Nobuhumi Nakamura

Glyoxal Oxidase from Phanerochaete chrysosporium Is a New Radical-Copper Oxidase

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (gav = 2.0055) characteristic of phenoxyl π-radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E1/2 = 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.

Characterization of the Native Lysine Tyrosylquinone Cofactor in Lysyl Oxidase by Raman Spectroscopy

Lysine tyrosylquinone (LTQ) recently has been identified as the active site cofactor in lysyl oxidase by isolation and characterization of a derivatized active site peptide. Reported in this study is the first characterization of the underivatized cofactor in native lysyl oxidase by resonance Raman (RR) spectrometry. The spectrum is characterized by a unique set of vibrational modes in the 1200 to 1700 cm−1 region. We show that the RR spectrum of lysyl oxidase closely matches that of a synthetic LTQ model compound, 4-n-butylamino-5-ethyl-1,2-benzoquinone, in aqueous solutions but differs significantly from those of other topa quinone-containing amine oxidases under similar conditions. Furthermore, we have observed the same 18O shift of the C=O stretch in both the lysyl oxidase enzyme and the LTQ cofactor model compound. The RR spectra of different model compounds and their D shifts give additional evidence for the protonation state of LTQ cofactor in the enzyme. The overall similarity of these spectra and their shifts shows that the lysyl oxidase cofactor and the model LTQ compound have the same structure and properties. These data provide strong and independent support for the new cofactor structure, unambiguously ruling out the possibility that the structure originally reported had been derived from a spurious side reaction during the derivatization of the protein and isolation of the active site peptide.

Characterization of Carbohydrate-Binding Cytochrome b562 from the White-Rot Fungus Phanerochaete chrysosporium

ABSTRACT cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b562 (CBCyt. b562). The resonance Raman spectrum suggested that the heme of CBCyt. b562 is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b562 may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b562 was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.

Catalytic and Structural Properties of Surfactant‐Horseradish Peroxidase Complex in Organic Media

A surfactant‐horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water‐in‐oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant‐HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV‐visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.

Heme–Cu complexes as oxygen-activating functional models for the active site of cytochrome c oxidase

Tri(2-pyridylmethyl)amineCu complex-linked iron meso-tetraphenylporphyine derivatives were prepared to model the active site of cytochrome c oxidase. Exposure to oxygen converted the reduced forms of the complexes to the corresponding stable mu-peroxo species in spite of the presence of three coordination sites, two on the heme and one on the Cu. The oxy forms were characterized spectroscopically. Kinetic analyses of the oxygenation reactions of the reduced forms suggests that preferential O2 binding occurs at the Cu site over the heme. This mechanism is also supported by examination of the redox potentials of the two metal ions. Since the peroxy complexes of the models exhibit a structure similar to that of the previously reported fully-oxidized form, the relevance of the model chemistry to the enzyme reaction is discussed.

STRUCTURES AND PROPERTIES OF TERNARY COPPER(II) COMPLEXES WITH 4,5-DIHYDROXYPHENYLALANINE OR 2,4,5-TRIHYDROXYPHENYLALANINE AND AROMATIC DIAMINES

Abstract Structural and spectroscopic studies were carried out on ternary Cu(II) complexes, Cu(DA)(AA), with tyrosine derivatives having hydroxyl substituents (AA = 4,5-dihydroxyphenylalanine (dopa) or 2,4,5-trihydroxyphenylalanine (topa)) and aromatic diamines (DA = 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen)). The charge transfer (CT) absorption bands observed as difference absorption spectra in the near-UV region indicate an effective stacking interaction between DA and the side-chain aromatic ring of AA in the ternary Cu(II) complexes. Three complexes, [Cu( dl -topa)(bpy)(H 2 O)] + (complex 1 ), [Cu( l -dopa)(phen)] + [Cu( l -dopa)(CH 3 CH 2 CH 2 OH)(phen)] + (complex 2 ), and [Cu( dl -dopa)(bpy)] + (complex 3 ), were isolated as single crystals, and their structures were determined by X-ray analysis. The central Cu(II) ions in complexes 1 and 3 have square-pyramidal and square-planar geometries respectively. Complex 2 contains two independent molecules having each of the two geometries. All the molecular structures reveal intramolecular aromatic ring stacking between DA and AA in correspondence with the solution spectral observations. The stacking with the topa ring having three hydroxyl groups was found to be slightly stronger than that with the dopa ring having two hydroxyl groups, which is consistent with the more intense CT absorption bands of the topa complex compared with the dopa complex in aqueous solution. Moreover, the oxidative deamination of benzylamine with the ternary Cu(II) complex containing dopa or topa was investigated under aerobic conditions.

Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2.

Methylomonas sp. strain J gives rise to two azurins (Az-iso1 and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616u2009nm in the visible absorption spectra respectively, being revealed at 620−630u2009nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E1/2) of the two azurins and the intermolecular electron-transfer rate constants (kET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E1/2 values of Az-iso1 and Az-iso2 are +321 and +278u2009mV vs NHE at pHu20097.0, respectively. The kET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6u2009Å X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH.

Electron transfer reaction of poly(ethylene oxide)-modified azurin in poly(ethylene oxide) oligomers

Abstract The native azurin (Az) was chemically modified with activated poly(ethylene oxide) (PEO) to solubilize in the PEO oligomers. PEO 2000 (average molecular weight of 2000)-modified Az (PEO 2000 -Az) was dissolved without denaturation in PEO 200 (average molecular weight of 200). The electronic absorption spectrometry exhibited no detectable conformational change of PEO 2000 -Az in phosphate buffer. The electron paramagnetic resonance (EPR) spectrum of PEO 2000 -Az is also quite similar to that of native Az in water. This result confirms that the chemical modification with PEO 2000 does not cause any structural change around the copper site. Moreover, since no changes in the EPR spectrum are observed in PEO 200 , the copper coordination geometry must be maintained even in PEO 200 . The redox activity of PEO 2000 -Az in PEO 200 (containing 0.5 mol dm −3 NaClO 4 ) was investigated by cyclic voltammetry. When we use the PEO 350 -SH (α-methoxy-ω-mercapto-poly(ethylene oxide); PEO with average molecular weight of 350) modified gold electrode as a working electrode, the redox response was observed in PEO 200 . The result of the measurements for the scan rate dependence suggests that PEO 2000 -Az could be immobilized on the PEO 350 -SH modified electrode.

Thermal stability and electron transfer reaction of modified myoglobin immobilized on a carbon electrode in poly(ethylene oxide) oligomers

Abstract Poly(ethylene oxide) (PEO)-modified myoglobin (PEO–Mb) was immobilized on a carbon electrode with the aid of polyelectrolytes, and the redox response was successively analyzed in salt-containing PEO oligomers. Complexation of PEO–Mb with poly- l -lysine was found to be quite effective in maintaining a constant peak current in cyclic voltammograms for more than 20 h, but the peak current gradually decreased at 50°C. Addition of poly- l -glutamic acid or their polyion complex was less effective to fix the PEO–Mb on the electrode. PEO–Mb was fixed through electrostatic interaction between PEO–Mb and poly- l -lysine. In order to achieve more stable redox activity of Mb in PEO oligomers, covalent immobilization of Mb to electrode surface was attempted. In this work, the maleic anhydride unit in the PEO–maleic anhydride copolymer was initially used to fix the Mb, followed by an ester formation between newly generated carboxylic groups and hydroxyl groups on the electrode surface. A stable redox response was obtained, and this effective immobilization of Mb on the electrode has yielded thermally stable redox response for 30 h even at 80°C without denaturation nor elution of Mb into PEO oligomers.

Elucidation of the factors affecting the oxidative activity of Acremonium sp. HI-25 ascorbate oxidase by an electrochemical approach.

Steady-state kinetics of Acremonium sp. HI-25 ascorbate oxidase toward p-hydroquinone derivatives have been examined by using an electrochemical analysis based on the theory of steady-state bioelectrocatalysis. The electrochemical technique has enabled one to examine the influence of electronic and chemical properties of substrates on the activity. It was proven that the oxidative activity of ascorbate oxidase was dominated by the highly selective substrate-binding affinity based on electrostatic interaction beyond the one-electron redox potential difference between ascorbate oxidases type 1 copper site and substrate.