Kazuya Yamaguchi

Structure–function relationships of copper-containing nitrite reductases

Abstract Dissimilatory nitrite reductase (NIR) is a key enzyme in the anaerobic respiratory pathway of denitrifying bacteria. There are two types of NIR, one of which contains copper and the other heme. Cu-containing NIR (Cu NIR) has the trimeric structure with one type 1 Cu (blue copper) atom and one type 2 Cu (nonblue copper) atom in each subunit. The type 1 Cu atom bound by 2His, Cys, and Met accepts one electron from an electron donor protein and shows an intense color, blue or green. The type 2 Cu atom bound by 3His and a solvent (H 2 O or OH − ) is a reduction center of nitrite to NO. The intramolecular long-range electron transfer process is observed from the type 1 site to the type 2 Cu site with a half-life period of ca. 0.3 ms. The present review deals with (i) spectroscopic characterization of Cu NIR’s, (ii) structures of Cu NIR’s, and (iii) functions of Cu NIR’s (intermolecular electron transfer process, intramolecular electron transfer process, and reduction of nitrite ion).

Structural basis of inter-protein electron transfer for nitrite reduction in denitrification

Recent earth science studies have pointed out that massive acceleration of the global nitrogen cycle by anthropogenic addition of bio-available nitrogen has led to a host of environmental problems. Nitrous oxide (N2O) is a greenhouse gas that is an intermediate during the biological process known as denitrification. Copper-containing nitrite reductase (CuNIR) is a key enzyme in the process; it produces a precursor for N2O by catalysing the one-electron reduction of nitrite () to nitric oxide (NO). The reduction step is performed by an efficient electron-transfer reaction with a redox-partner protein. However, details of the mechanism during the electron-transfer reaction are still unknown. Here we show the high-resolution crystal structure of the electron-transfer complex for CuNIR with its cognate cytochrome c as the electron donor. The hydrophobic electron-transfer path is formed at the docking interface by desolvation owing to close contact between the two proteins. Structural analysis of the interface highlights an essential role for the loop region with a hydrophobic patch for protein–protein recognition; it also shows how interface construction allows the variation in atomic components to achieve diverse biological electron transfers.

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

Abstractu2002Native 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 (t1/2u2009=u2009ca. 0.5u2009ms) at pHu20097.0. 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 - +0.28u2009V, 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 pHu20097.0, 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.

Catalyst design of hydrotalcite compounds for efficient oxidations

Various Mg-Al type hydrotalcites were examined as catalysts for the epoxidation of olefins and N-oxidation of pyridines using hydrogen peroxide. The catalytic activity of hydrotalcites increased with increasing the basicity of their surface. Adding cationic surfactants, e.g., n-dodecyltrimethylammonium bromide, to the above system remarkably accelerated the reaction rate. The hydrotalcites, into which were introduced both Ru and Co cations in the Brucite layers, were found to be good catalysts for the oxidation of various alcohols in the presence of molecular oxygen. Moreover, these hydrotalcites could smoothly catalyze also the oxygenation of diphenylmethane, fluorene, and xanthene at benzylic position with excellent yields. The hydrotalcite catalysts could be easily separated from the reaction mixture and reused with retention of their high catalytic performance for the above oxidations.

Catalysis of a hydroxyapatite-bound Ru complex:efficient heterogeneous oxidation of primary amines to nitriles in thepresence of molecular oxygen

A hydroxyapatite-bound Ru complex could efficiently catalyze nthe aerobic oxidation of various primary amines to nitriles which nwere further hydrated to amides in the presence of water.

Electron paramagnetic resonance studies of ferric cytochrome c′ from photosynthetic bacteria

Electronic ground nature of ferric cytochromes c isolated from five photosynthetic bacteria. Chromatium vinosum ATCC 17899, Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum molischianum ATCC 14031, and Rhodospirillum rubrum ATCC 11170 has been investigated by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra indicate that the electronic ground state of five ferric cytochromes c is a quantum mechanical admixed-spin state of a high spin (S = 5/2) and an intermediate spin (S = 3/2) at pH 7.2 and is high-spin state at pH 11.0. At physiological pH, however, the content of an intermediate spin state differs with the bacterial source of the protein: approximately 50%, Chromatium vinosum; approximately 40%, Rhodobacter capsulatus and Rhodopseudomonas palustris; approximately 10%, Rhodospirillum molischianum and Rhodospirillum rubrum. Computer simulation of the spectra supports this diversity of the contribution of an intermediate spin state. Model studies of the ferric porphyrin complexes suggest that the correlation between content of an intermediate spin state and heme iron displacement from the mean heme plane. Therefore, the variation of the content of an intermediate spin state observed in the present study reflects the subtle difference in the degree of heme iron displacement among the proteins.

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.

Spectroscopic characterization of nitrosylheme in nitric oxide complexes of ferric and ferrous cytochrome c′ from photosynthetic bacteria

Reactions of ferric and ferrous cytochromes c from four photosynthetic bacteria (Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum rubrum ATCC 11170, and Chromatium vinosum ATCC 17899) with nitric oxide have been investigated by electronic absorption and electron paramagnetic resonance spectroscopies. The heme iron(III) of these ferric cytochromes c has been recently reported to be in a quantum mechanically admixed (S = 5/2, 3/2) state [Fujii, S., Yoshimura, T., Kamada, H., Yamaguchi, K., Suzuki, S., Shidara, S. and Takakuwa, S. (1995) Biochim. Biophys. Acta 1251, 161-169]. The affinity of ferric cytochromes c for NO among these bacterial species (C. vinosum > Rps. palustris approximately Rb. capsulatus >> R. rubrum) was apparently related to the S = 3/2 content in the or der. In the reaction of ferrous cytochrome c with NO, six- and five-coordinated nitrosylhemes, which represent species with and without a ligand at the axial position trans to nitrosyl group, have been formed. The content of six-coordinated nitrosylheme in NO-ferrous cytochrome c has been determined to be Rb. capsulatus approximately Rps. palustris > C. vinosum < R rubrum, suggesting that a stability of iron-to-histidine bond decreases with this order. The NO reactions of ferric and ferrous cytochromes c from photosynthetic bacteria have been compared with those of cytochromes c from denitrifying bacteria.

Structure-based Engineering of Alcaligenes xylosoxidans Copper-containing Nitrite Reductase Enhances Intermolecular Electron Transfer Reaction with Pseudoazurin

The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (kET) are considerably lower between AcPAZ and AxNIR (Km = 1.34 mm and kET = 0.87 × 105 m-1 s-1) than between AcPAZ and its cognate nitrite reductase (AcNIR) (Km = 20 μm and kET = 7.3 × 105 m-1 s-1). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The kET values of these mutants become progressively larger as the number of mutated residues increases. The Km and kET values of A83D/A191E/G198E (Km = 88 μm and kET = 4.1 × 105 m-1 s-1) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.

Hydrolysis of phosphodiester with hydroxo- or carboxylate-bridged dinuclear Ni(II) and Cu(II) complexes

A hydroxo- or carboxylate-bridged dinuclear Ni(II) ncomplexes with nN,N,N′,N′-tetrakis{(6-methyl-2-pyridyl)methyl n}-1,3-diaminopropan-2-ol has been synthesized as models for nNi(II)-substituted phosphotriesterase, which are more active ncatalysts for hydrolysis of phosphodiester than the corresponding dinuclear nCu(II) and Zn(II) complexes.