Shun Hirota

Studies on galactose oxidase active site model complexes: effects of ring substituents on Cu(II)-phenoxyl radical formation

Copper(II) complexes of new N 3 O- and N 2 O 2 -donor tripodal ligands bearing one or two o -substituted phenol moieties have been synthesized as models for the galactose oxidase active site. The complexes of 2-[ N -(1-methyl-2′-imidazolylmethyl)- N -(6″-methyl-2″-pyridylmethyl)-aminomethyl)]-4-methyl-6-methylthiophenol (MeSL), [Cu(MeSL)Cl], and N -(6-methyl-2-pyridylmethyl)- N , N -bis(2′-hydroxy-3′,5′-di- tert -butylbenzyl)amine (t-buL2mepy), [Cu(t-buL2mepy)(H 2 O)], have been revealed by X-ray structural analysis to have a square-pyramidal structure with one and two phenolate oxygens in the basal plane, respectively. [Cu(MeSL)Cl] was converted into a Cu(II)- o -methylthiophenoxyl radical species by electrochemical or Ce(IV) oxidation. An o -methoxyphenoxyl radical in a similar complex was considerably more stable than the 2,4-di( tert -butyl)phenoxyl radical. While t-buL2mepy reacted with Cu(ClO 4 ) 2 to give [Cu(t-buL2mepy)(H 2 O)] without disproportionation, an N2O2-donor ligand containing an o -methoxyphenol, a 2,4-di( tert- butyl)phenol, and an N -methylimidazole moiety gave a phenoxyl radical complex exhibiting the characteristic absorption peak at 478 nm as a reddish powder by the reaction with Cu(ClO 4 ) 2 as a result of spontaneous disproportionation. It exhibited a quasi-reversible redox wave at E 1/2 =0.34 V (vs. Ag/AgCl) in CH 3 CN, which is lower than the potentials of the copper complexes of various N 3 O-donor ligands, and oxidized ethanol to acetaldehyde with a low turnover number.

Analysis of the active sites of copper/topa quinone-containing amine oxidases fromLathyrus odoratus andL. sativus seedlings

Amine oxidases from Lathyrus odoratus and L. sativus were isolated and, following a three step purification, gave pink coloured proteins (λmax 500 nm) which consisted of dimers of 72 kDa units each. Rabbit antiserum against the enzyme from L. odoratus cross-reacted with the enzyme from L. sativus. N-Terminal amino acid sequences of both enzymes show a high degree of similarity to other plant and microbial copper-containing amine oxidases. Aliphatic diamines and some polyamines were the best substrates, whereas substrate analogues, copper complexing agents and alkaloids were inhibitors of both amine oxidases. Differential pulse polarography indicated the existence of a copper–quinone redox system at the active site of the enzymes. Electron paramagnetic resonance (EPR) spectra confirmed the presence of Cu(II) ions coordinated in a tetragonal ligand field. Mn(II) ions were clearly detected, which are supposed partially to occupy another metal site in the enzyme. Spectrophotometric titrations with phenylhydrazines demonstrated one reactive carbonyl group per subunit of the enzymes. The pH shift of the absorption maximum of p-nitrophenylhydrazones of the enzymes and resonance Raman spectroscopy strongly suggest topa quinone as the organic cofactor.

The CoCH3 bond in Shiff base B12 models: influence of the trans and equatorial ligands as assessed by Fourier transform Raman spectroscopy

Properties of the CoC bond in organocobalt methyl Schiff base B12 model compounds in CHCl3 solution were probed using near-IR Fourier transform Raman spectroscopy. The influence of the trans ligand (L) on the CoCH3 stretching (νCoCH3) frequency was investigated by varying the trans ligand of [LCo(salen)(CH3)]. The influence of L is related to the ground state CoC bond strength and correlates empirically with the CoC bond length in closely related models. The νCoCH3, frequency decreased from 518 to 500 cm− with L in the order methanol 1-methylimidazole>4-dimethylaminopyridine ≽ pyridine ≽ piperidine > quinuclidine > triethylphosphine. This decrease reflects the greater electron in the 2eg orbital which results from the expected increase in the electron-donating character of L. The νCoCH3, frequency of the L = 2-methylpyridine (2-Mepy) derivative was detected at 518 cm−1, higher than the value of 512 cm−1 for the L = py derivative. This result is attributed to repulsion between the 2-Me group and the equatorial ligand, steric interactions making the coordination of the trans 2-Mepy ligand weaker. The νCoCH3 frequency for the putative five-coordinate [Co(salen)(CH3)] compound was detected at 513 cm−1 (491cm−1 for the CD3 analog), close to frequencies found for the six coordinate salen complexes studied. The νCoCH3 frequency of pyridine derivatives of several well known B12 model compounds was found to increase with changing equatorial ligands in the order (DO)(DOH)pn < (DH)2 < saloph < salen2. This trend suggests the ground state CoC bond experiences an apparent strengthening that could be attributed to a decrease in electron occupation of the anti-bonding 2eg orbital. We hypothesize that this orbital increases in energy across the series.

Effects of charged peptides on electron transfer from [Fe(CN)6]4– to cytochrome c or plastocyanin

Abstract Interactions of charged peptides, such as aspartic acid peptides (Aspptds) and lysine peptides (Lysptds), with cytochrome c (cyt c) or plastocyanin (PC) have been studied by measuring electron transfer between [Fe(CN)6]4– and cyt c or PC in the presence of these peptides. Aspptds, up to penta-aspartic acid, served as competitive inhibitors of electron transfer from [Fe(CN)6]4– to oxidized cyt c, while Lysptds, up to penta-lysine, promoted electron transfer from [Fe(CN)6]4– to oxidized PC. The electron transfer inhibitory effects of Aspptds are explained as competitive inhibition due to neutralization of the positively charged amino acid residues at the surface of cyt c by electrostatic interactions, whereas the electron transfer promoting effects of Lysptds may be due to formation of PC·Lysptd or Lysptd·[Fe(CN)6]4– complexes subsequently forming an electron transferring complex, PC·Lysptd·[Fe(CN)6]4–, without repulsion of the negative charges. The inhibitory effect of Aspptds and promotional effect of Lysptds became significant as the net charge or concentration of the peptides increased. The promotional effects of Lysptds decreased as the net charge of the PC negative patch was decreased by mutagenesis. Thus, charged peptides may serve as a probe for investigation of the molecular recognition character of proteins.

Kinetic studies on the oxidation of cytochrome b 5 Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes

Abstract. To illustrate the functions of the aromatic residue Phe35 of cytochrome b5 and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b5, we studied electron transfer reactions of cytochrome b5 and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)]–, [Fe(CDTA)]– and [Ru(NH3)6]3+. The changes at Phe35 of cytochrome b5 and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.