Search

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed and studied, as well as the corresponding O,O-derivatives, with a view to their potential pharmacological applications as xanthine oxidase (XO) inhibitors. The biological assays revealed that the O,S-ligands present high inhibitory activity towards XO (nanomolar order, close to that of the pharmaceutical drug allopurinol), in contrast to the corresponding O,O-analogues. Due to the biomedical relevance of this molybdenum-containing enzyme, the corresponding Mo(VI) complexes were studied both in solution and in the solid state, aimed at identifying the source of the biological properties. The solution studies showed that, in comparison with the O,O-analogues, the Mo(VI) complexes with the O,S-ligands present some stabilization, which is even more pronounced for the reduced Mo(IV) species. The crystal structures of the Mo(VI) complexes with the hydroxythiopyrone revealed good flexibility of the coordination modes, with two structural isomers and two polymorphic forms for a mononuclear and a binuclear species, respectively. These results give some support to mechanistic proposals for the XO inhibition involving the interaction of the thione group with the molybdenum cofactor, thus indicating a role of the sulfur atom in the XO inhibition.

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOXand xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. [ABSTRACT FROM AUTHOR]

Abstract: The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe–4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C4mim]Cl−. In addition, UV–Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date. [Copyright &y& Elsevier]