Alberto C. Rizzi

Kinetic, structural, and EPR studies reveal that aldehyde oxidoreductase from Desulfovibrio gigas does not need a sulfido ligand for catalysis and give evidence for a direct Mo-C interaction in a biological system.

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family 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. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH(2) ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

Structure and single crystal EPR study of Cu(II)(l-threonine)2·H2O

Abstract We report the structure and single crystal EPR studies at 9.8 and 33.3 GHz of the title compound, [Cu(C4H8NO3)2]·H2O. The Cu(II) ion is in an elongated octahedral environment equatorially trans-coordinated by two oxygen atoms and two nitrogen atoms of threonine molecules which act as bidentate ligands (mean d(CuO)=1.95(1) and d(CuN)=1.98(1) A). It is axially coordinated by carboxylic oxygen atoms belonging to a pair of molecules translated in ±b (CuO distances of 2.478(8) and 2.972(3) A). This axial O⋯Cu⋯O interaction gives rise to infinite copper ion chains along the b crystal axis. Neighboring chains are coupled through complex chemical paths including H-bonds and the amino acid side chain. Single crystal EPR spectra show a single, exchange-collapsed resonance at both microwave frequencies for any magnetic field orientation. We evaluated the crystal and the molecular g-tensors from the angular variation of the EPR line position. The results (g⊥=2.060(2) and g∣∣=2.240(3)) indicate that the electronic ground orbital of the Cu ions is mainly of the 3dx2−y2 type. The analysis of the angular variation of the EPR linewidth allows us to estimate a mean exchange coupling constant ∣J/k∣=1.45(5) K for the inter-chain chemical paths.

New homobinuclear carboxylate-bridged gadolinium(III) complexes

Abstract Two novel gadolinium(III) trans-2-butenoate dimeric compounds [Gd2(O2CCHCHCH3)6(H2O)4]·2H2O (1) and [Gd2(O2CCHCHCH3)6(phen)2]·2H2O (2) have been synthesized via hydrothermal routes. Compound 1 consists of two independent dimers in the unit cell, one is doubly bridged (η2:η1:μ2) and the other quadruply bridged (two η1:η1:μ2 and two η2:η1:μ2). Compound 2 is dimeric with a quadruply bridged core (two η1:η1:μ2 and two η2:η1:μ2). Magnetic measurements for 2 show that there is a weak antiferromagnetic intramolecular Gd⋯Gd interaction.

Structural, MALDI-TOF-MS, magnetic and spectroscopic studies of new dinuclear copper(II), cobalt(II) and zinc(II) complexes containing a biomimicking μ-OH bridge.

The Py(2)N(4)S(2) octadentate coordinating ligand afforded dinuclear cobalt, copper and zinc complexes and the corresponding mixed metal compounds. The overall geometry and bonding modes have been deduced on the basis of elemental analysis data, MALDI-TOF-MS, IR, UV-vis and EPR spectroscopies, single-crystal X-Ray diffraction, conductivity and magnetic susceptibility measurements. In the copper and zinc complexes, a μ-hydroxo bridge links the two metal ions. In both cases, the coordination geometry is distorted octahedral. Magnetic and EPR data reveal weakly antiferromagnetic high spin Co(II) ions, compatible with a dinuclear structure. The magnetic characterization of the dinuclear Cu(II) compound indicates a ferromagnetically coupled dimer with weak antiferromagnetic intermolecular interactions. The intra-dimer ferromagnetic behaviour was unexpected for a Cu(II) dimer with such μ-hydroxo bridging topology. We discuss the influence on the magnetic properties of non-covalent interactions between the bridging moiety and the lattice free water molecules.

Ligand Binding to Chlorite Dismutase from Magnetospirillum Sp.

Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.

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.

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.

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: an application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates.

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T(1) of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T(1) is longer, no modulation of the coupling between metal centers can be detected.

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.