Keiichi Tsukahara

Ionic strength effect on the ascorbate reduction of sperm whale and horse heart metmyoglobins

Abstract Kinetics of the reduction of sperm whale skeletal muscle and horse heart metmyoglobins (metMb) by ascorbic acid (H 2 A) were studied in a nitrogen atmosphere at 25 °C, at different ionic strengths (NaCl) and between pH 7.29 and 8.29. The second order rate constants were determined for the reductions of metMb(H 2 O) by ascorbate (HA − and A 2− ). It was found that the ascorbate reduction is faster for horse heart metMb than for sperm whale metMb at a higher ionic strength, and that at a lower ionic strength similar reactivities are observed. The logarithm of the rate constants correlates to the square root of the ionic strength. This is explained by a simple outer-sphere electron transfer mechanism. It was found that the effective charge of sperm whale metMb is higher than that of horse heart metMb and that there is no specific interaction of ascorbate with metMb.

Photoinduced electron-transfer reactions of zinc and magnesium myoglobins

Abstract Photoinduced electron-transfer (ET) reaction between zinc- or magnesium myoglobin and a variety of quenchers, such as [Ru(NH 3 ) 6 ] 3+ , cationic viologens, copper(II) protein (stellacyanin) and anlhraquinone-2-sulfonate has been studied in an aqueous degassed solution. Not only photoinduced ET but also thermal backward ET reactions were insensitive to the driving force of reactions, suggesting that the reactions are controlled by conformational changes in myoglobins.

Photoinduced electron-transfer reaction between cytochrome c and zinc myoglobin modified with diethylenetriaminepentaacetic acid

Abstract The kinetics and mechanism of the photoinduced electron-transfer reaction between cytochrome c and zinc-substituted myoglobin, the latter of which lysine residue was modified with diethylenetriaminepentaacetic acid, were studied in an aqueous solution at 25°C, pH = 7.0 (a 10 mM phosphate buffer) and ionic strengths from 0.02 to 0.32 M (NaCl). The excited triplet-state of the modified zinc myoglobin was efficiently quenched by the oxidized cytochrome c within a diprotein complex. Both the intracomplex electron-transfer quenching rate constant and the binding constant of the diprotein complex decreased with increasing ionic strengths.

Metal Ion-Dependent Fluorescent Dynamics of Photoexcited Zinc−Porphyrin and Zinc−Myoglobin Modified with Ethylenediaminetetraacetic Acid

The reconstituted zinc-myoglobin (ZnMb) dyads, ZnMb-[M(II)(edta)], have been prepared by incorporating a zinc-porphyrin (ZnP) cofactor modified with ethylenediaminetetraacetic acid (H(4)edta) into apo-Mb. In case of the monomeric ZnP(edta) cofactor coordinated by one pyridine molecule, ZnP(py)(edta), a spontaneous 1:1 complex with a transient metal ion was formed in an aqueous solvent, and the photoexcited singlet state of ZnP, (1)(ZnP)*, was quenched by the [Cu(II)(edta)] moiety through intramolecular photoinduced electron-transfer (ET) reaction. The rate constant for the intramolecular quenching ET (k(q)) at 25 degrees C was successfully obtained as k(q) = 5.1 x 10(9) s(-1). In the case of Co(2+), Ni(2+), and Mn(2+), intersystem crossing by paramagnetic effect was mainly considered between (1)(ZnP)* and the [M(II)(edta)] complex. For the ZnMb-[M(II)(edta)] systems, the intramolecular ET reaction between the excited singlet state of (1)(ZnMb)* and the [Cu(II)(edta)] moieties provided the slower quenching rate constant, k(q) = 2.1 x 10(8) s(-1), compared with that of the ZnP(py)(edta) one. Kinetic studies also presented the efficient fluorescence quenching of the (1)(ZnMb)*-[Co(II)(edta)] dyad. Our study clearly demonstrates that wrapping of the ZnP cofactor by the apoprotein matrix and synthetic manipulation at the Mb surface ensure metal ion-sensitive fluorescent dynamics of ZnMb and provides valuable information to elucidate the complicated mechanism of the biological photoinduced ET reactions of hemoproteins.

Synthesis and Spectroscopic Properties of Reconstituted ZincMyoglobin Appending a DNA‐Binding Platinum(II) Complex

A reconstituted zinc–myoglobin (ZnMb) dyad, ZnMb‐[Pt(bpy)(en)]2+, has been prepared by incorporating chemically‐modified zinc–porphyrin, being capable of DNA‐binding of the Pt complex, [Pt(bpy)(en)]2+, where bpy and en are 2,2′‐bipyridine and ethylenediamine, respectively. The steady‐state fluorescence of the cofactor, [Pt(bpy)(μ‐enPP)Zn]Cl2, in MeOH indicates that the excited singlet state of zincporphyrin was almost quenched, probably because of the strong hydrophobic and π–π stacking interactions between the [Pt(bpy)(μ‐enPP)Zn]2+ ions. In the reconstituted ZnMb‐[Pt(bpy)(en)]2+, the quenching reaction of 1(ZnMb)* with the [Pt(bpy)(en)]2+ moiety does not occur, indicating apo‐Mb matrix is essential. On the other hand, when the [Pt(bpy)(en)]2+ moiety was excited, the enhancement of the fluorescence from ZnMb unit was observed. It is suggested that the energy transfer from 1([Pt(bpy)(en)]2+)* to ZnMb occurs. The spectroscopic changes of ZnMb‐[Pt(bpy)(en)]2+ in the presence of calf‐thymus DNA were also provided. Soret band at 428 nm gradually decreased, and isosbestic points at 321, 414, and 432 nm were observed with increasing the DNA concentration. When the PtII moiety was excited at λex 321 nm, the fluorescence signal around 600 nm similarly decreased. The synthetic manipulation of ZnMb by using a DNA‐binding PtII complex demonstrates sensitive fluorescent signal for DNA and valuable information to study photoinduced electron transfer within a Mb–DNA complex.

Intramolecular electron-transfer reaction within a diprotein complex of cytochrome c with ferrylmyoglobin modified with diethylenetriaminepentaacetic acid

Abstract. Horse heart metmyoglobins modified with diethylenetriaminepentaacetic acid, metMb(DTPA)n (n=1, 2, 4, and 5), were characterized by a MALDI-TOF mass spectrometry, amino-acid sequence analysis, and UV-Vis and CD spectroscopies. The DTPA-binding sites on metMb were Lys47, Lys50, Lys87, Lys145, and Lys147 for metMb(DTPA)5, Lys47, Lys87, Lys145, and Lys147 for metMb(DTPA)4, Lys87 and Lys145 for metMb(DTPA)2, and Lys87 for metMbDTPA, respectively. The modified metMb(DTPA)n showed cytochrome c peroxidase-like activity more efficiently than native metMb: metMb(DTPA)5>metMb(DTPA)4>metMb(DTPA)2>metMbDTPA≈native metMb. The first-order rate constants for the reactions of ferrylMb(DTPA)n (n=2, 4, and 5) with reduced cytochrome c [cyt c(II)] were saturated with concentrations of cyt c(II), suggesting that the electron transfer (ET) occurs within a diprotein complex. The intramolecular ET rate constants in the diprotein complex increased with increasing the number of DTPA ions. The reactions of native ferrylMb and ferrylMbDTPA with cyt c(II) obeyed a second-order rate law. A possible ET mechanism is proposed; cyt c(II) binds the DTPA-linked anionic patch around Lys87, Lys145, and Lys147 region of ferrylMb(DTPA)n.

Photoinduced electron transfer from zinc myoglobin to benzoquinone studied by FT-EPR

A Fourier transform (FT) EPR technique has been used to study the photoinduced electron transfer from zinc-substituted myoglobin (ZnMb) to 1,4-benzoquinone (BQ). Both porphyrin cation and BQ anion radicals (BQ - ) were observed, which provide direct evidence of an electron transfer occurring between a porphyrin moiety (ZnPP) in myoglobin and BQ. Electron transfer rate constants, spin–lattice relaxation times of triplet porphyrin and BQ - and magnitudes of chemically induced dynamic electron polarization (CIDEP) due to the triplet mechanism (TM) and the radical pair mechanism (RPM) were evaluated from decay analyses of the FT-EPR signals of BQ - . These results were compared with those for a [5,10,15,20-tetrakis(4-sulfonatophenyl)porphinato]zinc(II) (ZnTPPS)–BQ system and discussed in terms of protein effects in ZnMb. We concluded that electron transfer occurs from the ZnPP moiety to BQ which is located at the surface of myoglobin.

Charge-transfer interaction between bipyridinium and naphthyl groups of optically active viologens in an aqueous solution

Abstract Optically active viologens containing naphthyl group(s) show an intermolecular charge-transfer interaction between bipyridinium and naphthyl groups in an aqueous solution. Association constants are dependent on the chirality of the viologens.

A nickel(II) Schiff base complex linked to viologen via number six of the methylene groups

Abstract A nickel(II) Schiff base complex linked to viologen via number six of the methylene groups was prepared and characterized. The characteristic absorption band of a methylviologen radical appeared with the reduction of the derivative by sodium dithionite in a DMFH 2 O solution, and then disappeared with aeration. Bromoalkyl derivatives of the complex with a varying number ( n = 2–5) of methylene groups were also prepared.

The rate of autoxidation of oxymyoglobin in the presence of detergents

Abstract The rate of autoxidation of oxymoglobin was studied spectrophotometrically in the presence of detergents such as sodium laurate and 1-laurylpyridinium chloride. The rate was extremely accelerated near the critical micelle concentration of the detergents. It was only slightly dependent on the pH in the range from 8.5 to 10.5, but significantly dependent on ionic strength. After the fast denaturation of globin, caused by cluster formation around hydrophobic amino-acid residues of globin near the critical micelle concentration, autoxidation might occur with retention of a prosthetic group inside a heme pocket. It is proposed that the formation of clusters might shift both amino-acid residues and the prosthetic group from their normal positions inside the heme pocket, resulting in an increase of the rate of autoxidation.