Synthesis, structure, and characterisation of a ferromagnetically coupled dinuclear complex containing Co(II) ions in a high spin configuration and thiodiacetate and phenanthroline as ligands and of a series of isomorphous heterodinuclear complexes containing different Co : Zn ratios.

We report the synthesis, crystal structure, and characterisation of a dinuclear Co(II) compound with thiodiacetate (tda) and phenanthroline (phen) as ligands (1), and of a series of metal complexes isomorphous to 1 with different Co : Zn ratios (2, 4 : 1; 3, 1 : 1; 4, 1 : 4; 5, 1 : 10). General characterisation methodologies and X-ray data showed that all the synthesised complexes are isomorphous to Zn(II) and Cu(II) analogues (CSD codes: DUHXEL and BEBQII). 1 consists of centrosymmetric Co(II) ion dimers in which the ions are 3.214 Å apart, linked by two µ-O bridges. Each cobalt atom is in a distorted octahedral environment of the N2O3S type. UV-vis spectra of 1 and 5 are in line with high spin (S = 3/2) Co(II) ions in octahedral coordination and indicate that the electronic structure of both Co(II) ions in the dinuclear unit does not significantly change relative to that of the magnetically isolated Co(II) ion. EPR spectra of powder samples of 5 (Co : Zn ratio of 1 : 10) together with spectral simulation indicated high spin Co(II) ions with high rhombic distortion of the zfs [E/D = 0.31(1), D > 0]. DC magnetic susceptibility experiments on 1 and analysis of the data constraining the E/D value obtained by EPR yielded g = 2.595(7), |D| = 61(1) cm-1, and an intradimer ferromagnetic exchange coupling of J = 1.39(4) cm-1. EPR spectra as a function of Co : Zn ratio for both powder and single crystal samples confirmed that they result from two effective S' = 1/2 spins that interact through dipolar and isotropic exchange interactions to yield magnetically isolated S' = 1 centres and that interdimeric exchange interactions, putatively mediated by hydrophobic interactions between phen moieties, are negligible. The latter observation contrasts with that observed in the Cu(II) analogue, where a transition from S = 1 to S' = 1/2 was observed. Computational calculations indicated that the absence of the interdimeric exchange interaction in 1 is due to a lower Co(II) ion spin density delocalisation towards the metal ligands.

Molecular and kinetic properties of copper nitrite reductase from Sinorhizobium meliloti 2011 upon substituting the interfacial histidine ligand coordinated to the type 2 copper active site for glycine.

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a âˆ¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

Solid-State Characterization of Acetylpyridine Copper Complexes for the Activation of H2 O2 in Advanced Oxidation Processes.

This work describes the synthesis of 4-(4-AcPy) and 3-acetylpyridine (3-AcPy) copper soluble complexes for the activation of hydrogen peroxide and the concomitant generation of reactive oxygen species (ROS). Given the paramagnetic effects of copper ions in the Nuclear Magnetic Resonance (NMR) lines, we aimed at demonstrating that the combination of high-resolution 2D solid-state NMR experiments, Electron Paramagnetic Resonance (EPR), single-crystal X-ray crystallography and Density Functional Theory (DFT) calculations allows a detailed study of the chemical structure of the ligands and the surrounding metal ions. The copper complexes synthesized with CuCl2 were useful for the activation of H2 O2 during which the only ROS was the hydroxyl one, as demonstrated by EPR experiments. A removal of methyl orange (MO) azo-dye higher than 85 % was achieved in 200 minutes, combining 1.7 mM of copper complexes with 60 mM of H2 O2 and 40 µM of MO.

Paramagnetic solid-state NMR assignment and novel chemical conversion of the aldehyde group to dihydrogen ortho ester and hemiacetal moieties in copper(ii)- and cobalt(ii)-pyridinecarboxaldehyde complexes.

The complex chemical functionalization of aldehyde moieties in Cu(ii)- and Co(ii)-pyridinecarboxaldehyde complexes was studied. X-ray studies demonstrated that the aldehyde group (RCHO) of the four pyridine molecules is converted to dihydrogen ortho ester (RC(OCH3)(OH)2) and hemiacetal (RCH(OH)(OCH3)) moieties in both 4-pyridinecarboxaldehyde copper and cobalt complexes. In contrast, the aldehyde group is retained when the 3-pyridinecarboxaldehyde ligand is complexed with cobalt. In the different copper complexes, similar paramagnetic 1H resonance lines were obtained in the solid state; however, the connectivity with the carbon structure and the 1H vicinities were done with 2D 1H-13C HETCOR, 1H-1H SQ/DQ and proton spin diffusion (PSD) experiments. The strong paramagnetic effect exerted by the cobalt center prevented the observation of 13C NMR signals and chemical information could only be obtained from X-ray experiments. 2D PSD experiments in the solid state were useful for the proton assignments in both Cu(ii) complexes. The combination of X-ray crystallography experiments with DFT calculations together with the experimental results obtained from EPR and solid-state NMR allowed the assignment of NMR signals in pyridinecarboxaldehyde ligands coordinated with copper ions. In cases where the crystallographic information was not available, as in the case of the 3-pyridinecarboxaldehyde Cu(ii) complex, the combination of these techniques allowed not only the assignment of NMR signals but also the study of the functionalization of the substituent group.

Heterologous production and functional characterization of Bradyrhizobium japonicum copper-containing nitrite reductase and its physiological redox partner cytochrome c550.

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions.

The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine.

Azanone (HNO, nitroxyl) is a highly reactive molecule that, in the past few years, has drawn significant interest because of its pharmacological properties. However, the understanding of how, when, and where endogenous HNO is produced remains a matter of discussion. In this study, we examined the ability of myoglobin to produce HNO via the peroxidation of hydroxylamine with H2O2 using both experimental and computational approaches. The production of HNO was confirmed using an azanone selective electrochemical method and by the detection of N2O using FTIR. The catalytic capacity of myoglobin was characterized by the determination of the turnover number. The reaction kinetics of the hydroxylamine peroxidation were studied by both electrochemical and UV-vis methods. Further evidence about the reaction mechanism was obtained by EPR spectroscopy. Additionally, quantum mechanical/molecular mechanics experiments were performed to calculate the energy barrier for HNO production and to gain insight into the reaction mechanism. Our results confirm that myoglobin produces HNO via the peroxidation of hydroxylamine with a great catalytic capacity. In addition, our mechanistic study allows us to state that the Mb ferryl state is the most likely intermediate that reacts with hydroxylamine, yielding important evidence for endogenous HNO generation.

Synthesis, structural elucidation and antiradical activity of a copper (II) naringenin complex.

Coupling the extraction and derivatization of flavonoids to the Citrus processing industry is attractive from both the environmental and economic points of view. In the present work, the flavonoid naringin, obtained by "green" extraction with a water:ethanol mixture from waste grapefruit industry, was hydrolyzed to obtain naringenin. This flavonoid was used to synthesize the complex trans-di(aqua) bis(7-hydroxy-2-(4-hydroxyphenyl)-4-oxo-5-chromanolato) copper (II). This compound was characterized by spectroscopic techniques (UV/Vis, IR, Raman, NMR and EPR), and by thermal analysis (TG and DSC). Then, a monocrystal of the complex obtained by dissolution and recrystallization in DMF was analyzed by single crystal X-ray diffraction. This is the first report of the crystal structure of a Citrus flavonoid complex. Additionally, its antiradical activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined and compared with that for naringenin, demonstrating that coordination to copper enhances the antiradicalar activity of naringenin. According to the Mulliken population analysis conducted, by copper favors the delocalization and stabilization of the produced radical, since it acts as an electronic density acceptor.

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods.

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s-1) lower than that of wt SmNir (kcat=240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.

Nitric oxide is reduced to HNO by proton-coupled nucleophilic attack by ascorbate, tyrosine, and other alcohols. A new route to HNO in biological media?

The role of NO in biology is well established. However, an increasing body of evidence suggests that azanone (HNO), could also be involved in biological processes, some of which are attributed to NO. In this context, one of the most important and yet unanswered questions is whether and how HNO is produced in vivo. A possible route concerns the chemical or enzymatic reduction of NO. In the present work, we have taken advantage of a selective HNO sensing method, to show that NO is reduced to HNO by biologically relevant alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent decomposition of the so-produced radical to yield HNO and an alkoxyl radical.

Isotropic exchange interaction between Mo and the proximal FeS center in the xanthine oxidase family member aldehyde oxidoreductase from Desulfovibrio gigas on native and polyalcohol inhibited samples: an EPR and QM/MM study.

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a homodimeric molybdenum-containing protein that catalyzes the hydroxylation of aldehydes to carboxylic acids and contains a Mo-pyranopterin active site and two FeS centers called FeS 1 and FeS 2. The electron transfer reaction inside DgAOR is proposed to be performed through a chemical pathway linking Mo and the two FeS clusters involving the pyranopterin ligand. EPR studies performed on reduced as-prepared DgAOR showed that this pathway is able to transmit very weak exchange interactions between Mo(V) and reduced FeS 1. Similar EPR studies but performed on DgAOR samples inhibited with glycerol and ethylene glycol showed that the value of the exchange coupling constant J increases ~2 times upon alcohol inhibition. Structural studies in these DgAOR samples have demonstrated that the Mo-FeS 1 bridging pathway does not show significant differences, confirming that the changes in J observed upon inhibition cannot be ascribed to structural changes associated neither with pyranopterin and FeS 1 nor with changes in the electronic structure of FeS 1, as its EPR properties remain unchanged. Theoretical calculations indicate that the changes in J detected by EPR are related to changes in the electronic structure of Mo(V) determined by the replacement of the OHx labile ligand for an alcohol molecule. Since the relationship between electron transfer rate and isotropic exchange interaction, the present results suggest that the intraenzyme electron transfer process mediated by the pyranopterin moiety is governed by a Mo ligand-based regulatory mechanism.

Aromatic aldehydes at the active site of aldehyde oxidoreductase from Desulfovibrio gigas: reactivity and molecular details of the enzyme-substrate and enzyme-product interaction.

Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using (13)C-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe425 and Tyr535 are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data.

One electron reduced square planar bis(benzene-1,2-dithiolato) copper dianionic complex and redox switch by O2/HO(-).

The complex [Ph4P]2[Cu(bdt)2] (1(red)) was synthesized by the reaction of [Ph4P]2[S2MoS2CuCl] with H2bdt (bdt = benzene-1,2-dithiolate) in basic medium. 1(red) is highly susceptible toward dioxygen, affording the one electron oxidized diamagnetic compound [Ph4P][Cu(bdt)2] (1(ox)). The interconversion between these two oxidation states can be switched by addition of O2 or base (Et4NOH = tetraethylammonium hydroxide), as demonstrated by cyclic voltammetry and UV-visible and EPR spectroscopies. Thiomolybdates, in free or complex forms with copper ions, play an important role in the stability of 1(red) during its synthesis, since in its absence, 1(ox) is isolated. Both 1(red) and 1(ox) were structurally characterized by X-ray crystallography. EPR experiments showed that 1(red) is a Cu(II)-sulfur complex and revealed strong covalency on the copper-sulfur bonds. DFT calculations confirmed the spin density delocalization over the four sulfur atoms (76%) and copper (24%) atom, suggesting that 1(red) has a "thiyl radical character". Time dependent DFT calculations identified such ligand to ligand charge transfer transitions. Accordingly, 1(red) is better described by the two isoelectronic structures [Cu(I)(bdt2, 4S(3-,)*)](2-) ↔ [Cu(II)(bdt2, 4S(4-))](2-). On thermodynamic grounds, oxidation of 1(red) (doublet state) leads to 1(ox) singlet state, [Cu(III)(bdt2, 4S(4-))](1-).

Pseudoazurin from Sinorhizobium meliloti as an electron donor to copper-containing nitrite reductase: influence of the redox partner on the reduction potentials of the enzyme copper centers.

Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 (-) to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.

Magnetic properties of weakly exchange-coupled high spin Co(II) ions in pseudooctahedral coordination evaluated by single crystal X-band EPR spectroscopy and magnetic measurements.

We report single-crystal X-band EPR and magnetic measurements of the coordination polymer catena-(trans-(µ2-fumarato)tetraaquacobalt(II)), 1, and the Co(II)-doped Zn(II) analogue, 2, in different Zn:Co ratios. 1 presents two magnetically inequivalent high spin S = 3/2 Co(II) ions per unit cell, named A and B, in a distorted octahedral environment coordinated to four water oxygen atoms and trans coordinated to two carboxylic oxygen atoms from the fumarate anions, in which the Co(II) ions are linked by hydrogen bonds and fumarate molecules. Magnetic susceptibility and magnetization measurements of 1 indicate weak antiferromagnetic exchange interactions between the S = 3/2 spins of the Co(II) ions in the crystal lattice. Oriented single crystal EPR experiments of 1 and 2 were used to evaluate the molecular g-tensor and the different exchange coupling constants between the Co(II) ions, assuming an effective spin S'= 1/2. Unexpectedly, the eigenvectors of the molecular g-tensor were not lying along any preferential bond direction, indicating that, in high spin Co(II) ions in roughly octahedral geometry with approximately axial EPR signals, the presence of molecular pseudo axes in the metal site does not determine preferential directions for the molecular g-tensor. The EPR experiment and magnetic measurements, together with a theoretical analysis relating the coupling constants obtained from both techniques, allowed us to evaluate selectively the exchange coupling constant associated with hydrogen bonds that connect magnetically inequivalent Co(II) ions (|JAB(1/2)| = 0.055(2) cm(­1)) and the exchange coupling constant associated with a fumarate bridge connecting equivalent Co(II) ions (|JAA(1/2)| ≈ 0.25 (1) cm(­1)), in good agreement with the average J(3/2) value determined from magnetic measurements.

Copper-substituted forms of the wild type and C42A variant of rubredoxin.

In order to gain insights into the interplay between Cu(I) and Cu(II) in sulfur-rich protein environments, the first preparation and characterization of copper-substituted forms of the wild-type rubredoxin (Rd) from Desulfovibrio vulgaris Hildenborough are reported, as well as those of its variant C42A-Rd. The initial products appear to be tetrahedral Cu(I)(S-Cys)n species for the wild type (n=4) and the variant C42A (n=3, with an additional unidentified ligand). These species are unstable to aerial oxidation to products, whose properties are consistent with square planar Cu(II)(S-Cys)n species. These Cu(II) intermediates are susceptible to auto-reduction by ligand S-Cys to produce stable Cu(I) final products. The original Cu(I) center in the wild-type system can be regenerated by reduction, suggesting that the active site can accommodate Cu(I)(S-Cys)2 and Cys-S-S-Cys fragments in the final product. The absence of one S-Cys ligand prevents similar regeneration in the C42A-Rd system. These results emphasize the redox instability of Cu(II)-(S-Cys)n centers.

Kinetic and structural studies of aldehyde oxidoreductase from Desulfovibrio gigas reveal a dithiolene-based chemistry for enzyme activation and inhibition by H(2)O(2).

Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) fashion inhibiting the enzyme activity.

Single crystal EPR of the mixed-ligand complex of copper(II) with L-glutamic acid and 1,10-phenanthroline: a study on the narrowing of the hyperfine structure by exchange.

We report an EPR study at X- and Q-bands of polycrystalline and single crystal samples of the mixed copper(II) complex with L-glutamic acid (glu) and 1,10-phenantroline (phen), [Cu(glu)(phen)(H(2)O)](+) NO(3)(-)·2(H(2)O). The polycrystalline sample spectrum at Q-band showed well resolved g(∥ )and g(⊥) features and partially solved hyperfine structure at g(∥), typical for weakly exchange coupled systems. This is confirmed from the angular variation of the EPR spectra which shows for certain magnetic field orientations a partially solved hyperfine structure characteristic of weak exchange, whereas a single Lorentzian line corresponding to strong exchange is observed for others. Analysis and simulation of the single crystal EPR spectra were performed using the random frequency modulation model of Anderson. Numerical simulations of the angular variation of the EPR spectra showed that the narrowing of the hyperfine structure is due to an exchange-mediated mechanism in which transitions between any pair of lines are equally likely. The exchange interaction responsible for this process is mediated by hydrophobic interactions between two phen molecules and a mixed chemical path of the type CuA-O(ap)H···O-C-O(eq)-CuB, for which we evaluated an average isotropic exchange parameter |J| ≈ 25 × 10(-4) cm(-1).

Overexpression, purification, and biochemical and spectroscopic characterization of copper-containing nitrite reductase from Sinorhizobium meliloti 2011. Study of the interaction of the catalytic copper center with nitrite and NO.

The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis-Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs.

Substrate-dependent modulation of the enzymatic catalytic activity: reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.