Photoinduced Intramolecular Electron-Transfer Reactions of

Osamu Ito,‡ and Keiichi Tsukahara†. Department of Chemistry, Faculty of Science, Nara Women's UniVersity, Nara, 630-8506 Japan and Institute of...
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J. Phys. Chem. B 2006, 110, 26413-26423

26413

Photoinduced Intramolecular Electron-Transfer Reactions of Reconstituted Met- and Zinc-Myoglobins Appending Acridine and Methylacridinium Ion as DNA-Binders Hiroshi Takashima,*,† Chisako Tara,† Sachiko Namikawa,† Tomoko Kato,† Yasuyuki Araki,‡ Osamu Ito,‡ and Keiichi Tsukahara† Department of Chemistry, Faculty of Science, Nara Women’s UniVersity, Nara, 630-8506 Japan and Institute of Multidisciplinary Research for AdVanced Materials, Tohoku UniVersity, Katahira, Aoba-ku, Sendai, 980-8577 Japan ReceiVed: August 28, 2006; In Final Form: October 18, 2006

Three types of reconstituted met- and zinc-myoglobin (metMb and ZnMb) dyads, ZnMbAc(4)Me+, ZnMbAc(6)Me+, and metMbAc(6) have been prepared by incorporating chemically modified metalloporphyrin cofactor appending an acridine (Ac) or a methylacridinium ion ([AcMe]+) into apo-Mb. In the bimolecular system between ZnMb and [AcMe]+, the photoexcited triplet state of ZnMb, 3(ZnMb)*, was successfully quenched by [AcMe]+ to form the radical pair of ZnMb cation (ZnMb•+) and reduced methylacridine ([AcMe]•), followed by a thermal back ET reaction. The rate constants for the intermolecular quenching ET (kq) and the back ET reaction (kb) at 25 °C were successfully obtained as kq ) (8.8 ( 0.4) × 107 M-1 s-1 and kb ) (1.2 ( 0.1) × 108 M-1 s-1, respectively. On the other hand, in case of the intramolecular photoinduced ET reactions of ZnMbAc(4)Me+ and ZnMbAc(6)Me+ dyads, the first-order quenching rate constants (kET) of 3(ZnMb)* by [AcMe]+ moiety were determined to be kET ) 2.6 × 103 and 2.5 × 103 s-1, respectively. When such ET occurs along the alkyl spacer via through-bond mechanism at the surface of Mb, the obtained kET is reasonable to provide decay constant of β (1.0-1.3 Å-1). Upon photoirradiation of [AcMe]+ moiety, kinetic studies also presented the intramolecular quenching reactions from the excited singlet state, 1([AcMe]+)*, whose likely process is the photoinduced energy-transfer reaction. For metMbAc(6) dyad, steady-state fluorescence was almost quenched, while the signal around 440 nm gradually appeared in the presence of various concentrations of DNA. Our study implies that synthetic manipulation at the Mb surface, by using an artificial DNA-binder coupled with photoinduced reaction, may provide valuable information to construct new Mb-DNA complex and sensitive fluorescent for DNA.

Introduction Photoinduced electron-transfer (ET) and energy-transfer (ENT) reactions of hemoprotein, containing hemes as cofactors, to transport the electron and energy initiated by the light has received considerable attention in the fields of both chemistry and biochemistry.1-6 Since the cofactor is stable in the heme pocket at physiological conditions, chemical modifications have been employed to make artificial photoinduced ET systems of hemoproteins, such as (1) metal-substitution of the heme,7 (2) amino acid residue modification by photosensitizer,8 and (3) heme replacement with a functionalized metalloporphyrin.9,10 To date, much research on intermolecular photoinduced ET reactions by zinc-substituted hemoproteins has been carried out,11-13 because their photoexcited triplet states can act as strong reductants, having lifetimes of several milliseconds. A series of typical organic and inorganic quenchers, such as methylviologen (MV2+),14 quinones,15 and inorganic complexes16 were utilized for studying bimolecular photoinduced ET and several mechanisms, including conformational gating,17 have been suggested for zinc cytochrome c,18 hemoglobin,19 and myoglobin (ZnMb).5,14,16Along this line, since an obvious property of the protein surface is chirality, stereoselective * To whom correspondence should be addressed. Phone: +81-742-203391. Fax: +81-742-20-3395. E-mail: [email protected]. † Nara Women’s University. ‡ Tohoku University.

bimolecular photoinduced ET of ZnMb with chiral organic agents has been discussed to date.20 Instead of the above metal substitution strategies, the semisynthetic reconstitution of an artificial cofactor into apo-Mb has been a topic of interest in recent years to functionalize Mb.21-24 Although there are only limited successful examples studied on the photoinduced ET reactions, modification of a heme propionate, as well as the amino acid residue at the surface of Mb, with a redox-active photosensitizer produces artificial intramolecular photoinduced ET systems.9,25,26 The reconstituted ZnMb, containing a covalently linked quinone or MV2+,27,28 confers the donor-acceptor dyad for the intramolecular photoinduced ET reaction from the 1(ZnMb)* or 3(ZnMb)*. Incorporation of a [Ru(bpy)3]2+ (bpy ) 2, 2′-bipyridine) and a noncovalently linked viologen catenane into a Zn-porphyrin cofactor can provide donor-sensitizer-acceptor triad on ZnMb surface.29 This realizes multistep photoinduced ET reaction and long-lived charge separation in the model system.30 A negatively charged domain constructed on ZnMb surface can form a stable complex with MV2+ and demonstrate the fast singlet photoinduced ET via an electrostatic interaction.31 These are successful, but some crucial and important problems still remain in the longrange photoinduced ET reaction mechanisms of such artificial Mbs: (1) Does the ET kinetics depend on the distance? and (2) Is an electron transferred via through-space or through-bond pathway at the surface of reconstituted Mb? No detailed

10.1021/jp0655571 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/05/2006

26414 J. Phys. Chem. B, Vol. 110, No. 51, 2006 CHART 1 Structures of the Cofactors Presented in This Paper.

discussions to answer these questions have been carried out. One of the reasons is the lack of the systematic investigation of the photoinduced ET reactions of chemically modified ZnMbs. With the objective of elucidating the intermolecular versus intramolecular photoinduced ET mechanisms of ZnMb dyads, we recently constructed ZnMb-methylacridinium ([AcMe]+) systems because of the interest photophysical and redox properties of [AcMe]+. It is well-known that [AcMe]+ shows a strong fluorescence in the visible region overlaps with the Soret absorption band of Mb. Moreover, based on the one-electron redox potential (-0.46 V vs SCE),32 it should be a good electron acceptor for the excited-state of ZnMb in the photoinduced ET reaction, leading to the charge separated state. In this study, we prepare new zinc(II)- and iron(III)-protoporphyrin IX containing an acridine derivative connected by amide linkages, [ZnPPAc(4, 6)Me]X (X ) OAc- or I-) and FeClPPAc(6), as shown in Chart 1. These three cofactors are successfully reconstituted into apo-Mb. By conducting steady-state and transient spectroscopic measurements, the photoinduced ET kinetics between ZnMb and [AcMe]+ and their reaction mechanisms and pathways are discussed. Another purpose of our investigation is to examine the DNA intercalating and groove binding properties of the acridine moiety at the surface of Mb. The construction of artificial DNA-Mb complexes is one of the recent significant subjects to functionalize Mb as a new biomaterial in the field of genetic research and biocatalysis study.33 However, previously reported Mbs, utilized for the complexation with DNA, are covalently conjugated with the designed peptide or the complementary polynucleotide at the heme propionates.34,35 In this regard, we design a new Mb-DNA complex using noncovalent interactions by hydrophobic and π-π interactions between DNA and DNAbinder, such as acridine (Ac), in an aqueous solution. These preferential interactions can regulate the intramolecular photoinduced ET reactions in Ac-modified Mb dyad, i. e., metMbAc(6), whose fluorescent signal is sensitive to DNA. Experimental Section Materials. 1, 6-Diaminohexane, 1, 4-diaminobutane, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), triethylamine, 2, 6-lutidine, FeCl2•nH2O, and Zn(OAc)2•2H2O were purchased from Wako Chemicals and used as received. N, N′-Diisopropylethylamine (DIEA) and thionyl chloride were obtained from Nacalai Tesque Co.

Takashima et al. Iodomethane was purchased from Tokyo Kasei Co. Acridine9-carboxylic acid was purchased from Aldrich Chemical Co. and used without further purification. Protoporphyrin IX monomethyl ester (PPMe) and [AcMe]I were prepared according to the literature methods.32 To conduct the kinetic measurements in an aqueous solution, the counter anions of [AcMe]I were converted to the Cl- form by column chromatography on Dowex1-X8 (Cl- form, φ 2.5 × 20 cm, MeOH). Column chromatography on silica gel was carried out by using Wakogel C-200 (Wako). Metmyoglobin from horse heart muscle (Sigma) was purified as previously described.66 CT-DNA was purchased from Sigma Chemical Co. and its concentration, expressed in base pairs, was determined spectrophotometrically using 260 ) 1.31 × 104 M-1 cm-1.67 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. Syntheses of Acridine-9-carbamoylaminobutylamine Hydrochloride {Ac(4)} and Acridine-9-carbamoylaminohexylamine Hydrochloride {Ac(6)}. Acridine-9-carboxylic acid (0.22 g, 1.0 × 10-3 mol) was suspended in 10 mL of thionyl chloride and stirred at 70 °C for 12 h. After cooling to room temperature, unreacted thionyl chloride was removed under reduced pressure and dried in vacuo. The yellow residue was then dissolved 50 mL of CH2Cl2. To this solution, 50 mL solution of 1, 4-diaminobutane (0.14 g, 1.5 × 10-3 mol) in CH2Cl2 was added dropwise and reacted for 48 h at room temperature. The reaction mixture was filtered off and the filtrate was evaporated to dryness. The white-yellow solid product was obtained with a yield of 0.28 g (97%). ESI-MS (MeOH, m/z) 294.19 ([M + H]+). 1H NMR (400 MHz, CDCl3, 300 K, TMS): δ/ppm ) 1.25 (m, 2H, methylene), 1.61 (m, 2H, methylene), 1.83 (m, 2H, methylene), 2.75 (t, 2H, 6 ) 6.6 Hz, NH2), 3.70 (q, 2H, J ) 6.6 Hz, methylene), 7.53 (t, 2H, J ) 7.6 Hz, Acridine-H3), 7.74 (t, 2H, J ) 7.3 Hz, Acridine-H2), 8.00 (d, 2H, J ) 8.7 Hz, Acridine-H4), 8.15 (d, 2H, J ) 8.7 Hz, Acridine-H1). Anal. Calcd for C18H19IN3O•2HCl•3CH3OH: C, 54.54; H, 7.19; N, 9. 08%. Found: C, 53.99; H, 6.89; N, 8.87%. Ac(6) was prepared according to similar method for Ac(4) using 1, 6-diaminohexane. Yield 0.59 g (92%). ESI-MS (MeOH, m/z) 322.20 ([M + H]+). Syntheses of 2-{2-[6-(2-Acridineaminocarboxy)butylcarbamoylamino]ethyl}-18-[2-(2-methoxycarbonyl)ethyl]-8, 13divinyl-3, 7, 12, 17-tetramethylporphine {PPMeAc(4)} and 2-{2-[6-(2-Acridineaminocarboxy)hexylcarbamoylamino]ethyl}-18-[2-(2-methoxycarbonyl)ethyl]-8, 13-divinyl-3, 7, 12, 17-tetramethylporphine {PPMeAc(6)}. To a solution of PPMe (0.10 g, 1.7 × 10-4 mol) in 40 mL N, N-dimethylformamide (DMF), DIEA (0.10 g, 7.7 × 10-4 mol) and BOP (0.25 g, 5.5 × 10-4 mol) were added and stirred at room temperature for 60 min. Then, 30 mL solution of Ac(4) (0.076 g, 2.5 × 10-4 mol) in DMF was added dropwise and reacted for 24 h. After removal of the solvent in vacuo, the crude mixture was dissolved in 100 mL of CH2Cl2. The solution was washed with 50 mL of water three times, and the organic solvent was evaporated to dryness. The residue was subjected to a column chromatography on silica gel (φ 2.5 × 20 cm, CH2Cl2-MeOH ) 100:1-30: 1(v/v)). The purple band was collected and was evaporated to yield desired compound, 0.15 g (98%). ESI-MS (MeOH : CH2Cl2 ) 1:1, m/z) 852.43 ([M + H]+), 874.63([M + Na]+). 1H NMR (400 MHz, CDCl3, 300 K, TMS): δ/ppm ) 1.25 (m, 2H, methylene in -(CH2)4-), 1.43 (m, 2H, -CH2CH2CONH-), 1.89 (m, 2H, methylene in -(CH2)4-), 2.65 (m, 2H, methylene in -(CH2)4-), 3.22 (m, 2H, methylene in -(CH2)4-

Reconstituted Myoglobins Appending Acridine and Methylacridinum Ion ), 3.32 (m, 2H, methylene in -(CH2)4-), 3.48 (m, 12H, each, 3, 7, 12, 17, methyl), 3.66 (m, 4H, -CH2CH2CONH- and -CH2CH2COOH), 4.44 (m, 2H, -CH2CH2COOH), 6.10 (m, 4H, -CH ) CH2), 7.23-7.31 (m, 4H, Acridine-H2, H3), 7.83 (m, 2H, -CH ) CH2), 8.07 (d, 2H, J ) 8.0 Hz, Acridine-H4), 8.38 (d, 2H, J ) 8.8 Hz, Acridine-H1), 9.28-1 0.26 (m, 4H, mesoH). PPMeAc(6) was prepared according to similar method for PPMeAc(4). Yield 0.49 g (45%). ESI-MS (MeOH, m/z) 880.66 ([M + H]+). 1H NMR (400 MHz, CDCl3, 300 K, TMS): δ/ppm ) 0.96 (m, 6H, methylene in -(CH2)6-), 1.46 (m, 2H, -CH2CH2CONH-), 2.52 (m, 4H, methylene in -(CH2)6-), 2.64 (m, 2H, methylene in -(CH2)4-), 3.21 (m, 12H, each, 3, 7, 12, 17, methyl), 3.62 (m, 4H, -CH2CH2CONH- and -CH2CH2COOH), 3.70 (m, 2H, -CH2CH2COOH), 6.28 (m, 4H, -CH ) CH2), 7.46 (m, 4H, Acridine-H2, H3), 7.86 (m, 2H, -CH ) CH2), 8.03 (d, 2H, J ) 8.8 Hz, Acridine-H4), 8.23 (d, 2H, J ) 8.8 Hz, Acridine-H1), 9.37-9.81 (m, 4H, meso-H). Anal. Calcd for C55H57N7O4•CH2Cl2•2CH3OH: C, 67.69; H, 6.56; N, 9.53%. Found: C, 67.70; H, 6.89; N, 9.17%. Syntheses of 2-{2-[6-(2-Acridineaminocarboxy)butylcarbamoylamino]ethyl}-18-propionic acid-8, 13-divinyl-3, 7, 12, 17-tetramethylporphine {PPAc(4)} and 2-{2-[6-(2-Acridineaminocarboxy)hexylcarbamoylamino]ethyl}-18-propionic acid8, 13-divinyl-3, 7, 12, 17-tetramethylporphine {PPAc(6)}. PPMeAc(4) (0.37 g, 4.4 × 10-4 mol) was dissolved in 120 mL of MeOH : THF ) 1 : 1(v/v). 10 mL of 1.0 M NaOHaq. was added, and the reaction mixture was stirred for 72 h at room temperature. The solution poured into 20 mL of distilled water and extracted with 40 mL of CH2Cl2-MeOH ) 3 : 1 (v/v) several times. The organic phase was collected and dried over Na2SO4. The solvent was evaporated and a desired product (0.34 g) was obtained in 93% yield. ESI-MS (CH2Cl2-MeOH ) 1 : 1, m/z) 866.32 ([M + H]+). 1H NMR (400 MHz, MeOH-d4, 300 K, TMS): δ/ppm ) 1.21 (m, 2H, methylene in -(CH2)4-), 1.28 (m, 2H, -CH2CH2CONH-), 2.47 (m, 2H, methylene in -(CH2)4-), 3.11 (m, 2H, methylene in -(CH2)4-), 3.19 (m, 2H, methylene in -(CH2)4-), 3.43 (m, 12H, each, 3, 7, 12, 17, methyl), 4.27 (m, 4H, -CH2CH2CONH- and -CH2CH2COOH), 5.51 (m, 2H, -CH2CH2COOH), 6.10 (m, 4H, -CH ) CH2), 7.56-7.72 (m, 4H, Acridine-H2, H3), 7.86 (t, 2H, J ) 6.8 Hz, -CH ) CH2), 8.07 (d, 2H, J ) 8.8 Hz, Acridine-H4), 8.18 (d, 2H, J ) 8.8 Hz, Acridine-H1), 9.37-9.81 (m, 4H, mesoH). UV-vis (CH2Cl2-MeOH ) 1 : 1, λ/nm) 252 (Ac), 404 (Soret band), 508, 543, 575, 629 (Q-band). PPAc(6) was prepared according to similar method for PPAc(4). Yield 0.19 g (88%). ESI-MS (MeOH, m/z) 866.42 ([M + H]+). 1H NMR (400 MHz, CDCl3, 300 K, TMS): δ/ppm ) 0.96 (m, 6H, methylene in -(CH2)6-), 1.42 (m, 2H, -CH2CH2CONH-), 2.42 (m, 4H, methylene in -(CH2)6-), 2.60 (m, 2H, methylene in -(CH2)6-), 3.41 (m, 12H, each, 3, 7, 12, 17, methyl), 3.723.90 (m, 4H, -CH2CH2CONH- and -CH2CH2COOH), 4.25 (m, 2H, -CH2CH2COOH), 6.25 (m, 4H, -CH ) CH2), 7.22 (m, 4H, Acridine-H2, H3), 7.60 (m, 2H, -CH ) CH2), 8.03 (d, 2H, J ) 8.8 Hz, Acridine-H4), 8.33 (d, 2H, J ) 8.8 Hz, Acridine-H1). UV-vis (CH2Cl2-MeOH ) 3 : 1, λ/nm) 252 (Ac), 360 (Ac), 404 (Soret band), 506, 542, 575, 630 (Q-band). Synthesis of 2-{2-[6-(2-(Methylacridine)aminocarboxy)butylcarbamoylamino]ethyl}-18-propionic acid-8, 13-divinyl3, 7, 12, 17-tetramethylporphine Iodide {[PPAc(4)Me]I}. PPAc(4) (0.040 g, 4.1 × 10-5 mol) was dissolved in 40 mL of MeOH and then reacted with 1.0 mL of methyl iodide for 24 h at 55 °C. The reaction mixture was poured into diethyl ether and the precipitate was collected by filtration. The black

J. Phys. Chem. B, Vol. 110, No. 51, 2006 26415

precipitate was washed with diethyl ether and the product (0.036 g) was obtained in 77% yield. ESI-MS (MeOH, m/z) 852.34 ([M]+). Synthesis of 2-{2-[6-(2-(Methylacridine)aminocarboxy)hexylcarbamoylamino]ethyl}-18-propionic acid-8, 13-divinyl3, 7, 12, 17-tetramethylporphine Iodide {[PPAc(6)Me]I}. PPAc(6) (0.053 g, 6.1 × 10-5 mol) was dissolved in 5 mL of DMF and then reacted with 0.1 mL of methyl iodide for 24 h at 55 °C. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved 20 mL of MeOH and reprecipitated by 100 mL of diethyl ether. The black precipitate was collected by filtration. This was washed with diethyl ether several times and the product (0.057 g) was obtained in 93% yield. ESI-MS (MeOH, m/z) 880.78 ([M]+). Synthesis of 2-{2-[6-(2-(Methylacridine)aminocarboxy)butylcarbamoylamino]ethyl}-18-propionic acid-8, 13-divinyl3, 7, 12, 17-tetramethylporphinatozinc(II) Acetate {[ZnPPAc(4)Me]OAc}. The free base [PPAc(4)Me]I (5.2 mg, 5.3 × 10-6 mol) and 2, 6-lutidine (1.2 mg, 5.3 × 10-6 mol) were dissolved in 5 mL of MeOH:CH2Cl2 ) 1 : 1 (V/V). Zn(OAc)2•2H2O (6.5 mg, 2.9 × 10-5 mol) in 5 mL of MeOH:CH2Cl2 ) 1 : 1 (V/V) was added and stirred for 2 h at room temperature. To this solution, 10 mL of CH2Cl2 was added and washed with 20 mL of water three times. The organic layer was collected and the solvent was evaporated off. The purple residue was dissolved in 15 mL of MeOH : CH2Cl2 ) 1:1 (V/V) and reprecipitated by 100 mL of diethyl ether. The precipitation was collected by filtration and dried to yield a solid compound, 4.9 mg (95%). ESI-MS (MeOH, m/z) 915.41 ([M]+), 938.39 ([M - H + Na]+). UV-vis (CH2Cl2-MeOH ) 3:1, λ / nm) 252 (Ac), 360 (Ac), 417 (Soret band), 548, 585 (Q-band). Anal. Calcd for C55H55N7O4Zn•2CH3OH•2H2O: C, 63.65; H, 6.27; N, 9.12%. Found: C, 63.96; H, 5.82; N, 8.76%. Synthesis of 2-{2-[6-(2-(Methylacridine)aminocarboxy)hexylcarbamoylamino]ethyl}-18-propionic acid-8, 13-divinyl3, 7, 12, 17-tetramethylporphinatozinc(II) Iodide{[ZnPPAc(6)Me]I}. The free base [PPAc(6)Me]I (0.031 g, 3.1 × 10-5 mol) and Zn(OAc)2•2H2O (0.033 g, 1.5 × 10-4 mol) were dissolved in 10 mL of MeOH : CH2Cl2 ) 1 : 1 (v/v). The reaction solution stirred for 2 h at room temperature. To this solution, 20 mL of CH2Cl2 was added and washed with 30 mL of water three times. The organic layer was collected and the solvent was removed. The purple residue was collected and dried to yield a solid compound, 0.020 g (55%). ESI-MS (CH2Cl2MeOH ) 1 : 1, m/z) 942.30 ([M]+). UV-vis (CH2Cl2-MeOH ) 3:1, λ / nm) 252 (Ac), 360 (Ac), 415 (Soret band), 546, 584 (Q-band). Anal. Calcd for C55H56IN7O4Zn•4CH2Cl2: C, 50.01; H, 4.73; N, 6.48%. Found: C, 49.96; H, 4.81; N, 7.10%. Synthesis of 2-{2-[6-(2-Acridineaminocarboxy)hexylcarbamoylamino]ethyl}-18-propionic acid-8, 13-divinyl-3, 7, 12, 17-tetramethylporphinatoiron(III) Chloride {FeClPPAc(6)}. The free base PPAc(6) (0.086 g, 1.0 × 10-4 mol) and FeCl2•nH2O (0.15 g, 1.2 × 10-3 mol) were dissolved in 20 mL of DMF and stirred at 65 °C for 12 h. After the reaction, the solvent was removed under reduced pressure and the residue was dissolved in 100 mL of CH2Cl2-MeOH ) 10:1 (V/V). This was washed with saturated NaClaq. several times and was evaporated to dryness. The target product (0.046 g) was obtained in 51% yield. ESI-MS (CH2Cl2-MeOH ) 3:1, m/z) 919.59 ([M - Cl-]+). UV-vis (CH2Cl2-MeOH ) 3:1, λ / nm) 360 (Ac), 404 (Soret band), 500, 589 (Q-band). Anal. Calcd for C54H53N7O4FeCl: C, 67.67; H, 5.98; N, 9.60%. Found: C, 67.59; H, 5.98; N, 9.50%.

26416 J. Phys. Chem. B, Vol. 110, No. 51, 2006 Reconstitution of Apo-Mb. The apo-Mb was prepared from horse heart Mb by acid/butanone procedure.16c To apo-Mb solution (0.010 M Tris/HCl buffer, pH 8.6) was added [ZnPPAc(4)Me]OAc, [ZnPPAc(6)Me]I, and FeClPPAc(6) in pyridine solution. The mixture was incubated at 4 °C for 4 h and dialyzed against 0.010 M phosphate buffer (pH 7.0, several times) for 48 h. After removal of the precipitates, the clear supernatant was loaded on CM-52 cellulose column chromatography (equilibrated with a 0.010 M phosphate buffer at pH 7.0) at 4 °C. All of the reconstituted Mbs was eluted with 0.020 M phosphate buffer at pH 7.0. The concentrations of metMbAc(6) and ZnMbs were determined spectrophotometrically (409 ) 1.88 × 105 M-1 cm-1 for metMbAc(6) and 428 ) 1.53 × 105 M-1 cm-1 for ZnMbs based on the native metMb and ZnMb, respectively).68 The native ZnMb solution, whose absorption ratio A428/A280 is greater than 9.5, was prepared according to the published method and used for kinetic measurements.16c Yield : ZnMbAc(4)Me+ 13%; ZnMbAc(6)Me+ 4%; metMbAc(6) 5%. Kinetic Measurements. Procedure for the kinetics was the same as that reported previously.20b,69 All the sample solutions were gently purged with Ar gas and then carefully degassed by freeze-pump-thaw cycles. Time-resolved fluorescence spectra were measured by a single-photon counting method using a second harmonic generation (SHG) of a Ti:sapphire laser (Spectra-Physics, Tsunami 3950-L2S, 1.5 ps fwhm) as an excitation source.69 Lifetimes of 1([AcMe]+)* and 1(ZnMb)* were evaluated with software attached to the equipment by monitoring decay at 400 and 600 nm, respectively. Nanosecond transient absorption measurements were carried out using the SHG (532 nm) of a Nd: YAG laser (Spectra-Physics, QuantaRay GCR-130, 6 ns fwhm) as an excitation source. For transient absorption spectra in the near-IR region (600-1000 nm), monitoring light from a pulsed Xe-lamp was detected with a Ge-APD (Hamamatsu Photonics, B2834).69 For spectra in the visible region (400-600 nm), a Si-PIN photodiode (Hamamatsu Photonics, S1722-02) was used as a detector.69 A single flash photolysis was done in the deaerated solutions containing ZnMbs (5.0 × 10-6 M) and quenchers (0 - 6.0 × 10-5 M) at 25 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M, using a Photal RA-412 pulse flash apparatus with a 30 µs pulsewidth Xe lamp (λ > 450 nm; a Toshiba Y-47 glass filter).20b Absorption spectral changes during the reaction were monitored at 460 nm for the decay of 3(ZnMb)*, and 680 nm for the formation and decay of the radical cation ZnMb•+. Other Measurements. UV-vis, fluorescence, and ESI-Mass spectra were measured with Shimadzu UV-2550, Shimadzu RF5300, and Applied Biosystems Mariner, respectively. All 1H NMR spectra were recorded on a JEOL JNM-AL400 FT-NMR. 1H NMR chemical shift values are reported in ppm as reference to the internal standard TMS. The pHs of the solutions were measured on a Hitachi-Horiba F-14RS pH meter. Results and Discussion Cofactor Syntheses and Reconstituions into Apo-Mb. The synthetic pathways of the Ac and the [AcMe]+ modified protoporphyrins, [ZnPPAc(4)Me]X (X ) OAc- or I-) and FeClPPAc(6), were developed by modification of the previous methods (Scheme 1).29,30 In this study, we used 9-acridine carboxylic acid as a starting material. To obtain Ac-appended free base protoporphyrins, this was first reacted with a diamine spacer, 1, 4-diaminobutane or 1, 6-diaminohexane, in the presence of thionyl chloride. The remaining amino group in the corresponding spacer was then condensed with a propionate

Takashima et al. SCHEME 1 Synthetic Routes of [ZnPPAc(4, 6)Me]X (X ) OAc- or I-) and FeClPPAc(6)a

a Reagents and Conditions: (i) Ac(4) or Ac(6), BOP, DIEA, DMF, r. t., 24 h. (ii) 1 M NaOHaq., THF-MeOH, r. t., 72 h. (iii) CH3I, DMF, 55 °C, 24 h. (iv) Zn(OAc)2•2H2O, 2, 6-lutidine, CH2Cl2-MeOH, r. t., 2 h. (v) FeCl2•nH2O, DMF, 65 °C, 12 h.

group of the PPMe by using BOP in DMF to yield PPMeAc(4, 6). Purification by a column chromatography on silica gel and the subsequent ester hydrolysis at room-temperature gave free base PPAc(4, 6) in good yield. Treatment of PPAc(4, 6) with methyl iodide in DMF at 55 °C afforded PPAc(4, 6)Me as an iodide form. The zinc(II) complexes, [ZnPPAc(4)Me]OAc and [ZnPPAc(6)Me]I, were produced from [PPAc(4, 6)Me]I by using Zn(OAc)2•2H2O as a metal source. The iron(III) insertion was conducted for PPAc(6) by FeCl2•nH2O to obtain FeClPPAc(6) as a chloride form. The UV-vis and ESI-MS spectra and elemental analysis clearly support the formation of these porphyrins and all signals were reasonably assigned (see the Experimental Section). The porphyrin regioisomers may exist by our synthetic procedure, and these have not been separated in this study. Reconstitution of apo-Mb was carried out at 4 °C in the dark using a method that has been previously published.16c A detailed procedure is described in the Experimental Section. Briefly, a small amount of pyridine solution containing a synthetic cofactor was used for the reconstitution.30,36 The mixture was incubated at 4 °C and dialyzed against phosphate buffer. After removal of the precipitates, the clear supernatant was loaded on a CM52 cellulose column by using a 0.010 M phosphate buffer at pH 7.0 and the reconstituted Mb was eluted with a 0.020 M phosphate buffer (pH 7.0) as a single band. UV and CD Spectra of Reconstituted Mbs. The absorption spectrum of ZnMbAc(6)Me+ show a strong sharp Soret band at 428 nm, two Q-bands at 553 and 596 nm, and a sharp band at 252 nm due to the [AcMe]+ unit (Figure 1). This spectrum is nearly identical to that of the 1:1 mixture solution of ZnMb and [AcMe]+ using 10-methylacridinium iodide ([AcMe]I). These data confirms the successful reconstitution of [ZnPPAc-

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J. Phys. Chem. B, Vol. 110, No. 51, 2006 26417

SCHEME 2 Photoinduced Intermolecular ET Reaction between ZnMb and [AcMe]+

Figure 1. UV-vis absorption spectra of ZnMbAc(6)Me+ (bold line), ZnMb (solid line), and the 1:1 mixture of ZnMb and [AcMe]+(dashed line) (5.0 × 10-6 M) at 25 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M.

(4)Me]OAc, and [ZnPPAc(6)Me]I with apo-Mb and the absence of a strong ground-state electronic interaction between the ZnMb and the [AcMe]+ chromophores. As shown in Table 1, the UVvis spectrum of metMbAc(6) (ferric state, Fe(III)) displays a strong Soret band (409 nm), two Q-bands (504 and 629 nm), and additional peaks at 252 nm (Ac). This spectrum is also similar to the sum of the spectra of metMb and Ac (data not shown). Moreover, reduction of the met-form of metMbAc(6) with Na2S2O4 and the ligand-exchange reaction by NaN3 gave absorption spectra of Soret bands corresponding the deoxy- (434 nm) and azide- (425 nm) forms, respectively. These properties are essentially identical to those of the native Mb and the semisynthetic reconstituted Mbs, indicating that FeClPPAc(6) was satisfactorily reconstituted into the heme pocket.25b,37 Table 1 also summarizes the CD spectral data for reconstituted Mbs in 0.010 M phosphate buffer (pH 7.0). The negative signs at 222 and 209 nm were obtained for ZnMbAc(4, 6)Me+ and metMbAc(6). The magnitudes of peak intensities both of these wavelengths for reconstituted Mbs were nearly the same as those of the corresponding native Mbs. This result clearly indicates that the conformations of ZnMbAc(4, 6)Me+ and metMbAc(6) have been preserved from any structural changes in the native Mb backbone. Intermolecular Photoinduced ET Quenching Reactions of 3(ZnMb)* by [AcMe]+. The photoexcited 3(ZnMb)* was generally known to be a strong reductant for several electron acceptors.5 First, we investigate the intermolecular photoinduced ET quenching reaction between 3(ZnMb)* and [AcMe]+ (Scheme 2). The ET quenching reaction of 3(ZnMb)* by [AcMe]+ is exothermic, ∆G0 ) -0.34 eV, based on the redox potentials. Under the present experimental conditions, in which [AcMe]+ ion is used in excess over 3(ZnMb)*, with concentration ([3(ZnMb)*]0) of 3.0 × 10-6 M, the electron-transfer quenching was almost completed (more than 99%).20f Figure 2a displays a first-order decay of the transient absorption of 3(ZnMb)* was monitored at 460 nm in the presence of [AcMe]+, at 25 °C, pH 7.0 (0.010 M phosphate buffer), and the ionic strength of I ) 0.020 M. The rate constant for the quenching reaction of 3(ZnMb)*, kobsd, increased linearly with increasing the concentra-

tion of [AcMe]+ (Figure 2a, inset), indicating no appreciable complex was formed between 3(ZnMb)* and [AcMe]+. The quenching ET process in Scheme 2 contains the elementary steps including a pre-equilibrium of binding of the [AcMe]+ with the 3(ZnMb)*, where the quenching and the ET rate constants are represented as kq and kET, respectively. When the 3(ZnMb)* loosely associated with the [AcMe]+, the quenching rate constant, kq, corresponds to kETk1/k-1. The intermolecular quenching rate constant (kq) was thus obtained from the slope of the plots of kobsd as a function of the concentration of [AcMe]+ and determined as kq ) (8.8 ( 0.4) × 107 M-1 s-1 at 25 °C (Table 2). Previous results for the intermolecular quenching reactions of 3(ZnMb)* revealed that the rate constants (kq) for the ET quenching reactions by several viologens and quinolinium ions at 25 °C were determined as about 107 M-1 s-1 and 105 - 106 M-1 s-1, respectively.20 The quenching rate constant between 3(ZnMb)* and [AcMe]+ is similar to those of viologens. In the case of quinolinium ions, the quenching reactions were controlled by an ET step, not by the conformational gating of ZnMb, according to the driving force dependence of the ET rate constants.20f, 38 This may partly arise because the driving force of the reaction is slightly exothermic, and the ET rate is not fast enough to be controlled by the conformational change in ZnMb. In order to evaluate the present quenching mechanism between 3(ZnMb)* and [AcMe]+, we calculated the rate constants using the following Marcus theory (eq 1):39

k12 ) (k11k22f12K12)1/2.

(1)

where, k12 is the rate constant for the cross reaction, k11 and k22 are those for the self-exchange reactions of the donor and acceptor, respectively, K12 is the equilibrium constant for the cross reaction, and f12 is given by

lnf12 ) (lnK12)2/4ln(k11k22/1022). Equations 1 and 2 are also represented by

lnk12 ) 25.3 - (λ12 + ∆G0)2/4λ12RT

ZnMb ZnMbAc(4)Me+ ZnMbAc(6)Me+ metMbAc(6)

UV-vis : λmax/nm (/104 M-1 cm-1) 280 (1.6) 252 (3.7) 252 (3.7) 252 (4.4)

428 (15.3) 428 (15.3) 428 (15.3) 409 (18.8)

553 (1.0) 553 (1.0) 553 (1.0) 504 (1.1)

(3)

where λ12 is the reorganization energy for the reaction, and equals the average of those of the donor and acceptor, (λ11 + λ22)/2. By using the data k11 ) 2.6 × 105 M-1 s-1 for ZnMb•+/

TABLE 1: UV-vis and CD Spectral Data of ZnMb, ZnMbAc(4)Me+, ZnMbAc(6)Me+, and metMbAc(6) at pH 7.0 (0.010 M Phosphate Buffer) and I ) 0.020 M Mbs

(2)

CD : λmax/nm (∆/102 M-1 cm-1) 596 (0.8) 596 (0.8) 596 (0.8) 629 (0.4)

222 (-5.4) 222 (-5.5) 222 (-5.5) 222 (-5.3)

209 (-5.2) 209 (-5.1) 209 (-5.1) 209 (-5.0)

26418 J. Phys. Chem. B, Vol. 110, No. 51, 2006

Takashima et al. SCHEME 3 Energy Diagram of ZnMbAcMe+ Dyad and the Major ET Pathways

Figure 2. Absorbance changes after irradiation of ZnMb with a Xe flash lamp in the presence of [AcMe]+ (3.9 × 10-5 M) at 25 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M. (a) Decay of 3(ZnMb)* at 460 nm. Plots of kobsd vs [AcMe+]0 are shown in the inset. (b) Decay of ZnMb•+ at 680 nm. A bold line is fitted to eq 4.

(λ11 ) 1.32 eV),39 k22 ) 1.0 × 108 M-1 s-1 for [AcMe]+/[AcMe]• couple (λ22 ) 0.71 eV),41,42 and K12 ) 5.8 × 105 for [AcMe]+ (the redox potential of ZnMb•+/3(ZnMb)* is 0.98 V and ∆G0 ) -0.34 eV),43 the calculated rate constant, k12, is 1.3 × 109 M-1 s-1 (f12 ) 0.11) for [AcMe]+. The calculated rate constant is different from the observed ones. We propose that the present ET quenching system is controlled by the conformational gating of ZnMb within the precursor complex (Scheme 2),17 as can be similarly expected for a typical ZnMb-viologen system.4,14b,16b,c,19b,20a,b,44 Thermal Back ET Reactions between ZnMb•+ and [AcMe]•. Since the ET quenching reaction, followed by prompt appearance of absorption band at 680 nm and bleaching of 3(ZnMb)* absorption at 460 nm, has been observed, we also monitored the kinetic trace at 680 nm for the formation and decay of the radical cation of ZnMb•+.20, 45 The transient absorption kinetics of the back ET in Figure 2b obeyed a secondorder rate law, suggesting that an equimolar amount of ZnMb•+ with a [AcMe]• radical formed. The rate constant of the intermolecular back ET reaction (kb) was evaluated at the latter portion of the decay of ZnMb•+ at 680 nm after the quenching of 3(ZnMb)* was completed based on the following eq 4: 3(ZnMb)*

At ) (A0 + kb[A]0A∞t)/(1 + kb[A]0t)

(4)

Here A0, At, and A∞ are the absorbances at time 0, t, and infinity, TABLE 2: Rate Constants Determined for the Intermolecular Quenching Reactions of 3(ZnMb)* by [AcMe]+ and the Back ET Reactions at pH 7.0 (0.010 M Phosphate Buffer), 25 °C, and I ) 0.020 M [AcMe+]0/10-5 M

kobsd/103 s-1

kb[A]0/102 s-1

kb/108 M-1 s-1

0 1.9 3.9 4.7 6.0

0.083 1.8 3.6 4.0 5.4

3.3 3.6 3.5 3.3

1.1 1.2 1.2 1.1

respectively, and [A]0 is an initial concentration of ZnMb•+. Three unknown parameters, A0, kb[A]0, and A∞ were simultaneously estimated. Then, the value of the second-order rate constants (kb) was determined as kb ) (1.2 ( 0.1) × 108 M-1 s-1 at 25 °C (Table 2) by using the value of which was calculated from the concentration of 3(ZnMb)* (∆428 ) (ground) - (triplet) ) 1.00 × 105 M-1 cm-1).46 Similarly, we obtained the calculated rate constant for the thermal back ET reaction, k12 ) 1.7 × 1010 M-1 s-1 (f12 ) 4.7 × 10-18) for [AcMe]• using the data k11 ) 2.6 × 105 M-1 s-1 for ZnMb•+/ZnMb (λ11 ) 1.32 eV), and K12 ) 2.5 × 1024 for [AcMe]• (∆G0 ) -1.44 eV). This is also much different for the observed ones, indicating that the thermal back ET reaction is not controlled by the ET step, as mentioned above in the previous section. Since the one-electron reduced radical, [AcMe]•, generated from quenching of the 3(ZnMb)* becomes a neutral molecule, we suggest that the charge effect on the acridinium ion moiety is as important as the conformational effect to explain the mechanism of the thermal back ET. Intramolecular Photoinduced ET Reactions of ZnMbAc(4, 6)Me+ by Excitation of ZnMb. We next examined photoinduced ET reactions of ZnMbAc(4, 6)Me+ dyads on the selective excitation of the ZnMb moiety. The shapes and intensities of the steady-state fluorescent signals of ZnMbAc(4)Me+ and ZnMbAc(6)Me+ (λex ) 428 nm), are the same compared to those of ZnMb and [AcMe]+ (1 :1) (Figure S1a in the Supporting Information). The time-resolved fluorescent decay profile of the excited singlet state 1(ZnMb)* measured at 600 nm (λex ) 400 nm) gave a lifetime of τ ) 2.2 ns (Figure S1b, single exponential, and k0 in Scheme 3), which is equal to the ZnMb one in the N2-saturated buffer solution at 25 °C (Table 3). Thus, intramolecular ET from 1(ZnMb)* to [AcMe]+moiety can be ruled out. Figure 3a shows the transient absorption spectrum of ZnMbAc(4)Me+ recorded 100 ns after selective excitation at 532 nm. Absorption bands at 460 and 840 nm promptly appeared, TABLE 3: Fluorescent Spectral Data, Lifetimes, and the Quenching Rate Constants for ZnMb, 1:1 Mixture of ZnMb and [AcMe]+, ZnMbAc(4)Me+, and ZnMbAc(6)Me+ at pH 7.0 (0.010 M Phosphate Buffer) and I ) 0.020 M Mbs

λem/nm (τ/ns)

ZnMb ZnMb+[AcMe]+ 440 (33)b (1 : 1) ZnMbAc(4)Me+ 440 {0.084 (74%), 1.6 (26%)} ZnMbAc(6)Me+ 440 {0.10 (83%), 1.9 (17%)}

kq(AcMe)/1010 s-1a

600 (2.2) 650 600 (2.2) 650 600 (2.2) 650

1.2

600 (2.2) 650

0.98

a Calculated from k ) 1/τ - 1/τ . b The lifetime of 1([AcMe]+)* in 0 an aqueous solvent (ref 56).

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J. Phys. Chem. B, Vol. 110, No. 51, 2006 26419

TABLE 4: Rate Constants for Intramolecular ET Quenching Reactions of ZnMb, ZnMbAc(4)Me+, ZnMbAc(6)Me+ at pH 7.0 (0.010 M Phosphate Buffer), 25 °C, and I ) 0.020 M Mbs

3

(ZnMb)* τ/ms

3 (ZnMb)* kqtriplet/103 s-1

r-r0/Åa

β/Å-1

ZnMb ZnMbAc(4)Me+ ZnMbAc(6)Me+

12 0.38 0.40

0.08 2.6 2.5

16.3 20.9

1.3 1.0

a

Figure 3. (a) Transient absorption spectrum of ZnMbAc(6)Me+ (5.0 × 10-6 M) recorded at 100 ns after irradiation by the 532 nm laser at 25 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M. (b) Time profile of 3(ZnMb)* at 460 nm.

accompanied by the bleaching of Soret and Q-bands (428, 553, and 596 nm). These absorption bands at 460 and 840 nm could be assigned to the excited triplet state, 3(ZnMb)*-[AcMe]+, and the decay of the T-T absorption of 3(ZnMb)*-[AcMe]+ was of first order (Figure 3b).28-30,47 The lifetime of the 3(ZnMb)*[AcMe]+ was determined to be τ ) 0.38 ms and was much faster than those of ZnMb and the intermolecular ZnMb + [AcMe]+ (1:1) system (τ ) 12 ms in a degassed buffer solution at 25 °C), indicating that the direct intramolecular ET reaction from 3(ZnMb)* to [AcMe]+ moiety occurred as shown in Scheme 3. On the basis of the one-electron redox potential of [AcMe]+, E0([AcMe]+/[AcMe]•) ) -0.46 V,32 and the oneelectron oxidation potential of 3(ZnMb)*, E0(ZnMb•+/3(ZnMb)*) ) -0.80 V,43 the energy difference ∆G0(ZnMb•+-[AcMe]•/3(ZnMb)*-[AcMe]+) ) -0.34 eV is estimated and allows ET quenching in the dyad. Therefore, intramolecular ET rate constant was evaluated to be kET ) 2.6 × 103 s-1 for ZnMbAc(4)Me+ according to the following eq 5,

kET ) kobsd - k′0

(5)

where k′0 is the rate constant for the natural decay (k′0 ) 83 s-1). In case of ZnMbAc(6)Me+, a similar decay of 3(ZnMb)*[AcMe]+ was observed with the ET rate constant of kET ) 2.5 × 103 s-1 (Table 4). The dependence of the distance on the intramolecular ET rate of hemoproteins had been proposed as the electronic coupling of spacers by Larsson;48 thereafter, the following eq 6 was presented for the intramolecular ET rate constant:49,50

kET ) 1013 • exp[ - β(r-r0)] • exp[ - (∆G0 + λ)2/4λRT] (6) Here, r is the distance between the donor and acceptor centers, r0 is the contact distance, β is the decay constant which describes the sensitivity of the coupling to changes in distance, ∆G0 is

Estimated as through-bond edge-to-edge distance.

the driving force, λ is the reorganization energy which equals the mean value of the contribution from donor and acceptor, (λD + λA)/2, R is the gas constant, and T is the absolute temperature. If the ET from Zn-porphyrin to [AcMe]+ moiety occurs along the methylene spacer by a through-bond interaction, the edge-to-edge distances of ZnMbAc(4)Me+ and ZnMbAc(6)Me+ are estimated to be 16.3 and 20.9 Å, respectively. By using the parameters λ ) 1.02 eV (the values of λ are 1.32 and 0.71 eV for 3(ZnMb)* and [AcMe]+, respectively),40,41 ∆G0 ) -0.34 eV, r-r0 ) 16.3 or 20.9 Å, and kET ) 2.6 × 103 or 2.5 × 103 s-1, we obtained β values of 1.3 and 1.0 Å-1 for ZnMbAc(4)Me+ and ZnMbAc(6)Me+, respectively (Table 4). These values lie within 0.8 Å-1 e β e 1.3 Å-1 for the previously reported ET systems of Mbs.6,40,50,51 Therefore, the present intramolecular photoinduced ET reactions may be explained by the through-bond mechanism over 16.3 Å for ZnMbAc(4)Me+ and 20.9 Å for ZnMbAc(6)Me+. We note that the conformation of the alkyl spacer is flexible at the surface of ZnMb. If the ET reactions occur by through-space interaction, the edge-to-edge distances for ZnMbAc(4)Me+ and ZnMbAc(6)Me+ are estimated to be 12.8 and 15.2 Å, respectively. In such cases, the calculated β values are 1.5-1.7 Å-1 and are apparently larger than the previously reported ones (0.8 Å-1 e β e 1.3 Å-1). The calculated driving force for the back ET reaction is ∆G0(ZnMb•+-AcMe•/ZnMb-AcMe+) ) -1.44 eV based on the redox potential of ZnMb, E0(ZnMb•+/ZnMb) ) 0.98 V, and is energetically favorable (Scheme 3). In case of the intramolecular ZnMbAc(4)Me+ and ZnMbAc(6)Me+ systems, the kinetic behavior of the charge separated state, ZnMb•+-AcMe•, was also monitored by the transient absorption and its signal change in 680 nm which is characteristic of ZnMb•+ cation. After laser flash, however, the time course spectral changes did not show any formation and decay signals of ZnMb•+-AcMe•. This was probably due to the immediate disappearance of these signals. We assumed the rate for the presumed intramolecular back ET might be faster than the forward one, according to the large difference in the driving force energy: ∆G0 ) -1.44 eV for the back ET and -0.34 eV for the forward ET quenching. [AcMe]+ Excitation. As mentioned above, the fluorescent signals of ZnMb, ZnMbAc(4)Me+ and ZnMbAc(6)Me+ at 600 nm (λex ) 428 nm) and their lifetimes (2.2 ns) clearly indicate that the excited singlet state of 1(ZnMb)* is not quenched by [AcMe]+ moiety (Figure S1). In case of ZnMbAc(4)Me+ and ZnMbAc(6)Me+ dyads, we could consider two fluorescence signals around 440 and 600 nm generated from the excited singlet state of acridinium ion, 1([AcMe]+)*, and 1(ZnMb)*, respectively. Figure 4a displays the partial fluorescence spectra of ZnMb, ZnMbAc(6)Me, and intermolecular 1:1 dyad (ZnMb + [AcMe]+) upon excitation of the acridinium moiety (λex ) 252 nm) under the same concentrations in buffer solution (0.010 M phosphate, pH 7.0). Each fluorescent signal was corrected according to the absorption intensity at the excited wavelength of 252 nm. Data comparison between intramolecular and

26420 J. Phys. Chem. B, Vol. 110, No. 51, 2006

Takashima et al. fluorescence intensity (It), time (t), lifetime (τ), and the fractional contribution (A) by the following equation.

It ) A1exp( - t/τ1) + A2exp( - t/τ2)

(7)

This yielded a short lifetime component of 102 ps (83%) and a long lifetime component of 1.9 ns (17%) for ZnMbAc(6)Me+.55 These lifetimes can be assigned to the fluorescence from 1([AcMe]+)* and the quenching rate constants (k ([AcMe]+)) q for both of ZnMbAc(4)Me+ and ZnMbAc(6)Me+ are calculated from eq 8,

kq([AcMe]+) ) 1/τ - 1/τ0

(8)

where τ and τ0 are the lifetimes of ZnMbAc(4)Me+ or ZnMbAc(6)Me+ and [AcMe]+ (33 ns), respectively.56 Table 3 summarizes the fluorescent spectral data, lifetimes, and the kq([AcMe]+) for the intramolecular quenching of ZnMbAc(4)Me+ and ZnMbAc(6)Me+, where kq([AcMe]+) equals to k′′0 + k′ET + kEN in Scheme 3. If the singlet-to-singlet ENT from 1([AcMe]+)* occurs, the rate constant of the long-range ENT reaction (kEN) is often written in the form derived by Fo¨rster:57

kEN ) (1/τ0)(R0/R)6 Figure 4. (a) Fluorescence spectra of ZnMbAc(6)Me+ (bold line), ZnMb (solid line), and the 1:1 mixture of ZnMb and [AcMe]+ (dashed line) (5.0 × 10-6 M, λex ) 252 nm) at 25 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M. The inset provides from 400 to 700 nm. (b) Fluorescence decay of 1([AcMe]+)* in the ZnMbAc(6)Me+ measured at 440 nm. A bold line is fitted to eq 7.

intermolecular dyads showed that about 95% of the acridinium fluorescence (λem ) 440 nm) in the ZnMbAc(6)Me+ dyad was quenched, while the ZnMb one at 600 nm increased. About 2.1 and 2.2-fold enhancement in the ZnMb fluorescence were observed for ZnMbAc(4)Me+ and ZnMbAc(6)Me+, respectively, based on the intermolecular ZnMb and [AcMe]+ (1:1) reference. The likely processes are the photoinduced singletsinglet ENT reaction from 1(AcMe)* to ZnMb moiety and the photoinduced ET quenching reaction of 1([AcMe]+)* with ZnMb as represented in Scheme 3. Both of these reactions are energetically favorable based on the ∆G0 ) -0.22 and -0.15 eV for the photoinduced ET and ENT reactions, respectively. To our knowledge, no intramolecular ENT reaction systems have been constructed by using ZnMb-based dyads. However, in the model systems of molecular dyads, comprising a metalloporphyrin and a photosensitizer, there are some reports described on the photoinduced energy transfer reactions, where the metalloporphyrin accepts the excited energy. A covalently linked azulene and zinc-tetraphenylporphyrin (ZnTPP) dyad allows the singlet-singlet ENT reaction from the excited-state of azulene to ZnTPP, since azulene has the absorption band in the visible region and exhibits special S2-S0 fluorescence.52 The intramolecular ENT reactions from the S2 state of azulene to ZnTPP have been investigated.52b Other examples are ZnTPP-[Ru(bpy)3]2+ and palladium-TPP-acridine orange dyads. They exhibit photoinduced triplet-to-triplet and singlet-to-singlet ENT reactions upon excitation of [Ru(bpy)3]2+ and acridine orange, respectively.53,54 The kinetic detail in the fluorescence quenching reaction of 1([AcMe]+)* was studied by the time-resolved lifetime measurement. Figure 4b displays the fluorescence decay of ZnMbAc(6)Me+ measured at 440 nm by exciting at 256 nm. The decay profile was analyzed as a sum of two exponentials using the

(9)

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 9 can be replaced by eq 10 by using the efficiency (E),

E ) 1 - I/I0 ) 1 - τ/τ0 ) {1 + (R/R0)6}-1.

(10)

The calculated ENT efficiencies for both ZnMbAc(4)Me+ and ZnMbAc(6)Me+ dyads by using the obtained τ and τ0 (Table 3) are about 0.99, being similar to those obtained from the steady-state emission intensities, I and I0, of the 1([AcMe]+)*. If the present ENT efficiency is adopted for eq 10 by using the edge-to-edge distances as estimated in the above section, R ) 12.8 and 15.2 Å for ZnMbAc(4)Me+ and ZnMbAc(6)Me+, respectively,58 the ENT critical distance of R0 is calculated to be in 34 - 41 Å. This large R0 value indicates that efficient quenching reaction of 1([AcMe]+)* by ZnMb may occur by the shorter distance via through-space mechanism. In these systems, the observed singlet-to-singlet ENT efficiency and the rate constant are almost independent of the methylene number (n) in the alkyl spacer. Therefore, several conformations and orientations of [AcMe]+ are provided by the flexible spacer at the surface of ZnMb in solution. Photochemical Property of MetMbAc(6) and Interaction with CT-DNA. The Ac molecule is a photosensitizer as well as the [AcMe]+ and the excited singlet state of Ac (1Ac*) can be a good reductant for the various electron acceptors, such as MV2+, leading to the charge separated state.59 We herein carried out fluorescence measurements for the reconstituted metMbAc(6) dyad and the 1 : 1 mixture of metMb and Ac (intermolecular system) in a 0.010 M phosphate buffer (pH 7.0). As seen in Figure 5a (dashed line) for the intermolecular system, Ac exhibits strong fluorescence around 440 nm (λex ) 252 nm), which is clearly attributable to the 1Ac*. On the other hand, after covalent attachment of Ac to the heme propionate of metMb (intramolecular system), almost no emission was detected (solid line), indicating the intramolecular efficient ET quenching reaction occurs. It is noteworthy that such intramolecular quenching did not occur for FeClPPAc itself (inset of Figure 5a) in CH2Cl2:MeOH ) 1:3 solvent, indicating apoMb

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J. Phys. Chem. B, Vol. 110, No. 51, 2006 26421

including determination of the binding constant, for example, by means of calorimetric titration.33 However, our DNAregulated ET system based on the metMb scaffold is an interesting biomimetic model for the photoinduced ET reaction within a protein-DNA complex.65 Conclusions

Figure 5. (a) Fluorescence spectra of metMbAc(6) (solid line) and the 1:1 mixture of metMb and Ac (dashed line) (5.0 × 10-6 M, λex ) 252 nm) at 25 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M. The inset displays the fluorescence spectrum of FeClPPAc in CH2Cl2:MeOH ) 1:3. (b) Fluorescence spectral changes of metMbAc(6) (λex ) 355 nm) in the presence of the CT-DNA at 20 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M.

matrix is essential. Since the one-electron redox potential of metMb/deoxyMb couple is + 0.06 V (vs NHE),60 intramolecular photoinduced ET from 1Ac* {E0(Ac•+/1Ac*) ) -1.60 V and the singlet energy E00(1Ac*) ) 2.82 eV}59a to iron(III) center of metMb is thermodynamically favorable (∆G0 ) -1.66 eV). The fluorescence decay of metMbAc(6) was then obtained after laser excitation (λex ) 256 nm) monitored at 440 nm in N2saturated buffer solution (pH 7.0, 0.010 M phosphate). Two lifetimes of τ1 ) 99 ps (87%) and τ2 ) 1.6 ns (13%) were obtained and the first-order rate constant of the intramolecular ET quenching reaction was calculated to be 1.0 × 1010 s-1. It is also well-known that Ac possesses binding properties with DNA, such as groove binding and intercalation into base pairs, because noncovalent hydrophobic and π-π interactions can increase DNA affinity in an aqueous solution.61 Once it binds with DNA strand, its strong fluorescent intensity in a bulk solution decreased arisen from the ET quenching by DNA bases (preferable from guanine)62 of the 1(Ac)*.61,63 Then, we study the noncovalent binding reactions between metMbAc(6) and calf thymus DNA (CT-DNA) by fluorescence titration measurements. The steady-state fluorescence spectra of the metMbAc(6) in the presence of the CT-DNA at 20 °C, pH 7.0 (0.010 M phosphate buffer), and I ) 0.020 M are displayed in Figure 5b. Interestingly, the fluorescent signal around 440 nm (λex ) 355 nm) gradually appeared with increasing the CT-DNA concentration (0-8 eq.). This was obviously induced by noncovalent interactions between Ac moiety and CT-DNA, followed by conformational change of Ac at the surface of metMb, where the donor-acceptor distance was probably elongated by CTDNA. Owing to the low sample solubility and stability in buffer solutions, the spectroscopic titration at higher DNA concentrations could not be conducted.64 Further studies are necessary to investigate the metMbAc(6)-DNA interactions in detail,

In conclusion, we have demonstrated intermolecular and intramolecular photoinduced ET reactions between ZnMb and [AcMe]+ by preparing new reconstituted ZnMb-[AcMe]+ dyads. In the bimolecular system between ZnMb and [AcMe]+, the photoexcited triplet state of ZnMb, 3(ZnMb)*, was successfully quenched by [AcMe]+ to form the radical pair of ZnMb cation (ZnMb•+) and reduced methylacridine ([AcMe]•), followed by a thermal back ET reaction. In the intramolecular ZnMbAc(4)Me+ and ZnMbAc(6)Me+ dyads, the quenching ET rate constants of the 3(ZnMb)* by the [AcMe]+ moiety provided the reasonable β values, if the ET occurs along the alkyl spacer at the surface of Mb via through-bond mechanism. Kinetic studies also presented the intramolecular quenching of 1([AcMe]+)* in ZnMbAc(4, 6)Me+, where the likely process is the intramolecular photoinduced ENT reaction. Finally, the steady-state fluorescence measurements of metMbAc(6) were investigated in the absence and the presence of CT-DNA. It reveals that the noncovalent recognition of Ac moiety with CTDNA in an aqueous solution is important to form artificial MbDNA complex and display DNA-sensitive fluorescence changes. We believe further synthetic manipulations at the Mb surface, by using several DNA-binders coupled with the photoinduced reactions, may provide valuable information to elucidate the complicated Acknowledgment. This research was partly supported by Grant-in-Aid for Encouragement of Young Scientists no. 14750697 from the Ministry of Education, Culture, Sports, and Science and Technology (MEXT) of Japanese Government and Nara Women’s University Intramural Grant for Project Research. We thank Professor Kuninobu Kasuga of Shimane University for elemental analyses. Supporting Information Available: A PDF file containing fluorescent spectral data for kinetics. This material is available free of charge via the Internet at http://pubs. acs. org. References and Notes (1) (a) Moore, G. R.; Pettigrew, G. W. Cytochrome c. EVolutionary, Structural and Physicochemical Aspects; Springer-Verlag: Berlin, 1990. (b) Kosti′c, N. M. Metal Ions in Biological Systems; Siegel, H., Siegel, A., Eds.; Marcel Dekker: New York, 1991; Vol. 27, pp 129-182. (c) Protein Electron Transfer. Bendall, D. S., Ed.; BIOS Scientific Publishers Ltd: Oxford, 1996. (2) Zhou, J. S.; Granda, E. S. V.; Leontis, N. B.; Rodgers, M. A. J. J. Am. Chem. Soc. 1990, 112, 5074-5080. (3) McLendon, G.; Hake, R. Chem. ReV. 1992, 92, 481-490. (4) Tsukahara, K.; Okada, M.; Asami, S.; Nishikawa, Y.; Sawai, N.; Sakurai, T. Coord. Chem. ReV. 1994, 132, 223-228. (5) 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. (6) Gray, H. B.; Winkler, J. R. Electron Transfer in Chemistry; Balzani, V., Ed.; Wiley-VCH: Weinheim, 2001; Vol. 3, pp 3-23. (7) (a) Paulson, D. R.; Addison, A. W.; Dolphin, D.; James, B. R. J. Biol. Chem. 1979, 254, 7002-7006. (b) Hoffman, B. M. The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1979; Vol VII, pp 403404. (c) McLendon, G.; Murphy, P. J. Biol. Chem. 1980, 255, 4035-4039. (d) Cowan, J. A.; Gray, H. B. Inorg. Chem. 1989, 28, 2074-2078. (8) (a) Pan, L. P.; Durham, B.; Wolinska, J.; Millett, F. Biochemistry 1988, 27, 7180-7184. (b) Durham, B.; Pan, L. P.; Long, J. E.; Millett, F. Biochemistry 1989, 28, 8659-8665. (c) Geren, L.; Hahm, S.; Durham, B. Millet, F. Biochemistry 1991, 30, 9450-9457. (d) Hahm, S.; Durham, B.;

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