Search

Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis ( AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz. [ABSTRACT FROM AUTHOR]

Bifunctional copper nitrite reductase (CuNIR) catalyzes nitrite reduction to nitric oxide and dioxygen reduction to hydrogen peroxide. In contrast to the well-researched nitrite reduction mechanism, the oxygen reduction mechanism in CuNIR has been totally unknown, because mononuclear copper–oxygen complexes decompose so readily that their visualization has been challenging. Here, we provide spectroscopic evidence that a foreign ligand binds to the catalytic copper (T2Cu) site of CuNIR, and determine CuNIR structures displaying a diatomic molecule on T2Cu. This unknown ligand can be interpreted as dioxygen and may provide insights into the oxygen reduction mechanism of CuNIR. [ABSTRACT FROM AUTHOR]

The 1.55 Å resolution thermostable copper nitrite reductase structure at 320 K displayed a near-bidentate binding mode of nitrite distinct from a monodentate mode in a cryogenic structure. To our knowledge, this is the first case in which the difference in substrate binding modes between cryogenic and high-temperature structures has been visualized by crystallography. [ABSTRACT FROM AUTHOR]

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3Å and 1.8Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c 551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c 551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition. [ABSTRACT FROM AUTHOR]

Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO−2) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate-bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique η1-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH–O hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen-bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. On the basis of these observations, the reaction mechanism of GtNIR through the η1-O coordination manner is proposed. [ABSTRACT FROM AUTHOR]

Copper-containing nitrite reductases (CuNiRs), which catalyse the reversible one-electron reduction of nitrite to nitric oxide, are members of a large family of multi-copper enzymes that require an interprotein electron transfer (ET) reaction with redox partner proteins. Here, we show that the naturally fused type of CuNiR tethering a cytochrome c (Cyt c) at the C-terminus folds as a unique trimeric domain-swapped structure and has a self-sufficient electron flow system. The C-terminal Cyt c domain is located at the surface of the type 1 copper (T1Cu) site in the N-terminal CuNiR domain from the adjacent subunit, the heme-to-Cu distance (10.6 Å) of which is comparable to the transient ET complex of normal CuNiR with Cyt c. The structural aspects for the domain–domain interface and the ET kinetics indicate that the Cyt c–CuNiR domain interaction should be highly transient. The further electrochemical analysis of the interprotein ET reaction with a cognate redox partner protein suggested that an electron is directly transferred from the partner to the T1Cu. Structural and mechanistic comparisons of Cyt c–CuNiR with another cupredoxin-tethering CuNiR highlight the behaviours of extra domains on the fusion types of CuNiRs required for ET through proteins. [ABSTRACT FROM AUTHOR]