Publications

• A new CuZ active form in the catalytic reduction of N(2)O by nitrous oxide reductase from Pseudomonas nautica.

  Dell'Acqua S., Pauleta S.R., Paes de Sousa P.M., Monzani E., Casella L., Moura J.J., Moura I.

  The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of ... >> More

  The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/-0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed. << Less

  J Biol Inorg Chem 15:967-976(2010) [PubMed] [EuropePMC]

• Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina. Purification and properties of a novel multicopper enzyme.

  Coyle C.L., Zumft W.G., Kroneck P.M., Korner H., Jakob W.

  Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 kDa and was composed of two presumably identical subunits. The isoelectric point was 5.1. Several spectroscopically di ... >> More

  Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 kDa and was composed of two presumably identical subunits. The isoelectric point was 5.1. Several spectroscopically distinct forms of the enzyme were identified. A 'pink' form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous-oxide-dependent oxidation of photochemically reduced benzyl viologen. A 'purple' form of the enzyme, whose catalytic activity was 2-5-fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2-(N-cyclohexylamino)ethanesulfonate buffer at pH approximately equal to 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A 'blue', enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate. Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E'0,540 of +260 mV (vs SHE, 25 degrees C, Tris/HCl buffer, pH 7.5). Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g parallel region consisted of a seven-line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g parallel = 2.18 (A parallel = 3.83 mT) and g perpendicular = 2.03 (A perpendicular = 2.8 mT). Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive. Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell-free extracts. << Less

  Eur. J. Biochem. 153:459-467(1985) [PubMed] [EuropePMC]

• Respiratory transformation of nitrous oxide (N2O) to dinitrogen by Bacteria and Archaea.

  Zumft W.G., Kroneck P.M.

  N2O is a potent greenhouse gas and stratospheric reactant that has been steadily on the rise since the beginning of industrialization. It is an obligatory inorganic metabolite of denitrifying bacteria, and some production of N2O is also found in nitrifying and methanotrophic bacteria. We focus thi ... >> More

  N2O is a potent greenhouse gas and stratospheric reactant that has been steadily on the rise since the beginning of industrialization. It is an obligatory inorganic metabolite of denitrifying bacteria, and some production of N2O is also found in nitrifying and methanotrophic bacteria. We focus this review on the respiratory aspect of N2O transformation catalysed by the multicopper enzyme nitrous oxide reductase (N2OR) that provides the bacterial cell with an electron sink for anaerobic growth. Two types of Cu centres discovered in N2OR were both novel structures among the Cu proteins: the mixed-valent dinuclear Cu(A) species at the electron entry site of the enzyme, and the tetranuclear Cu(Z) centre as the first catalytically active Cu-sulfur complex known. Several accessory proteins function as Cu chaperone and ABC transporter systems for the biogenesis of the catalytic centre. We describe here the paradigm of Z-type N2OR, whose characteristics have been studied in most detail in the genera Pseudomonas and Paracoccus. Sequenced bacterial genomes now provide an invaluable additional source of information. New strains harbouring nos genes and capability of N2O utilization are being uncovered. This reveals previously unknown relationships and allows pattern recognition and predictions. The core nos genes, nosZDFYL, share a common phylogeny. Most principal taxonomic lineages follow the same biochemical and genetic pattern and share the Z-type enzyme. A modified N2OR is found in Wolinella succinogenes, and circumstantial evidence also indicates for certain Archaea another type of N2OR. The current picture supports the view of evolution of N2O respiration prior to the separation of the domains Bacteria and Archaea. Lateral nos gene transfer from an epsilon-proteobacterium as donor is suggested for Magnetospirillum magnetotacticum and Dechloromonas aromatica. In a few cases, nos gene clusters are plasmid borne. Inorganic N2O metabolism is associated with a diversity of physiological traits and biochemically challenging metabolic modes or habitats, including halorespiration, diazotrophy, symbiosis, pathogenicity, psychrophily, thermophily, extreme halophily and the marine habitat down to the greatest depth. Components for N2O respiration cover topologically the periplasm and the inner and outer membranes. The Sec and Tat translocons share the task of exporting Nos components to their functional sites. Electron donation to N2OR follows pathways with modifications depending on the host organism. A short chronology of the field is also presented. << Less

  Adv Microb Physiol 52:107-227(2007) [PubMed] [EuropePMC]