Complex formation of silver(I) ions with a glucosinolate derivative: structural and mechanistic insights into myrosinase-mimicking C-S bond cleavage.

The crystal structure of an unprecedented silver complex of O-acetylsinigrin has shown chelating bonds of the sulfated thiohydroximate and an η2-bond of the ethylene moiety with Ag(i). Mechanistic studies on the formation and decomposition of the complex by the 1H NMR measurements and theoretical calculations with the DFT method indicated relevance to the glucosinolate degradation in biological systems.

Identification of productive and futile encounters in an electron transfer protein complex.

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.

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 ß-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one ß-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.

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 Šresolution and the nitrite-bound form of the C135A mutant at 1.90 Šresolution. 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.

The structure of the cytochrome p450cam-putidaredoxin complex determined by paramagnetic NMR spectroscopy and crystallography.

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.

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.

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).

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.

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). The reaction rate of the photoreduction of nitrite depended upon the concentration of the excited Ru(II) moiety in the complex. The photoinduced intramolecular electron transfer rate constants between the Ru(II) and Cu(II) moieties (*Ru(II)-Cu(II) --> Ru(III)-Cu(I) and Ru(III)-Cu(I) --> Ru(II)-Cu(II)) in the dinuclear complex were calculated to be 2.3 x 10(9) and 8.3 x 10(7) s(-1), respectively, by laser flash photolysis.

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). 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.

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).

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.

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 14.5 kDa protein and contains one copper atom in the molecule. The electronic absorption spectrum of HdBCP exhibits two absorption maxima near 450 and 750 nm comparable with the intense 600 nm band (epsilon(450)/epsilon(600) = ca. 0.9). The rhombic electron paramagnetic resonance spectrum shows clearly that the copper centre is a 'perturbed' type 1 copper geometry. Stopped-flow kinetics indicates that HdBCP accepts efficiently an electron from cytochrome c(L) (k(2) = 4.0 x 10(6) M(-1) s(-1) at 25.0 degrees C), which is a physiological electron acceptor for methanol dehydrogenase. According to cloning and DNA sequencing of the structural gene, the deduced amino acid sequence shows significant similarities with pseudoazurins, which are a physiological electron donor for Cu-containing nitrite reductase from the denitrifying bacteria. Based on these results, we discuss the role of HdBCP in the electron-flow system, which link 'methanol oxidation' and 'denitrification' together.

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. The overall structure of this H. denitrificans NIR reveals a trigonal prism-shaped molecule in which a monomer consisting of 447 residues and three Cu atoms is organized into a unique hexamer (i.e., a tightly associated dimer of trimers). Each monomer is composed of an N-terminal region containing a Greek key beta-barrel folding domain, cupredoxin domain I, and a C-terminal region containing cupredoxin domains II and III. Both cupredoxin domains I and II bind one type 1 Cu and are combined with a long loop comprising 31 amino acid residues. The type 2 Cu is ligated at the interface between domain II of one monomer and domain III of an adjacent monomer. Between the two trimeric C-terminal regions are three interfaces formed by an interaction between the domains I, and the type 1 Cu in the domain is required for dimerization of the trimer. The type 1 Cu in domain II functions as an electron acceptor from an electron donor protein and then transfers an electron to the type 2 Cu, binding the substrate to reduce nitrite to NO. The discussion of the intermolecular electron transfer process from cytochrome c(550) to the H. denitrificans NIR is based on x-ray crystallographic and kinetic results.

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. To explain the mechanism for electron transfer between these proteins, the dependence of MDH activity on the concentration of Hd-Cyt c(L) has been investigated at pH 4.5-7.0. The Michaelis constant for Hd-Cyt c(L) shows the smallest value (approximately 0.3 microM) at pH 5.5. The pseudo-first-order rate constant (k(obs)) of the reduction of Hd-Cyt c(L) exhibits a hyperbolic concentration dependence of Hd-MDH at pH 5.5, although k(obs) linearly increases at pH 6.5. These findings indicate formation of a transient complex between these proteins during an electron transfer event. Hd-MDH (148 kDa) is a large tetrameric protein with an alpha(2)beta(2) subunit composition, showing a high degree of structural similarity with other MDHs. Hd-Cyt c(L) (19 kDa) exhibiting the alpha-band at 550.7 nm has a unique C-terminal region involving a disulfide bond between Cys47 and Cys165. Moreover, there is a pair of Hd-Cyt c(L) monomers related with a pseudo-2-fold axis of symmetry in the asymmetric unit, and the two monomers tightly interact with each other through three hydrogen bonds. This configuration is the first example in the studies of cytochrome c as the physiological electron acceptor for MDH. The docking simulation between the coupled Hd-Cyt c(L) molecules and the heterotetrameric Hd-MDH molecule has been carried out.

Thiocyanate hydrolase is a cobalt-containing metalloenzyme with a cysteine-sulfinic acid ligand.

Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.

Thermal equilibrium of two conformations in photosensitive nitrile hydratase probed by the FTIR band of nitric oxide bound to the non-heme iron center.

Nitrile hydratase (NHase) from Rhodococcus N-771 is a novel enzyme that is inactive in the dark due to an enodogenous nitric oxide (NO) molecule bound to the non-heme iron center, and is activated by its photodissociation. FTIR spectra in the NO stretching region of the dark-inactive NHase were recorded in the temperature range of 270-80 K. Two NO peaks were observed at 1854 and 1846 cm-1 at 270 K, and both frequencies upshifted as the temperature was lowered, retaining the peak separation of 8-9 cm-1. The relative intensity of the lower-frequency peak increased with decreasing temperature up to ~120 K, whereas it was mostly unchanged below this temperature. This observation indicates that two distinct conformations with slightly different NO structures are thermally equilibrated in the dark-inactive NHase above ~120 K, and the interconversion is frozen-in at lower temperatures. The intensity ratio of the NO bands changed gradually upon increasing the pH from 5.5 to 11.0, but no specific pKa value was found. This result, together with the comparison of the light-induced FTIR difference spectra measured at pH 6.5 and 9.0, suggests that the protonation/deprotonation of a specific amino acid group in the active site of NHase is not a direct cause of the occurrence of the two conformations, although several protonatable groups in the protein may influence the energetics of the two conformers. From the previous observation that the isolated alpha subunit of NHase exhibited a single broad NO peak, it is suggested that interaction of the beta subunit forming the reactive cavity is essential for the double-minimum potential of the active-site structure. The frequencies and widths of the two NO bands changed upon addition of propionamide, 1,4-dioxane, and cyclohexyl isocyanide, indicating that these compounds are bound to the active pocket and change the interactions of the iron center or the dielectric environments around the NO molecule. Thus, the NO bands of NHase can also be a useful probe to monitor the binding of substrates and their analogues to the active pocket.

Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy.

Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.