Association of antitumor antibiotic Mithramycin with Mn2+ and the potential cellular targets of Mithramycin after association with Mn2+

Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn2+). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn2+ in the mole ratio of 2:1 [MTR: Mn2+] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)2 Mn2+] has a right-handed twist conformation similar in structure with the complexes reported for Mg2+ and Zn2+. This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)2 Mn2+] complex binds to double-stranded DNA with an apparent dissociation constant of 32 μM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298 μM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn2+ containing metalloenzyme manganese superoxide dismutase from Escherichia coli.


Introduction
Mithramycin (plicamycin, MTR) is a naturally occurring anticancer antibiotic isolated from Streptomyces plicatus (Calabresi, Chabner, Hardman, & Limbard, 1991). It belongs to the aureolic family of drugs, which consists of a chromomycinone moiety, either side of which is linked to sugar residues via O-glycosidic linkages ( Figure 1) (Wohlert et al., 1999). MTR is established as an antibiotic for Gram-positive bacteria. It has been used clinically for many years to treat testicular carcinoma and several types of cancer like leukemia, as well as hypercalcemia in patients with metastatic bone lesions (Lumachi, Brunello, Roma, & Basso, 2008). The anticancer properties were ascribed to the inhibitory effect of MTR on DNA replication and transcription during macromolecular biosynthesis (Goldberg & Friedman, 1971). The ability of MTR to bind via DNA minor groove and thereby regulating gene expression is the rationale for using it in these pathological cases (Chakraborty, Devi, Sarkar, & Dasgupta, 2008;Devi, Chakraborty, & Dasgupta, 2009;Goldberg & Friedman, 1971;Wohlert et al., 1999). MTR was found to inhibit the binding of transcription factor Sp1 to its promoter, which in turn leads to gene transcription modulation of different genes, including c-myc, and haras, as well as antiapoptotic genes (Campbell et al., 1994;Jones, Cui, & Miller, 1995). Moreover, MTR has been found to cross the blood-brain barrier and is in its preclinical trials in Huntington's disease (Ferrante et al., 2004). Structural analysis revealed that, MTR binds to minor groove of DNA around GC-rich sequences in the presence of bivalent metal ions such as Mg 2+ , Zn 2+ , Co 2+ , Fe 2+ etc (Aich & Dasgupta, 1995;Devi, Pal, Banerjee, & Dasgupta, 2007;Gochin, 1998;Hou & Wang, 2005;Lu, Wang, Yuann, Huang, & Hou, 2009). Previous studies from our laboratory have shown that MTR binds to different types of bivalent metal ions like Mg 2+ , Zn 2+ , and Cu 2+ , of which Mg 2+ belongs to the main group element, whereas Zn 2+ and Cu 2+ are transition metal ions (Aich & Dasgupta, 1995;Devi et al., 2007;Lahiri et al., 2012).
Considering the complex-formation ability of MTR with the metal ions, we have studied its binding ability with a Mn 2+ , an essential micronutrient. Manganese is an important trace element required by all organisms in various cellular processes, including metabolism and oxidative stress defense (Chandra & Shukla, 2006;Mena, Marin, Fuenzalida, & Cotzias, 1967;Montes, Alcaraz-Zubeldia, Muriel, & Rıós, 2001). The most important of them is that it protects against reactive oxygen species (ROS) and increases the fitness of cells by minimizing energy expenditure on the synthesis of a defense mechanism. The manganese superoxide dismutase (MnSOD), a metalloenzyme, is a typical example of such defense mechanism against oxidative damage (Fridovich, 1975(Fridovich, , 1997Zheng, Domsic, Cabelli, McKenna, & Silverman, 2007). It catalyzes the degradation of toxic superoxide radicals to molecular oxygen and hydrogen peroxide. MnSOD is found in both prokaryotes and mitochondrial matrices.
In human body a very high concentration of Mn 2+ is present in saliva whereas in blood the concentration is in nanomolar range (Horsburgh, Wharton, Karavolos, & Foster, 2002). Thus, manganese ions become a potential signal by which bacteria can identify a shift from a mucosal environment to a more invasive site. The homeostasis of Mn 2+ is important, as excess of Mn 2+ is toxic. For instance, in humans, exposure to manganese has been associated with a neurological syndrome called 'manganism,' whose symptoms resemble those of Parkinson's disease (Olanow, 2004). Recently it has been shown that Mn 2+ accumulates in brains of cirrhotic patients who also present motor abnormalities. Mn 2+ ion alters dopaminergic transmission promoting an increase in the turnover of dopamine (Pal, Samii, & Calne, 1999). Small molecules with the ability to sequester Mn 2+ from the cell with a relatively high affinity might be putative therapeutic agents for these cases to regulate cellular manganese homeostasis pathway.
Considering the complex-formation ability of MTR with the metal ions, we have studied by means of different spectroscopic and calorimetric techniques its binding ability with Mn 2+ in view of the important biological roles of the metal ion. These studies have led us to understand its mechanism of binding with Mn 2+ . We have also examined plausible targets of the resultant antibiotic: Mn 2+ complex inside the cell. With the above objectives, we have studied the ability of binding [MTR: Mn 2+ ] complex to bind double helical DNA as well as chromatin, the protein-DNA complex keeping in view the earlier reports of [(MTR) 2 : metal ion] complexes as active DNA-binding agents. Earlier we have shown the ability of MTR to bind Zn(II) containing metalloenzymes such as alcohol dehydrogenase (Devi et al., 2009

Absorption spectroscopy
Absorbance spectra were recorded in a Cecil CE7500 spectrophotometer. Concentration of MTR was calculated from a known molar absorption coefficient of 10,000 M −1 cm −1 at 400 nm wavelength. Similarly, concentrations of the polynucleotides were measured from the molar extinction coefficients, which are 8400 M −1 cm −1 at 254 nm for poly (dG-dC) and 6800 M −1 cm −1 at 260 nm poly (dA-dT) (Wells, Larson, Grant, Shortle, & Cantor, 1970). All the spectroscopic studies were carried out in 20 mM Tris-HCl buffer pH 8.0 at 25°C.

Fluorescence spectroscopy
Fluorescence spectra were recorded in a Perkin-Elmer LS55 luminescence spectrometer using a quartz cuvette of 1 cm path length. Reported spectra were an average of four runs and corrected for buffer contribution. In order to avoid photo-degradation of MTR, the fluorescence excitation wavelength was 470 nm instead of the absorption maximum and the emission was recorded over the range of 500-700 nm (Aich & Dasgupta, 1995).

Circular dichroism (CD) spectroscopy
The Circular dichroism (CD) spectra were recorded in a Jasco J720 spectropolarimeter. CD spectra in the near UV and visible region were recorded on the addition of increasing concentration of Mn 2+ to a fixed concentration of MTR (20 μM). Reported spectra were an average of four scans. The observed data points were smoothened by Jasco CD Standard Analysis Software.

Isothermal titration calorimetry (ITC)
The enthalpy changes resulting from the association of MTR with Mn 2+ was measured in a VP-ITC microcalorimeter (MicroCal Inc., USA.) using the inbuilt VP viewer 2000 software with Origin 7.0. The single injection mode (SIM) was used to determine the apparent binding constant for the association of MTR with Mn 2+ . A single shot of 250 μl Mn 2+ solution (2.5 mM) was injected over a span of 500 s into the cell containing 75 μM of MTR and the resultant enthalpy change was monitored for 2000 s. The stirring speed of the syringe was 546 rpm. For the control experiments, the same amount of Mn 2+ was added to the buffer (20 mM Tris-HCl, pH 8.0) present in the cell. Corrected enthalpy changes were determined by subtracting the enthalpy change upon addition of Mn 2+ into buffer from the enthalpy change due to addition of Mn 2+ into the same buffer Mn 2+ -containing MTR (75 μM). The resultant enthalpy changes were then analyzed using 'one set of sites' model in the inbuilt MicroCal LLC SIM software to obtain the apparent dissociation constant, K app d , and other thermodynamic parameters (ΔH and ΔS).

Preparation of chromatin and chromosomal DNA
Soluble chromatin was isolated from chicken liver using the method reported earlier by Majumder and Dasgupta (2011). In brief, chicken liver nuclei were isolated as described by Blobel and Potter (1966), and chromatin was prepared by partial digestion with Micrococcal nuclease. Chromosomal DNA was isolated from soluble chromatin by phenol-chloroform-isoamyl alcohol extraction followed by precipitation with isopropanol. Soluble chromatin and chromosomal DNA thus obtained, were dialyzed extensively against 20 mM Tris-HCl (pH 8.0) and concentrations of the samples were determined spectrophotometrically using the molar extinction coefficient (ε 260 ) of 6600 M −1 cm −1 at 260 nm.

Determination of binding stoichiometry of MTR with Mn 2+
The method of continuous variation was employed to determine the stoichiometry of the complex between MTR and Mn 2+ . Absorbance of MTR at 400 nm wavelength was recorded at 25°C for the reaction mixtures, in which the number of moles of Mn 2+ and MTR were varied in fixed volume keeping the total number of moles of Mn 2+ and MTR constant. Under these conditions, the absorbance at 400 nm (A 400 ) was plotted against the mole fraction of MTR in the respective solutions. The break point in the resulting plot corresponds to the mole fraction of MTR in the MTR: Mn 2+ complex. The stoichiometry was obtained in terms of MTR: Mn 2+ [ χ ligand : (1 − χ ligand )], where χ ligand is the mole fraction of MTR calculated as the ratio of molar concentration of antibiotics to the total molar concentration of MTR and Mn 2+ .

Evaluation of dissociation constant (K app d ) of the complex between MTR and Mn 2+
The dissociation constant for the complex between MTR and Mn 2+ was determined by fluorescence, absorbance, and circular dichroism method. In all these methods, fixed concentration of MTR (20 μM) in a cuvette (path length of 1 cm) was titrated with increasing concentration of Mn 2+ . All samples were dissolved in 20 mM Tris-HCl pH 8.0. We employed two methods to determine the affinity constant for association of MTR and Mn 2+ , as described in earlier reports from our laboratory (Devi et al., 2007;Lahiri et al., 2012). In one method, apparent dissociation constant (K app d ) was obtained from the concentration of Mn 2+ required for 50% binding of MTR, i.e. the half saturation value obtained from the plot of fraction bound against the input concentration of Mn 2+ . The other method was used to determine the true dissociation constant (K D ), which takes into account the 2:1 stoichiometry (in terms of MTR: Mn 2+ ) of the complex.

Determination of kinetic parameters for the association of MTR with Mn 2+
The kinetics of association of MTR with Mn 2+ was followed by different spectroscopic properties, such as absorbance at 440 nm, fluorescence at 540 nm, and CD at 275 nm. In all these methods, a fixed concentration of Mn 2+ is mixed with different concentrations of MTR at 25°C. The formation of the complex with time is monitored by change in absorbance intensity at 440 nm, as well as change in fluorescence intensity at 540 nm (λ ex = 470 nm). To determine the rate constant 'k', four independent runs are accumulated in the final data analysis. The association kinetics of MTR and Mn 2+ was also observed from the change in ellipticity at 275 nm. The rate constant of the complexation reaction was determined by fitting the observed kinetic traces to a curve generated on the basis of pseudo-first-order rate equation (1) where S 0 , S t , and S ∞ denote the spectroscopic property at the start of the reaction, after time 't' (in seconds) and at the end of the reaction, respectively. 'k' denotes the rate constant of the reaction. Measurement of change in the spectroscopic property (S t ) started after an initial time lag of 10 − 12 s.

Determination of activation energy for the association of MTR with Mn 2+
The activation energy (E a ) for the association of MTR with Mn 2+ was determined using Arrhenius equation (2) as follows From the slope of Arrhenius plot (ln k vs. 1/T ), the value of activation energy (E a ) was calculated.

Determination of the order of the reaction between MTR and Mn 2+
The methods of isolation and initial rates were used to determine the order of the association reaction between MTR and Mn 2+ . The initial rates were obtained from the slope of the linear portion of the kinetic trace for the first 50 s of the association reaction. To obtain the order of the reaction, one of the reactant concentration was kept constant and in large excess. At different concentrations of the other reactant (R), if the reaction has an order 'n' with respect to R; then the rate of the reaction (r) can be written as equations (3) and (4) where k obs = k [X] and [X] is the reactant whose concentration is kept constant. A double-logarithmic plot of ln r vs. ln [R] gives a straight line of slope 'n', which is the order of the reaction.

Analysis of binding for the association of [(MTR) 2 Mn 2+ ] complex with chromosomal DNA and chromatin
The dissociation constant (K app d ) for association of [(MTR) 2 Mn 2+ ] with chromosomal DNA was obtained by monitoring the change in absorbance, fluorescence, and CD spectra of [(MTR) 2 Mn 2+ ] upon addition of chromosomal DNA using the method described in the Section 2.3.2. Results from absorbance titration to study association of [(MTR) 2 Mn 2+ ] with chromosomal DNA and chromatin were also analyzed by the Scatchard plot (1949) (eq. [5]).
where r = C b /C p (C b is the concentration of bound drug and C p the concentration of DNA/chromatin); n is the binding stoichiometry in terms of bound drug per nucleotide; C f is the molar concentration of the free ligand, and K′ is the intrinsic binding constant.

Enzymatic assay of MnSOD
Inhibition of pyrogallol autoxidation (Marklund & Marklund, 1974) by MnSOD has been used to determine the activity of MnSOD in presence and absence of MTR. The autoxidation was followed from the increase in absorbance of pyrogallol at 420 nm for 30 min in 50 mM Tris-cacodylic acid buffer, pH 8.20 at 25°C. To study the effect of MTR upon the enzyme activity of MnSOD, the solution containing MnSOD (100 nM) was preincubated with different concentrations of MTR (10, 20, and 50 μM) for 1 h at 25°C and mixed with 0.2 mM pyrogallol.

Characterization of MTR: Mn 2+ complex
Association of MTR with Mn 2+ was demonstrated by absorption, fluorescence, and CD spectroscopy. Upon addition of Mn 2+ there is a decrease in absorbance at 400 nm. As shown in Figure 2

Determination of binding stoichiometry
The plot obtained from the method of continuous variation for the formation of MTR: Mn 2+ complex is shown in Figure 3. The inflection point occurs at χ ligand = 0.71 suggesting a stoichiometry of 2.4:1 with respect to MTR: Mn 2+ . Therefore, the composition of the complex is [(MTR) 2 Mn 2+ ].

Binding parameters for the association of MTR with Mn 2+
The stability constant for the complex of MTR with Mn 2+ was evaluated using the method described in Section 2.3.2. Figure 3 represents the plot of the fraction bound against the input concentration of Mn 2+ . A mean curve fits the data satisfactorily. The apparent dissociation constant, K app d , evaluated from the spectroscopic methods is summarized in Table 1. The table also contains the K D values, which are the 'true dissociation constants.' 3.4. Kinetics of association of MTR with Mn 2 + Association of MTR with Mn 2+ follows first-order kinetics with comparable rate constants obtained from the time-dependent changes in absorbance at 440 nm, fluorescence emission intensity at 540 nm, and ellipticity at 275 nm (Figure 4(a)). This is further corroborated from the linear plot for determining the order of the reaction (using to Equation (4)), which was found to be one with respect to MTR (Figure 4(b)) and zero order with respect to Mn 2+ (Figure 4(c)). These results indicate that the same structural alteration step of the reaction is monitored by all the three spectroscopic methods. The relevant kinetic data are summarized in Table 2. Figure S1 (supplementary data) shows a linear plot of ln k vs. 1/T, which suggests a single-step reaction for the association of MTR with Mn 2+ . Energy of activation for the association of MTR with Mn 2+ [obtained from Arrhenius plot, Equation (2)] is 20.73 ± 0.37 kcal mol −1 K −1 .

Thermodynamic parameters for the complex formation between MTR and Mn 2+
Isothermal titration calorimetry (ITC) profile for the association of MTR with Mn 2+ is shown in Figure 5. The thermodynamic parameters for the association are summarized in Table 3. Apparent dissociation constant obtained from  Table 4. Linearity of the Scatchard plot indicates that the binding is non-cooperative.

Association of [(MTR) 2 Mn 2+ ] complex with chromatin
Change in absorption spectra of [(MTR) 2 Mn 2+ ] upon binding to chromatin is shown in Figure 7(a). The binding affinity of [(MTR) 2 Mn 2+ ] for chromatin is lower than that for chromosomal DNA as evident from the dissociation constants (Table 4 and Figure 7(b)).

Interaction of MTR with MnSOD
Spectroscopic studies (absorption, fluorescence, and CD) suggest association of MTR with MnSOD as shown in Figure 8(a-c). Contrary to the quenching of fluorescence intensity of MTR upon binding to Mn 2+ , the emission intensity of MTR increases on association with MnSOD. The pyrogallol autoxidation assay shows that the enzymatic activity of MnSOD remains unaltered upon binding to MTR (Figure 8(d)).

Discussion
The current report proposes the mechanism of association of MTR with the essential micronutrient, Mn 2+ . The biological significance of this association has been demonstrated in the chromatin context. Like other bivalent metal ions Zn 2+ , Fe 2+ , and Co 2+ , MTR forms only one type of complex with Mn 2+ with the stoichiometry of 2:1 (MTR: Mn 2+ ) (Devi et al., 2007;Hou, Lu, Huang, Fan, & Yuann, 2009;Hou & Wang, 2005). However, the association of MTR with Mg 2+ is different from these metal ions (Zn 2+ , Fe 2+ , and Co 2+ ) (Aich & Dasgupta, 1995;Devi et al., 2007;Hou et al., 2009;Hou & Wang, 2005 (Yavuz, Altunkaynak, & Güzel, 2003). It has variable coordination number and prefers to form coordination bond with oxygen/nitrogen atoms of incoming ligands (Millonig, Pous, Gouyette, Subirana, & Campos, 2009   electronic environment of the chromophore (chromomycinone moiety) present in MTR. Due to the complexation with Mn 2+ , π * orbital of the chromomycinone moiety might be stabilized via charge transfer to the positively charged metal center (ligand to metal charge transfer, LMCT), thereby reducing the energy gap between π → π * transition (Huheey, Keiter, & Keiter, 1993). Broadening of both absorption and fluorescence emission spectra might occur due to the distortion of chromophore, which perturbs the energy levels of the molecule. Quenching of the emission spectrum could be an outcome of the deactivation of the singlet exited state  Dissociation constants (K app d ) were determined from spectrophtometric titration using non-linear curve-fitting analysis as stated in Materials and methods. b n values are obtained from Scatchard plot described in Materials and methods.  of the chromomycinone moiety (Aich & Dasgupta, 1995). Similar changes in CD spectra for Mg 2+ , Zn 2+ , Mn 2+ , and Fe 2+ suggests a single type of conformational change of MTR due to complexation (Aich & Dasgupta, 1995;Devi et al., 2007); Hou and Wang 2005. The kinetic mechanism for the formation of [(MTR) 2 Mn 2+ ] has been followed by different spectroscopic methods, like absorption, fluorescence, and CD. From all these methods, we see that the reaction follows a firstorder kinetics with respect to both MTR and Mn 2+ , similar to the kinetics of [(MTR) 2 Zn 2+ ] (Devi et al., 2007). Association of two bulky antibiotics with one metal center in a single step might face steric hindrance. So, we propose that the formation of [(MTR) 2 Mn 2+ ] might go through the following steps: First and third steps of the proposed mechanism are either spectroscopically silent or too fast to be monitored by the spectroscopic methods employed. Linear nature of the Arrhenius plot favors the first explanation. In the second step, structural alteration of the complex takes place. A transient species [(MTR) Mn 2+ ] * is formed, which is monitored spectroscopically. This step is comparatively slower and therefore the rate-determining step of the reaction. Once the conformational alteration has occurred, the second antibiotic binds rapidly to the transient species to form [(MTR) 2 Mn 2+ ]. The overall rate for the above mechanism can be written as: The concentration of Mn 2+ was kept in large excess (300 μM) such that the contribution of (k −1 + k 2 )/k 1 becomes negligible. Under this condition, the overall rate for the complexation reduces to: This is consistent with the observed kinetic data obtained from different spectroscopic methods employed in this study.
Paramagnetic Mn 2+ leads to line broadening in 1 H NMR spectrum. This prevented us from obtaining clean   Devi et al., 2009;Hou et al., 2009). In all these complexes, the metal ion is octahedrally coordinated to O1 and O9 of the chromophore, which serves as a bidentate ligand and the two water molecules act as the fifth and sixth ligand. To comment upon the probable coordination geometry of the metal center in [(MTR) 2 Mn 2+ ], we have compared the CD spectral features of [(MTR) 2 Mn 2+ ] with the previously reported MTR-metal complexes. The CD spectra of all these complexes are characterized by the appearance of a broad band around 450 nm and a negative peak around 274 nm. Available reports have suggested that these spectral features are evidences for an octahedral coordination sphere around the metal ion which have also been validated from the NMR spectra of the complexes Hou & Wang, 2005). We can thus propose that the metal center in [(MTR) 2 Mn 2+ ] has an octahedral coordination. The positive value of the difference in the amplitudes of the observed Cotton effects (A = Δε 1 − Δε 2 ) suggests that this octahedral geometry has a right-handed screw conformation (Demicheli, Albertini, & Garnier-Suillerot, 1991;Lu et al., 2009).
Having characterized the association of MTR with Mn 2+ and the structure of the complex we went on to study the interaction of [(MTR) 2 Mn 2+ ] with chromatin and chromosomal DNA. In eukaryotes, DNA is wrapped with histones to form a nucleoprotein complex, chromatin. [(MTR) 2 Mn 2+ ] binds to chromatin with a relatively lower affinity and lower binding stoichiometry (in terms of ligand/nucleotide) compared to chromosomal DNA (Table 4). This may be ascribed to the histone(s) present in chromatin, which hinders the accessibility of DNA minor groove to [(MTR) 2 Mn 2+ ]. The association of the [(MTR) 2 Mg 2+ ] and [(MTR) 2 Zn 2+ ] with chromatin and chromosomal DNA follow a similar trend (Das & Dasgupta, 2005;Mir, Das, & Dasgupta, 2004;Mir & Dasgupta, 2001). The right-handed twist conformation of [(MTR) 2 Mn 2+ ] facilitates the binding of the complex via DNA minor groove. The binding affinity and stoichiometry for the association of chromatin and chromosomal DNA with [(MTR) 2 Mn 2+ ] is comparable to the earlier reported values of their interaction with other MTR-metal complexes. The red shift of the absorption peaks of [(MTR) 2 Mn 2+ ] upon binding to chromatin and chromosomal DNA could be ascribed to perturbation of the π → π * transition by the chromomycinone ring upon interaction with DNA.
Fluorescence intensity of [(MTR) 2 Mn 2+ ] is quenched in the presence of DNA. On the contrary, the fluorescence intensity of other MTR-metal (Mg 2+ and Zn 2+ ) complexes is enhanced upon binding to DNA. This dif-ference could be attributed to the inherent properties of the metal ions. Affinity of a metal ion for a specific site on DNA is a general function of its charge, hydration-free energy, coordination geometry, and coordinate bond-forming capacity (Subirana & Soler-López, 2003). In aqueous medium, the transition metal ion Mn 2+ has a loosely bound hydration sphere with a distorted octahedral geometry. In the inner hydration sphere, the distance between water and Mn 2+ is 2.3 Å (Millonig et al., 2009). It has been reported that Mn 2+ interacts with DNA through major groove by forming direct bonds with N7 and O6 atoms of guanine bases at GG and GC steps (van de Sande, McIntosh, & Jovin, 1982). It can also form direct bonds with N7 of guanine and O atoms of phosphate backbone. Thus, it can either unwind the DNA helix or change the water structure around the helix. Therefore, the change in conformation of DNA mixed with 1 mM Mn 2+ , may not lead to the excited state proton transfer to [(MTR) 2 Mn 2+ ] bound in the minor groove (Davey & Richmond, 2002). Upon association with DNA, [(MTR) 2 Mn 2+ ] may lose any of the two coordinated water molecules present in the complex to form a direct bond to nitrogen atom present in the nearby guanine base, without any radical alteration in the structure of DNA. In this condition, the central metal ion becomes more electron deficient, which, in turn, can induce LMCT. This phenomenon might lead to the quenching of fluorescence intensity of [(MTR) 2 Mn 2+ ] upon binding to DNA.
In order to check the sequence selectivity of binding of [(MTR) 2 Mn 2+ ] if any, we have determined the binding affinity of [(MTR) 2 Mn 2+ ] with poly (dG-dC) and poly(dA-dT) ( Figure S4). Results indicate that [(MTR) 2 Mn 2+ ] has a higher affinity towards poly (dG-dC) than poly(dA-dT). It has been reported that [(MTR) 2 Mg 2+ ] also binds specifically to the GC-rich sequence of DNA (Goldberg & Friedman, 1971). The molecular basis of sequence specific recognition of DNA was attributed to the H-bonding of phenolic group of MTR and the amino group present in the guanine base in the minor groove of DNA (Banville, Keniry, Kam, & Shafer, 1990;Keniry, Banville, Simmonds, & Shafer, 1993;Keniry, Brown, Berman, & Shafer, 1987;Sastry, Fiala, & Patel, 1995;Sastry & Patel, 1993). This suggests that change in the metal center of the dimeric complex of MTR does not alter sequence specificity of the complex.
To investigate whether the Mn 2+ binding ability of MTR alters the activity of Mn 2+ -containing metalloenzymes, we have studied the effect of MTR on MnSOD using various spectroscopic methods. MnSOD is a metalloenzyme which deals with the defense mechanism against the toxic effects of reactive oxygen species by catalyzing the dismutation of the superoxide radical anion ðO ÁÀ 2 Þ into O 2 and H 2 O 2 (Zheng et al., 2007). MnSOD from E. coli as well as from many prokaryotes is a dimer (Lah et al., 1995;Smith & Doolittle, 1992). The active site of MnSOD contains one Mn 2+ which is coordinated in trigonal bipyramidal geometry by three histidines, one aspartate, and a bound solvent molecule (Fridovich, 1975;Bannister, Bannister, & Rotilio, 1987;Yamakura et al., 1998). Change in absorption spectrum and increase in fluorescence quantum yield can be attributed to the change in transition dipole of the chromophore of MTR due to hydrophobic environment within the protein. This arises as a result of binding of MTR in the hydrophobic backbone of MnSOD. A close inspection of CD spectra of MTR in the presence of MnSOD shows certain similarity in its feature with the CD spectrum of [(MTR) 2 Mn 2+ ]. Binding of MTR to the Mn 2+ in MnSOD could be a plausible reason for the similarity. However, the notable feature is that the association of the antibiotic with MnSOD does not alter its activity.
The affinity of MTR towards various bivalent metal ions may have important roles in different pathological conditions. Mn 2+ is an important trace element required by all organisms in various cellular processes. It is a transition metal with variable oxidation states (Mn 2+ and Mn 3+ ). Therefore, it can participate in disproportionation reaction with the reactive oxygen species (ROS) produced in several biochemical reactions. But excess of free Mn 2+ is toxic, as it gets accumulated in basal ganglia of brain leading to a diseased condition. Removal of excess Mn 2+ is thus necessary. MTR can cross the blood-brain barrier and specifically bind to Mn 2+ present there (Ferrante et al., 2004;Olanow, 2004;Pal et al., 1999). In the current report, we have shown that MTR does not inhibit the free radical scavenging activity of MnSOD upon binding to it. MTR might be used in the treatment of various brain pathologies as a metal ion chelator with reduced toxicity (Jamuar & Lai, 2012). Moreover, the complex-forming ability of MTR with Mn 2+ under physiological conditions and the association of [(MTR) 2 Mn 2+ ] with DNA, may provide an additional pathway for the antitumor activity of MTR in brain tumor cells. Thus, MTR has the potential as an anticancer agent with low toxicity as compared to other metalbased chemotherapeutics.

Supplementary material
The supplementary material for this paper is available online at http://dx.doi.10.1080/07391102.2014.887031.