Hiroaki Shinohara

ELECTROCHEMICAL CHARACTERIZATION OF LIGNIN PEROXIDASE FROM THE WHITE-ROT BASIDIOMYCETE PHANEROCHAETE CHRYSOSPORIUM

Abstract Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126xa0mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pKa values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pKa value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.

Addition of redox function to streptavidin by chemical conversion of the tyrosine residues to 3-aminotyrosine residues

Tyrosine residues of streptavidin were chemically converted to 3-aminotyrosine residues by nitration followed by reduction. Although the biotin binding activity of the modified streptavidin reduced by about a half, no conformational change was observed in CD spectroscopy. Furthermore, electron transfer between the modified streptavidin with 3-aminotyrosine residues and the biotin-modified gold electrode was detected by a voltammetry technique. This chemical conversion method will be a general and promising method for providing an electron transfer function to various proteins.

Electrochemical Oxidation of Biological Amines on the Electrodes Modified with Hydrophobic Monolayers

The electrodes modified with hydrophobic monolayers were applied to detect electrochemically an indicator hormone for biological rhythm, melatonin, without separation preocesses. It was demonstrated that octanol-modified glassy carbon electrode could be available to oxidize melatonin selectively to a certain degree against the interfering substances and that the modified layer was stable under the high positive potential application for the detection of melatonin.

Polypeptides Endowed with Artificial Redox Functions

Polypeptides carrying 2-anthraquinonyl groups or ferrocenyl groups were synthesized. Poly(l-2-anthraquinonylalanine) was found to form left-handed a-helical main chain and the anthraquinonyl side groups were arranged helically along the main chain. The anthraquinonyl polypeptide showed a unique redox property that was attributed to fast electron migrations along the anthraquinonyl side groups. Poly(l-ferrocenylalanine) was also synthesized. The latter polypeptide was found to take randomly-coiled conformations.

Nanospace interface for the electrochemical communication between redox-active biomolecules and metal oxide electrodes

Oriented assembly and interfacial electron transfer of flavin coenzymes on titanium dioxide (TiO2) electrodes have been studied to develop the smart enzyme sensors or reactors. It was demonstrated that FMN and FAD were chemically adsorbed via phosphate moiety on TiO2 surface in weak acidic solutions to make monolayers. Quasi reversible slow electron transfer was observed on the FMN or FAD-adsorbed TiO2 electrodes. It was further demonstrated that the FMN-assembled TiO2 electrode electrochemically catalyzed the oxidation of NADH. The FMN-assembled TiO2 was then combined with some dehydrogenases and NADH to perform amperometric sensing for enzyme substrates. The results suggest that the assembled flavin coenzyme might be promising for a nanospace interface to achieve electrochemical communication between redox active biomolecules and the metal oxide electrodes.

Design of conductive nanospace interfaces for the smooth electrochemical communication between redox enzyme proteins and electrodes

Nanoscale layers of conducting polymers were prepared around redox enzyme molecules on electrode surfaces and were applied to electric interfaces to perform the smooth electron transfer between the redox proteins and electrodes. The interface design might be useful to bind the functions of both biocatalysts and electronic materials and to develop intelligent devices.