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The pH-dependence of the ability of coproporphyrin (CP) and uroporphyrin (UP) to photogenerate hydrogen peroxide (H2O2) in aqueous solution was investigated, with special attention to the structure-activity relationship related to the aggregation of the porphyrins. It was found that the efficiency was strongly dependent on the aggregation of CP and UP mediated by changes in the pH of the solution, and a dimeric form had a weak ability to produce H2O2, while a highly aggregated form had a good ability. The increased efficiency of the highly aggregated porphyrin to produce H2O2 was further demonstrated using a different type of aggregate formed by the electrostatic interaction of cationic tetrakis-5,10,15,20-(N-methyl-4-pyridyl)porphin (TMPyP) with anionic tetrakis-5,10,15,20-(4-sulfonatophenyl)porphin (TSPP). The present results demonstrated the importance of the state of aggregation of porphyrin to photogenerate H2O2, and the results may help to develop a new type of medicine for photodynamic therapy.

The roles of Phe-120 and Glu-222 in the oxidation of chiral substrates bunitrolol (BTL) and bufuralol (BF) by CYP2D6 are discussed. Wild-type CYP2D6 (CYP2D6-WT) oxidized BTL to 4-hydroxybunitrolol (4-OH-BTL) with substrate enantioselectivity of (R)-(+)-BTL > (S)-(-)-BTL. The same enzyme converted BF into 1''-hydroxybufuralol with substrate enantioselectivity of (R)-BF >> (S)-BF and metabolite diastereoselectivity of (1''R)-OH < (1''S)-OH. The substitution of Phe-120 by alanine markedly increased the apparent K(m) and V(max) values for enantiomeric BTL 4-hydroxylation by CYP2D6. In contrast, the same substitution caused an increase only in V(max) values of (S)-BF 1''-hydroxylation without changing apparent K(m) values, while kinetic parameters (K(m) and V(max) values) for (R)-BF 1''-hydroxylation remained unchanged. Furthermore, the substitution of Glu-222 as well as Glu-216 by alanine remarkably decreased both the apparent K(m) and V(max) values without changing substrate enantioselectivity or metabolite diastereoselectivity. A computer-assisted simulation study using energy minimization and molecular dynamics techniques indicated that the hydrophobic interaction of an aromatic moiety of the substrate with Phe-120 and the ionic interaction of a basic nitrogen atom of the substrate with Glu-222 in combination with Glu-216 play important roles in the binding of BF and BTL by CYP2D6 and the orientation of these substrates in the active-site cavity. This modeling yielded a convincing explanation for the reversal of substrate enantioselectivity in BTL 4-hydroxylation between CYP2D6-WT and CYP2D6-V374M having methionine in place of Val-374, which supports the validity of this modeling., (Copyright 2006 Wiley-Liss, Inc.)

Electrochemical formation of H2O2 and the subsequent ferryl porphyrin were examined by measuring luminol chemiluminescence and absorption spectrum using flow-injection method. Emission was observed under the cathodic potential (0.05 V at pH 2.0 and -0.3 V at pH 11.0) by the electrochemical reduction of buffer electrolytes solution but no emission was observed at anodic potentials. Fe(III)TMPyP solution was added at the down stream of the working electrode and was essential for the emission. Removal of dissolved O2 resulted in the decrease of emission intensity by more than 70%. In order to examine the lifetime of reduced active species, delay tubes were used in between working electrode and Fe(III)TMPyP inlet. Experimental results suggested the active species were stable for quite long. The emission was quenched considerably (>90%) when hydroperoxy catalase was added at the down stream of the working electrode whereas SOD had little effect. Significant inhibition of the emission by the addition of alkene at the down stream of the Fe(III)TMPyP inlet was considered as evidence of oxo-ferryl formation. The spectra at reduction potential under aerated condition were shifted to the longer wavelength (>430 nm) compared to the original spectrum of Fe(III)TMPyP (422 nm). All the spectra were perfectly reproduced by a combination of Fe(III)TMPyP and O=Fe(IV)TMPyP (438 nm) spectra. These observations lead to the conclusion that H2O2 was produced first by electrochemical reduction of O2, which then converted Fe(III)TMPyP into O=Fe(IV)TMPyP to activate luminol. The current efficiencies for the formation of H2O2 were estimated as about 30-65% in all over the pH.

Electrochemical formation of ferryl porphyrin was examined by electrocatalytic oxidation of alkene by measuring luminol chemiluminescence using a flow-injection method. Emission was observed both below the reduction potential of Fe(III)TMPyP (-0.08 V at pH 11, -0.02 V at pH 7 and 0.15 V at pH 3) and above the oxidation potential (0.6 V at pH 11, 0.75 V at pH 7 and 1.1 V at pH 3). However, both anodic and cathodic emissions were inhibited significantly by the addition of alkene (cyclopent-2-ene-1-acetic acid) solutions downstream of the working electrode. Further, the spectra at both anodic and cathodic sides shifted to the longer wavelength (>424 nm) compared to the original spectrum of Fe(III)TMPyP (422 nm), which was not observed with the addition of alkene solution. Therefore, the results suggest that the electrochemically generated oxo-ferryl species have been engaged in catalytic oxidation of alkene before the flow reaches the observation cell.

The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1''-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1''-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF >> S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1''R-OH-BF << 1''-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1''-hydroxylation by CYP2D6., (Copyright 2004 Wiley-Liss, Inc.)

High-valent oxo-iron(IV) species are commonly proposed as the key intermediates in the catalytic mechanisms of iron enzymes. Water-soluble iron(III) tetrakis-5,10,15,20-(N-methyl-4-pyridyl)porphyrin (Fe(III)TMPyP) has been used as a model of heme-enzyme to catalyse the hydrogen peroxide (H(2)O(2)) oxidation of various organic compounds. However, the mechanism of the reaction of Fe(III)TMPyP with H(2)O(2) has not been fully established. In this study, we have explored the kinetic simulation of the reaction of Fe(III)TMPyP with H(2)O(2) and of the catalytic reactivity of FeTMPyP in the luminescent peroxidation of luminol. According to the mechanism that has been established in this work, Fe(III)TMPyP is oxidized by H(2)O(2) to produce (TMPyP)(*+)Fe(IV)[double bond]O (k1 = 4.5 x 10(4)/mol/L/s) as a precursor of TMPyPFe(IV)[double bond]O. The intermediate, (TMPyP)(*+)Fe(IV)[double bond]O, represented nearly 2% of Fe(III)TMPyP but it does not accumulate in sufficient concentration to be detected because its decay rate is too fast. Kinetic simulations showed that the proposed scheme is capable of reproducing the observed time courses of FeTMPyP in various oxidation states and the decay profiles of the luminol chemiluminescence. It also shows that (TMPyP)(*+)Fe(IV)[double bond]O is 100 times more reactive than TMPyPFe(IV)[double bond]O in most of the reactions. These two species are responsible for the initial sharp and the sustained luminol emissions, respectively., (Copyright 2003 John Wiley & Sons, Ltd.)

Bufuralol (BF), a nonselective beta-adrenoceptor blocking agent, has a chiral center in its molecule, yielding the enantiomers 1'R-BF and 1'S-BF. beta-Adrenoceptor blocking potency is much higher in 1'S-BF than in 1'R-BF. One of the metabolic pathways of BF is 1"-hydroxylation of an ethyl group attached at the aromatic 7-position forming a carbinol metabolite (1"-hydroxybufuralol, 1"-OH-BF), and further oxidation (or dehydrogenation) produces a ketone metabolite (1-oxobufuralol, 1"-Oxo-BF). Both 1"-OH-BF and 1"-Oxo-BF are known to have beta-adrenoceptor blocking activities comparable to or higher than those of the parent drug. The 1"-hydroxylation introduces another chiral center into the BF molecule and four 1"-OH-BF diastereomers are formed from BF racemate in mammals, including humans, making elucidation of the metabolic profiles complicated. HPLC methods employing derivatization, reversed phase, or chiral columns have been developed to efficiently separate the four 1"-OH-BF diastereomers formed from BF enantiomers or racemate. Accumulated in vitro experimental results revealed that 1'R-BF is a much more preferential substrate than 1'S-BR for BF 1"-hydroxylation in human liver microsomes. Kinetic studies using recombinant human cytochrome P450 (CYP) enzymes indicate that CYP2D6 serves as a major BF 1"-hydroxylase and that CYP1A2 and CYP2C19 also contribute to BF 1"-hydroxylation in human livers. This mini-review summarizes the knowledge reported so far on the pharmacology of BF and its metabolites and the profiles of BF metabolism, especially focusing on the stereoselectivity in the oxidation of BF mainly in human livers and recombinant CYP enzymes., (Copyright 2003 Wiley-Liss, Inc.)

The reaction of iron(III) tetrakis-5,10,15,20-(N-methyl-4-pyridyl)porphyrin (Fe(III)TMPyP) with hydrogen peroxide (H(2)O(2)) and the catalytic activity of the reaction intermediates on the luminescent peroxidation of luminol in aqueous solution were studied by using a double-mixing stopped-flow system. The observed luminescence intensities showed biphasic decay depending on the conditions. The initial flashlight decayed within <1 s followed by a sustained emission for more than 30 s. Computer deconvolution of the time-resolved absorption spectra under the same conditions revealed that the initial flashlight appeared during the formation of the oxo-iron(IV) porphyrin, TMPyPFe(IV) = O, which is responsible for the sustained emission. The absorption spectra 0.0-0.5 s did not reproduce well by a simple combination of the two spectra of Fe(III)TMPyP and TMPyPFe(IV) = O, indicating that transient species was formed at the initial stage. Addition of uric acid (UA) caused a significant delay in the initiation of the luminol emission as well as in the formation of the TMPyPFe(IV) = O. Both of them were completely diminished in the presence of UA equimolar with H(2)O(2), while mannitol had no effect at all. The delay of the light emission as well as the appearance of TMPyPFe(IV) = O was directly proportional to the [UA](0) but other kinetic profiles were not changed significantly. Based on these observations and the kinetic analysis, we confirmed the involvement of the oxo-iron(IV) porphyrin radical cation, (TMPyP)(.+)Fe(IV) = O, as an obligatory intermediate in the rate-determining step of the overall reaction, Fe(III)TMPyP + H(2)O(2) --> TMPyPFe(IV) = O, with a rate constant of k = 4.3 x 10(4)/mol/L/s. The rate constants for the reaction between the (TMPyP)(.+)Fe(IV) = O and luminol, and between the TMPyPFe(IV) = O and luminol were estimated to be 3.6 x 10(6)/mol/L/s and 1.31 x 10(4)/mol/L/s, respectively., (Copyright 2003 John Wiley & Sons, Ltd.)

The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and bunitrolol (BTL) 4-hydroxylations as indices of drug-metabolizing enzymes. The substitution with hydrophobic amino acid residues such as valine and phenylalanine did not affect the enzymatic properties such as reduced CO-difference spectra, microsomal CYP contents and oxidation activities towards DB and BTL. The substitution of glycine-42 with a polar but noncharged amino acid residue (serine) exhibited a similar reduced CO-different spectrum, but the substitution with a charged basic (lysine and arginine) or acidic (glutamic acid) amino acid residue commonly produced a peak at 420 nm in addition to a Soret peak at 450 nm. Cytochrome P450 contents and microsomal contents of G42S, G42K, G42R and G42E estimated spectrophotometrically and estimated by Western blot analysis, respectively, were lower than those of the wild-type. Kinetic analysis revealed that the substitution of glycine-42 with charged amino acid residues such as lysine, arginine and glutamic acid markedly increased the apparent K(m) values for DB and BTL oxidations without remarkable changes in the V(max) values. The subsitution with noncharged amino acid residues such as serine, valine and phenylalanine did not cause such a marked change in the K(m) values. Efficiencies (V(max)/K(m)) as DB and BTL 4-hydroxylases of CYP2D6 mutant proteins having charged amino acid residues were found to be decreased mainly by increasing their K(m) values. These results indicate that the properties of amino acid residues at position 42 affect the behavior of CYP2D6 proteins such as anchoring into ER membranes, conversion of P450 to P420 and incorporation of heme into apoproteins.

A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

We have been investigated the possibility of B-Z transition in ZnTMPyP-DNA interaction based on the observation of spectroscopic data. In this study, we found drastic change in the AFM image of supercoiled plasmid DNA when it was interacted with TMPyPs indicating that the considerable amount of unwinding of double helix or B-Z transition is induced by the drug-DNA interaction. Such phenomena were not observed for other cationic drugs examined.

The breakdown of methyllinoleate hydroperoxide (LOOH) and peroxidation of methyllinoleate (LH) catalyzed by hemin and hematin were studied in 98% methanol. Spectrophotometry was used to follow the breakdown of LOOH. The peroxidation process of LH was monitored by the oxygen consumption. The pKa for the conversion of hemin to hematin was determined as 7.3 in 98% methanol. The catalytic rates for both processes were found to reach a maximum at pH 8 where about 80% of hemin was in the hematin form. This could be accounted for by the acceleration of the breakdown of LOOH due to binding of hydroxide or methoxide ion to the hemin iron. The reduction in the catalytic activity at a higher pH, where 100% of hemin was in the hematin form, however, suggested that H+ was also necessary. We propose a new scheme which shows the role of H+ and OH- (or CH3O-) together with ferryl iron in the catalytic process. The breakdown of hemin itself was also observed and its catalytic cycle number was estimated as 9. The gradual decomposition of hemin suggests involvement of a Fenton-type mechanism as a minor catalytic process.