Sohsuke Shidara

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.

Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

Abstract Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying bacteria (Achromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015, and Alcaligenes xylosoxidans GIFU 1051) have been characterized by electronic absorption, circular dichroism, and electron paramagnetic resonance spectra. The characteristic visible absorption spectra of these NIRs are due to the type 1 Cu centers, while the type 2 Cu centers hardly contribute in the same region. The intramolecular electron transfer (ET) process from the type 1 Cu to the type 2 Cu in native NIRs has been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in T2D NIRs exhibits the same reoxidation. The ET process obeys first-order kinetics, and observed rate constants are 1400–1900 s–1 (t1/2 = ca. 0.5 ms) at pH 7.0. In the presence of nitrite, the ET process also obeys first-order kinetics, with rate constants decreased by factors of 1/12–1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V, close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite also would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased. Pulse radiolysis experiments on T2D NIRs in the presence of nitrite demonstrate that the type 1 Cu(I) site is slowly oxidized with a first-order rate constant of 0.03 s–1 at pH 7.0, suggesting that nitrite bound to the protein accepts an electron from the type 1 Cu. This result is in accord with the finding that T2D NIRs show enzymatic activities, although they are lower than those of the native enzymes.

Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase

The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.

Five coordinated nitrosylhemoprotein in the whole cells of denitrifying bacterium, Achromobacter xylosoxidans NCIB 11015

The denitrifying bacterium, Achromobacter xylosoxidans NCIB 11015, was cultivated in meat extract-peptone medium and in Mn-free synthetic medium under denitrifying or non-denitrifying conditions. Electron paramagnetic resonance (EPR) spectra for the whole cells of the bacteria thus obtained were measured at 77K. The characteristic three-line signal was observed in the whole cells of the bacteria under denitrifying conditions, but not under non-denitrifying conditions. The three-line signal was more distinctly observed in the cells cultured in Mn-free medium. This signal could be assigned to nitrosylcytochrome c′ containing a five-coordinated nitrosylheme. The elemental composition in these cells is also discussed.

Effects of nutrients and arbuscular mycorrhizal colonization on the growth of Salix gracilistyla seedlings in a nutrient-poor fluvial bar

A field survey and a pot culture experiment were conducted to examine the effects of nutrients (N and P) and arbuscular mycorrhizal (AM) fungi on the growth of Salix gracilistyla, a pioneer plant in riparian habitats. The plants growing in the field were colonized by AM and/or ectomycorrhizal fungi. However, the direct effect of AM colonization on seedling growth was not detected in the pot culture experiment. In contrast, N application significantly promoted plant growth, suggesting that the growth of S. gracilistyla seedlings is largely limited by the availability of N in the field.

Novel spectroscopic aspects of type I copper in Hyphomicrobium nitrite reductase

Copper-containing nitrite reductase isolated from Hyphomicrobium sp. A 3151 has been characterized by electronic absorption, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopies. The visible absorption spectrum of Type I copper (blue copper) in the enzyme especially indicates novel features compared with those of Type I coppers in not only several nitrite reductases already reported but also small blue copper proteins. The EPR spectrum of Type I copper exhibits an axial symmetry.

Spectral properties of carbon monoxide or cyanide complexes of cytochromes c′ from denitrifying bacteria

Abstract The CO and CN − derivatives of cytochromes c ′ from three denitrifying bacteria ( Alcaligenes sp. NCIB 11015 and Achromobacter xylosoxidans GIFU 543 and 1051) have been investigated by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at room temperature. The spectral data have been characterized and compared with those of the corresponding derivatives of cytochromes c ′ from photosynthetic bacteria, cytochrome c , and hemoproteins containing b-type heme such as myoglobin, horseradish peroxidase, and indoleamine 2,3-dioxygenase. The absorption and MCD spectra of the CO-cytochromes c ′ complexes from the above denitrifying bacteria show low-spin ferrous chromophores with extra weak bands at around 435 nm, which are assigned to the unreacted ferrous species (high-spin). The yields of the unreacted species are 6–18%. Complete formation of the CO complex of Alcaligenes cytochrome c ′ takes place in the presence of n-propanol (50% n-propanol/50% 0.05 M phosphate buffer, pH 7.2). Since cytochromes c ′ from most photosynthetic bacteria give the 100% CO complexes, their CO affinities seem to be generally higher than those of cytochromes c ′ from denitrifying bacteria. The binding of cyanide to Alcaligenes ferric cytochrome c ′ is spectroscopically demonstrated as the second CN − complex (low-spin) reported after the CN − -cytochrome c ′ complex from Chromatium vinosum (a photosynthetic bacterium) reported already, although anionic ligands were earlier reported not to bind to the ferric cytochromes c −. The treatment of the CN − complex with Na 2 S 2 O 4 results in formation of the ferrous CN − complex (low-spin) with a small amount of the free ferrous species (high-spin).

Spectroscopic distinction between two Co(II) ions substituted for types 1 and 2 Cu in nitrite reductase

The Co(II) substitution of nitrite reductases (NIRs) containing types 1 and 2 Cu from two denitrifying bacteria (Achromobactercycloclasters IAM 1013 and Alcaligenes xylosoxidans GIFU 1051) has been carried out. The Co(II) derivatives have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopies. The tetrahedral Co(II) centers replaced type 1 Cu show the MCD bands at 300–500 nm (CysS−aCo(II) charge transfer transitions) and 600–700 nm (d-d transitions of Co(II), which are quite similar to those of Co(II)-substituted plastocyanin. The Co(II) ion will have a donor set of 2His, Cys and Met residues. The enzyme-substituted Co(II) ions for type 2 Cu exhibit the characteristic MCD bands due to d-d transitions in the region of 500–600 nm, which are quite similar to those of half Co(II)-substituted hemocyanin and suggest a tetrahedral Co(II) center bound to three histidyl imidazoles and one water molecule. The two types of Co(II) in NIRs are easily distinguishable by MCD spectroscopy. Moreover, the electronic absorption bands of Co(II) NIRs are assigned to types 1 and 2 Co on the basis of the MCD data.