Photophysical and DNA-Binding Properties of Cytochrome c Modified

Oct 11, 2010 - Cytochrome c (cyt c) derivatives modified with a platinum(II) complex at the lysine residue, cyt c(III)-[Pt(bpy)(dapap)]1 {bpy = 2,2′...
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J. Phys. Chem. B 2010, 114, 13889–13896

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Photophysical and DNA-Binding Properties of Cytochrome c Modified with a Platinum(II) Complex Hiroshi Takashima,*,† Miho Kitano,† Chiharu Hirai,† Hiroshi Murakami,‡ and Keiichi Tsukahara*,† Department of Chemistry, Faculty of Science, Nara Women’s UniVersity, Nara, 630-8506 Japan, and Kansai Photon Research Institute, Japan Atomic Energy Agency, Kizugawa, Kyoto, 619-0215 Japan ReceiVed: July 2, 2010; ReVised Manuscript ReceiVed: September 3, 2010

Cytochrome c (cyt c) derivatives modified with a platinum(II) complex at the lysine residue, cyt c(III)[Pt(bpy)(dapap)]1 {bpy ) 2,2′-bipyridine, and dapap ) 3-(2,3-diaminopropionylamino)propionic acid}, have been prepared. The modified residues are Lys8, Lys13, Lys55, Lys60, Lys73, and Lys88. In the case of the cyt c(III)-[Pt(bpy)(dapap)]1 dyad, the photoexcited singlet state of 1([Pt(bpy)(dapap)]1)* was quenched by the heme Fe(III) moiety through the intramolecular photoinduced energy-transfer reaction via a through-space mechanism. Next, in the presence of calf thymus (CT)-DNA, the DNA-responsive fluorescence properties of cyt c(III)-[Pt(bpy)(dapap)]1 isomers were investigated. The order of the obtained binding constants between the cyt c(III)-[Pt(bpy)(dapap)]1 isomer and CT-DNA in an aqueous solution suggested that the electrostatic interaction is one of the important factors to stabilize the cyt c-DNA complex. Finally, we discussed the rotational motion of the [Pt(bpy)(dapap)]2+ moiety at the surface of cyt c by fluorescence anisotropy measurement. The increase in the anisotropy parameter, r, for each cyt c isomer clearly revealed that the noncovalent recognition of the [Pt(bpy)(dapap)]2+ moiety by CT-DNA is an essential event in the formation of the cyt c-DNA complex and generation of DNA-sensitive fluorescence signals. Introduction Photoinduced long-range electron transfer (ET) and energy transfer (ENT) reactions within protein-DNA matrixes to transport electrons and energy initiated by light have recently received considerable attention in the fields of both chemistry and biology.1-4 In natural systems, DNA photolyase, containing a reduced flavin adenine dinucleotide (FADH-) as a cofactor, is responsible for the repair of cyclobutane pyrimidine dimer lesions by using blue or near-UV light.5 After photon absorption, the excited FADH- in photolyase protein transfers an electron to the pyrimidine dimer lesion in the duplex DNA.6 As a model system, Carell et al. reported a photoinduced DNA-repair reaction from a flavin donor to a thymine dimer acceptor introduced in an oligonucleotide or a defined DNA scaffold as photolyase mimics.7 In addition, another type of cofactor, redoxactive [4Fe4S] cluster, is also found in DNA repair proteins.8,9 Barton et al. demonstrated DNA-mediated ET between the [4Fe4S] cluster in two of these proteins, MutY and endonuclease III, and the guanine radicals.10 These results indicate that the presence of the [4Fe4S] cluster allowed DNA repair proteins to search for damaged bases in DNA.11 Similarly, DNA-binding proteins containing cysteine residues are redox active and can be oxidized at a distance through DNA-mediated ET.12 In order to elucidate the complicated mechanisms of photoinduced reactions within protein-DNA matrixes, a study of protein-regulated DNA ET reactions using a variety of naturally occurring DNA-binding proteins is important.13 Another promising way is the construction of artificial protein-DNA systems * To whom correspondence should be addressed. Phone: +81-742-203391; fax: +81-742-20-3395; e-mail: (H.T.) [email protected], (K.T.) [email protected]. † Nara Women’s University. ‡ Japan Atomic Energy Agency (JAEA).

by using metalloproteins containing a redox-active cofactor. To this end, semisyntheses of hemoproteins can be utilized, because of their known structures and ET properties.14-16 Since the cofactor is stable in the heme pocket under physiological conditions, chemical modifications have been employed to make artificial photoinduced reaction systems,17 such as those involving (1) metal substitution of the heme,18-20 (2) amino acid residue modification by a redox-active photosensitizer,21 and (3) heme replacement with a functionalized metalloporphyrin.22,23 Also, the semisynthetic reconstitution of an artificial cofactor into apo-myoglobin (Mb) has been a topic of interest;24,25 in particular, modification of a heme propionate produces artificial intramolecular photoinduced ET systems of Mb.26,27 Reconstituted zinc-Mb (ZnMb), containing a covalently linked quinone or methylviologen,28,29 confers the donor-acceptor dyad for the intramolecular photoinduced ET reaction from 1(ZnMb)* or 3 (ZnMb)*. Incorporation of a [Ru(bpy)3]2+ (bpy ) 2,2′bipyridine) and a noncovalently linked viologen catenane into a zinc-porphyrin cofactor can provide a donor-sensitizeracceptor triad on the ZnMb surface.30 As previously reported, Mb has been covalently conjugated to a designed peptide or a DNA complementary polynucleotide at the heme propionate groups for the purpose of complexation with DNA.31,32 Also, reconstituted cytochrome b562 with the hemins tethering the polyamine moiety provide a strong binding affinity for DNA.33 To date, we have developed zinc- and metMb modified with acridine derivatives as DNA-binding small molecules to form stable Mb-DNA complexes using noncovalent interactions by hydrophobic and π-π interactions.34 In addition, we have recently designed zinc(II)-protoporphyrin IX appended to a [Pt(bpy)(en)]2+ (en ) ethylenediamine) complex, because of the interesting photophysical and DNA-binding properties of [Pt(bpy)(en)]2+.35 This cofactor is successfully reconstituted into apo-Mb, and the fluorescence signal from the excited singlet

10.1021/jp106121n  2010 American Chemical Society Published on Web 10/11/2010

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SCHEME 1: Synthetic Routes to cyt c(III)-[Pt(bpy)(dapap)]1a

a Reagents and conditions: (a) HSDU, TEA, DMF, r.t., 1 h. (b) TFA, CH2Cl2, r.t., 20 h. (c) [PtCl2(bpy)], AgPF6, DMF, r.t., 32 h. (d) cyt c, Tris/HCl buffer, pH 8.6, 4 °C.

state of1(ZnMb)* is sensitive to the DNA binding. However, there is an experimental problem for these reconstituted Mbs, due to heme or zinc-porphyrin dissociation from the Mb pocket upon complexation with DNA. In this study, we thus prepare cytochrome c (cyt c) modified with a [Pt(bpy)(dapap)]2+ {dapap ) 3-(2,3-diaminopropionylamino)propionic acid} complex at the lysine residue, cyt c(III)-[Pt(bpy)(dapap)]1, as shown in Scheme 1. The obtained cyt c(III)-[Pt(bpy)(dapap)]1 possesses one covalently bound heme via two thioether linkages and one [Pt(bpy)(dapap)]2+ complex at a selected surface lysine residue site. These isomers demonstrate intramolecular photoinduced ET or ENT reactions from the excited singlet state of 1([Pt(bpy)(dapap)]1)* to the heme iron(III) with through-bond and van der Waals interactions. Moreover, because of the preferential noncovalent interactions between DNA and [Pt(bpy)(dapap)]2+ in an aqueous solution, we can study the photophysical and DNA-binding properties of cyt c(III)-[Pt(bpy)(dapap)]1. The detailed mechanisms are discussed for each isomer. Results and Discussion Modification of cyt c by Using the [Pt(bpy)(dapaps)]Cl2 Complex. The synthetic pathway to the platinum(II) complex, 2,2′-bipyridine[3-(2,3-diaminopropionylamino)propionic acid succinimidyl ester]platinum(II) chloride {[Pt(bpy)(dapaps)]Cl2}, was developed according to our previous report (Scheme 1).36 In that study, we used racemic 3-[2,3-bis(tert-butoxycarbonylamino)]propionic acid (1) as a starting material. To produce the succinimidyl ester (2), 1 was first reacted with O-succinimidyl-1,3-dimethyl-1,3-trimethyleneuronium hexafluorophosphate (HSDU) in the presence of triethylamine (TEA) in N,Ndimethylformamide (DMF). After removal of the Boc-group from compound 2 by trifluoroacetic acid (TFA), the resulting 3 was combined with a platinum(II) complex, [PtCl2(bpy)], treated with AgPF6 in DMF. The insoluble materials were then removed by centrifugation, and the desired platinum(II) complex, [Pt(bpy)(dapaps)]Cl2, was obtained. The 1H NMR, ESI-MS, UV-vis spectra, and elemental analysis clearly support the formation of these compounds, and all signals were reasonably assigned (see Supporting Information). The modification of cyt c was carried out at 4 °C. The detailed procedure is described in Experimental Section. Briefly, a cyt c solution in 0.010 M Tris/HCl buffer (pH 8.6) was treated with

Figure 1. Elution behavior of cyt c-[Pt(bpy)(dapap)]n on a CM cellulose column, eluted with 0.10, 0.15, and 0.20 M NaCl solutions containing 0.010 M phosphate buffer at pH 7.0.

Figure 2. UV-vis absorption spectra of cyt c(III)-[Pt(bpy)(dapap)]1 (solid line) and native cyt c(III) (dashed line) in 0.010 M phosphate buffer at concentrations of 1.0 × 10-5 M and at pH 7.0 and 25 °C.

a small amount of DMF solution containing a 4-fold excess of [Pt(bpy)(dapaps)]Cl2. After the solution was dialyzed with water, the supernatant was loaded on a CM-52 cellulose column, equilibrated with 0.010 M phosphate buffer at pH 7.0. The modified cyt c species were eluted with 0.10, 0.15, and 0.20 M NaCl solutions containing 0.010 M phosphate buffer at pH 7.0, and four fractions (1, 2, 3, and 4) were obtained as displayed in Figure 1. Characterization of cyt c(III)-[Pt(bpy)(dapap)]1. The number of [Pt(bpy)(dapaps)]2+ complexes, n, attached on the cyt c surface was determined by UV-vis spectroscopy and ESI-MS. The absorption spectrum of fraction 1 showed a strong Soret and two Q-bands at 415 and 520, 550 nm, respectively, indicating unmodified cyt c(II). For fraction 2, the ratio A409/ A280 in the absorption spectrum was 4.61 and consistent to that for native cyt c(III) (Soret and Q-bands at 409 and 528 nm, respectively, broken line in Figure 2). On the other hand, the UV-vis spectrum of fraction 3 gave a sharp peak at 318 nm as shown in Figure 2 (solid line) and was identical to the sum of the cyt c(III) and the [Pt(bpy)(dapaps)]2+ complex (1:1). The m/z value of ESI-MS analysis of fraction 3 was 12861.0 after deconvolution (Figure S1, Supporting Information). Since the m/z values for native cyt c and [Pt(bpy)(dapaps)]2+ are 12354.5 and 526.5, respectively, fraction 3 was found to be singly modified cyt c(III)-[Pt(bpy)(dapap)]1 (calcd for 12862.9). We note that the yield of cyt c(III)-[Pt(bpy)(dapap)]1 was 17%. Finally, fraction 4 was identified as doubly modified cyt c(III)-

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J. Phys. Chem. B, Vol. 114, No. 43, 2010 13891 TABLE 1: ESI-MS Peak Assignment of Digested Peptides c-h

Figure 3. HPLC profile of cyt c(III)-[Pt(bpy)(dapap)]1 on a CM-5PW column, eluted with a linear gradient from 0.010 M phosphate buffer at pH 7.0 to 0.010 M phosphate buffer containing 0.50 M NaCl at pH 7.0. The inset is a typical profile for the second cycle separation under the same conditions.

HPLC fraction

observed mass

cyt c digested peptide

calculated mass

Lys number

c (I) d (II) e (III) f (IV) g (V) h (VI)

862.77 986.21 590.34 656.37 634.92 919.02

56-72 54-73 80-99 73-79 8-13 9-22

863.29 986.62 590.88 656.74 634.74 919.65

Lys60 Lys55 Lys88 Lys73 Lys8 Lys13

new fragment peak appeared as peak I, whose ESI-MS spectrum gave m/z at 862.77, 863.11, and 863.43 as z ) +3 (bottom spectrum in Figure 4). These observed values are in good agreement with the calculated value for the amino acid sequence from Glu56 to Lys72, GITWKEETLMEYLENPK, appended to the [Pt(bpy)(dapap)]2+ complex, i.e., 863.29. From this result, it is concluded that the platinum(II) complex in fraction c binds the Lys60-amino group. The other HPLC profiles for the enzymatic digestion of fractions d, e, f, g, and h are summarized in Figure S3, Supporting Information. New fragment peaks appeared as II, III, IV, V, and IV, respectively, and these peptides could be successfully characterized by ESI-MS analysis. Here, we represent the peak assignment of digested peptides in Table 1, and the modified Lys residues in the cyt c sequence are found to be 60, 55, 88, 73, 8, and 13 for fractions c, d, e, f, g, and h, respectively. Fluorescence Spectra and Lifetime Measurements. Next, we demonstrate steady-state fluorescence of cyt c(III)-[Pt(bpy)(dapap)]1 (Lys8, Lys13, Lys55, Lys60, Lys73, and Lys88) and a 1:1 mixture of cyt c(III) and [Pt(bpy)(dapap)]2+, measured with the excitation wavelength λex ) 318 nm in 0.010 M phosphate buffer at pH 7.0 and 25 °C (Figure 5). All cyt c(III)[Pt(bpy)(dapap)]1 isomers display broad fluorescence around 430 nm, and the ratios (I/I0) of their intensities based on the I0 for the 1:1 mixture of cyt c(III) and [Pt(bpy)(dapap)]2+ are summarized in Table 2. It is suggested that an intramolecular quenching reaction take place for the cyt c(III)-[Pt(bpy)(dapap)]1 system, and the I/I0 values of six isomers decrease in the order of Lys60, 88, 13, 55, 8, and 73, respectively. Then, we also determined the fluorescence lifetime (τ) for a series of cyt c(III)[Pt(bpy)(dapap)]1 and 1:1 mixture of cyt c(III) and [Pt(bpy)-

Figure 4. HPLC profile for the peptide fragments of fraction c, cyt c(III)-[Pt(bpy)(dapap)]1, digested with lysyl endopeptidase along with that for native cyt c on an ODS column, eluted with a linear gradient from 0.1% aqueous TFA to a mixed solution of aqueous TFA with acetonitrile (1:1) (top). ESI-MS spectrum of the peptide fragments (I) different from those of native cyt c (bottom).

[Pt(bpy)(dapap)]2 by using similar UV-vis and ESI-MS procedures (m/z ) 13370.9 calcd. for 13371.2, Figure S2, Supporting Information). Figure 3 demonstrates the HPLC separation of cyt c(III)[Pt(bpy)(dapap)]1 isomers. Eight fractions, a, b, c, d, e, f, g, and h were obtained for the first separation cycle by HPLC. Fractions a and b appeared to be unmodified cyt c(II) and cyt c(III), respectively. Further purification for fractions c, d, e, f, g, and h was conducted for the second cycle under the same conditions, and we have successfully obtained six cyt c(III)[Pt(bpy)(dapap)]1 isomers. Determination of the Modified Site of cyt c(III)-[Pt(bpy)(dapap)]1. Figure 4 shows typical HPLC profile for the peptide fragments of fraction c, cyt c(III)-[Pt(bpy)(dapap)]1, digested with lysyl endopeptidase along with that for native cyt c. A

Figure 5. Fluorescence spectra of (a) a 1:1 mixture of cyt c(III) and [Pt(bpy)(dapap)]2+ and the six cyt c(III)-[Pt(bpy)(dapap)]1 isomers (b) Lys60, (c) Lys88, (d) Lys13, (e) Lys55, (f) Lys8, and (g) Lys73, measured with the excitation wavelength λex ) 318 nm in 0.010 M phosphate buffer at concentrations of 2.4 × 10-6 M and at pH 7.0 and 25 °C.

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TABLE 2: Ratios of Fluorescence Intensities and Lifetimes for cyt c(III)-[Pt(bpy)(dapap)]1 I/I0 τ1/ns (A1/%)a τ2/ns (A2/%)a τ/τ0b cyt c(III) + [Pt(bpy)(dapap)]2+ Lys60 Lys88 Lys13 Lys55 Lys8 Lys73

1.0 0.60 0.45 0.39 0.36 0.32 0.27

8.2 (100) 0.6 (35) 0.5 (31) 0.4 (25) 0.5 (30) 0.4 (25) 0.4 (32)

5.4 (65) 5.0 (69) 4.7 (75) 4.7 (70) 4.5 (75) 4.2 (68)

1.0 0.45 0.44 0.44 0.42 0.42 0.36

Determined by It ) A1 exp(-t/τ1) + A2 exp(-t/τ2). b τ0 is the lifetime for 1:1 mixture of cyt c(III) and [Pt(bpy)(dapap)]2+. a

Figure 6. Fluorescence decay of cyt c(III)-[Pt(bpy)(dapap)]1 (Lys73, solid line) measured with the excitation wavelength λex ) 266 nm in 0.010 M phosphate buffer at concentrations of 2.4 × 10-6 M and at pH 7.0 and 25 °C. A thin line is the pulse decay, and a dashed line fits to a double exponential decay function.

(dapap)]2+, measured with the excitation wavelength λex ) 266 nm using a third harmonic generation from a mode-locked Ti: Sapphire laser in 0.010 M phosphate buffer at pH 7.0 and 25 °C. Figure 6 shows a typical decay for the Lys73 isomer by photon counting at 450 nm. The ratios of τ/τ0, where τ0 is the lifetime for a 1:1 mixture of cyt c(III) and [Pt(bpy)(dapap)]2+ and τ is the averaged lifetime of cyt c(III)-[Pt(bpy)(dapap)]1, are also presented in Table 2. The order of the τ/τ0 ratios is found to be comparable with that of I/I0. In order to elucidate the mechanism of fluorescence quenching, we prepared reduced cyt c(II)-[Pt(bpy)(dapap)]1 by using sodium ascorbate as a reducing agent. The fluorescence spectra of cyt c(II)-[Pt(bpy)(dapap)]1 and cyt c(III)-[Pt(bpy)(dapap)]1 excited with the same absorbance at 318 nm are given in Figure S4, Supporting Information. These spectra are almost the same, indicating that fluorescent quenching in cyt c(III)-[Pt(bpy)(dapap)]1 may occur by the ENT mechanism, not by ET, from the excited singlet state of 1([Pt(bpy)(dapap)]1)* to the Fe(III) moiety. If the singlet-to-singlet ENT from 1([Pt(bpy)(dapap)]1)* occurs, the rate constant of the long-range ENT reaction (kEN) is often written in the form derived by Fo¨rster:37

kEN ) (1/τ0)(R0/R)6

(1)

where R is the distance between the donor and the acceptor, and R0 is the critical distance where the transfer probability equals the emission probability. Equation 1 can be replaced by eq 2 by using the fluorescent lifetimes,

kEN ) 1/τ1-1/τ0

(2)

TABLE 3: Intramolecular Energy-Transfer Rate Constants and Distances between Heme Iron and Platinum Centers of cyt c(III)-[Pt(bpy)(dapap)]1 through-space through-space through-bond kEN/109 s-1 a R/Å (R0 ) 43 Å)b R’/Åc R/Åd Lys60 Lys88 Lys13 Lys55 Lys8 Lys73

1.5 1.9 2.4 1.9 2.4 2.4

28.3 27.2 26.2 27.2 26.2 26.2

25.8-29.4 28.2-34.0 19.7-22.6 21.1-24.0 25.8-28.0 20.8-26.4

31.7 20.7 32.7 51.7 37.5 44.5

a Calculated by kEN ) 1/τ1 - 1/τ0. b Calculated by kEN ) (1/ τ0)(R0/R)6 where R0 ) 43 Å. c Estimated from the several conformers of cyt c(III)-[Pt(bpy)(dapap)]1. d Estimated from center-to-center distance.

The calculated kEN values for the six isomers of cyt c(III)[Pt(bpy)(dapap)]1 using the obtained τ1 and τ0 values are summarized in the first column of Table 3. If the present ENT is adopted for eq 1 by using τ0, kEN, and the ENT critical distance of R0 ) 43 Å, the center-to-center through-space distance of R for the cyt c(III)-[Pt(bpy)(dapap)]1 isomers can be calculated (shown in the second column, Table 3). These values, except for Lys13 and Lys55, are in agreement with those estimated, R′ (third column), from their several conformers. In these systems, the calculated kEN clearly depends on the estimated through-space distances of R, not on the through-bond distances, as summarized in the third and fourth columns. Thus, the intramolecular quenching reaction of 1([Pt(bpy)(dapap)]1)* by cyt c(III) may occur via through-space ENT mechanism. Thus, intramolecular quenching reaction of 1([Pt(bpy)(dapap)]1)* by cyt c(III) may occur via through-space ENT mechanism. In the case of Lys13 and Lys55 isomers, the flexible spacer at the surface of cyt c(III) may provide some orientations of 1([Pt(bpy)(dapap)]1)* in solution. Therefore, their quenching mechanisms are still unclear at this stage. Interactions Between cyt c(III)-[Pt(bpy)(dapap)]1 and CTDNA. It is also well-known that [Pt(bpy)(en)]2+ and its related square planar mixed-ligand platinum(II) complexes possess binding properties with DNA, such as groove binding, and can be good probes for monitoring interactions with DNA.38 At first, we carried out UV spectral titration for the [Pt(bpy)(dapap)]2+ complex on the addition of CT-DNA (0-10 equiv) at 25 °C in 0.010 M phosphate buffer (pH 7.0) and 0.10 M NaCl. The absorbance at 317 nm decreased with increasing concentrations of CT-DNA, with isosbestic points at 302 and 321 nm (Figure S5, Supporting Information). Therefore, it is suggested that a complex between [Pt(bpy)(dapap)]2+ and CT-DNA is formed. Plots of the absorbance changes at 317 nm versus CT-DNA concentrations in Figure S5 also gave a binding constant (K) of (0.31 ( 0.03) × 103 M-1 by using a nonlinear least-squares method. Next, we investigated the binding property of cyt c(III)[Pt(bpy)(dapap)]1 with CT-DNA in 0.010 M phosphate buffer (pH 7.0) and 0.10 M NaCl. However, the Soret band of cyt c(III)-[Pt(bpy)(dapap)]1 at 409 nm gradually increased in the presence of CT-DNA (0-7 equiv), because of the formation of precipitates of the cyt c(III)-DNA complex (data not shown). A previous report on the binding of native cyt c with CT-DNA in 0.050 M Tris-HCl buffer (pH 8.0, 0.12 M NaCl, 0.020 M KCl) revealed that addition (0.03%) of the nonionic detergent, Triton X-100, can stabilize the cyt c-DNA complex in an aqueous solution without any precipitation.39 Thus, further experiments are conducted by using the following buffer conditions: 0.010 M phosphate buffer, pH 7.0, 0.10 M NaCl, and 0.03% Triton X-100 (buffer A).

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Figure 7. Fluorescence spectral changes of cyt c(III)-[Pt(bpy)(dapap)]1 (Lys60, 7.0 × 10-6 M, λex ) 321 nm) after addition of CT-DNA (0-10 equiv) at 25 °C in 0.010 M phosphate buffer, pH 7.0, 0.10 M NaCl, and 0.03% Triton X-100.

Figure 8. Fluorescence spectra of cyt c(III)-[Pt(bpy)(dapap)]1 (Lys60, 1.0 × 10-5 M) with the vertically polarized excitation wavelength of λex ) 321 nm at 25 °C in 0.010 M phosphate buffer, pH 7.0, 0.10 M NaCl, and 0.03% Triton X-100: (a) unpolarized emission, (b) vertical emission, (c) horizontal emission.

Figure S6, Supporting Information, shows the UV-vis spectral changes of cyt c(III)-[Pt(bpy)(dapap)]1 (Lys60) after addition of CT-DNA in buffer A solutions. No precipitates were formed, and the peak at 317 nm and the Soret band at 409 nm slightly decreased in intensity, with an isosbestic point at 321 nm with increasing CT-DNA concentration (0-10 equiv). Recently, Tan et al. reported that the K value for complexation between methemoglobin (Hb) and CT-DNA at pH 7.0 (0.020 M Tris buffer) was 4.9 × 105 M-1 by using caloriometric titration.40 However, the UV-vis spectral changes of Hb after addition of CT-DNA (up to 20 equiv) under the same conditions were too small (about 5% increase at Soret band) to observe. This minor change indicates that the interactions between the native Hb and DNA do not alter the heme environment to a significant extent. Therefore, the present absorption spectral changes indicate the interactions between the [Pt(bpy)(dapap)]2+ moiety and CT-DNA. We further investigated the steady-state fluorescence of cyt c(III)-[Pt(bpy)(dapap)]1 in the absence and the presence of CTDNA at 25 °C in buffer A. As shown in Figure 7, the fluorescence intensity around 450 nm (λex ) 321 nm) apparently increased. This is probably due to the conformational change of the [Pt(bpy)(dapap)]1 moiety at the surface of cyt c, where the donor-acceptor distance is elongated by CT-DNA, resulting in decreased intramolecular ENT efficiency. From these titrations, the binding constants, K, for the complexation between cyt c(III)-[Pt(bpy)(dapap)]1 isomers and CT-DNA could be determined by using a typical nonlinear least-squares analysis for the changes in the fluorescence intensity at 450 nm. Table 4 summarizes the comparison between the binding constant of

cyt c(III)-[Pt(bpy)(dapap)]1 with CT-DNA and the retention time of HPLC for cyt c(III)-[Pt(bpy)(dapap)]1. Since cyt c(III)[Pt(bpy)(dapap)]1 is cationic and CT-DNA is a polyanion under neutral buffer conditions (pH 7.0), the order of magnitude of the binding constants for the isomers, Lys60 > Lys55 > Lys73 > Lys8 > Lys13, correlates with their order of elution from a cation-exchange CM-5PW HPLC column. For example, the Lys60 isomer has the highest affinity for CT-DNA and has the shortest retention time (Table 4). Electrostatic interaction between the [Pt(bpy)(dapap)]2+ moiety and CT-DNA is one of the most important factors for stability of the cyt c-DNA complexes. Fluorescence Anisotropy Changes for cyt c(III)-[Pt(bpy)(dapap)]1 upon DNA Binding. In order to study the rotational motion of the [Pt(bpy)(dapap)]2+ moiety on the surface of cyt c(III), we performed fluorescence anisotropy measurements with or without CT-DNA. Figure 8 displays a typical steady-state fluorescence spectra of cyt c(III)-[Pt(bpy)(dapap)]1 (Lys60) in buffer A with a vertically polarized excitation wavelength of λex ) 321 nm. By using vertically and horizontally polarized fluorescence intensities and monitoring at 450 nm, we calculated the fluorescence anisotropy, r, and the angle between the absorption and emission transition moments, θ (see Experimental Section). Table 4 also summarizes the degree of fluorescence anisotropy and the binding constant with CT-DNA. In the absence of CT-DNA, the anisotropy parameters, r (θ), of [Pt(bpy)(dapap)]2+ were 0.08 (47) and 0.11 (44) in 0.010 M phosphate buffer (pH 7.0) and buffer A, respectively. In the case of the six cyt c(III)-[Pt(bpy)(dapap)]1 isomers, each r value is different under both phosphate buffer and buffer A conditions,

TABLE 4: Correlation between the Binding Constant (K) of cyt c(III)-[Pt(bpy)(dapap)]1 with CT-DNA, the Retention Time by HPLC, and the Degree of Fluorescence Anisotoropy (r) of cyt c(III)-[Pt(bpy)(dapap)]1 r ((0.02) (θ/degree) a

3

K /10 M [Pt(bpy)(dapap)]2+ cyt c(III) + [Pt(bpy)(dapap)]2+ (1:1) Lys13 Lys8 Lys73 Lys88 Lys55 Lys60 a

c

-1

0.31 ( 0.03b 0.65 ( 0.06 0.99 ( 0.09 1.1 ( 0.1 1.2 ( 0.1 -c 1.3 ( 0.1 1.3 ( 0.1

b

retention time/min

buffer

40.5 39.5 38.6 38.0 37.0 36.0

0.08 (47) 0.22 (33) 0.24 (31) 0.10 (45) 0.20 (35) 0.25 (30) 0.20 (35) 0.14 (41)

buffer Aa

buffer A with DNA (20 equiv)a

0.11 (44) 0.26 (29) 0.28 (27) 0.17 (38) 0.38 (10) 0.26 (29) 0.37 (14) 0.16 (39)

0.11 (44) 0.22 (34) 0.29 (25) 0.35 (17) 0.40 (4) 0.40 (3) 0.39 (6) 0.37 (13)

In 0.010 M phosphate buffer (pH 7.0), 0.10 M NaCl, and 0.03% Triton X-100. b In 0.010 M phosphate buffer (pH 7.0) and 0.10 M NaCl. Not determined.

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Takashima et al. cyt c scaffold is a new and interesting biomimetic model for the photoinduced ET or ENT reactions within a protein-DNA complex. Conclusions We have prepared a platinum(II) complex-bound cyt c, cyt c(III)-[Pt(bpy)(dapap)]1, whose modified Lys residue sequence is 8, 13, 55, 60, 73, and 88. The photoexcited singlet state of 1 ([Pt(bpy)(dapap)]1)* in the cyt c(III)-[Pt(bpy)(dapap)]1 dyad was quenched by the heme Fe(III) moiety through the intramolecular photoinduced ENT reaction via a through-space mechanism. In the presence of CT-DNA, the DNA-responsive fluorescence properties of cyt c(III)-[Pt(bpy)(dapap)]1 isomers were investigated. The order of magnitude of the obtained binding constants between the cyt c(III)-[Pt(bpy)(dapap)]1 isomer and CT-DNA in an aqueous solution, that is, Lys60 > Lys55 > Lys73 > Lys8 > Lys13, correlates with their order of elution from HPLC, which suggests that the electrostatic interaction between the [Pt(bpy)(dapap)]2+ moiety and CT-DNA is an important factors for the stability of the cyt c-DNA complex. Finally, we discussed the rotational motion of the [Pt(bpy)(dapap)]2+ moiety at the surface of cyt c as measured by fluorescence anisotropy with or without CT-DNA. The increase in the anisotropy parameter, r, for each cyt c isomer clearly revealed that the noncovalent recognition of the [Pt(bpy)(dapap)]2+ moiety by CT-DNA is essential for the formation of the cyt c-DNA complex and the generation of DNAsensitive fluorescence signals. We believe that further synthetic manipulation of the cyt c surface by using several small DNAbinders may provide valuable information to elucidate the complicated mechanism of biological photoinduced ET within a protein-DNA matrix. Experimental Section

Figure 9. Illustration of the surface cationic Lys, Arg, and His residues of cyt c by using PyMOL software, including Lys8 and Lys13 (top) and Lys55, Lys60, Lys73, and Lys88 (bottom) residues and heme.

depending on the modified position of the cyt c surface. This is probably because Triton X-100 can increase the viscosity of the buffer solution and regulate the rotational motion of the [Pt(bpy)(dapap)]1 moiety. In the presence of CT-DNA (20 equiv), the fluorescence anisotropy further increased by 0.01-0.21 in buffer A solution, indicating the formation of the cyt c-DNA complex. We herein note that the anisotropy parameters of cyt c(III)-[Pt(bpy)(dapap)]1 may correlate with the binding constant with CT-DNA (Table 4). In the natural systems, cyt c utilizes the multipoint electrostatic interactions to recognize its redox partners, such as cyt b5, flavodoxin, and cyt c peroxidase.41-44 Additionally, theoretical work has suggested that the surface cationic Lys residues of cyt c are crucial.43,45 Figure 9 illustrates the surface cationic Lys, Arg, and His residues of cyt c, including Lys8 and 13 (green) and Lys55, 60, 73, and 88 (blue) residues used in this study. Our experiments indicated that both the multipoint electrostatic interactions and the intercalation into DNA by the [Pt(bpy)(dapap)]2+ moiety at these cationic domains are the essential events to enable the formation of artificial cyt c-DNA complexes in water. The present model system based on the

Materials. DMF was purchased from Wako Chemicals and used as received. [Pt(bpy)(dapaps)]Cl2 was prepared according to Scheme 1 (see Supporting Information). CT-DNA was purchased from Sigma. Cyt c from horse heart (Sigma) was purified as previously described.46 CM-52 cellulose was purchased from Whatman Co. and used as received. All other reagents and solvents were of guaranteed grade. All aqueous solutions were prepared from redistilled water. The ionic strength, I, of the solution was adjusted with NaCl and KCl. Preparation of Pt(II) Complex-Modified cyt c (cyt c(III)[Pt(bpy)(dapap)]n). The following procedures were carried out at 4 °C. Horse heart cyt c in 0.010 M tris(hydroxymethyl)aminomethane (Tris)/HCl buffer (pH 8.6) was treated with a DMF solution of a 4-fold excess of [Pt(bpy)(dapaps)]Cl2. The pH of the mixture was adjusted to 8.5 by using aqueous 0.10 M NaOH. After the solution was dialyzed with water (1 h × 5), the supernatant was loaded on a CM-52 cellulose column (φ 2.2 × 12 cm, equilibrated with 0.010 M phosphate buffer at pH 7.0) at 4 °C. The modified cyt c species were eluted with 0.10, 0.15, and 0.20 M NaCl solutions containing 0.010 M phosphate buffer at pH 7.0, and four fractions were obtained. Each fraction was dialyzed with water and was loaded on a CM-52 cellulose column (φ 2.2 × 2 cm), equilibrated with 0.010 M phosphate buffer at pH 7.0. The concentrated cyt c was eluted with 0.010 M phosphate buffer containing a 1.0 M NaCl at pH 7.0. After the solution was dialyzed with water, the number, n, of modified [Pt(bpy)(dapaps)]Cl2 complexes was determined by UV-vis spectroscopy and ESI-MS. The concentrations of cyt c(III) were spectrophotometrically determined (ε409 ) 1.06 × 105 M-1 cm-1).47

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HPLC Separation of cyt c(III)-[Pt(bpy)(dapap)]1 Isomers. The singly modified cyt c(III)-[Pt(bpy)(dapap)]1 isomers were separated with a Tosoh CM-5PW column (φ 7.5 mm ×7.5 cm) in a Shimadzu LC-10 HPLC system.48 The elution conditions were as follows: a linear gradient from 0.010 M phosphate buffer at pH 7.0 to 0.010 M phosphate buffer containing 0.50 M NaCl at pH 7.0, a flow rate of 0.6 mL/min, and wavelength monitoring at 317 nm. Six fractions containing cyt c(III)-[Pt(bpy)(dapap)]1 were dialyzed with water (1 h × 5) and each was purified again by the same HPLC procedure. Enzymatic Digestion of cyt c(III)-[Pt(bpy)(dapap)]1. A solution of the purified cyt c(III)-[Pt(bpy)(dapap)]1 in 0.10 M Tris-HCl buffer at pH 9.0 was treated with lysyl endopeptidase from Achromobacter lyticus (Wako) at 35 °C for 8 h by a standard method (cyt c:enzyme ) 200:1).48,49 The peptide fragments were dissolved in aqueous 0.1% TFA and separated by reversed-phase chromatography with a Tosoh ODS 120T column (φ 4.6 mm × 25 cm) in a Shimadzu LC-10 HPLC system. The elution conditions were as follows: a linear gradient from 0.1% aqueous TFA to a mixed solution of aqueous TFA with acetonitrile (1:1), a flow rate of 1.0 mL/min, and wavelength monitoring at 317 nm. Peptide fragments different from those of native cyt c were identified by ESI-MS using an Applied Biosystems Mariner. Fluorescence Measurements. Steady-state fluorescence spectra were recorded on a Shimadzu RF-5300 fluorometer. The steady-state fluorescence anisotropy, r, was calculated according to eq 3. Emission intensities were measured on a Perkin-Elmer LS55 fluorometer equipped with a polarizer:

r ) (IVV - GIVH)/(IVV + 2GIVH)

(3)

where IVV and IVH are the corresponding vertically polarized and horizontally polarized emission intensities elicited by vertically polarized excitation, respectively. G is a correction factor with the experimental setup and can be calculated from

G ) IHV/IHH

(4)

where IHV and IHH are the corresponding vertically polarized and horizontally polarized emission intensities elicited by horizontally polarized excitation, respectively. If IHV equals IHH, the maximum theoretical anisotropy (or fundamental anisotropy, r0) can be determined as r0 ) 0.4. Also, r0 can be determined by

r ) r0{(3 cos2 θ - 1)/2}

(5)

where θ is the angle between the absorption and emission transition moments.50 By using eqs 3-5, we evaluated r and θ values from the steady-state fluorescence intensities, monitoring at 450 nm. Fluorescence Lifetime Measurements. All the sample solutions were gently and carefully purged with Ar gas for the fluorescence lifetime measurements. Time-resolved fluorescence spectra were measured by a single-photon counting method using a third harmonic generation (THG, 266 nm) from a modelocked Ti:Sapphire laser (Clark-MXR NJA-5) using 0.8-mmthick type I β-barium borate (BBO) crystal (CASIX) as an excitation source according to our previous report (see Supporting Information).51 The lifetime of the photoexcited cyt

c(III)-1{[Pt(bpy)(dapap)]1}* was evaluated with software attached to this equipment by monitoring decay at 450 nm. Other Measurements. UV-vis and CD spectra were measured with Shimadzu UV-2550 and Jasco J-720 spectrometers, respectively. ESI-mass spectra were measured with a JEOL JMS-T100LC AccuTOF apparatus. All 1H NMR spectra were recorded on a JEOL JNM-AL400 FT-NMR spectrometer. 1H NMR chemical shift values are reported in ppm referenced to the internal standard TMS. The pH of the solutions was measured on a Hitachi-Horiba F-14RS pH meter. Acknowledgment. This research was partly supported by Grant-in-Aid for Scientific Research No. 19550064 from the Ministry of Education, Culture, Sports, and Science and Technology (MEXT) of the Japanese Government and Nara Women’s University Intramural Grant for Project Research. This work was performed under the Common-Use Facility Program of JAEA. The authors thank Professor Itaru Hamachi of Kyoto University for the measurement of fluorescence anisotropy and Professor Makoto Handa of Shimane University for elemental analyses. Supporting Information Available: Partial experimental data and ESI-MS, HPLC, and steady-state fluorescence spectral data of modified cyt c, and UV-vis spectra from DNA-binding experiments. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Cullis, P. M.; Jones, G. D. D.; Symons, M. C. R.; Lea, J. S. Nature 1987, 330, 773–774. (2) (a) Stemp, E. D. A.; Barton, J. K. Inorg. Chem. 2000, 39, 3868– 3874. (b) Nguyen, K. L.; Steryo, M.; Kurbanyan, K.; Nowitzki, K. M.; Butterfield, S. M.; Ward, S. R.; Stemp, E. D. A. J. Am. Chem. Soc. 2000, 122, 3585–3594. (3) Bjorklund, C. C.; Davis, W. B. Biochemistry 2007, 46, 10745– 10755. (4) (a) Merino, E. J.; Boal, A. K.; Barton, J. K. Curr. Opin. Chem. Biol. 2008, 12, 229–237. (b) Gorodetsky, A. A.; Buzzeo, M. C.; Barton, J. K. Bioconjugate Chem. 2008, 19, 2285–2296. (c) Genereux, J. C.; Boal, A. K.; Barton, J. K. J. Am. Chem. Soc. 2010, 132, 891–905. (5) Mees, A.; Klar, T.; Gnau, P.; Hennecke, U.; Eker, A. P. M.; Carell, T.; Essen, L.-O. Science 2004, 306, 1789–1793. (6) Sancar, A. Chem. ReV. 2003, 103, 2203–2238. (7) (a) Behrens, C.; Burgdorf, L. T.; Schwo¨gler, A.; Carell, T. Angew. Chem., Int. Ed. 2002, 41, 1763–1766. (b) Breeger, S.; Hennecke, U.; Carell, T. J. Am. Chem. Soc. 2004, 126, 1302–1303. (c) Pieck, J. C.; Kuch, D.; Grolle, F.; Linne, U.; Haas, C.; Carell, T. J. Am. Chem. Soc. 2006, 128, 1404–1405. (8) (a) Fromme, J. C.; Verdine, G. L. EMBO J. 2003, 22, 3461–3471. (b) Fromme, J. C.; Banerjee, A.; Huang, S. J.; Verdine, G. L. Nature 2004, 427, 652–656. (9) Cunningham, R. P.; Asahara, H.; Bank, J. F.; Scholes, C. P.; Salerno, J. C.; Surerus, K.; Munck, E.; McCracken, J.; Peisach, J.; Emptage, M. H. Biochemistry 1989, 28, 4450–4455. (10) (a) Boon, E. M.; Livingston, A. L.; Chmiel, N. H.; Davis, S. S.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12543–12547. (b) Boal, A. K.; Yavin, E.; Lukianova, O. A.; O’Shea, V. L.; Davis, S. S.; Barton, J. K. Biochemistry 2005, 44, 8397–8407. (c) Yavin, E.; Boal, A. K.; Stemp, E. D. A.; Boon, E. M.; Livingston, A. L.; O’Shea, V. L.; David, S. S.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3546–3551. (11) Gorodetsky, A. A.; Boal, A. K.; Barton, J. K. J. Am. Chem. Soc. 2006, 128, 12082–12083. (12) Augustyn, K. E.; Merino, E. J.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 18907–18912. (13) (a) Rajski, S. R.; Kumar, S.; Roberts, R. J.; Barton, J. K. J. Am. Chem. Soc. 1999, 121, 5615–5616. (b) Wagenknecht, H. A.; Rajski, S. R.; Pascaly, M.; Stemp, E. D. A.; Barton, J. K. J. Am. Chem. Soc. 2001, 123, 4400–4407. (c) Rajski, S. R.; Barton, J. K. Biochemistry 2001, 40, 5556– 5564. (d) Boon, E. M.; Salas, J. E.; Barton, J. K. Nat. Biotechnol. 2002, 20, 282–286. (14) McLendon, G.; Hake, R. Chem. ReV. 1992, 92, 481–490. (15) Nocek, J. M.; Zhou, J. S.; Forest, S. D.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem. ReV. 1996, 96, 2459–2490. (16) Gray, H. B.; Winkler, J. R. Q. ReV. Biophys. 2003, 36, 341–372.

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