Shinnichiro Suzuki

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.

Formation of amino acids from elemental carbon by contact glow discharge electrolysis

THE formation of amino acids in various inferred prebiotic conditions has been widely studied1,2. Recently we reported syntheses of amino acids from aliphatic carboxylic acids and ammonia3,4, and from aliphatic amines and formic acid4,5 by means of contact glow discharge electrolysis (CODE)6. We describe here the formation of amino acids and urea from elemental carbon in the presence of aqueous ammonia by CODE. By this reaction and the following process, the elemental carbon of the elctrode was converted into the bio-organic compounds.

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)=2.05, A(//)=16.4mT) of a tetragonal Cu(2+) chromophore. The redox potential, which is related to the Cu(+)/Cu(2+) couple, was -63mV (E=72mV) at pH 5.5. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of CuMe(2)bpaCl(2) in the collagen matrix. Upon reaching -300mV, a linear generation of NO was observed for the CuMe(2)bpaCl(2)/collagen film-coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed using the Michaelis-Menten equation, where V(max)=3.16nM s(-1) and K(m)=1.1mM at pH 5.5. The current increase and the reaction rate were dependent on the pH of the solution. The mechanism of nitrite reduction by the copper complex in the collagen matrix was the same mechanism as that of the enzyme in aqueous solution.

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 1.3u2005Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 115.1, c = 87.5u2005Å. Preliminary studies and molecular-replacement calculations reveal the presence of one subunit of the homotrimeric structure in the asymmetric unit; this corresponds to a V(M) value of 3.14u2005Å(3)u2005Da(-1).

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 0.135 and 0.169, respectively. Visualization of the anisotropic displacement parameters as thermal ellipsoids provided insight into the atomic motion within the perturbed type 1 Cu site. The asymmetric unit includes three HdPAz molecules which are tightly packed by head-to-head cupredoxin dimer formation. The shape of the Cu-atom ellipsoid implies significant vibrational motion diagonal to the equatorial xy plane defined by the three ligands (two His and one Cys). The geometric parameters of the type 1 Cu site in the HdPAz structure differ unambiguously from those of other pseudoazurins. It is demonstrated that their structural aspects are consistent with the unique visible absorption spectrum.

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. The X-ray diffraction experiment was carried out at SPring-8 and diffraction data were collected to 3.3 A resolution. The crystals were tetragonal (space group P4(1)2(1)2), with unit-cell parameters a = b = 130.39, c = 505.55 A. Preliminary analysis indicated that there was one HdNIR and at least two HdPAz molecules in the asymmetric unit of the crystal.

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 2.4 A. The crystal belonged to space group P2(1)3, with unit-cell parameters a = b = c = 108.63 A. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V(M) of 2.1 A(3) Da(-1).