Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of GK0767, the copper-containing nitrite reductase from Geobacillus kaustophilus.

The soluble region (residues 32-354) of GK0767, a copper-containing nitrite reductase from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426, has been cloned and overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 1.

The first example of photochemical reduction of nitrite into nitrogen monoxide by a dinuclear Ru(II)-Cu(II) complex and photoinduced intramolecular electron transfer reaction between Ru(II) and Cu(II) moieties.

The photochemical reduction of nitrite to NO by the dinuclear Ru(II)-Cu(II) complex ([Ru(bpy)(2)(Mebpy-COOC(3)H(6))Me(2)bpaCu(H(2)O)(ClO(4))](ClO(4)).(PF(6))(2).(H(2)O) was observed in the absence of sacrificial electron donor reagents in CH(2)Cl(2).

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 (N(2)O) 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 N(2)O by catalysing the one-electron reduction of nitrite (NO2-) to nitric oxide (NO).

Electroreduction of nitrite to nitrogen oxide by a copper-containing nitrite reductase model complex incorporated into collagen film.

The electrocatalytic reduction of nitrite to NO by CuMe(2)bpaCl(2), which is a model for the active site of copper-containing nitrite reductase, incorporated into collagen film was investigated. The 77-K EPR spectrum of CuMe(2)bpaCl(2) in the collagen matrix revealed the typical axial signals (g(//)=2.26, g( perpendicular)=2.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnes.

The soluble domain (residues 483-913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.

Crystallization and preliminary X-ray diffraction analysis of a complex between the electron-transfer partners hexameric Cu-containing nitrite reductase and pseudoazurin.

The complex between Cu-containing nitrite reductase (HdNIR) and its electron-donor protein pseudoazurin (HdPAz) from Hyphomicrobium denitrificans has been crystallized. The crystals were obtained from a mixture of the two proteins using the hanging-drop vapour-diffusion method in the presence of polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) as precipitants. SDS-PAGE analysis demonstrated that the crystals contained both proteins.

Atomic resolution structure of pseudoazurin from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans: structural insights into its spectroscopic properties.

The crystal structure of native pseudoazurin (HdPAz) from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans has been determined at a resolution of 1.18 A. After refinement with SHELX employing anisotropic displacement parameters and riding H atoms, R(work) and R(free) were 0.

Identification of a blue copper protein from Hyphomicrobium denitrificans and its functions in the periplasm.

It has been known that the methylotrophic denitrifying bacteria have the specific electron transfer chains, involving in 'methanol oxidation' and 'denitrification', in the periplasm. Recently, a unique blue copper protein (HdBCP) has been isolated from the methanol-grown methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans. HdBCP is a 14.

Structure and function of a hexameric copper-containing nitrite reductase.

Dissimilatory nitrite reductase (NIR) is a key enzyme in denitrification, catalyzing the first step that leads to gaseous products (NO, N(2)O, and N(2)). We have determined the crystal structure of a Cu-containing NIR from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans, at 2.2-A resolution.

Significant enhancement of monooxygenase activity of oxygen carrier protein hemocyanin by urea.

Oxygenation of a series of p-substituted phenols to the corresponding catechols (phenolase activity) by the (mu-eta2:eta2-peroxo)dicopper(II) species of Octopus hemocyanin has been directly examined for the first time by using a UV-vis spectroscopic method in a 0.5 M borate buffer solution containing 8 M urea under anaerobic conditions. Preliminary kinetic studies have indicated that the reaction involves an electrophilic aromatic substitution mechanism as in the case of phenolase reaction of tyrosinase.

Crystal structures of cytochrome c(L) and methanol dehydrogenase from Hyphomicrobium denitrificans: structural and mechanistic insights into interactions between the two proteins.

Methanol dehydrogenase (Hd-MDH) and its physiological electron acceptor, cytochrome c(L) (Hd-Cyt c(L)), isolated from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151, have been kinetically and structurally characterized; the X-ray structures of Hd-MDH and Hd-Cyt c(L) have been determined using molecular replacement at 2.5 and 2.0 A resolution, respectively.

Electroreduction of nitrite on gold electrode modified with Cu-containing nitrite reductase model complex.

A gold electrode modified with copper complexes containing a tridentate aromatic amine compound (bis(6-methyl-2-pyridylmethyl)amine ethyl sulfide, which is a model for the nitrite reduction centre of copper-containing nitrite reductase, catalyzed electrochemically the reduction of nitrite to nitrogen monoxide under acidic conditions.

Roles of Trp144 and Tyr203 in copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013.

The roles of the Trp144 and Tyr203 residues near the type 1 Cu site of Achromobacter cycloclastes nitrite reductase (AcNIR) have been examined with mutants of AcNIR. Tyr203 is located on the protein surface near the type 1 Cu site of AcNIR, and Trp144 is between the Tyr203 and the type 1 Cu center in AcNIR. Single mutation of Trp144 or Tyr203 in AcNIR to Leu resulted in decreased rate constants of intermolecular electron transfer from its cognate pseudoazurin (AcPAZ) (k(ET)=1.

Structural reorganization of the copper binding site involving Thr15 of mavicyanin from Cucurbita pepo medullosa (zucchini) upon reduction.

Mavicyanin, a glycosylated protein isolated from Cucurbita pepo medullosa (zucchini), is a member of the phytocyanin subfamily containing one polypeptide chain of 109 amino residues and an unusual type-I Cu site in which the copper ligands are His45, Cys86, His91, and Gln96. The crystal structures of oxidized and reduced mavicyanin were determined at 1.6 and 1.

Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151.

Dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151 (HdNIR) is a novel copper-containing nitrite reductase (CuNIR) composed of six identical subunits. One plastocyanin-like domain and one green CuNIR-like domain are connected to each other, suggesting that the HdNIR subunit structure resembles a complex of green CuNIR and pseudoazurin (or azurin). Recombinant HdNIR protein was crystallized using the hanging-drop vapour-diffusion method with PEG 4000 as the precipitant at pH 8.

Characterization of two type 1 Cu sites of Hyphomicrobium denitrificans nitrite reductase: a new class of copper-containing nitrite reductases.

We report (1) the amino acid sequence of Hyphomicrobium denitrificans nitrite reductase (HdNIR), containing two type 1 Cu sites and one type 2 Cu site; (2) the expression and preparation of wild-type HdNIR and two mutants replacing the Cys ligand of each type 1 Cu with Ala; and (3) their spectroscopic and functional characterization. The open-reading frame of 50-kDa HdNIR is composed of the 15-kDa N-terminal domain having a type 1 Cu-binding motif like cupredoxins and the 35-kDa C-terminal domain having type 1 Cu-binding and type 2 Cu-binding motifs such as common nitrite reductases (NIRs). Moreover, the amino acid sequences of the N- and C-terminal domains are homologous to those of plastocyanins and NIRs, respectively.

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 (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.

Characterization of nitrous oxide reductase from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151.

A Cu-containing nitrous oxide reductase (HdN2OR) from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151, has been aerobically prepared and spectroscopically characterized. Purple and blue forms of HdN2OR have been isolated. Each form is a homodimer comprising monomers with a molecular mass of 65 kDa.

The significance of the flexible loop in the azurin (Az-iso2) from the obligate methylotroph Methylomonas sp. strain J.

The obligate methylotroph Methylomonas sp. strain J produces two azurins (Az-iso1 and Az-iso2) as candidates for electron acceptor from methylamine dehydrogenase (MADH) in the electron-transfer process involving the oxidation of methylamine to formaldehyde and ammonia. The X-ray crystallographic study indicated that Az-iso2 gives two types of crystals (form I and form II) with polyethylene glycol (PEG4000) and ammonium sulfate as the precipitants, respectively.

Crystallization and preliminary X-ray crystallographic studies of mavicyanin from Cucurbita pepo medullosa.

Mavicyanin isolated from Cucurbita pepo medullosa is a glycosylated protein containing a single polypeptide chain of 109 amino-acid residues and is a member of the phytocyanin subclass of cupredoxins. Non-glycosylated recombinant mavicyanin, which was expressed in Escherichia coli, was crystallized by the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant at pH 5.5.

Characterization and function of Met150Gln mutant of copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013.

The mutant (M150Q-NIR) replacing the Met150 ligand of the type 1 Cu center in Achromobacter cycloclastes nitrite reductase (AcNIR) with Gln has been physicochemically and functionally characterized. The electronic absorption and CD spectra of M150Q-NIR are similar to those of mavicyanin and stellacyanin having the 2His, Cys, and Gln ligands, but the EPR signal has an axial character, although their blue copper proteins show rhombic EPR signals. The mutant has about 80% catalytic activity of AcNIR.

Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes.

The role of the active site Cu(2+) of phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been studied by substitution with other divalent cations, where we were able to remove >99.5% of Cu(2+) from the active site. The enzymes reconstituted with Co(2+) and Ni(2+) (Co- and Ni-AGAO) exhibited 2.

Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase.

The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin. The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site. The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR.

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).