Yusaku Ikegami

Mechanism of O−2 generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems

O2- generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q10 oxidoreductase system, was examined using a model system, NADPH-coenzyme Q1-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q1 produces coenzyme Q1-. and O2- during enzyme-catalyzed reduction and O2+ coenzyme Q1-. are in equilibrium with O2- + coenzyme Q1 in the presence of enough O2. The coenzyme Q1-. produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q10 radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O2. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q1 by the rapid dismutation of O2- generated, thereby preventing the reduction of coenzyme Q1 by O2-. The enzymatic reduction of coenzyme Q1 to coenzyme Q1H2 via coenzyme Q1-. is smoothly achieved under anaerobic conditions. The rate of coenzyme Q1H2 autoxidation is extremely slow, i.e., second-order constant for [O2][coenzyme Q1H2] = 1.5 M-1.s-1 at 258 microM O2, pH 7.5 and 25 degrees C.

Generation of hydroxyl radicals during the enzymatic reductions of the Fe3+-ADP-Phosphate-adriamycin and Fe3+-ADP-EDTA systems: Less involvement of hydroxyl radical and a great importance of proposed perferryl ion complexes in lipid peroxidation

A system which contains NADPH, purified cytochrome P-450 reductase (enzyme) and Fe3+-ADP-adriamycin complex in Tris-HCl buffer does not produce hydroxyl radical, but possesses a strong lipid peroxidation activity on exogenously added phospholipid micelles. Fe3+-ADP-adriamycin complex, a tightly coordinated complex in Tris-HCl buffer, could be dissociated to Fe3+-ADP-phosphate complex and adriamycin in phosphate buffer. Hydroxyl radical, which can be detected by a spin trapping method using N-tert-butyl-alpha-phenylnitrone, is produced during the enzymatic reduction of a mixture of Fe3+-ADP-phosphate complex and adriamycin or of Fe3+-ADP-EDTA complex while it is not involved in phospholipid peroxidation under the conditions used. With hydroxyl radical-generating systems, little or no quenching of hydroxyl radical in Tris-HCl buffer could be demonstrated. The oxidative cleavage of phospholipid is initiated by the proposed perferryl ion complex, which may be generated by the interaction of Fe2+-ADP-adriamycin complex with O2. A similar perferryl ion complex is also produced during the enzymatic reduction of Fe3+-ADP-EDTA complex with a molar ratio of 2 for [Fe3+]/[EDTA] in the presence of air. This is also able to catalyze lipid peroxidation.

Generation of free radicals and initiation of radical reactions in nitrones — Fe2+ - phosphate buffer systems

Abstract Stability of spin trapping reagents, phenyl-tert-butylnitrone (PBN) and 5,5-dimethyl-pyrroline-N-oxide (DMPO), in Fe2+-phosphate buffer (pH 7.8 and 7.0) was examined. Both nitrones generate the spin adducts without substrate. It was supposed that PBN and DMPO produce the benzoyl and hydroxyl spin adducts, respectively, by the decomposition of themselves in the system. The presence of alcohols causes the initiation of radical reaction and leads to strong ESR signals due to hydroxyalkyl spin adducts.

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.

Anisotropic hyperfine interaction in the electron spin resonance spectrum of the methyl radical trapped in CH3COONa⋅3D2O crystal at low temperatures

The electron spin resonance spectrum of the methyl radical produced by γ irradiation of CH3COONa⋅3D2O single crystal at 77 K has been measured at low temperatures. The spectrum observed at below 24 K consisted of eight lines with approximately equal intensities, instead of the four lines (1:3:3:1) at higher temperatures, due to the quantum tunneling of the rotational states. The transitions at low temperatures were angular dependent upon the applied field. From the analyses of the angular dependence of the spectrum with the aid of the theoretical calculation of the transition field for the tunneling methyl radical, the following anisotropic hyperfine tensor for the proton was determined (MHz): Axx=−31.4, Ayy=−93.7, Azz=−60.3, a (iso) =−61.8. These parameters provide the first experimental assessment of the well‐known theoretical results on the proton hyperfine anisotropy in the free methyl radical.

Time-resolved EPR observation of the short-lived excited triplet states of diamagnetic metallophthalocyanines in a rigid glassy matrix

Abstract Time-resolved EPR spectra were first observed for the lowest excited triplet state of metallophthalocyanines (MPc: M2Na, Mg, and Zn) in a rigid glassy matrix at 77 K. The zero-field splitting | D | parameters are smaller compared with those of metalloporphyrins. Introduction of the Zn atom to Pc induced drastic change in the intersystem crossing process, while the effect on the ZFS parameters was small.

Semiautomatic mutual inductance bridge for magnetic susceptibility measurements

A semiautomatic Hartshorn bridge has been designed to improve the complicated adjusting procedures of the bridge in the measurements of magnetic susceptibilities. The bridge is useful for a continuous recording of the susceptibility of a sample, especially when it varies with time.

Proton tunneling of tropolone in durene single crystal as studied by time-resolved EPR detected excitation spectroscopy

We have measured the excitation spectra for tropolone–OH in durene single crystal and tropolone–OD in deuterated durene using a time-resolved electron paramagnetic resonance (TREPR) detection method that makes possible to separate the signals due to magnetically different sites. The tunneling doublet with 3 cm−1 was observed in the sharp zero-phonon line. The small splitting indicates that the crystal field increases the barrier of double-minimum potential for the proton tunneling in the S1 state. Moderately asymmetric potentials of the S0 and S1 states, where the energetic imbalance between two wells in the S1 state potential is opposite the S0 state potential, reasonably explained the observed unusual intensity ratio of the tunneling doublet (01+<01−). A well-resolved progression of a phonon band with a 15 cm−1 separation was also obtained in durene crystal at very low temperature. From a Franck–Condon analysis of the relative intensity of the phonon band, it was clarified that the stable configuration ...

1,1'-(P- OR M-PHENYLENE)BIS(2,4,6-TRIPHENYLPYRIDINYL) DIRADICALS: GROUND STATE IN A HETEROATOM-CONTAINING SYSTEM IN RELATION TO THE TOPOLOGY RULE

Abstract 1,1′-( p - or m -Phenylene)bis(2,4,6-triphenylpyridinyl) diradical were prepared by the reduction of the corresponding dications. The plots of the ESR signal intensity vs. 1/T show that both the p - and m -diradicals are singlet in the ground state with a singlet-triplet energy gap of 51 cal/mol for the p -isomer and 44 cal/mol for the m -isomer.

The Thermal and Photochemical Behaviors of the Cyclomers of Ethylenebis- and Trimethylenebis(pyridinyl) Diradicals

Pyridinyl diradicals of the type Py . -(CH 2 ) n -Py . (Py . = pyridinyl, n=2,3), which possess two pyridinyl monoradicals connected through a methylene chain, exist usually in equilibrium with the meso and dl cyclomers formed by intramolecular cyclization of the diradical. The equilibrium between diradical and cyclomers tends overwhelmingly toward the cyclomers in the dark and the cyclomers photodissociate to generate the diradical. This paper concentrates our recent results on the preparation, structure, and thermal and photochemical behaviors of the diradicals and the cyclomers.