Structural Insights into the Binding of Uranyl with ... - ACS Publications

Department of Natural Sciences, University of Houston—Downtown, Houston, Texas 77002, College of Science, California State University, East Bay, Hay...
0 downloads 0 Views 2MB Size
Download PDF
Chem. Res. Toxicol. 2009, 22, 1613–1621

1613

Structural Insights into the Binding of Uranyl with Human Serum Protein Apotransferrin Structure and Spectra of Protein-Uranyl Interactions Maria G. Benavides-Garcia† and Krishnan Balasubramanian*,‡,§,| Department of Natural Sciences, UniVersity of HoustonsDowntown, Houston, Texas 77002, College of Science, California State UniVersity, East Bay, Hayward, California 94542, Chemistry and Material Science Directorate, Lawrence LiVermore National Laboratory, LiVermore, California 94550, and Nuclear Science DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed June 1, 2009

Ab initio quantum mechanical computational studies for the structure and IR spectra of the uranyl complex with human serum apotransferrin (TF) protein are carried out to model uranyl intake into the human cell through endocytosis and formation of a coordination complex with the protein binding sites. The computed IR spectra and structure of the uranyl-protein complex facilitate interpretation of the observed spectra and confirm the primary binding sites of the transferrin protein with the uranyl ion. Our computed equilibrium geometry and the IR spectra of the uranyl-TF complex reveal that uranyl ion is bound to two tyrosines, one aspartate group, and one carbonate ion. Our IR spectra indicate that histidine is not involved in binding to uranyl with transferrin protein. Our computations reveal a short, strong hydrogen bond, which could play an important role in the stabilization and formation of the uranyl-TF complex. Computed Laplacian charge plots indicate high chemical reactivity on this complex as both an electrophile and a nucleophile, facilitating binding to different receptors and thus entry into a number of target organs and the blood-brain barrier. The Mulliken charge density plots and the three-dimensional charge density plots suggest a donor-acceptor mechanism in the complex formation. Introduction Uranium entry into human cells appears to be primarily governed by its coordination chemistry to proteins, and thus, a number of studies have focused on uranyl-protein interactions as a means to understand the toxicity of uranium and its harmful effects on target organs such as lungs, liver, kidneys, bone, brain, and muscle (1-16). Recent studies have revealed surprising findings that the brain is also a target organ because uranium is capable of crossing the blood-brain barrier (BBB) (3-6). Consequently, there is an increasing concern about the types of adverse effects that could result from uranium toxicity. The concern has been accentuated by the fact that actinide contamination could be caused by both groundwater transport and deliberate deployments by terrorists (17). Estimation of the risks and effects associated with environmental exposure to uranium is critical to the improvement of human health risk assessment; it is also critical for the development of effective response strategies and policies to address nuclear terrorism. A solid understanding into how uranium enters the human cells is paramount to develop better ways to treat uranium exposure. The mechanism by which uranium enters target organs remains poorly understood because very little is known about uranium-protein interactions. Uranium binding sites in particular are very difficult to predict and to study because uranium has a strong tendency to hydrolyze in aqueous medium and forms a hexavalent uranyl ion, UO22+, which exhibits rather * To whom correspondence should be addressed. † University of HoustonsDowntown. ‡ California State University. § Lawrence Livermore National Laboratory. | Lawrence Berkeley National Laboratory.

fluxional complexes in solvated form (18). Taylor (7) has also demonstrated that actinides have strong tendency to hydrolyze in mammalian blood, and thus, their behavior in human proteins is complicated. Human serum proteins have been screened for uranium binding (8), and among the studied proteins, apotransferrin commonly known as transferrin (TF) was identified as a key protein exhibiting affinity for uranium. Review articles by Harris (9) and Sun et al. (10) describe how TF binds to a number of metal ions, and the former review includes a list of about 40 metal ions known to bind TF; the list includes various actinide ions, one of which is uranyl ion. As noted by Sun et al. (10), there is very little understanding into the mechanism of actinide binding with TF, and further work is needed. Human serum TF is known to be one of the major iron carriers in charge of iron regulation in human cells. It is a monomeric glycoprotein of about 80 kDa, and its crystal structure shows that it consists of a polypeptide chain folded into two homologous lobes connected by a short polypeptide, with each lobe containing a metal binding site; each lobe is identified as either N-terminal (N-lobe) or C-terminal (C-lobe), and it is divided into two domains by a cleft (11). In a recent study, Vidaud et al. (12) have suggested that TF plays an important role in the entry of uranium into human cells by proposing a model that involves the binding of two uranyl ions to the two metal binding sites of TF (one uranyl ion per metal binding site). The crystal structure of the N-lobe of human serum TF bound to Fe3+ ion (Figure 1) shows the iron ion is octahedrally coordinated by two tyrosines (Tyr95 and Tyr188), one monodentate aspartate (Asp63), one histidine (His249), and a bidentate carbonate ion (13). When iron binds to TF, the Fe3+-TF complex adopts a “closed” conformation, and upon iron release, TF adopts an “open” conformation.

10.1021/tx900184r CCC: $40.75  2009 American Chemical Society Published on Web 08/13/2009

1614

Chem. Res. Toxicol., Vol. 22, No. 9, 2009

BenaVides-Garcia and Balasubramanian

Figure 1. Recombinant N-terminal lobe of human serum TF bound to Fe3+; PDB ID 1a8e.

Vidaud et al. (12) have postulated that the same metal binding site in TF that coordinates Fe3+ is involved in the formation of the uranyl-TF complex. Therefore, some of the ligands that coordinate to Fe3+ ion are also expected to bind to the uranyl ion upon complex formation. The model shows that the coordination environment around the uranyl ion consists of two tyrosines (Tyr95 and Tyr188), one monodentate aspartate (Asp63), and a monodentate carbonate ion coordinated to the uranyl in the equatorial plane. The FTIR spectra obtained by the Vidaud et al. (12) suggest that His249, which is coordinated to Fe3+ ion, does not appear to be coordinated to the uranyl ion. In addition, Vidaud et al. (12) have mentioned in their study some very interesting conformational differences between these two complexes based on the results that they have obtained using various analytical techniques; they indicated that while the Fe3+-TF complex exists in a “closed” conformation, the uranyl-TF exists in an “semiopen” conformation. As can be seen from the above survey, there are many questions concerning uranyl-protein interactions that remain unanswered. A purely experimental approach to gain structural insights into how uranyl binds TF can be very challenging because uranyl has a strong tendency to undergo complex chemical speciation under physiological conditions (14), thus complicating definitive assignments. Although a number of computational approaches such as hierarchical quantitative structure-activity relationship (QSAR) methods have been employed to address chemotoxicity, in general (19-25), thus far, uranyl-protein complexes have not been studied using a quantum mechanical approach, and no experimental structural parameters are available on the structure of the complex. Pible et al. (26) have modeled uranyl bound to TF, but this study was carried out using a low-level molecular mechanical approach and thus does not provide information on the quantum molecular properties nor the electronic structure or the IR spectra

Figure 2. (a) Equilibrium geometry of the uranyl-TF complex that represents the metal binding site of the uranyl-TF complex. (b) Coordination environment around the uranyl ion. The atoms are labeled according to the atom-labeling scheme used during all calculations.

of the complex. To shed further light into how uranyl binds to TF, we have tackled this very important question concerning human toxicology by using a high-level quantum mechanical computational approach. Computational methods are very powerful tools because they allow predictions of molecular structures, molecular energies, and other chemical and thermodynamic properties, especially for actinide complexes for which experimental studies are challenging due to safety and security concerns. In addition, our computational models predict structural and electronic properties at atomistic level and provide new structural insights into protein-toxic metal ion interactions. Moreover, as there is no crystal structure reported for the uranyl-TF protein complex, the current study can be very valuable. We have modeled the uranyl-TF complex to represent the binding site of the uranyl-TF complex by having the uranyl ion coordinated by two tyrosines (Tyr), one monodentate aspartate (Asp), and one monodentate carbonate ion in the equatorial plane (Figure 2). Thus, the present study provides insights into the structural parameters of the uranyl-TF complex and the role of relativistic effects on the uranyl-amino acid cluster interactions.

Materials and Methods We have employed an ab initio quantum chemical model for the geometry optimization of the uranium-protein complex under consideration. To provide a tractable model for such high level computations, we have modeled the complex by explicit quantum chemical treatment of the uranyl and all of the amino acids that are directly bound to the uranyl in the protein. The resulting uranyl-TF local cluster that we shall refer to as the uranyl-TF

Binding of Uranyl with Human Serum Protein TF Table 1. Bond Distances and Bond Lengths of the Oxygen Atoms Directly Coordinated to the Central Atom, Uranium, in the Uranyl-TF Complexa U-Ox

bond length (Å)

equatorial U-O2 U-O22 U-O42 U-O59

2.50 (2.47)b 2.36 (2.34)b 2.48 (2.49)b 2.13 (2.15)b

axial U-O54 U-O55

1.79 (1.79)b 1.79 (1.79)b

Ox-U-Oy

bond angle (°)

O2-U-O54 O2-U-O55 O22-U-O54 O22-U-O55 O42-U-O54 O42-U-O55 O59-U-O54 O59-U-O55 O54-U-O55

89.78 (89.32)b 87.61 (87.83)b 91.66 (91.20)b 90.41 (90.99)b 87.49 (88.02)b 86.24 (86.50)b 92.59 (92.68)b 93.68 (92.78)b 173.35 (174.04)b

a The labels of the atoms are consistent with the atomic labeling scheme used in all calculations. The HOMO-LUMO energy gap ) 1.90 eV, the dipole moment of the complex is 7.79 D, and the complex has an overall charge of -3. b Larger basis sets augmented with diffuse Cartesian Gaussian functions: U atom, one 6s and one 7s (Rs ) 0.01096) and one 6p (ap ) 0.0093); O atom, one 2s (Rs ) 0.098) and one 2p (Rp ) 0.0622).

complex was fully optimized to represent the binding site of the uranyl-TF protein by coordinating the uranyl with two tyrosines, one monodentate aspartate, and one monodentate carbonate ion in the equatorial plane as can be seen from Figure 2. We have optimized the equilibrium geometry of the uranyl-TF complex followed by a frequency calculation. From the computed harmonic vibrational frequencies, we have generated the IR spectrum of the uranyl-TF complex. As the modeling techniques used here are all standard and well-known quantum chemical methods, the readers are referred to a number of references available in the literature for a detailed description of these methods (26-34). All calculations were carried out using the DFT (density functional theory) (27) approach that utilized Becke’s three-parameter functional (28) with Vosko et al.’s (29) local correlation part abbreviated as B3LYP (30). Relativistic effects are quite important for uranium because of its very large nuclear charge, and thus, electrons tend to move at speeds comparable to speed of light (31). Consequently, our present calculations include the use of relativistic effective core potentials (RECPs) (32) to replace the core electrons of uranium. The 6s and 6p core electrons and the 5f, 6d, and 7s valence electrons were explicitly treated using the reported Gaussian basis sets for uranium (32). Although oxygen, carbon, and nitrogen do not exhibit significant relativistic effects, to maintain consistency, their 1s2 core electrons were replaced by ECPs (33) while the 2s and 2p shells were treated explicitly by using Gaussian basis sets that were augmented with sets of six Cartesian 3d Gaussian functions (oxygen, Rd ) 0.85; nitrogen, Rd ) 0.80; and carbon, Rd ) 0.70) adopted from Dunning and Hay (34). We have employed van Duijneveldt’s basis sets (35) for the hydrogen atoms. As the complex has an overall -3 charge, diffuse functions on both uranium and oxygen could play an important role in determining the molecular properties. Consequently, we have also carried out computations using a larger basis set that included diffuse Gaussian functions for the U atom and the O atoms. The basis set was augmented further with diffuse function on uranium with Rs ) 0.01096 and Rp ) 0.0093. We also augmented the oxygen basis sets with diffuse functions with exponents Rs ) 0.098 and Rp ) 0.0622. The results of the two computations are compared (see Table 1), and it is shown that the added diffuse functions did not make substantial differences and thus validating our computational models. The computational models that we have employed here have been validated previously to gauge the accuracy of the predicted equilibrium geometries, molecular properties, and IR spectra of uranyl ion and uranium complexes as compared with experimental data (36-38). As the complex considered here is closer to carbonate complexes considered before, we are providing here a critical comparison of our modeling results and experiment. In a previous study, Majumdar et al. (38) have used the same basis sets and techniques as the ones used here to compute the equilibrium geometries of uranyl-carbonate complexes. Their calculated axial

Chem. Res. Toxicol., Vol. 22, No. 9, 2009 1615 UdO bond distance for uranyl ion (1.77 Å) compared well with the experimental value (1.79 Å); the experimental results are from the EXAFS data obtained by Allen et al. (39) The computed U-Ceq and U-Oeq bond distances (2.29 and 2.81 Å, respectively) were compared to experimental EXAFS results of 2.45 and 2.90 Å, respectively. Moreover, Cao and Balasubramanian’s (36) computed IR spectra were in very good agreement with experimental IR spectra as illustrated by the their calculated vibrational frequencies for the symmetric stretching and asymmetric stretching modes of uranyl ion (891 and 990 cm-1, respectively), which compared very well with the experimental values (869 and 961 cm-1) (36, 40). On the basis of these comparisons, we conclude that the present computational models should provide accuracy of 0.02-0.04 Å in bond lengths, and the spectra reported here should have sufficient qualitative accuracy to deduce the presence or absence of an amino acid in the complex. We have determined the equilibrium geometries of the free amino acids Tyr, Asp, and His. We have also computed the IR spectra of these species with two frequency calculations for every deprotonated amino acid, first one with the carboxylic group protonated (overall charge -1) and on the second one with the carboxylic group deprotonated (overall charge -2). All calculations were carried out using the same DFT/B3LYP method and basis sets that were used in the uranyl-TF complex calculations. All of the calculations described here were carried out using Gaussian 03 suite of codes (41).

Results and Discussion Equilibrium Geometries. Figure 2a shows the optimized equilibrium geometry of the uranyl complex with a local cluster of uranyl and amino acids accentuated that represents the metal binding site of the uranyl-TF complex. This computationally intensive geometry optimization was carried out by using Livermore Lawrence National Lab’s UP supercomputer. The geometry of the complex was fully optimized at the DFT/ B3LYP level, and the second derivatives of energies were also computed to generate the computed IR spectrum of the complex to facilitate assignment of the observed spectrum. It should be emphasized that our calculations included the large relativistic effects on the uranium atom through the use of RECPs for U. Figure 2b shows a magnified region of the geometry around the uranium atom so that one could get a more detailed perspective of the geometry near the uranyl moiety. We have also shown the labels for the various atoms in this vicinity in accordance to the atom-labeling scheme used in all calculations. As can be seen from both Figure 2a,b, the coordination environment around the uranium is octahedral, although uranium is known to adopt higher order coordination geometries both in aqueous solutions and in gas phase (36). Each axial position has an oxygen bound to uranium through a double bond (O54 and O55), while in the equatorial positions, there are four singly bonded oxygens: O2 and O22 from two tyrosines, O42 from an aspartate group, and O59 from the carbonate ion. As can be seen from Figure 2b, it is very intriguing to observe a hydrogen bond (O2-H57) between the hydrogen (H57) on the amino group of the aspartate and the oxygen (O2) in the phenolic group in tyrosine. This hydrogen bonding is facilitated by the presence of uranyl as the two amino acids have to approach in that orientation close enough to bind to the uranyl. As it is wellknown, hydrogen bonding plays a central role in the stabilization of proteins and protein complexes, and it is very interesting that our computed equilibrium geometry indicates the presence of this intramolecular interaction. The hydrogen bond distance is 2.36 Å, which corresponds to a low-barrier hydrogen bond (LBHB), and it is thus biochemically quite significant. According to Cleland and Krevoy (42), such hydrogen bond distances

1616

Chem. Res. Toxicol., Vol. 22, No. 9, 2009

are characterized as short and thus represent strong hydrogen bonding. These strong hydrogen bonds play a key role in enzyme catalysis reactions, and they can lower the activation energy barriers between 10 and 20 kcal/mol to facilitate otherwise difficult enzymatic reactions. Consequently, we predict that this LBHB plays a central role during the formation of the uranyl-TF complex and that this kind of hydrogen bonding could play an important role in the binding of actinides to proteins, in general. Table 1 shows more details on the electronic structure of the uranyl complex such as bond lengths and bond angles of the bonds formed between the axial and equatorial oxygen atoms and the uranium atom. The U-O bond lengths in the axial positions are significantly shorter (1.79 Å) as compared to the U-O bonds lengths in the equatorial positions (2.13-2.50 Å) because the bonds formed between the axial oxygen atoms (O54 and O55) and the uranium are double bonds, while the bonds formed between the equatorial oxygen atoms (O2, O22, O42, and O59) and the uranium atom correspond to single bonds. There is an excellent agreement between the uranium-oxygen double bond distances that we have determined from the equilibrium geometry (1.79 Å) and those experimentally measured by EXAFS methods (1.78 Å) for uranyl ion and uranyl hydroxide complexes (40). There is also good agreement between the range of bond distances for the bonds formed between equatorial oxygen atoms and uranium atom (2.13-2.50 Å) and those found experimentally in the literature (40, 43) (2.41 Å). The uranyl-TF complex has a charge of -3 near the uranyl-amino acid moiety and thus possesses a significant dipole moment (7.79 D), and it has an overall singlet electronic state characterized as 1A. The highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) energy gap, which is a qualitative measure of the extent of stability, is 1.90 eV, suggesting that the complex is quite stable. All bond angles between the oxygen atoms and the uranium atom deviate from 90° because this is not a symmetrical octahedral complex as a consequence of different ligand bulky groups. Moreover, the Oax-U-Oax axial bond angle exhibits a small deviation from linearity (173.36°) due as well to the fact that the uranyl-TF complex is not symmetrical, and thus, different ligands can cause different extents of charge transfers between the uranyl group and the amino acids. However, in solvated form, we expect this bond to approach linearity as solvent molecules would have the effect of stabilizing the complex and canceling differences in the electronic pulls of different amino acids. Overall, the uranyl-TF complex is formed through charge transfer from the amino acid ligands to the uranyl and back transfer of electronic charge from the uranyl through dπ-pπ interactions between uranium and oxygens. We also note that the 5f orbitals of uranium are not involved substantially in the bonding as they are not occupied in the uranyl ion. This could differ for other actinides, which have occupied 5f orbitals. Table 1 compares the U-O bond distances and the bond angles between the U atom and the O atoms in the equatorial and axial positions to those obtained using larger basis sets with diffuse functions indicated in parentheses. The results in parentheses were obtained from an optimization of the uranyl-TF complex using a larger basis set that included diffuse Gaussian functions for the U atom and the O atoms. The basis set was augmented further with diffuse function on uranium with Rs ) 0.01096 and Rp ) 0.0093. We also augmented the oxygen basis sets with diffuse functions with exponents Rs ) 0.098 and Rp ) 0.0622. A critical comparison of the bond distances and bond angles listed in Table 1 indicates that addition of diffuse

BenaVides-Garcia and Balasubramanian

Gaussian functions to the basis sets of U and O atoms did not result in significant changes in the equilibrium geometry. Vibrational Frequencies and IR Spectra. To compare with experiment and assign the observed IR spectrum of the uranyl-TF protein complex, we have computed the second derivatives of the energies and carried out frequency computations at the DFT/B3LYP level. Figure 3a shows the computed IR spectrum for the uranyl-TF complex based on the computed harmonic vibrational frequencies. In the same figure, we have also shown the IR spectra of uncomplexed Tyr and Asp amino acids. We have also shown the computed IR spectrum of deprotonated His (histidine) amino acid, as there is a question to the involvement of histidine with the uranyl-TF complex. In Table 2, we have shown select harmonic vibrational frequencies corresponding to the bending and stretching modes of the axial and equatorial oxygen atoms of the amino acids/carbonate bonded to the uranium atom. These vibrational modes are concerted with other motions (bending and/or stretching) simultaneously occurring at the amino acids and/or carbonate ion sites. It is very interesting to notice that the vibrational frequency for the symmetrical stretch (ss) mode between the uranium atom and the two axial oxygen atoms is in excellent agreement (868.70 cm-1) with the vibrational frequency of the symmetrical stretch mode in the uranyl ion in aqueous solution as experimentally determined by Toth et al. (44) (869 cm-1). The highest frequency bands (around 3700 cm-1) correspond to stretching of the hydroxo group in the carboxylic ends of the aspartate group and the tyrosines. The high frequency bands (around 3500 cm-1) correspond to asymmetrical stretching (as) in the amino groups, while the bands around 3400 cm-1 correspond to symmetrical stretching (ss) in the amino groups. The bands in the range 3000-3200 cm-1 correspond to stretching modes of the hydrogens in the aromatic rings of the tyrosines. The midfrequency bands around 1800 cm-1 correspond to bending modes in the amino groups, while the bands around 1700 cm-1 correspond to the out-of-plane mode in the carbonate ion. Bands in the 1550-1675 cm-1 range corresponding to the carboxylic groups will be discussed further, as they are the most important bands that need to be discussed for the uranyl-TF complex. We have also found vibrational bands in the region from 1643 to 1536 cm-1 that correspond to rocking modes in the aromatic rings in the tyrosines. Bands in the region from 1485 to 1466 cm-1 correspond to rocking modes in the methylene groups, whereas those between 1457 to around 918 cm-1 correspond to rocking and wagging modes in the tyrosines and aspartate group. Bands in the region of 895 to 724 cm-1 correspond to concerted stretching modes between the uranium atom and the oxygen atoms directly coordinated to uranium and rocking and wagging modes in the tyrosines, the aspartate group, and the carbonate ion. Lower frequency bands in the region between 713 and about 300 cm-1 correspond to rocking and wagging modes in the tyrosines, aspartate groups, and carbonate ion. Bands in the region between 300 and 241 cm-1 include bending modes of the uranium atom and the axial and equatorial oxygen atoms that are concerted with rocking and wagging in other parts of the uranyl-TF complex. There is a question concerning participation of His249 in the uranyl-TF complex. It has been noted that although His249 participates in complex formation with Fe3+ and TF, Vidaud et al. (12) have suggested that His249 is not involved in the uranyl-TF complex. Their inference is based on the absence of a band near 1105 cm-1 attributed to His249 side chain. Whereas this band was observed for the Fe3+-TF complex, the corresponding band was not observed for the uranyl-TF

Binding of Uranyl with Human Serum Protein TF

Chem. Res. Toxicol., Vol. 22, No. 9, 2009 1617

Figure 3. (a) Uranyl-TF complex, (b) histidine, (c) aspartate, and (d) tyrosine. Computed IR spectrum generated from a frequency calculation based on the equilibrium geometry of the uranyl-TF complex representing the metal binding of the uranyl-TF complex (a). Computed IR spectra of deprotonated histidine (b), aspartate (c), and tyrosine (d).

1618

Chem. Res. Toxicol., Vol. 22, No. 9, 2009

BenaVides-Garcia and Balasubramanian

Table 2. Harmonic Vibrational Frequencies and Modes Corresponding to the First Coordination Sphere of the Uranium Complexa system

vibrational mode

frequency (cm-1)

IR intensity

O55-U-O59 O54-U-O59 O54-U-O55 O54-U-O55 O54-U-O55 O54-U-O55, U-O59 O54-U-O55, U-O59 O54-U-O55, U-O59 O54-U-O55 O54-U-O55 U-O42 O2-U-O22

bending bending bending bending bending symmetric stretch asymmetric stretch symmetric stretch asymmetric stretch asymmetric stretch stretch asymmetric stretch

241.1 248.8 278.8 294.8 300.3 724.1 795.8 868.7 890.1 895.6 918.7 1374.5

5.33 13.66 95.79 57.50 70.56 37.32 163.97 257.61 198.87 221.02 37.43 727.30

a The labels of the atoms correspond to the atomic labeling scheme used in all calculations.

Figure 4. Plot of Mulliken charge distribution (hydrogens not shown). Green colors indicate atoms with positive charges, red colors designate atoms with negative charges, and black colors signify neutral atoms. The complex thus has both hydrophobic and hydrophilic regions.

complex, thus eliminating the possibility of His249 being involved in the uranyl-TF complex. As can be seen from Figure 3a, there are several features of His IR spectrum that are not present in the IR spectrum observed by Vidaud et al. (12) or the ones that we have computed for the complex. The IR spectral feature around 1105 cm-1 that Vidaud et al. (12) attributed to His249 side chain is computed in our study as 1075 cm-1 for His in an uncomplexed form (Figure 4b). The other prominent features of His are the intense bands at 1150, 1825, and 2900 cm-1. These bands are not observed experimentally for the uranyl-TF complex nor did our computations exhibit any prominent features. The absence of these prominent signatures of His eliminate this amino acid being involved in the uranyl-TF complex, thus confirming Vidaud et al. (12). The IR spectral features that we see for the uranyl-TF complex in Figure 3a in the region of 1500-1600 cm-1 warrant further discussion as these correspond to the vibrations of carboxylate group bound to the uranyl ion. These modes are expected to be red-shifted as the carboxylate groups of the amino acid bind to the uranyl ion in comparison to the free amino acids shown in Figure 3c,d. First, we consider the amino acid anions in their free forms for comparison. The Asp has a carboxylate group that coordinates the uranium, and this group has a symmetric stretch (ss) mode of 1375.6 cm-1 and an asymmetric stretch (as) mode of 1723.9 cm-1. When both amino

and carboxylic groups are deprotonated (with overall charge -2), we obtain νss and νas 1203.1 and 1830.1 cm-1 for His, 1190.0 and 1824.2 cm-1 for Tyr, and 1322.5 and 1811.3 cm-1 for Asp. All of these modes are expected to be red-shifted when bound to the uranyl and as seen from Figure 3b to d; the most intense band at 1550 cm-1 as well as bands in the 1650-1700 cm-1 region are attributed to the νas asymmetric stretches of the carboxylate groups bound to the uranyl ion. However, we note one difference between Vidaud et al.’s (12) interpretation and ours. That is, Vidaud et al. (12) have suggested that the carbonate ion may not be involved in the complex formation with uranyl although they have not excluded the possibility, but we find that the carbonate ion binds to the uranyl group with two Tyr and one Asp as seen from Figure 2a. The asymmetric carboxylate group stretches seen in Figure 3a are combinations of vibrational modes of the amino acid and the carbonate ion. Clearly, the carboxylate group stretches are redshifted in the complex as compared to the free amino acid frequencies consistent with the observed spectra and complex formation with the uranyl ion. The sharp IR spectral features in the 1350-1400 cm-1 region for the uranyl-TF complex are clearly due to the symmetric carboxylate groups stretching frequencies of the bound amino acids to the uranyl and are redshifted in relation to the corresponding frequencies of the amino acids in their free uncomplexed forms. Molecular Orbitals, Laplacian and Electron Density Plots, and Mulliken Charge Analysis. We have carried out Mulliken population analysis calculations as well as molecular orbital electron density analysis to obtain information regarding the distribution of the partial atomic charges, the electron density, and the molecular orbitals in the uranyl complex. Figure 4 shows the plot of the Mulliken charge distribution on the atoms close to the uranyl of the uranyl-TF complex (hydrogen atoms not shown). The green color indicates electronic charge deficiency or positive charge, while the red color in Figure 4 indicates electronic charge concentration. The uranium atom has an overall charge depletion of 0.935, while the oxygen atoms directly coordinated to U have excess charges that range from -0.240 to -0.357. These partial charges result from electron transfer from the amino acid groups to U and back transfer from uranyl to amino acid. Thus, the charges on carboxylate groups are reduced substantially from unity for the free uncomplexed anions. The bonding between uranyl and amino acids is dative in nature where electron-rich anions donate considerable charge density to U. We also note that the uncomplexed uranyl ion would have two positive charges on U, which are reduced to 0.935 as a consequence of complex formation. However, back transfer is indicated as uranyl would have gained more electronic charge if each amino acid anion and carbonate contributed a single electron to U. This is clearly suggestive of dπ-pπ back transfer of electronic charge from U to the amino acids bound to it. Figure 5a shows the three-dimensional (3D) plot of the electron density of the uranyl-TF complex, and it can be seen that there are areas of significant electron density along the entire uranyl-TF complex, particularly around the coordination environment around the uranium atom, as well as in the aromatic rings of the two tyrosines. The various peaks in Figure 5a suggest delocalization of the electron charge density consistent with the presence of aromatic rings in the complex as well as electron donor-acceptor mechanisms operating in the complex. Figure 5b shows the 3D plot of the Laplacian of the electron density of uranyl-TF complex, and it shows some very interesting features. Laplacians are second gradients of electron

Binding of Uranyl with Human Serum Protein TF

Figure 5. (a) Three-dimensional electronic density plots of the uranyl-TF complex. (b) Three-dimensional plot of the Laplacian of the charge density of the uranyl-TF complex.

charge densities as they contrast the electrophilic and nucleophilic regions in the complex. The peaks in the Laplacian plots represent regions of electron depletion, while the valleys represent electron accumulation or nucleophilic regions. Thus, 3D Laplacian plots serve as excellent indicators of chemical reactivity because they show where the charge is concentrated (Lewis base) represented by a negative valley and where there is charge depletion (Lewis acid) represented by positive peaks. The Laplacian plot shows a positive peak located on the uranium atom, which indicates that there is charge depletion on the uranium atom, which is consistent with the positive charge (0.936) determined in the Mulliken population analysis (Figure 4). We can also notice that the equatorial and axial oxygen atoms exhibit negative valleys, which indicate that these oxygen atoms have negative charges concentrated on them, also in agreement with the negative charges determined in the Mulliken population analysis (-0.240 to -0.357). The positive peak on the uranium atom indicates that this is a Lewis acid (electron acceptor) even in the complexed form, while the axial and equatorial oxygen atoms act as Lewis bases (electron donors). Another very interesting observation is that there is a large positive peak located on the H atom (H57) that participates in the LBHB between the phenolic group in one tyrosine and the amino group in the aspartate group. Figure 6a,b show the HOMO and the LUMO, respectively. The HOMO orbital consists mainly of contributions from the 3p orbitals from the O atoms in the carbonate ion, while the LUMO orbital consists mostly of contributions from the atoms in the tyrosine that form a hydrogen bond with the aspartate group, H (2s orbital), C atoms (2s, 4p orbitals), N atoms (2s orbital), and O atoms (3p, 4p orbitals). The HOMO-LUMO

Chem. Res. Toxicol., Vol. 22, No. 9, 2009 1619

Figure 6. (a) HOMO of the uranyl-TF complex. (b) LUMO (lowest occupied molecular orbital) of the uranyl-TF complex.

energy gap corresponds to 1.90 eV; this energy gap is a good indicator of chemical reactivity because if the energy gap is medium-small, like in this case, then the complex is expected to be reactive. Thus, the complex can accept electrons from strong donors at the uranium site and donate electrons further to acceptors stronger than uranium, thus acting in a dual manner. This may explain how uranyl complexes have the capability to enter the BBB through binding to receptors, as the complex exhibits desired characteristics of both hydrophobic and hydrophilic features that are required to bind to receptors that facilitate entry into the BBB.

Conclusions We have optimized the equilibrium geometry for the local environment of the uranyl-TF complex that is formed when uranium binds to TF protein. We have also obtained the computed IR spectra of the complex and corresponding free amino acids that are in their free uncomplexed forms. Our optimized geometry of the uranyl-TF complex reveals that uranyl is bound primarily to two Tyr, one Asp, and a carbonate ion in the complex. Moreover, we have found a short, strong hydrogen bond formed between the aspartate group and one of the tyrosines, which could play an important role in the stabilization and formation of the uranyl-TF complex. The computed IR spectra indicate that the uranyl-TF complex is formed by complexation of two tyrosines (Tyr95 and Tyr188), one aspartate group (Asp63), and one carbonate ion. His (His249) is not involved in the formation for the complex unlike the corresponding complex with Fe3+, where His249 is involved in the complex. Our computed IR spectra are also consistent with the ones observed by Vidaud et al. (12) in that we have

1620

Chem. Res. Toxicol., Vol. 22, No. 9, 2009

also found red shifts in the symmetric and asymmetric COOstretching frequencies of the complexed amino acids as compared to the free amino acids. These features were noted in the region of 1350-1400 cm-1 for the symmetric stretches and 1550-1700 cm-1 for the asymmetric stretches. The His prominent bands in the 1150, 1825, and 2900 cm-1 were missing in the complex observed experimentally and the one that we computed, suggesting that His is not present in the complex. Our computed Laplacian charge plots indicate high chemical activity in this complex both as an electrophile and as a nucelophile, facilitating binding to a number of receptors that facilitate entry into a number of target organs and the BBB. Lipophilicity, H-bond donor acidity, and H-bond acceptor basicity have been considered as important molecular properties in QSAR models of BBB permeation. These properties are intimately connected to the electrophilic and nuclephilic regions present in the complex (45, 46), which in turn provide for binding of suitable receptors for entry into BBB. The Mulliken charge density plots and the 3D charge density plots reveal the operation of a donor-acceptor mechanism in the complex formation as electronic charge is donated form the amino acid anions to the uranyl and back-donation of charge from the uranium to the amino acid through dπ-pπ dative interaction. Acknowledgment. This research was supported by the U.S. Department of Energy under Grant DE-FG02-05ER15657. The work at LLNL was performed under the auspices of U.S. Department of Energy. We acknowledge computational support on Lawrence Livermore’s supercomputer comprising 992 processors supported by the DOE’s accelerated supercomputing initiative program.

References (1) Taylor, D. M., and Taylor, S. K. (1997) Environmental uranium and human health. ReV. EnViron. Health 12, 147–157. (2) Mordasheva, V. V. (2004) Long-term intake of uranium and its distribution in human organs and tissues. Med. Radiol. Radiats. Bezop. 49, 5–12. (3) Ghosh, S., Kumar, A., Pandey, B. N., and Mishra, K. P. (2007) Acute exposure of uranyl nitrate causes lipid peroxidation and histopathological damage in brain and bone of Wister rat. J. EnViron. Pathol., Toxicol. Oncol. 26, 255–261. (4) Lestaevel, P., Houpert, P., Bussy, C., Dhieux, B., Gourmelon, P., and Paquet, F. (2005) The brain is a target organ after acute exposure to depleted uranium. Toxicology 212, 219–226. (5) Lemercier, V., Millot, X., Ansoborlo, E., Menetrier, F., Flury-Herard, A., Rousselle, Ch., and Scherrmann, J. M. (2003) Study of uranium transfer across the blood-brain barrier. Radiat. Prot. Dosim. 105, 243– 245. (6) Houpert, P., Frelon, S., Monleau, M., Bussy, C., Chazel, V., and Paquet, F. (2007) Homogeneous accumulation of uranium in the brain of rats. Radiat. Prot. Dosim. 127, 84–89. (7) Taylor, D. M. (1998) The bioinorganic chemistry of actinides in blood. J. Alloys Compd. 271-273, 6–10. (8) Vidaud, C., Dedieu, A., Basset, C., Plantevin, S., Dany, I., Pible, O., and Que´me´meur, E. (2005) Screening of human serum proteins for uranium binding. Chem. Res. Toxicol. 18, 946–953. (9) Harris, W. R. (1998) Binding and transport of nonferrous metals by serum transferrin. Struct. Bonding (Berlin) 92, 7919–7928. (10) Sun, H., Li, H., and Sadler, P. J. (1999) Transferrin as a metal ion mediator. Chem. ReV. 99, 2817–2842. (11) Wally, J., Hallbrooks, P. J., Vonrhein, C., Rould, M. A., Everse, S. J., Mason, A. B., and Buchanan, S. K. (2006) The crystal structure of iron-free human serum transferring provides insight into inter-lobe communication and receptor binding. J. Biol. Chem. 281, 24934– 24944. (12) Vidaud, C., Gourion-Arsiquaud, A., Rollin-Genetet, F., Torne-Celer, C., Plantevin, S., Pible, O., Berthomieu, C., and Que´me´meur, E. (2007) Structural consequences of binding UO22+ to apotransferrin: Can this protein account for entry of uranium into human cells. Biochemistry 46, 2214–2226.

BenaVides-Garcia and Balasubramanian (13) MacGillivray, R. T., Moore, S. A., Chen, J., Anderson, B. F., Baker, H., Luo, T., Bewley, M., Smith, C. A., Murphy, M. E., Wang, T., Mason, A. B., Woodworth, R. C., Brayer, G. D., and Baker, E. N. (1998) Two high-resolution crystal structures of the recombinant N-lobe of human transferrin reveal a structural change implicated in iron release. Biochemistry 37, 7919–7928. (14) Sutton, M., and Burastero, S. R. (2004) Uranium (IV) solubility and speciation in simulated elemental biological fluids. Chem. Res. Toxicol. 17, 1468–1480. (15) Grossmann, J. G., Crawley, J. B., Strange, R. W., Patel, K. J., Murphy, L. M., Neu, M., Evans, R. W., and Hasnain, S. S. (1998) The nature of ligand induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants. J. Mol. Biol. 279, 461–472. (16) Scapolan, S., Ansoborlo, Moulin, C., and Madic, C. (1998) Investigations by time-resolved laser-induced fluorescence and capillary electrophoresis of the uranyl-phosphate species: application to blood serum. J. Alloys Compd. 271-273, 106–111. (17) Barnett, D. J., Parker, C. L., Blodgett, D. W., Wierzba, R. K., and Links, J. M. (2006) Understanding radiologic and nuclear terrorism as public health threats: preparedness and response perspectives. J. Nucl. Med. 47, 1653–1661. (18) Balasubramanian, K., and Cao, Z. (2007) Fluxional motions and internal rotational barriers of water molecules bound to UO22+, NpO22+, and PuO22+. Chem. Phys. Lett. 432, 259–263. (19) Balasubramanian, K., Khokhani, K., and Basak, S. C. (2006) Complex graph matrix representations and characterizations of proteomic maps and chemically induced changes to proteomes. J. Proteome Res. 5 (5), 1133–1142. (20) Gute, B. D., Balasubramanian, K., Geiss, K. T., and Basak, S. C. (2004) Prediction of halocarbon toxicity from structure: A hierarchical QSAR approach. EnViron. Toxicol. Pharmacol. 16 (1-2), 121–129. (21) Basak, S. C., Grunwald, G. D., Gute, B. D., Balasubramanian, K., and Opitz, D. (2000) Use of statistical and neural net approaches in predicting toxicity of chemicals. J. Chem. Inf. Comput. Sci. 40 (4), 885–890. (22) Basak, S. C., Mills, D., Gute, B. D., and Hawkins, D. M. (2003) Predicting mutagenicity of congeneric and diverse sets of chemicals using computed molecular descriptors: A hierarchical approach. In QuantitatiVe Structure-ActiVity Relationship (QSAR) Models of Mutagens and Carcinogens (Benigni, R., Ed.) Chapter 7, pp 207234, CRC Press, Boca Raton, FL. (23) Basak, S. C., Mills, D., Gute, B. D., Grunwald, G. D., and Balaban, A. T. (2002) Applications of topological indices in the property/ bioactivity/ toxicity prediction of chemicals. In Topology in Chemistry: Discrete Mathematics of Molecules (Rouvray, D. H., King, R. B., Eds.) pp 113-184, Horwood Publishing Ltd., Chichester, West Sussex, United Kingdom. (24) Randic, M., Zupan, J., and Balaban, A. T. (2004) Unique graphical representation of protein sequences based on nucleotide triplet codons. Chem. Phys. Lett. 397, 247–252. (25) Ilies, M. A., Seitz, W. A., Ghiviriga, I., Johnson, B. H., Miller, A., Thompson, E. B., and Balaban, A. T. (2004) Pyridinium cationic lipids in gene delivery: A structure-activity correlation study. J. Med. Chem. 47, 3744–3754. (26) Pible, O., Guilbaud, P., Pellequer, J.-L., Vidaud, C., and Que´me´neur, E. (2006) Structural insights into protein-uranyl interactions: Towards an in silico detection method. Biochimie 88, 1631–1638. (27) Parr, R. G., and Yang, W. (1989) Density Functional Theory of Atoms and Molecules, Oxford University Press, New York. (28) Becke, A. D. (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652. (29) Vosko, S. H., Wilk, L., and Nusair, M. (1980) Accurate spin-dependent electron liquid correlation energies for local spin density calculations: A critical analysis. Can. J. Phys. 58, 1200–1211. (30) Stephens, P. J., Devlin, I. J., Chabalowski, C. F., and Frish, M. J. (1994) Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields. J. Phys. Chem. 98, 11623–11627. (31) Balasubramanian, K. (1997) RelatiVistic Effects in Chemistry. Parts A & B, Wiley-Interscience, New York, NY. (32) Ermler, W. C., Ross, R. B., and Christiansen, P. A. (1991) Ab initio relativistic effective core potentials with spin-orbit operators. VI. Fr through Pu. Int. J. Quantum Chem. 40, 829–846. (33) Pacios, L. F., and Christiansen, P. A. (1985) Ab initio relativistic effective core potentials with spin-orbit operators. I. Li through Ar. J. Chem. Phys. 82, 2664–2671. (34) Dunning, T. H., and Hay, P. J. (1977) Methods of Electronic Structure Theory, Phenum, New York. (35) Van Duijneveldt, F. B. (1971) IBM Tech. Res. Rep. RF1971, 945. (36) Cao, Z., and Balasubramanian, K. (2005) Theoretical studies of UO2(H2O)n2+, NpO2(H2O)n2+, and PuO2(H2O)n2+ complexes (n ) 46) in aqueous solution and gas phase. J. Chem. Phys. 123, 114309.

Binding of Uranyl with Human Serum Protein TF (37) Majumdar, D., and Balasubramanian, K. (2005) Theoretical studies on the electronic structures of UO2(CO3)22- and its metal salts: M2UO2(CO3)2 (M ) Li+ and Na+). Mol. Phys. 103, 931–938. (38) Majumdar, D., Balasubramanian, K., and Nitsche, H. (2003) Theoretical study of aqueous uranyl carbonate (UO2CO3) and its hydrated complexes: UO2CO3. nH2O (n ) 1-3). Chem. Phys. Lett. 372, 232– 241. (39) Allen, P. G., Bucher, J. J., Clark, D. L., Edelstein, N. M., Ekberg, S. A., Gohdes., J. W., Hudson, E. A., Kaltsoyannis, N., and Lukens, W. W. (1995) Multinuclear NMR, Raman, EXAFS, and X-ray diffraction studies of uranyl carbonate complexes in near-neutral aqueous solution. X-ray structure of [C(NH2)3]6[(UO2)3(CO3)6].6.5H2O. Inorg. Chem. 34, 4797–4807. (40) Wahlgreen, U., Moll, H., Grenthe, I., Schimmelpfennig, B., Maron, L., Vallet, V., and Gropen, O. (1999) Structure of unarium (VI) in strong alkaline solutions. A combined theoretical and experimental investigation. J. Phys. Chem. A 103, 8257–8264.

Chem. Res. Toxicol., Vol. 22, No. 9, 2009 1621 (41) Frisch, M. J., et al. (2003) GAUSSIAN 03, revision C.02, Gaussian, Inc., Pittsburgh, PA. (42) Cleland, W. W., and Krevoy, M. M. (1994) Low-barrier hydrogen bond and enzymic catalysis. Science 242, 1887–1890. (43) Clark, D. L., Hobart, D. E., and Neu, M. P. (1995) Actinide carbonate complexes and their importance in actinide environmental chemistry. Chem. ReV. 95, 25–48. (44) Toth, L. M., and Begun, G. M. (1981) Raman spectra of uranyl ion and its hydrolysis products in aqueous nitric acid. J. Phys. Chem. 85, 547–549. (45) Adenot, M., and Lahana, R. (2004) Blood-brain barrier permeation models: Discrimination between potential CNS and non_CNS drugs including P-glycoprotein substrates. J. Chem. Inf. Comput. Sci. 44, 239–248. (46) Rozas, I., Du, Q., and Arteca, G. A. (1995) Interrelation between electrostatic and lipophilicity potentials on molecular surfaces. J. Mol. Graphics 13, 98–108.

TX900184R