992) Phys Rev B 45:13244–13249

36. Perdew JP (1986) Phys Rev B 33:8822–8824

37. Perdew JP (1986) Phys Rev B 34:7406–7406

38. Krishnan R, Binkley JS, Seeger R, Pople JA (1980) J Chem Phys

72:650–654

39. Miertus S, Scrocco E, Tomasi J (1981) Chem Phys 55:117–129

40. Miertus S, Tomasi J (1982) Chem Phys 65:239–245

41. Cossi M, Barone V, Cammi R, Tomasi J (1996) Chem Phys Lett

255:327–335

42. Drago RS (1977) Physical methods in chemistry. Saunders,

Philadelphia

43. Wertz JE, Bolton JR (1986) Electron spin resonance: elementary

theory and practical applications. Chapman and Hall, New York

44. Munzarova ML, Kaupp M (2001) J Phys Chem B

105:12644–12652

45. Saladino AC, Larsen SC (2003) J Phys Chem A 107:1872–1878

46. Neese F (2003) J Chem Phys 118:3939–3948

47. Micera G, Garribba E (2009) Dalton Trans 1914–1918

48. Micera G, Garribba E (2011) J Comput Chem 32:2822–2835

49. Buhl M, Kabrede H (2006) J Chem Theory Comput

2:1282–1290

50. Buhl M, Reimann C, Pantazis DA, Bredow T, Neese F (2008) J

Chem Theory Comput 4:1449–1459

51. De Munno G, Bazzicalupi C, Faus J, Lloret F, Julve M (1994) J

Chem Soc Dalton Trans 1879–1884

52. Shuter E, Rettig SJ, Orvig C (1995) Acta Crystallogr C

51:12–14

53. Caravan P, Gelmini L, Glover N, Herring FG, Li H, McNeill JH,

Rettig SJ, Setyawati IA, Shuter E, Sun Y, Tracey AS, Yuen VG,

Orvig C (1995) J Am Chem Soc 117:12759–12770

54. Riley PE, Pecoraro VL, Carrano CJ, Bonadies JA, Raymond KN

(1986) Inorg Chem 25:154–160

55. Cornman CR, Zovinka EP, Boyajina YD, Geisre-Bush KM,

Boyle PD, Singh P (1995) Inorg Chem 34:4213–4219

56. Vlahos AT, Tolis EI, Raptopoulou CP, Tsohos A, Sigalas MP,

Terzis A, Kabanos TA (2000) Inorg Chem 39:2977–2985

57. Monga V, Thompson KH, Yuen VG, Sharma V, Patrick BO,

McNeill JH, Orvig C (2005) Inorg Chem 44:2678–2688

58. Collison D, Mabbs FE, Temperley J, Christou G, Huffman JC

(1988) J Chem Soc Dalton Trans 309–314

59. Wiggins RW, Huffman JC, Christou G (1983) J Chem Soc

Chem Comm 1313–1315

60. Wuthrich K (1965) Helv Chim Acta 48:1012–1017

61. Chasteen ND (1981) In: Berliner LJ, Reuben J (eds) Biological

magnetic resonance, vol 3. Plenum, New York, pp 53–119

62. Smith TS II, LoBrutto R, Pecoraro VL (2002) Coord Chem Rev

228:1–18

J Biol Inorg Chem (2012) 17:773–790 789

123

63. Liboiron BD, Thompson KH, Hanson GR, Lam E, Aebischer N,

Orvig C (2005) J Am Chem Soc 127:5104–5115

64. Kaupp M, Buhl M, Malkin VG (eds) (2004) Calculation of

NMR and EPR parameters theory and applications. Wiley-VCH,

Weinheim

65. Garribba E, Micera G, Sanna D (2006) Inorg Chim Acta

359:4470–4476

66. Bonadies JA, Carrano CJ (1986) J Am Chem Soc

108:4088–4095

67. Sanna D, Micera G, Strinna Erre L, Molinu MG, Garribba E

(1996) J Chem Res (S) 41–42

68. Munzarova ML, Kubacek P, Kaupp M (2000) J Am Chem Soc

122:11900–11913

69. Schweiger A (1991) Angew Chem Int Ed Engl 30:265–292

70. Dikanov SA, Tsvetkov YD (1992) Electron spin echo envelope

modulation (ESEEM) spectroscopy. CRC, Boca Raton

71. Deligiannakis Y, Louloudi M, Hadjiliadis N (2000) Coord Chem

Rev 204:1–112

72. Fukui K, Ohya-Nishiguchi H, Kamada H (1997) Inorg Chem

36:5518–5529

73. Fukui K, Ohya-Nishiguchi H, Kamada H, Iwaizumi M, Xu Y

(1998) Bull Chem Soc Jpn 71:2787–2796

74. LoBrutto R, Hamstra BJ, Colpas GJ, Pecoraro VL, Frasch WD

(1998) J Am Chem Soc 120:4410–4416

75. Neese F (2001) J Phys Chem A 105:4290–4299

76. Ames WM, Larsen SC (2009) Phys Chem Chem Phys

11:8266–8274

77. Saladino AC, Larsen SC (2003) J Phys Chem A 107:4735–4740

78. Fukui K, Fujii H, Ohya-Nishiguchi H, Kamada H (2000) Chem

Lett 198–199

79. Reijerse EJ, Shane J, de Boer E, Hofer P, Collison D (1991) In:

Yordanov ND (ed) Electron magnetic resonance of disordered

systems. World Scientific, Singapore, pp 253–271

80. Smith TS II, Root CA, Kampf JW, Rasmussen PG, Pecoraro VL

(2000) J Am Chem Soc 122:767–775

81. Saladino AC, Larsen SC (2002) J Phys Chem A

106:10444–10451

82. Micera G, Pecoraro VL, Garribba E (2009) Inorg Chem

48:5790–5796

83. DeKoch RJ, West DJ, Cannon JC, Chasteen ND (1974) Bio-

chemistry 13:4347–4354

84. Rees DC, Lewis M, Lipscomb WN (1983) J Mol Biol

168:367–387

85. Gorelsky S, Micera G, Garribba E (2010) Chem Eur J

16:8167–8180

86. Buglyo P, Kiss E, Fabian I, Kiss T, Sanna D, Garribba E, Micera

G (2000) Inorg Chim Acta 306:174–183

87. Rangel M, Leite A, Amorim MJ, Garribba E, Micera G, Lodyga-

Chruscinska E (2006) Inorg Chem 45:8086–8097

88. Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Nature

370:621–628

89. Houseman ALP, Morgan L, LoBrutto R, Frasch WD (1994)

Biochemistry 33:4910–4917

90. Houseman ALP, LoBrutto R, Frasch WD (1994) Biochemistry

33:10000–10006

91. Houseman ALP, LoBrutto R, Frasch WD (1995) Biochemistry

34:3277–3285

92. Mahroof-Tahir M, Keramidas AD, Goldfarb RB, Anderson OP,

Miller MM, Crans DC (1997) Inorg Chem 36:1657–1668

93. Hamstra BJ, Houseman ALP, Colpas GJ, Kampf JW, LoBrutto

R, Frasch WD, Pecoraro VL (1997) Inorg Chem 36:4866–4874

94. Vilter H, Glombitza K-W, Grawe A (1983) Bot Mar 26:331–340

95. Vilter H (1984) Phytochemistry 23:1387–1390

96. Christmann U, Dau H, Haumann M, Kiss E, Liebisch P, Rehder

D, Santoni G, Schulzke C (2004) Dalton Trans 2534–2540

97. Messerschmidt A, Prade L, Wever R (1997) Biol Chem

378:309–315

98. de Boer E, Boon K, Wever R (1988) Biochemistry

27:1629–1635

99. de Boer E, Keijzers CP, Klaassen AAK, Reijerse EJ, Collison D,

Garner CD, Wever R (1988) FEBS Lett 235:93–97

100. Arber JM, de Boer E, Garner CD, Hasnain SS, Wever R (1989)

Biochemistry 28:7968–7973

101. Kravitz JY, Pecoraro VL, Carlson HA (2005) J Chem Theory

Comput 1:1265–1274

102. Parker AR, Moore JA, Schwab JM, Davisson VJ (1995) J Am

Chem Soc 117:10605–10613

103. Hawkes TR, Thomas PG, Edwards LS, Wilkinson KW, Rice

DW (1995) Biochem J 306:385–397

104. Glynn SE, Baker PJ, Sedelnikova SE, Davies CL, Eadsforth TC,

Levy CW, Rodgers HF, Blackburn GM, Hawkes TR, Viner R,

Rice DW (2005) Structure 13:1809–1817

105. Petersen J, Hawkes TR, Lowe DJ (1997) J Biol Inorg Chem

2:308–319

106. Petersen J, Hawkes TR, Lowe DJ (1998) J Am Chem Soc

120:10978–10979

107. Petersen J, Hawkes TR, Lowe DJ (2000) J Inorg Biochem

80:161–168

790 J Biol Inorg Chem (2012) 17:773–790

123

Application of DFT methods to the study of the coordination environment of the VO2+ ion in...

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