The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase

The catalytic mechanism of nitrate reduction by periplasmic nitrate reductases has been investigated using theoretical and computational means. We have found that the nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the MoV species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with MoVI ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of MoV species. MoVI intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the MoVI species allow water molecule dissociation, and, the concomitant enzymatic turnover.Fil: Cerqueira, N.M.F.S.A.. Faculdade de Ciências E Tecnologia, Universidade Nova de Lisboa; PortugalFil: Gonzalez, P.J.. Faculdade de Ciências E Tecnologia, Universidade Nova de Lisboa; PortugalFil: Brondino, Carlos Dante. Universidad Nacional del Litoral; ArgentinaFil: Romão, M.J.. Faculdade de Ciências E Tecnologia, Universidade Nova de Lisboa; PortugalFil: Romão, C.C.. Instituto de Tecnologia Qu&Fil: Moura, I.. Faculdade de Ciências E Tecnologia, Universidade Nova de Lisboa; PortugalFil: Moura, J.J.G.. Faculdade de Ciências E Tecnologia, Universidade Nova de Lisboa; Portuga

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction

Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and -III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction.Fil: González, P.J.. Universidad Nacional del Litoral; ArgentinaFil: Correia, C.. Universidad Nacional del Litoral; ArgentinaFil: Moura, Isabel. Universidad Nacional del Litoral; ArgentinaFil: Brondino, Carlos Dante. Universidad Nacional del Litoral; ArgentinaFil: Moura, J.J.G.. Universidad Nacional del Litoral; Argentin

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans.

A two cluster (4Fe4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins. The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an absorbance ratio of A385 A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52°C. The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria

Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes / lipidic bilayer nanocomposite

An enzymatic biosensor based on nitric oxide reductase (NOR; purified from Marinobacter hydrocarbonoclasticus) was developed for nitric oxide (NO) detection. The biosensor was prepared by deposition onto a pyrolytic graphite electrode (PGE) of a nanocomposite constituted by carboxylated single-walled carbon nanotubes (SWCNTs), a lipidic bilayer [1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di-(9Zoctadecenoyl)-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolaminepolyethylene glycol (DSPE-PEG)] and NOR. NOR direct electron transfer and NO bioelectrocatalysis were characterized by several electrochemical techniques. The biosensor development was also followed by scanning electron microscopy and Fourier transform infrared spectroscopy. Improved enzyme stability and electron transfer (1.96 × 10−4 cm.s−1 apparent rate constant) was obtained with the optimum SWCNTs/(DOPE:DOTAP:DSPEPEG)/NOR) ratio of 4/2.5/4 (v/v/v), which biomimicked the NOR environment. The PGE/[SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR] biosensor exhibited a low Michaelis-Menten constant (4.3 μM), wide linear range (0.44–9.09 μM), low detection limit (0.13 μM), high repeatability (4.1% RSD), reproducibility (7.0% RSD), and stability (ca. 5weeks). Selectivity tests towards L-arginine, ascorbic acid, sodiumnitrate, sodiumnitrite and glucose showed that these compounds did not significantly interfere in NO biosensing (91.0 ± 9.3%–98.4 ± 5.3% recoveries). The proposed biosensor, by incorporating the benefits of biomimetic features of the phospholipid bilayer with SWCNT's inherent properties and NOR bioelectrocatalytic activity and selectivity, is a promising tool for NO.info:eu-repo/semantics/publishedVersio

“Study of the Spin-Spin Interactions between the Metal Centers of Desulfovibrio gigas Aldehyde Oxidoreductase: Identification of the Reducible Sites of the [2Fe-2S]1+,2+ Clusters”

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