ORIGINAL PAPER

Binding of VIVO2+ to the Fe binding sites of human serumtransferrin. A theoretical study

Goncalo C. Justino • Eugenio Garribba •

Joao Costa Pessoa

Received: 20 April 2013 / Accepted: 3 August 2013 / Published online: 20 August 2013

� SBIC 2013

Abstract The binding of VIVO2? to human serum trans-

ferrin (hTF) at the FeIII binding sites is addressed. Geometry

optimization calculations were performed for the binding of

VIVO2? to the N-terminal lobe of hTF (hTFN), and indicate

that in the presence of CO32- or HCO3

-, VIV is bound to five

atoms in a distorted geometry. The structures of VIVO–hTFN

species optimized at the semiempirical level were also used

to calculate the 51V and 14N A tensors by density functional

theory methods, and were compared with the reported

experimental values. Globally, of all the calculated VIVO–

hTF structures, the one that yields the lowest calculated

heats of formation and minimum deviations from the

experimental values of the 51V and 14N A tensor compo-

nents is the structure that includes CO32- as a synergistic

anion. In this structure the V=O bond length is approxi-

mately 1.6 A, and the vanadium atom is also coordinated to

the phenolate oxygen atom of Tyr188 (at approximately 1.9

A), the aspartate oxygen atom of Asp63 (at approximately

1.9 A), the His249 Ns atom (at approximately 2.1 A), and a

carbonate oxygen atom (at approximately 1.8 A). The

Tyr95 phenolic ocygen atom is approximately 3.3 A from

the metal center, and thus is very weakly bound to VIV. All

of these oxygen atoms are able to establish dipolar inter-

actions with groups of the protein.

Keywords Electron paramagnetic resonance �Density functional theory � Oxidovanadium(IV) �Transferrin � Geometry optimization

Abbreviations

DFT Density functional theory

ESEEM Electron spin echo envelope modulation

HF Hartree–Fock

hTF Human serum transferrin

hTFC C-terminal lobe of human serum transferrin

hTFN N-terminal lobe of human serum transferrin

mal- Maltolate

pic- Picolinate

Introduction

The presence of vanadium in biological systems, its pos-

sible physiological roles, its insulin-enhancing action [1–

3], its anticancer activity [4–6], and its antiparasitic activity

[7–10] have driven a considerable amount of research.

Particular interest has been given to the study of the

potential benefits of vanadium compounds as oral insulin

substitutes for the treatment of diabetes.

Most studies agree that in higher organisms the transport

and possibly the delivery of vanadium into cells can be

promoted by natural carriers such as plasma proteins:

human serum transferrin (hTF), human serum albumin, and

immunoglobulin G [11–24].

Transferrin is a single-chain glycoprotein containing

around 630 amino acids which are arranged in two similar

lobes, normally designated as the N-terminal (hTFN) and

C-terminal (hTFC) lobes. It binds reversibly two FeIII ions

and it is the main component in blood for the transport of

G. C. Justino � J. C. Pessoa (&)

Centro Quımica Estrutural,

Instituto Superior Tecnico, Universidade Tecnica de Lisboa,

Av. Rovisco Pais, 1049-001 Lisbon, Portugal

e-mail: joao.pessoa@ist.utl.pt

E. Garribba

Dipartimento di Chimica e Farmacia, and 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

123

J Biol Inorg Chem (2013) 18:803–813

DOI 10.1007/s00775-013-1029-x

FeIII [25–27]. Transferrin’s binding affinity is particularly

high for FeIII, but it can also bind other metal ions,

including BiIII, GaIII, InIII, AlIII, CuII, MnII, ZnII, NiII, and

RuIII [25–27]; binding by transferrin may have a significant

role in the transport and delivery of essential metal ions,

diagnostic radioisotopes, and toxic metal ions to cells [25].

Each of the two iron-binding sites of hTF is located in

clefts in the protein, one on the N-terminal side, the other

on the C-terminal side. FeIII binds to one nitrogen atom

from histidine, and three oxygen atoms from one aspartate

residue and two tyrosine residues (His249, Asp63, Tyr95,

and Tyr188 from the N-terminal lobe, and His585, Asp392,

Tyr426, and Tyr517 from the C-terminal lobe). The metal

atoms also interact with the synergistic carbonate anion,

which is anchored in place by electrostatic interactions

with positively charged arginines and by hydrogen bonding

to the protein helixes [27, 28]. Conformational changes

occur in transferrin that are associated with FeIII binding or

release. When iron is bound, each domain moves to form

what is designated as the ‘‘closed conformation’’ of hTF.

Similarly, on release of iron, the metal-binding domains

move apart. Once FeIII is bound to transferrin, this protein

changes to the ‘‘closed’’ form, which can be recognized by

the hTF cell receptors and it is internalized by the cell

through a process known as receptor-mediated endocytosis.

Transferrin is present in human blood plasma at a con-

centration of about 37 lM. In normal serum, only about

30 % of the total binding sites are occupied by iron, FeIII

being nonrandomly distributed between the two binding

sites of transferrin, a preferential occupancy of the N-ter-

minal site existing [28, 29]. This means that there are still

sites available for other metal ions, without needing to

replace the tightly bound FeIII. In fact, hTF can bind

strongly to a range of other metal ions, as is the case for

VIVO2? [11, 13, 27].

Strong evidence has been given indicating that most of

the vanadium in the serum is bound to hTF at the iron

binding sites [11, 13–24, 30–34], but the exact way the

VIVO2? cations are bound is not known. One suggested

binding set is given in Fig. 1 [7]. The EPR spectra indicate

that the binding sites in the C-terminal and N-terminal

lobes are distinct; the possible presence of water or

hydroxide has been inferred on the basis of electron spin

echo envelope modulation (ESEEM) studies, from the

observation of a proton with dipolar coupling to the VIV

center, at a distance of approximately 2.6 A. Alternatively,

a proton might be delivered, e.g., to carbonate or aspartate,

via the hydrogen-bonding network [7].

In this work we performed geometry optimization cal-

culations using all atoms of hTFN, on the basis of the

known X-ray diffraction structure of hTFN [35], to model

the binding of VIVO2? at the iron binding site of hTFN.

Additionally, density functional theory (DFT) methods

were used to calculate relevant EPR and ESEEM

parameters.

Materials and methods

Materials and instrumentation

The solutions containing apo-hTF and VIVOSO4 were

prepared in a buffer containing: 50 mM N-(2-hydroxy-

ethyl)piperazine-N0-ethanesulfonic acid, 25 mM carbonate

added as NaHCO3, 1 mM phosphate, 0.1 mM citrate,

1.5 mM lactate, and 0.20 mM KCl at pH 7.4. The EPR

spectra were recorded at 77 K (on glass made by freezing

solutions in liquid nitrogen) with a Bruker ESP 300E

X-band spectrometer.

Calculations

hTF optimizations

Geometry optimizations were performed at the semiem-

pirical level using the PM6 Hamiltonian [36] as imple-

mented in MOPAC2009 [37], replacing the conventional

self-consistent field procedure with a localized molecular

orbital method (MOZYME) and using the L-BFGS func-

tion minimizer for geometry optimization. This has been

previously used to model several protein systems regarding

both geometry and energetics [38, 39]). Briefly, the X-ray

structure of the closed holo-hTFN (with FeIII and CO32-)

was taken from the Protein Data Bank (ID 1A8E [35]) and

it was protonated at pH 7.4 using the empirical propKa

method [40–43] as implemented in pdb2pqr [44–46]. The

pKa values obtained are in agreement with those previously

published [47–49]. In particular, in the starting structures,

the coordinating residues Tyr188 and Tyr95 were in the

phenolic form and, for residue His249, calculations were

V

O (Tyr)

(Tyr) O

O

(Asp) O

(Arg)

(His) N

C

O

O

HO (Tyr)

(Arg)

O

Fig. 1 Proposed mode of binding of the VIVO2? ion to transferrin, as

suggested in [7]. VIV is considered to bind to the same binding site as

FeIII

804 J Biol Inorg Chem (2013) 18:803–813

123

done with both tautomeric forms of the aromatic ring in

order to ascertain the most relevant one, which is the one

with the imidazole nitrogen-bound hydrogen atom further

away from the vanadium atom. Calculations were per-

formed both in the gas phase and with a polarizable con-

tinuum model approach, the conductor-like screening

model, to mimic the effects of water as an implicit solvent.

The VIVO2? ion as well as carbonate anions were placed

in the FeIII binding site. Briefly, the VIVO2?–CO32- hTF

model was built by replacing the iron atom with a vana-

dium atom and attaching to it the oxido oxygen atom; from

this model, the carbonate ion was removed to obtain a

starting VIVO2?–hTF model, and the VIVO2?–HCO3-–

hTF model was derived from the VIVO2?–CO32-–hTF

model by including a hydrogen atom on each of the two

oxygen atoms pointing away from the metal; a hydrogen

atom was also initially placed on the coordinating oxygen

atom but this led to a fast and marked increased in the total

energy of the system. For geometry comparison, an octa-

hedral [VO(H2O)5]2? complex was also studied in the

same conditions as the protein systems. An initial optimi-

zation was performed only on the hydrogen atoms, fol-

lowed by an unrestricted optimization of the whole protein.

A cutoff value (interatomic distance where the neglect of

diatomic differential overlap approximation stops) of 9 A

was used for all optimizations; larger values (12, 18, and

24 A) were also used, but no geometry or energy

improvements were obtained. Geometries were considered

optimized when the heat of formation (as calculated by

MOPAC) changed by less than 0.1 kcal mol-1 for 20

consecutive optimization cycles. Heats of formation, bond

parameters, and molecular orbitals were obtained by a

single-point calculation on the optimized geometry in order

to reorthogonalize the localized molecular orbitals.

Heats of formation of the species involved in the for-

mation of complexes of maltolate (mal-) and picolinate

(pic-) with VIVO2?, VIVO(mal)2(H2O), and VIVO(-

pic)2(H2O), respectively [1, 2, 11, 13, 14, 16–18, 20, 21,

24, 33, 34], were also calculated in order to compare their

corresponding association energetics with those of the

VIVO2?–HCO3-–hTF and VIVO2?–CO3

2-–hTF systems.

DFT calculations

The calculations for 51V and 14N tensors (AV and AN) were

performed with the program Gaussian 09 (revision C.01) [50].

Of the structures optimized at the semiempirical level,

only the residues less than 10 A from vanadium were

considered. To the CO–NH bonds that were fragmented in

the process a hydrogen atom was added: thus, these resi-

dues start with NH2– and end with a –CONH2 group.

Subsequently, the position of the hydrogen added was

optimized at B3P86/6-311g level of theory [51], freezing

all the other atoms. For the resulting structures, 51V (ANiso,

AVx , AV

y , and AVz ) and 14N (AN

iso, ANx , AN

y , and ANz ) hyperfine

coupling constants were calculated in the gas-phase using

the spin-unrestricted formalism.

The method used was tested on the model where CO32-

is introduced in the VIVO2?–hTF system (model 2, see

later), considering in the gas phase (1) only residues less

than 12 A from vanadium and (2) only residues interacting

with vanadium (Asp63, Tyr95, Tyr188, His249, and

CO32-). The results were comparable, confirming that the

values of the 51V and 14N hyperfine coupling constants

depend mainly on the first-sphere coordination environ-

ment of vanadium [52]. This is in agreement with the

additivity relationship, largely used over the last 30 years

for the characterization of VIVO2? species, which allows

one to predict the value of Az from the contribution of the

four donors bound to vanadium in the equatorial plane,

independently of the ligand complexity [53, 54]. Thus, to

predict correctly the spectroscopic parameters, it is

important that the semiempirical simulation provide a

reasonable structure; in particular, the coordination envi-

ronment of vanadium must be adequately described.

The 51V hyperfine coupling constants were calculated

with BHandHLYP functional and the 6-311g(d,p) basis set,

according to the procedure established in the literature [55–

60], the half-and-half functional BHandHLYP being

incorporated in Gaussian 09. In the first-order approxima-

tion, the hyperfine coupling tensors Ai, where i is V or N,

have one isotropic contribution deriving from the Fermi

contact (Aiiso) and another from the dipolar hyperfine

interaction (tensor Ti): Ai ¼ Aiiso1þ Ti. As demonstrated in

the literature, DFT simulations are a valid tool to predict

EPR parameters of VIVO2? complexes [55–67]. It must

also be remembered that for a VIVO2? species the AVz value

is usually negative, but in the literature its absolute value is

usually reported. The 14N hyperfine coupling constants

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

[52], with the BHandH functional incorporated in the

Gaussian 09 package.

BHandHLYP and BHandH include a mixture of exact

Hartree–Fock (HF) and DFT methods to calculate the

exchange–correlation energy (EXC), and this seems to be

necessary to simulate correctly AViso and AN

iso, 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 þ0:5� EB

X þ ELYPC and BHandH is defined as 0:5� EHF

X þ0:5ELSDA

X þ ELYPC , where EHF

X ; ELSDAX ; EB

X , and ELYPC are

the energies due to the HF exchange, the local spin density

approximation exchange functional, the gradient-corrected

J Biol Inorg Chem (2013) 18:803–813 805

123

Becke 88 exchange functional, and the gradient-corrected

Lee–Yang–Parr correlation functional, respectively.

Results and discussion

Computational modeling of hTF–VIVO binding

The binding site responsible for accommodating the FeIII

and carbonate species is well described in the literature

[26–29]. In the X-ray structure used, iron is coordinated to

two oxygen atoms from the carbonate anion (at 2.1 and 2.2

A), by the phenolate oxygen atoms of Tyr95 and Tyr188 (at

2.0 and 1.8 A, respectively), by one carboxylate oxygen

from Asp63 (2.0 A) and by the Ns atom of His249 (2.0 A).

For the VIVO2? ion (no carbonate present, model 1), the

modeling results indicate that the vanadium atom is coor-

dinated to the same aspartate, tyrosine, and histidine resi-

dues, at distances of 2.0 A (Asp63 O atom), 2.2 A (Tyr95

O atom), 1.8 A (Tyr188 O atom), and 2.0 A (His249 Nsatom), and that the V=O bond has a length of 1.6 A (see

Table 1). Noticeably, Tyr188 is present in the phenolate

form, whereas Tyr95 remains in the phenolic form, and the

phenolic hydrogen is bonded to the terminal nitrogen atom

of Lys296, which is thus present in the protonated Lys–

NH3? form. The modeled structure shows C–O–V angles

of 96.9� for Tyr95, 126.9� for Tyr188, and 131.6� for

Asp63, whereas with FeIII the three C–O–Fe angles are in

the 132–146� range; the major difference for Tyr95 may be

due to the presence of the hydroxylic hydrogen atom.

The oxido oxygen atom is able to participate in a salt

bridge to the guanidinium Nd atom of Arg124

(rON = 3.1 A), and in a hydrogen bond with the Arg124

terminal NH2 group (rOH = 2.0 A, rON = 2.8 A,

hNHO = 133.9�). In ferric transferrin, Arg124 is bound to

the synergistic carbonate anion [68]. Oxido oxygen inter-

actions with the atoms coordinated to vanadium are also

possible, although at longer distances: 2.8 A to the car-

boxylate oxygen atom of Asp63, 3.0 and 2.7 A to the

phenolate oxygen atoms of Tyr95 and Tyr188, respectively,

and 2.8 A to the Ns atom of His249. The OAsp63OTyr95-

OTyr188V dihedral is 20.8� and the NsHis249OTyr95OoxidoV

dihedral is -10.2�. The VIVO2? interactions with the pro-

tein are summarized in Fig. 2a, where the relevant residues

and major interactions are depicted.

The potential role of CO32- and HCO3

- was also taken

into account by introducing each of these anions in the

system. Carbonate was modeled from an initial orientation

similar to that in the known X-ray structure of

hTF ? FeIII ? CO32-, where it coordinates FeIII in a

bidentate mode [69].

When CO32- is introduced in the VIVO–hTFN system, in

the modeling calculation (model 2) it assumes a position

where all oxygen atoms are able to establish dipolar

Table 1 Distances between atoms of VIVO2? and the most relevant atoms involved in its binding to human serum transferrin (hTF)

Model 1 (without carbonate) Model 2 (with CO32-) Model 3b (with HCO3

-)

V=O 1.55 (2.49) 1.58 (2.31) 1.56 (2.34)

V–N (His249) 2.00 (0.75) 2.13 (0.55) 2.11 (0.58)

V–O (Tyr95) 2.25 (OH) (0.43) 3.28 (OH)a (0.05) 2.69 (OH)a (0.17)

V–O (Tyr188) 1.80 (1.22) 1.92 (0.91) 1.86 (1.04)

V–O (Asp63) 1.98 (0.72) 1.92 (0.78) 2.00 (0.69)

V–O1 (carbonate) – 3.60 (0.03) 3.80 (0.02)

V–O2 (carbonate) – 1.79 (1.09) 3.90 (0.02)

V–O3 (carbonate) – 4.00 (0.02) 1.88 (0.88)

Ooxido–N (His249) 2.78 (0.02) 2.87 (0.01) 2.76 (0.01)

Ooxido–O (Tyr95) 2.97 (0.01) 4.86 (0.00) 4.23 (0.01)

Ooxido–O (Tyr188) 2.67 (0.02) 2.82 (0.03) 2.77 (0.03)

Ooxido–O (Asp63) 2.79 (0.01) 2.89 (0.02) 2.93 (0.01)

Ooxido–NH2 (Arg124) (H bond) rON = 2.85 rON = 2.63 rON = 2.66

rOH = 2.05 rOH = 2.17 rOH = 2.28

h = 133.9 h = 104.9 h = 100.0

All bond lengths are given in angstroms; in the case of the hydrogen bond between the oxido oxygen atom and the NH2 group of Arg124, the

angle is given in degrees. The bond orders as computed with MOPAC [37] for the various atom pairs are given in parenthesesa The V–L(axial) internuclear distances, L(axial) being an atom coordinated trans to the oxido oxygen donor, are normally significantly longer

than the V–L(equatorial) distances, being up to approximately 2.4 A. In, e.g., model 2, the O=V–V–OH(Tyr95) angle is 178�, but the V–

O(Tyr95) distance is 3.28 A, which corresponds to a bond order of 0.05 (thus strictly it should not be considered a bond). For V–OH(Tyr95) the

internuclear distances are 3.28 and 2.69 A (bond orders of 0.05 and 0.17, respectively); whereas the latter may be considered a very weak bond,

the internuclear distance of 3.28 A corresponds to a very weak interaction

806 J Biol Inorg Chem (2013) 18:803–813

123

interactions with groups of the protein (see Fig. 2b). The

V=O bond length remains at 1.6 A, the vanadium atom is

coordinated to an Asp63 carboxylate oxygen (1.9 A), to the

phenolate oxygen of Tyr188 (1.9 A), to Ns of His249

(2.1 A) and to the O3 atom of carbonate (1.8 A). The OH

group of Tyr95 is located at a distance of 3.3 A and it does

not participate in the coordination. CO32- is parallel to the

V=O bond, the dihedral angle between the V=O and C=O

directions being -5.6�, and the Ooxido–V–OH(Tyr95) angle

is 178�.

The oxido oxygen atom also interacts with Arg124

through both a dipolar interaction with the guanidinium Ndatom (3.2 A) and a hydrogen bond with a terminal NH

group (rOH = 2.1 A, rON = 2.6 A, hNHO = 104.9�); the

oxido oxygen atom also has rather long range interactions

with the vanadium-coordinating atoms from Asp63

(2.9 A), Tyr188 (2.8 A), and His249 (2.9 A); the phenolic

oxygen atom of Tyr95 is too distant (4.9 A) to allow

interactions to occur.

Although each FeIII in (Fe)2hTF is coordinated to two of

the carbonate oxygen atoms, the carbonate anion in VIVO-

bound transferrin in model 2 only coordinates to VIV

through one carbonate oxygen atom, and assumes a

geometry that is roughly 45� to that of the FeIII case; this

monodentate coordination of carbonate to VIV is in

agreement with previous experimental results for both

transferrin and lactoferrin and is also suggested in Fig. 1.

The angle between the aromatic ring of His249 and the

V=O bond is approximately 35�.

An HCO3- anion was also considered in the VIVO–hTF

system (model 3), and two possible locations for the

hydrogen atom were obtained. In one case, the hydrogen

atom is located on an oxygen atom close to VIVO (model

3a), and in the other case, it is located on the most distant

oxygen atom, pointing away from the VIVO2? ion (model

3b). In model 3a HCO3- is parallel to the V=O bond as in

model 2 (dihedral angle between the V=O and C=O

directions of -1.5�); in model 3b it is perpendicular

Fig. 2 Calculated structures of the human serum transferrin complex

with VIVO2?. The three different possibilities obtained are shown:

a in the absence of carbonate (model 1), b with CO32- (model 2), and

c with HCO3- (model 3b). A superposition of the three modeled

structures is shown in d, where the most relevant residues of model 1

are shown with a cyan carbon skeleton, the most relevant residues of

the model 2 are shown with a magenta carbon skeleton, and the most

relevant residues of the model 3b are shown with a yellow carbon

skeleton. The atoms of VIVO are shown as spheres at the center of a–

d (gray for vanadium and red for oxygen), and relevant residues are

depicted as sticks (Asp63, Tyr95, Tyr188, and His249) and as lines. In

a–c, the residues labeled in blue are those involved in iron

coordination in ferric transferrin, and residues labeled in orange

were also found to be relevant in VIV binding in this study. Vanadium

coordinating bonds are shown in orange, bonds to oxido oxygen are

shown in yellow, and bonds from CO32- or HCO3

- to the protein are

shown in red. Further possible bonds to vanadium in the absence of

carbonate are shown in magenta. The protein backbone is depicted in

a transparent cartoon mode. Bonding details are given in the text

J Biol Inorg Chem (2013) 18:803–813 807

123

(dihedral angle of 86.7�). Energetically, there is a

1.3 MJ mol-1 difference in favor of the latter owing to the

local rearrangement of atoms and to a higher number of

hydrogen bonds, three in model 3b and just one in

model 3a.

In the energetically favored case (model 3b; Fig. 2c), the

V=O bond length is 1.6 A and the vanadium atom is

coordinated to Asp63 (carboxylate O, at 2.0 A), Tyr188

(phenolate O, at 1.9 A), His249 (Ns atom, at 2.1 A), and an

HCO3- oxygen atom at 1.9 A (see Table 1). The phenolic

oxygen atom of Tyr95 is located at 2.7 A, thus corre-

sponding to a weak interaction.

The oxido oxygen atom may also interact with Arg124

through either a hydrogen bond with a terminal NH2 group

(rOH = 2.3 A, rNO = 2.7 A, hNHO = 100.0�) or a salt

bridge to the same nitrogen atom; a salt bridge to the Nd atom

of Arg124 (3.2 A) is also possible. There are also long-range

interactions with the vanadium-coordinating atoms: with

Asp63 (2.9 A), Tyr188 (2.8 A), His249 (2.8 A), and HCO3-

(2.7 A). In model 3b the OAsp63OTyr95OTyr188V dihedral is

1.9� and the NsHis249OTyr95OVOV dihedral is -55.2�. The

HCO3- ion interacts with the protein by various hydrogen

bonds and salt bridges (see Fig. 2c), in particular from

hydrogen of the HCO3- ion to the OH group of Thr120

(rOH = 1.6 A, rOO = 2.6 A, hOHO = 158.7�) and from the

noncoordinating oxygen of HCO3- to the amide groups of

Ala126 (rOH = 1.9 A, rNO = 2.8 A, hNHO = 147.2�) and

Gly127 (rOH = 2.1 A, rNO = 3.1 A, hNHO = 154.2�).

A summary of the most important interactions involving

VIVO2? are presented in Table 1, and Fig. 1 depicts some

of the modeled VIVO–hTFN binding sites.

The bond lengths and the distances between vanadium

and the coordinating atoms obtained in our study of the

VIVO–hTFN system, performed at the semiempirical level

with the PM6 Hamiltonian, are in agreement with the dis-

tances computed for analogous systems at the DFT level. In

VIVO2?, the V=O bond length is approximately 1.57 A at

the B3LYP/6-311g(d) level for VIVO complexes with

nitrogen and oxygen coordinating ligands [56] and ranges

from 1.59 to 1.62 A at the same level of theory for a set of

peptidic ligands for which the experimentally determined

V=O bond lengths range from 1.58 to 1.62 A [55]; in our

studies, we obteined a V=O bond length of 1.6 A. Micera

and Garribba determined V–O coordinating distances from

1.8 to 2.1 A [56, 58–60], and in the present cases they range

from 1.8 to 2.2 A. For the V–N coordinating distances, the

DFT values are in the ranges 1.97–2.08 A for an amide N-,

2.15–2.19 A for an amine NH2, and 2.05–2.14 A for an

imidazole nitrogen atom [55]; our values for the imidazole

nitrogen atom are between 2.0 and 2.1 A.

A study of the octahedral water–VIVO complex was

performed in the same conditions as for the protein studies;

the geometry obtained [V–Ooxido = 1.56 A, V–

O(H2O)equatorial = 2.00 (±0.05) A, V–O(H2O)axial =

2.21 A, average Ooxido–V–O(H2O)equatorial angle =

(103.22 ± 3.2)�] is similar to the DFT geometry at the

B3LYP/3-21G (1.57 A, 2.03 A, 2.17 A, 97.8�, respec-

tively) that accurately describes the electronic structure of

this species [67].

Smith et al. [70] proposed a model for VIVO2? binding to

the C-terminal lobe of human lactoferrin in the presence of

carbonate, where vanadium is coordinated to Asp60, Tyr92,

Tyr192, and His253 (which are the residues equivalent to

Asp63, Tyr95, Tyr188, and His249 from the N-terminal

lobe), and the oxido oxygen atom is considered to establish a

hydrogen bond with a side-chain NH2 of Arg121 (Arg124 in

hTFN), carbonate being bound to both the NH2 and the

guanidinium Ns hydrogen atom of the side chain of Arg121,

as well as to the hydroxylic hydrogen atom of Thr117

(Thr120 in hTFN) [70]. In our calculated model, the carbonate

anion (protonated and deprotonated) is closer to Ser125 than

to Arg124, and VIVO2? is located between the side-chain

nitrogen atoms of Arg124 and the synergistic ions.

Our models, as described above, are capable of

accommodating the VIVO2? ion with a synergistic CO32-

or HCO3- ion without inducing major structural changes in

the protein, with the exception of the side chain of the

Tyr188 residue, which moves about 1.7 A,, and the Asp63

residue, which moves around 1.4 A, both due to the steric

effect of the presence of the carbonate ion.

As determined from the X-ray structure, in (Fe)2hTF the

FeIII is coordinated to six atoms that form an octahedral

environment with low distortion. Overall, in the case of

transferrin-bound VIVO2? without HCO3- or CO3

2- added

(Fig. 2a), vanadium is bound to five atoms in a distorted

arrangement (model 1). We analyzed this coordination

geometry using the s trigonality parameter of Addison

et al. [71]; this parameter is 0 for a perfect square pyramid

and 1 for a perfect trigonal bipyramid; intermediate values

reflect the structural continuum between the two structures.

In the case of model 1, s = 0.06, indicating that the

coordination geometry is essentially a square pyramid.

In the presence of either CO32- or HCO3

-, vanadium is

also bound to five atoms, in a distorted octahedral geom-

etry; in both cases the amine groups of Arg124 are not

spatially oriented toward the vanadium atom. Taking the

oxido oxygen atom as forming the main bond, we find the

coordination geometry is again essentially square pyrami-

dal, although with a higher trigonal bipyramid distortion.

The calculated trigonality indexes are s = 0.23 for hTF/

VIVO/CO32- (model 2) and s = 0.32 for hTF/VIVO/

HCO3- (model 3b), indicating that these two structures

also have a predominantly square pyramidal character,

which is more evident in the CO32- case. Figure 3 depicts

a schematic of the binding of VIV to the residues of the iron

binding site of hTFN for this particular model.

808 J Biol Inorg Chem (2013) 18:803–813

123

Energetics

After geometry optimization, a single-point calculation was

performed to reorthogonalize the localized molecular

orbitals and to obtain the reaction energies of the various

systems, which are presented in Table 2. For comparison,

the same approach was applied to the formation of

VIVO2?–mal- [72] and VIVO2?–pic- [73] complexes,

which are systems previously characterized by potentio-

metric and spectroscopic studies [72, 73] and DFT calcu-

lations [18, 20]. The values obtained indicate that the

presence of the CO32- ion or HCO3

- ions favors VIVO2?

binding to transferrin, clearly indicating that the hTF–

VIVO2? system is stabilized by the presence of carbonate

anions. This confirms that carbonate also plays a syner-

gistic role in VIVO2? binding to hTF, similar to that

observed for FeIII binding. The protonation state of the

carbonate species is also important, as this stabilizing

effect is larger in the system with a bianionic CO32- than

with a monoanionic HCO3- (although this depends on the

hydrogen atom orientation). We emphasize that we based

our choices on the calculated heats of formation and not on

the free energy of binding. We also did not take into

account that in blood HCO3- is present in a much higher

concentration than CO32-. However, the values obtained

suggest the tendency for the preference of CO32-.

The use of heats of formation to establish favorable

forms in metal–protein complexes may be questionable,

and free energies of binding and proper consideration of

the relative concentrations of CO32- and HCO3

- in solu-

tion should have been taken into account to fully support

the conclusions. However, the heats of formation (and

DrE values) do reveal a tendency for the binding.

In fact, the DrE values obtained for VIVO(pic)2(H2O),

-0.1154 MJ mol-1, which corresponds to a formation

constant b2 = 1012.1 [73], and for VIVO(mal)2(H2O),

-1.3695 MJ mol-1, which corresponds to b2 = 1016.3

[72], do conform with this tendency.

The open and closed forms of hTFN are distinguishable

by specific bonds that are present in each case. It is

important to emphasize that since our modeling calculation

started from the closed conformation of ferric transferrin, it

is unlikely to obtain hTF in the open conformation. In fact,

we used energy minimization approaches that, unlike the

molecular dynamics methods, are not adequate to study

events such as protein hinge rotation that may open the

transferrin cleft. In our modeling calculations the modeled

system remains in the closed form on carbonate and VIVO

binding. Recent results obtained by some of us indicate that

hTF in hTF(VIVO)2 assumes a closed conformation [34].

Prediction of EPR and ESEEM spectra

The structures optimized at the semiempirical level were

used to calculate 51V and 14N tensors, AV and AN,

respectively (see ‘‘DFT calculations’’).

Figure 4 depicts the EPR spectra recorded for the

VIVO2?-hTF system, and the gz and AVz values simulated

using the computer program ROKI [74] are reported in

Table 3. The EPR spectrum of frozen aqueous solution

containing VIVO2? and hTF at physiological pH is composed

of two sets of resonances (A and B), corresponding to at least

two slightly different VIV environments whose relative

intensity is pH-dependent. The A and B resonances were

attributed to VIVO2? bound in the N-terminal (our simula-

tions) and C-terminal [75] sites, respectively. Various values

for AVz of A resonances have been reported in the literature,

ranging from 166 9 10-4 to 168.5 9 10-4 cm-1 [32, 53,

76–78], so a mean value around 168 9 10-4 cm-1 can be

used as a reference. No reliable accurate measure of AVx and

AVy exists, owing to the contemporaneous presence of EPR

resonances belonging to the VIV–hTFN and VIV–hTFC sites.

The DFT methods used for such calculations were val-

idated recently [52, 55]. Among several functionals tested,

the order of accuracy in the prediction of 51V AVz is

BHandHLYP [ PBE0 � B3PW [ TPSSh [ B3LYP �BP86 [ VWN5 [55].1 The better performance of half-and-

half functionals such as BHandHLYP than the hybrid

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 [55, 56]. The spin polarization is difficult

to simulate with high accuracy, and is significantly

underestimated by most of the functionals [61, 63]. The

higher fraction of HF exchange in half-and-half functionals

V

O

(Tyr95) HO

(Tyr188) O

(His249) N

O

COO

Ser125Ala126

HO (Thr120)

O (Asp63)

Arg 124

Fig. 3 Binding of VIVO2? ion to the residues of the iron binding site

of the N-terminal lobe of human serum transferrin (hTF) as obtained

in computed model 2 (CO32- as a synergistic anion)

1 For the acronyms of the functionals and their meaning, the reader is

referred to [55].

J Biol Inorg Chem (2013) 18:803–813 809

123

such as BHandHLYP, which is available in Gaussian 09,

improves considerably the prediction of the Fermi contact

and, hence, that of AVz .

Our results for the calculation of the 51V AV tensor

components of the modeled structures of VIVO–hTFN are

presented in Table 4. They can be summarized as follows:

(1) the maximum value of the percent deviation is observed

when carbonate or hydrogen carbonate is not included in

the structure; (2) when carbonate or hydrogen carbonate is

taken into account in the calculations AVz

����calcd

increases

significantly and approaches AVz

����exptl

; (3) the structure that

yields the minimum deviation is model 2, with carbonate

coordinated to vanadium; (4) the deviation of 2.6 % for

model 2 is of the same order as the deviations obtained for

simple VIVO2? complexes and is in agreement with pre-

vious publications [52, 55, 57]; (5) models 3a and 3b, with

HCO3- included in the simulations, cannot be distin-

guished from these data.

The short relaxation times in EPR spectroscopy produce

lines that are considerably broad and in most of cases this

precludes the resolution of the coupling 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). Information on this super-

hyperfine coupling can be provided by ESEEM spectros-

copy, a variant of EPR spectroscopy [79–81]. The

superhyperfine coupling constant reported for 14N are in

the range 1–8 MHz and vary with the nature, position, and

orientation of the ligands [82–84]. In the studies on the

identification of nitrogen donors bound to vanadium in

VIVO species, both ANiso and AN

z are used. Very few com-

putational studies have been published in which the ligand

superhyperfine coupling constants for VIVO2? complexes

were simulated [52, 85]. A recent study showed that the

half-and-half hybrid BHandH, which is available in the

Gaussian package, performed better than the other func-

tionals tested. The order of accuracy is

BHandH & B3PW91 � B3P86 [ B3LYP [48]. It was

also demonstrated that the 14N AN tensor shows a func-

tional dependence on the dihedral angle h between the

Table 2 Reaction energies of the various systems studied, computed with the semiempirical PM6 Hamiltonian

System DrE (MJ mol-1)

VIVO H2Oð Þ2þ5 þ2pic��VIVO picð Þ2 H2Oð Þ þ 4H2O -0.1154

VIVO H2Oð Þ2þ5 þ2mal��VIVO malð Þ2 H2Oð Þ þ 4H2O -1.36951

VIVO H2Oð Þ2þ5 þhTFN�VIVO�hTFN þ 5H2O (model 1) -9.17391

VIVO H2Oð Þ2þ5 þhTFN þ CO2�3 �VIVO�hTFN�CO2�

3 þ 5H2O (model 2) -9.3776

VIVO H2Oð Þ2þ5 þhTFN þ HCO�3 �VIVO�hTFN�HCO�3 þ 5H2O (model 3a) -8.00596

VIVO H2Oð Þ2þ5 þhTFN þ HCO�3 �VIVO�hTFN�HCO�3 þ 5H2O (model 3b) -9.32996

DrE values were computed as the difference between the sum of the heats of formation of the products and those of the reactants. The

VIVO H2Oð Þ2þ5 reactant is considered to lose four water molecules to form the VIVO(pic)2(H2O) and VIVO(mal)2(H2O) complexes (the species

stable in aqueous solution; mal- is maltolate and pic- is picolinate) and five water molecules to form the N-terminal lobe of hTF (hTFN)

complexes

1.551.751.952.152.352.55

g-value

Fig. 4 First-derivative X-band frozen solution EPR spectra of

solutions containing apo-hTF (ChTF = 750 lM) and VIVO2? with

hTF to VIVO molar ratios of 1:1 (black line) and 1:2 (gray line)

Table 3 Spin Hamiltonian parameters obtained from the simulation

of the recorded EPR spectra

gz AVz � 104cm�1 Reference

VO–hTF species Aa 1.939 168.5 This work

1.937 168.3 [32]

1.938 168.0 [70]

1.938 168.0 [72]

1.940 166.8 [71]

VO–hTF species Ba 1.940 171.1 This work

1.941 170.3 [70]

1.937 172.4

1.934 170 [71]

1.941 170.5 [32]

1.935 171.8

a See the text for the assignment of species A and B

810 J Biol Inorg Chem (2013) 18:803–813

123

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

the two nitrogen atoms in the imidazole ring) and on the

angle u between the O=V and V–N bonds (where N is the

coordinated aromatic nitrogen atom) [52]. The data

obtained from the simulations are listed in Table 5.

From an examination of Table 5, it can be observed that

h and u for the four models studied are comparable. Thus,

the values of ANcalcdiso are very similar. Overall, the model that

gives the best results is the one with CO32- bound to

vanadium (model 2), followed by the models with HCO3-

(models 3a and 3b). This conclusion is again in line with the

results of EPR simulations, but the differences are so small

that they cannot be used to evaluate the best model. Only

the combined analysis of the energy and EPR data allows us

to suggest which of the four structures is the stablest.

Conclusions

The geometry optimization calculations performed indicate

that in the binding of VIVO2? to hTFN, in the presence of

CO32- or HCO3

-, vanadium is coordinated to five atoms,

in a distorted geometry; moreover, between the CO32- and

HCO3-, the DrE values (computed as the difference

between the sum of the heats of formation of the products

and those of the reactants; Table 2), obtained in the

modeling calculations suggest that the preferred synergistic

anion is CO32-. Free energies of binding and correct

consideration of the relative concentrations of CO32- and

HCO3- in solution would be required to fully support this

conclusion, but the DrE values do reveal a tendency for the

preference of CO32- as a synergistic anion.

Both VIVO2?–hTF–HCO3- and VIVO2?–hTF–CO3

2-

structures were modeled. For the latter, the V=O bond

length is approximately 1.6 A, and the vanadium atom is

also coordinated to the Tyr188 phenolate oxygen atom (at

approximately 1.9 A), the His249 Ns atom (at approxi-

mately 2.1 A), an Asp63 carboxylate oxygen atom (at

approximately 1.9 A), and a carbonate oxygen atom at

approximately 1.8 A. The Tyr95 phenolic oxygen atom is

approximately 3.3 A from the metal center, and thus at best

very weakly interacts with VIV. All oxygen atoms are able

to establish dipolar interactions with protein groups.

The structures optimized at the semiempirical level were

used to calculate 51V AV and 14N AN tensors by DFT

methods, and 51V AVz , 14N AN

iso, and 14N ANz were compared

with the reported experimental values. Of the calculated

VIVO–hTFN structures, the one that yields both the lowest

calculated heats of formation and the minimum deviations

from the experimental values of 51V AVz , 14N A AN

iso, and14N AN

z is the structure that includes CO32- as a synergistic

anion.

Table 4 Calculated 51V AV tensor components for the calculated structures of VIVO2?–hTF

Simulation AViso TV

x TVy TV

z AVcalcdx AVcalcd

y AVcalcdz AVexptl

zDeviation (%)a

Model 1 -88.0 33.7 36.1 -69.8 -54.3 -51.8 -157.8 -168.0 -6.1

Model 2 -92.8 32.2 38.6 -70.9 -60.6 -54.1 -163.6 -168.0 -2.6

Model 3a -90.1 34.4 36.5 -71.0 -55.7 -53.6 -161.0 -168.0 -4.1

Model 3b -90.1 33.3 37.9 -71.2 -56.9 -52.2 -161.3 -168.0 -4.0

All values are given in 10-4 cm-1

a Percent deviation of AVx

����calcd

from AVz

����exptl

, expressed as 100� AVz

����calcd� AV

z

����exptl

� �

= AVz

����exptl

Table 5 Calculated 14N AN tensor components for the calculated structures of VIVO2?–hTF

Simulation h u ANcalcdiso A

Nexptliso

a Deviation (%)bANexptl

zc ANexptl

zDeviation (%)d

Model 1 6.8 102.4 -6.2 -6.6 -6.1 -7.8 -7.1 9.9

Model 2 8.4 100.2 -6.3 -6.6 -4.5 -7.1 -7.1 0.0

Model 3a -4.7 100.7 -6.3 -6.6 -4.5 -7.6 -7.1 7.0

Model 3b 10.8 95.8 -6.1 -6.6 -7.6 -7.1 -7.1 -0.0

All values are given in megahertza Values taken from [86]

b Percent deviation of ANiso

��

��calcd

from ANiso

��

��exptl

, expressed as 100� ANiso

��

��calcd� AN

iso

��

��exptl

� �

= ANiso

��

��exptl

c Values taken from [87]

d Percent deviation of ANz

����calcd

from ANz

����exptl

, expressed as 100� ANz

����calcd� AN

z

����exptl

� �

= ANz

����exptl

J Biol Inorg Chem (2013) 18:803–813 811

123

The present modeling calculations taken together with the

results obtained recently by some of us [34], indicating that

on binding of VIVO2? to apo-hTF the protein closes its

conformation similarly to (FeIII)2hTF, suggest that

(VIVO)2hTF may be recognized by hTF cell receptors and

may thus be taken up by endocytosis (also similarly to FeIII).

Acknowledgments The authors thank the Portuguese Foundation

for Science and Technology and the FEDER and POCI programs,

namely, PEst-OE/QUI/UI0100/2013 and SFRH/BPD/68789/2010, for

financial support. G.C.J. acknowledges research grant SFRH/BPD/

27536/2006 from the Portuguese Foundation for Science and Tech-

nology. We also thank G. Goncalves for the measurement of the EPR

spectra in Fig. 4.

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