Yohta Fukuda

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Significance Copper nitrite reductase (CuNiR) is involved in denitrification of the nitrogen cycle. Synchrotron X-rays rapidly reduce copper sites and decompose the substrate complex structure, which has made crystallographic studies of CuNiR difficult. Using femtosecond X-ray free electron lasers, we determined intact structures of CuNiR with and without nitrite. Based on the obtained structures, we proposed a redox-coupled proton switch model, which provides an explanation for proton-coupled electron transfer (PCET) in CuNiR. PCET is widely distributed through biogenic processes including respiratory and photosynthetic systems and is highly expected to be incorporated into bioinspired molecular devices. Our study also establishes the foundation for future studies on PCET in other systems. Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography

Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR.

Structural insights into the function of a thermostable copper-containing nitrite reductase

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 η(1)-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 η(1)-O coordination manner is proposed.

Expression from engineered Escherichia coli chromosome and crystallographic study of archaeal N,N′‐diacetylchitobiose deacetylase

In order to develop a structure‐based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on N,N′‐diacetylchitobiose deacetylases (Dacs) from Pyrococcus horikoshii (Ph‐Dac) and Pyrococcus furiosus (Pf‐Dac). Neither Ph‐Dac nor Pf‐Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were active and thermostable up to 85 °C. The crystal structures of Ph‐Dac and Pf‐Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph‐Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N‐acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the Nε atom of His264 (from the adjacent polypeptide in the Ph‐Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn‐dependent deacetylases.

Structural and mechanistic insights into the electron flow through protein for cytochrome c-tethering copper nitrite reductase

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.

Structural and functional characterization of the Geobacillus copper nitrite reductase: involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

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 c551 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 c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.

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 1.3 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 115.1, c = 87.5 Å. 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 3.14 Å(3) Da(-1).

Insights into unknown foreign ligand in copper nitrite reductase.

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.

Structural insights into a secretory abundant heat‐soluble protein from an anhydrobiotic tardigrade, Ramazzottius varieornatus

Upon stopping metabolic processes, some tardigrades can undergo anhydrobiosis. Secretory abundant heat‐soluble (SAHS) proteins have been reported as candidates for anhydrobiosis‐related proteins in tardigrades, which seem to protect extracellular components and/or secretory organelles. We determined structures of a SAHS protein from Ramazzottius varieornatus (RvSAHS1), which is one of the toughest tardigrades. RvSAHS1 shows a β‐barrel structure similar to fatty acid‐binding proteins (FABPs), in which hydrophilic residues form peculiar hydrogen bond networks, which would provide RvSAHS1 with better tolerance against dehydration. We identified two putative ligand‐binding sites: one that superimposes on those of some FABPs and the other, unique to and conserved in SAHS proteins. These results indicate that SAHS proteins constitute a new FABP family.

The structure of hyperthermophilic β-N-acetylglucosaminidase reveals a novel dimer architecture associated with the active site

The β‐N‐acetylglucosaminidase from the hyperthermophilic bacteria Thermotoga maritima (NagA) hydrolyzes chitooligomers into monomer β‐N‐acetylglucosamine. Although NagA contains a highly conserved sequence motif found in glycoside hydrolase (GH) family 3, it can be distinguished from other GH family 3 β‐N‐acetylglucosaminidases by its substrate specificity and biological assembly. To investigate its unique structure around the active site, we determined the crystal structure of NagA at a resolution of 2.43 Å. The NagA forms a dimer structure in which the monomer structure consists of an N‐ and a C‐terminal domain. The dimer structure exhibits high solvation free energy for dimer formation. From mutagenesis analyses, the catalytic nucleophile and general acid–base residues were supposed to be Asp245 and His173, respectively. The most striking characteristic of NagA was that it forms the active site cleft from the N‐terminal domain and the C‐terminal domain of the next polypeptide chain, whereas the other two‐domain GH family 3 enzymes form the site within the same molecule. Another striking feature is that the loops located around the active site show high flexibility. One of the flexible loops contains the general acid–base His173 and was thought to be involved in substrate distortion during catalysis. In addition, a loop in close contact with the active site, which comes from the C‐terminal domain of the next polypeptide chain, contains a region of high B‐factor values, indicating the possibility that the C‐terminal domain is involved in catalysis. These results suggest that the dimer structure of NagA is important for its activity and thermostability.