Nobuya Nishio

Spectroscopic and Electrochemical Studies on Active-site Transitions of the Type 1 Copper Protein Pseudoazurin from Achromobacter cycloclastes

The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode. Measurements carried out at 25.0°C indicate a midpoint reduction potential of E = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of ΔE = 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 × 10 cm s and 3.7 × 10 cm s, respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 × 10 cm s, consistent with a slower process at the positively charged electrode surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure. The trigonal form has elongated axial bonding and an axial EPR spectrum. At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm are shifted to higher energy (10 cm), consistent with a stronger Cu-S bond.

Structure-Based de novo design of non-nucleoside adenosine deaminase inhibitors

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.

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 616 nm in the visible absorption spectra respectively, being revealed at 620−630 nm 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 +278 mV vs NHE at pH 7.0, 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 1.6 Å 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.

Crystallization and preliminary analysis of bovine adenosine deaminase

Adenosine deaminase (ADA) from bovine intestine was crystallized with purine riboside by vapour diffusion using ammonium sulfate as precipitant. The crystals are tetragonal and have unit-cell parameters a = b = 80.03, c = 141.68 A. They belong to space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to at least 2.0 A resolution. The structure is being solved by molecular replacement.

Crystallization and preliminary X-ray study of iso-2 azurin from the methylotrophic bacterium, Methylomonas J

The obligate methylotroph Methylomonas J possesses two distinct azurins. The iso-2 azurin, which functions as an electron acceptor for methylamine dehydrogenase, has been crystallized using two kinds of precipitants: PEG 4000 and ammonium sulfate. The crystals precipitated with PEG belong to the monoclinic system, space group P21, with unit-cell parameters a = 32.96, b = 33.67, c = 47.34 A and beta = 101.35 degrees. The crystals precipitated with ammonium sulfate belong to the orthorhombic system, space group C2221, with unit-cell parameters a = 31.52, b = 62.49 and c = 135.41 A. The crystals diffract to 1.6 and 1.9 A resolution, respectively, and were suitable for X-ray crystallographic studies. A Patterson search is being conducted using the recently reported structure of Alcaligenes xylosoxidans NCIMB 11015 as a starting model.